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ABSTRACT For sensing of biological events in vitro, detectors utilizing 129Xe NMR of trapped hyperpolarized baronial gas can go a manner provided the attack can traverse the spread in sensitiveness required. Here constructs based on the non selective grafting of cryptophane precursors on a protein interacting with cell surface receptors are proposed. The survey of their interaction with cell suspensions via fluorescence microscopy and 129Xe NMR indicates interesting characteristics such as a deficiency

KEYWORDS. Hyperpolarization ; Xenon ; Biosensor ; cell surface receptors.


Working on unrecorded cell suspensions through NMR is non an easy undertaking, as the needed figure of cells per volume unit limits their life-time. A figure of intracellular biological events could nevertheless be studied if the sensitiveness of the analysis method could be raised.

Hyperpolarized Xe is now widely recognized as a powerful MR detector of molecular or biological events, due non merely to the immense signal obtained by the anterior optical pumping or dynamic atomic polarisation measure, but besides to the broad chemical displacement scope spanned by this nucleus holding a large deformable negatron cloud. In 2001, an attack where the hyperpolarized baronial gas is vectorized toward biological receptors via functionalized host systems was proposed by the group of Alexander Pines.1 When encapsulated in the nucleus of such transporters made by cage-molecules2 or nanoparticles,3 xenon takes a specific chemical displacement, which enables its favoritism from free Xe in spectroscopic imaging.4 Whereas the proof-of-concept of this attack has been successfully achieved in isotropous solution by several groups,5-9 it has ne’er been tested in vitro ( and moreover in vivo ) by executing hyperpolarized 129Xe NMR experiments straight with cell suspensions.

Here we detect the interaction of a 129Xe NMR-based biosensor with transferrin receptors ( TfR ) via hyperpolarized 129Xe NMR on cell suspensions. This system has been chosen for the undermentioned grounds: I ) it is a good described system: so Transferrin ( Tf ) , a of course bing protein, has already received considerable attending in the country of drug aiming since it is biodegradable, non-toxic, and nonimmunogenic,10 two ) the biological marks are cell surface receptors, therefore easy approachable by dissolved xenon, three ) they can be abundant ( till 105 ) for some cell lines, four ) endocytosis is susceptible to farther increase the local denseness of xenon receptors. The affinity of holo-transferrin for the beta globulin receptor being 800 times larger than this of apo-transferrin, it seemed to us of import to work with the former, despite the relaxation induced on Xe by the paramagnetism. But if the state of affairs had become excessively critical ( excessively short life-time of the hyperpolarization ) , Fe atoms could hold been replaced by Ga or In atoms for a similar affinity.ref


Concept and synthesis of the TfR detectors

It is known that the molecular systems for which xenon exhibits the highest affinity are cryptophanes, aromatic cage-molecules made of two cyclotriveratrylene bowls joigned by aliphatic linkers.ref Brotin, Dutasta, Chem Rev Consequently they will represent the nucleus of our 129Xe NMR-based biosensor. The scheme we have chosen consists in foremost constructing precursors where a leash constituted by a polyethylene spacer ended by a succinidimic group is grafted on the cryptophane ( see Materials and Methods ) . The pick of four OCH2 groups to represent the spacer gives flexibleness to the cryptophane mediety, which is expected to be sufficient to avoid wide lines for encapsulated Xe that would be due to excessively fast cross relaxation.11 This spacer is besides expected to be long plenty to avoid multiplicity of the signals of encapsulated Xe for the racemic mixture.

The succinimidic group belonging to the aminoalkane reactive derived functions reacts to the aminoalkane groups of the lysine sidechains in a non specific coupling.ref Such a building presents many advantages: I ) the cryptophane-PEG-succinimide can be a precursor for many biosensors, i. e. it can be non-specifically grafted on a protein provided that this protein exhibits lysine residues on its external side ( and far from its active site ) , ii ) several cryptophane nucleuss can be grafted on a protein, multiplying the sites for Xe and therefore perchance increasing the signal/noise ratio ratio, three ) under a certain figure of cryptophanes grafted on the protein it is expected that the affinity of the assembly for the biological receptors is maintained. Indeed the grafting of a little entity on a large protein is a priori less susceptible to modify its affinity for these receptors.

Transferrin is a ball-shaped protein of molecular weight ~80,000. It possesses 54 Lys residues, 23 of them located on its external surface, and its holo signifier contains two Fe atoms ( insert of Figure 1 ) . Labeling of the protein with the cryptophane precursor was made utilizing the process detailed in Materials and Methods. In first measure different stoichiometries of the cryptophane: Tf mixtures were prepared in phosphate buffer saline ( PBS ) . Preservation of the protein folding was tested by 1H NMR. With simple unidimensional spectra ( needfully acquired during several proceedingss on a high-field spectrometer equipped with a cryocooled probehead ) , it was observed that a upper limit of 10 cryptophanes could be tethered on the beta globulin. Beyond this value, the protein unfolds. Obviously there is a statistical distribution of the figure of cryptophanes and of their locations on the protein. However the mass spectroscopy indicated a narrow distribution of the figure of grafted cryptophanes.

Finally we have chosen to utilize two concepts. In the first one, a 129Xe NMR-based biosensor made with a ratio of five cryptophanes per protein was built ( biosensor B1 ) . In the 2nd one, a ratio of 2:2:1 was used for the cryptophane, fluorescent investigation and protein, severally ( biosensor B2 ) . Proton translational diffusion experiments completed the mass spectroscopy to look into that the cryptophanes were covalently linked and non trapped inside anfractuosities of the protein.

Figure 1 displays the xenon spectra obtained at 11.7 T and xxxxK with B1 at 1.3 AµM in H2O. The first observation is that the Xe @ B1 line is alone and non wide. This constitutes a verification that the spacer between the cryptophane portion and the protein is long and flexible plenty. The sensing threshold per clip unit of the laser-polarized 129Xe NMR experiments with such concepts incorporating paramagnetic Fe3+ ions could be estimated. Given that the signal-to-noise ratio is 6.6 on the sub-spectrum obtained by insistent selective excitement of the Xe @ B1 signal ( direct observation method [ ref ] ) and that this experiment can be repeated at least 8 times with the gaseous Xe reservoir in the NMR tubing on top of the solution merely by agitating, this leads to a sensing threshold on the order of 100 nanometers. NON, A VIRER

Besides, we have compared the 129Xe longitudinal relaxation rates encountered with similar concentrations of apo- and holo-transferrin. Obviously fast exchange conditions on the T1 timescale are encountered, and a computation taking into history the fraction of encapsulated Xe has been done. The extracted values are thirty and thirty for the apo- and holo- signifiers, severally.

Survey of their interaction with biological cells

On a cell suspension, harmonizing to our process including shaking of the NMR tubing as a manner to present fresh hyperpolarized Xe into solution, it is hard to imagine a direct MR contrast. Furthermore even if on the 129Xe NMR spectrum a bantam alteration of the cryptophane environment is susceptible to give rise to a chemical displacement fluctuation of the Xe @ biosensor signal, [ refs ] here the line broadening due to the presence of a high figure of biological cells in suspension renders improbable the frequence separation between the signal of xenon caged in the biosensor free in solution ( Xe @ B_aq ) and this of Xe in the biosensor in the cytol ( Xe @ B_intracell ) . However Schroder and colleagues have shown that a chemical displacement difference of up to xxxx ppm can happen between Xe in the cryptophane in the aqueous stage and Xe in the cryptophane in a lipidic stage. [ ref ]

These troubles led us to conceive of the undermentioned ‘batch ‘ process, which outline is represented in Table 1:

a ) in a first measure, 120 1000000s K562 cells have been incubated during one hr at 37A°C with 400 AµL of a 5 AµM solution of B2 in PBS. Then the cells and the supernatant ( after three lavations ) have been separated by centrifugation ( inside informations in Materials and Methods ) . This has given rise to two solutions: ca ( cells a ) and sa ( supernatant a ) .

B ) in analogue, 120 1000000s K562 have been treated with pronase, a mixture of proteolytic enzymes designed to stamp down the beta globulin receptors. Precisely the same process than in a ) has so been applied: incubation during one hr at 37A°C with 400 AµL of a solution of B2 in PBS ( concluding concentration 5 AµM ) . The separation between the cells and the supernatant gave rise to solutions cb and antimony, severally. Pairwise comparing of the cell samples ca and cb through fluorescence microscopy and hyperpolarized 129Xe NMR could so be performed, every bit good as comparing between the supernatant samples sa and sb.refx As exchange of the beta globulin biosensor between the intra- and extra-cellular compartments is driven by thermodynamics Torahs, xxxx

Figure 2 displays the fluorescence microscopy consequences. After incubation of the cells with B2, the internalisation of the biosensor is clearly seeable. The observation of fluorescent sums inside the cells could be due to the formation of vacuoles. Besides the boundary line of some cells appears green, meaning that a portion of the biosensor lies in their plasmic membrane.

Figure 3 gives the corresponding 55-95 ppm part of the laser-polarized 129Xe NMR spectra, plus these of the supernatant samples. This part entirely corresponds to the signal of the baronial gas encapsulated in the cryptophane. The first comment is that no extremum in this part appears for the cells antecedently treated with pronase ( sample cb ) although the xenon polarisation degree is similar to this of the other cell sample ( Table 2 ) . This is absolutely consistent with the observations of fluorescence microscopy. On the 129Xe NMR spectrum of sample ca, 2 extremums appear in this part: in add-on to the extremum at 67 ppm, a 2nd extremum appears near 80 ppm ( raffiner les deplacements chimiques ) . A fast computation taking into history the figure of cells present in solution and the possible figure of transferrin receptors per cell shows that the 2nd extremum can non be due to xenon in the biosensor in the cell, whereas the first 1 remains the biosensor free in solution. Indeed a factor 0.001 for the current biosensor concentration should be found between the countries of these two extremums. Rather, as antecedently mentioned, the extremum at 80 ppm can be representative of the biosensor in the cell membrane.

These two extremums have similar strengths. This indicates that a large portion of the biosensor lies in a lipidic environment. Indeed whereas the left extremum represents Xe in the cell membranes, the right extremum should incorporate the part of Xe in the intra-cell aqueous compartment and in the free biosensor.

Appraisal of the specificity of the biosensor

UTILITE DE LA PROCEDURE. In this batch experiment, the net difference between the 129Xe NMR spectra of Xe in the biosensor interacting with the cells after intervention or non by pronase ( samples cb and ca ) indicates that the beta globulin receptors are required for a strong interaction of the biosensor with the cells via internalisation, i.e. an active crossing occurs between the beta globulin biosensor and the cells. The resemblance between the xenon spectra of the supernatant samples ( SA and antimony ) proves xxxx. The visual aspect of a 2nd extremum on the selective Xe spectra witnessing a big portion of the biosensor location in the membranes leads us nevertheless to turn to the inquiry of the specificity of the interaction. Indeed add-on of pronase may hold led to a destructuration of the cell membrane. In order to analyze this specificity, we have built another biosensor by grafting the cryptophane precursor on a ball-shaped protein, the bovine serum albumen ( BSA ) and the fluorescent investigation ( at a ratio of 2 cryptophanes and 2 Rhodamine Green medieties per protein, taking to the biosensor B3 ) . The same process than antecedently, californium. Table 1 ( samples milliliter and Sc ) has been performed. As there is no receptor of this protein on the surface of the K562 cells, the 129Xe spectrum of the milliliter sample should be exempt of any extremum in the 55-95 ppm part. Figure 4 displays the fluorescence microscopy image and the laser-polarized 129Xe NMR spectrum of the milliliter sample. Whereas the fluorescence image is less bright than this of the beta globulin biosensor B1, surprisingly once more two extremums appears. Even if the ratio between these extremums is more in favour of the cryptophane in the lipid portion than for B1 ( see Table 2 ) , this indicates a deficiency of specificity of the biosensor B1. Given that there is no known affinity of BSA for any surface receptor of the K562 cells, a hypothesis is that the hydrophobicity of the cryptophane medieties is responsible for the anchoring of the biosensors in the cell membranes.

In order to measure this point, two other experiments have been performed. First, cryptophane-A, the organo-soluble parent, has been introduced in the NMR tubing incorporating B3 and the K562 cells after separation from the supernatant ( i.e. sample milliliter ) . The 129Xe NMR spectrum displayed in Figure S1 of the Supporting Information shows that the country of the left extremum, antecedently assigned to xenon in the cryptophane located in a lipidic environment, additions, in understanding with our hypothesis about the leaning of the cage-molecule to be inserted in the cell membrane. Second, two new transferrin-based concepts have been built. In the first one, the fluorescent investigation has been grafted ( once more via the lysine sidechains ) on the apo-transferrin. In the 2nd one, the cryptophane precursor AND the fluorescent investigation have been grafted on the protein, at a ratio 2:2 for one protein. These concepts have been introduced in solutions incorporating elephantine unilamellar cysts. Comparison of the fluorescence microscopy images obtained in both instances is displayed in Figure S2 of the Supporting Information. Whereas xxxx, the location of the 2nd concept in the lipid bilayer is clearly seeable.

All these consequences seem to bespeak that the cryptophane-based biosensors have a big leaning to interact with plasmic and intracellular membranes, due to the hydrophobic character of the xenon host molecule which is nevertheless non strong plenty to blossom the protein.


a. Synthesis

Synthesis of cryptophanol. Pure iodotrimethylsilane ( 65 i?­L, 0.455 mmol ) was added in one part via syringe to a moved solution of cryptophane-A ( 400 milligram, 0.45 mmol ) in CH2Cl2 ( 8 milliliter ) . The solution was strirred in the dark for 16 hours under an Ar ambiance at room temperature. CH2Cl2 ( 10 milliliter ) was added to the solution and the solutrion was acidified with a HCl solution ( 1M, 6 milliliter ) . The organic bed was collected, washed with H2O and so dried over Na sulphate. After vaporizing the dissolver under decreased force per unit area the residue was purified on a column chromatography ( CH2Cl2/Et2O: 90/10 ) to give the cryptophanol as a white solid ( 144 milligram, 37 % ) . Spectroscopic informations are indistinguishable to those antecedently reported for this compound.

Synthesis of cryptophane X. The protected PEG X ( 254 milligram, 0.51 mmol, 1.5 combining weight. ) was introduced in a three cervixs flask incorporating caesium carbonate ( 167 milligram, 0.51 mmol ) , cryptophanol 1 ( 300 milligram, 0.34 mmol ) in newly distilled DMF ( 12 milliliter ) . The mixture was stirred for 16 hours under an Ar ambiance at 60A°C. CH2Cl2 ( 25 milliliter ) and seawater ( 10mL ) ware added to the mixture. The aqueous bed was extracted twice with CH2Cl2 ( 10 milliliter ) . The combined beds are so washed with seawater ( 5i‚? 10 milliliter ) to take DMF and dried over Na sulphate. The dissolver was removed under decreased force per unit area to give a residue, which was so purified on silicon oxide gel ( a gradient of solvent Et2O/AcOEt was used: 100/0 ; 80/20 ; 50/50 ; 0/100 ) . Vaporization of the dissolver gave compound 3 as a white formless solid ( 282 milligram, 69 % ) . 1H NMR ( 500 MHz, CDCl3, 25A°C ) i?¤ 7.33-7.32 ( m, 5H, Ar ) , 6.77 ( s, 1H ; Ar ) , 6.74 ( s, 1H ; Ar ) , 6.73 ( s, 1H ; Ar ) , 6.72 ( s, 1H ; Ar ) ; 6.71 ( s, 2H ; Ar ) , 6.67 ( s, 1H ; Ar ) ; 6.66 ( s, 1H ; Ar ) , 6.65 ( s, 2H ; Ar ) , 6.64 ( s, 1H ; Ar ) , 5.16 ( s, 2H ; OCH2Bn ) , 4.59-4.53 ( m, 6H ; Ha ) , 4.19-4.10 ( m, 15H, OCH2, OCH2COO ) ; 3.93 ( m, 1H ) ; 3.80-3.66 ( m, 30 H ; OCH2 and OCH3 ) , 3.39-3.35 ( m, 6H ; He ) . 13C NMR ( 125.7 MHz, CDCl3, 25A°C ) i?¤ 170.32, 162.50, 149.85, 149.73, 149.61, 149.56, 148.96, 147.20, 146.72, 146.67, 146.62, 146.59, 135.45, 134.31, 134.26, 134.12, 134.04, 133.99, 132.72, 131.83, 131.57, 131.54, 131.47, 131.36, 128.60, 128.42, 128.39, 122.30, 121.39, 121.26, 120.54, 120.47, 120.33, 117.03, 114.64, 113.70, 113.59, 70.94, 70.86, 70.75, 70.65, 69.88, 69.60, 69.53, 69.38, 69.29, 69.20, 68.73, 68.64, 68.48, 56.09, 55.73, 55.66, 55.64, 55.59, 36.46, 36.30. HRMS i?›M+Nai??+ calcd 1227.4949 found 1227.4935.

Synthesis of cryptophane X and X. H2 gas was introduced to 25 milliliters flask incorporating compound 3 ( 560 milligram, 0.465 mmol ) , CH2Cl2 ( 12 milliliter ) , Ethanol ( 2 milliliter ) , and Pd/C ( 70 milligram, 0.0658 mmol, 0.14 combining weight ) . The mixture was stirred at room temperature for 5 h. After completion of the reaction the mixture was filtrated and the solid residue was washed with CH2Cl2 ( 2 i‚? 10 milliliter ) . The dissolver was so removed under decreased force per unit area to give compound 4 ( 0.46 g ; 89 % ) . 1H NMR ( 500 MHz, CDCl3, 25A°C ) i?¤ 6.77 ( s, 1H ; Ar ) , 6.74 ( s, 1H ; Ar ) , 6.73 ( s, 3H ; Ar ) , 6.725 ( s, 1H ; Ar ) , 6.72 ( s, 2H ; Ar ) , 6.69 ( s, 1H ; Ar ) , 6.66 ( s, 1H ; Ar ) , 6.65 ( s, 3H ; Ar ) ; 4.59-4.53 ( m, 6H ; Ha ) ; 4.21-4.12 ( m, 15H ; OCH2 and OCH2COO ) , 3.95 ( m, 1H ) , 3.82 ( T, 2H, 3J ( H, H ) = 5.0 Hz ) , 3.78-3.73 ( m, 27 H, OCH2-OCH3 ) , 3.41-3.36 ( m, 6H, He ) . 13C NMR ( 125.7 MHz, CDCl3, 25A°C ) i?¤ 170.99, 149.83, 149.71, 149.63, 149.59, 149.55, 148.91, 147.21, 146.65, 146.60, 134.39, 134.26, 134.16, 134.09, 134.00, 132.75, 131.87, 131.59, 131.54, 131.40, 122.17, 121.28, 120.59, 120.54, 120.38, 117.12, 114.72, 113.72, 113.61, 71.59, 70.88, 70.76, 70.64, 70.37, 70.18, 69.85, 69.59, 69.53, 69.38, 69.31, 69.23, 69.13, 69.72, 56.16, 55.74, 55.66, J 55.64, 55.60, 36.22, 36.20, 36.15. HRMS i?›M+Nai??+ calcd 1137.4460 found 1137.4452. Activation of the acid map was performed by adding in a three cervixs flask N, N’-disuccinimidyl carbonate ( 58 milligram, 0.226 mmol, 2.1eq. ) to a solution of 4 ( 0.12 g, 0.108 mmol ) in acetonitrile ( 3 milliliter ) and pyridine ( 0.1 milliliter ) under an Ar atmosphere. The mixture was stirred. The mixture was stirred for 6h at room temperature. Then CH2Cl2 ( 10 milliliter ) and HCl 1M ( 3 milliliter ) were added. The organic bed was separated and dried over Na sulphate. The remotion of the solvent gives 5, which was used without farther purification.

Compound Ten: 11-tetrahydropyranyloxy-3,6,9-trioxaundecan-1-ol ( 5g, 18 mmol, 1eq. ) in THF ( 60 milliliter ) was added dropwise to a moved solution of NaH 60 % ( 1.8g, 45 mmol, 2.5 combining weight ) in THF ( 50 milliliter ) under an Ar ambiance. After complete add-on the mixture was stirred for an extra 1 hr. A solution of bromoacetic acid ( 2.8 g, 20 mmol, 1.1 combining weight ) in THF was so added dropwise. After add-on the solution was heated nightlong under reflux status. The solution was allowed to make room temperature and benzyl bromide ( 2.3 milliliter, 19.3 mmol, 1.1 combining weight. ) was added in one part. The solution was so heated nightlong under reflux status. After vaporizing the dissolver under decreased force per unit area, CH2Cl2 ( 250 milliliter ) and H2O ( 100 milliliter ) were added. The beds were separated and the aquous bed was extracted twice with CH2Cl2 ( 100 milliliter ) . The combined organic beds are so washed twice with H2O ( 50 milliliter ) and dried over Na sulphate. After vaporization of the dissolver the petroleum merchandise was purified by column chromatography ( AcOEt ) to give 5 as a colorless oil ( 5.75 g, 75 % ) . 1H NMR ( 500 MHz, CDCl3, 25A°C ) i?¤ 7.35-7.28 ( m, 5H ; Ar ) , 5.15 ( s, 2H ; OCH2Bn ) , 4.59 ( m, 1H, OCHO ) , 4.17 ( s, 2H, OCH2COO ) , 3.85-3.80 ( m, 2H ; OCH2 ) , 3.71-3.69 ( m, 2H, OCH2 ) , 3.66-3.60 ( m, 12H, OCH2 ) , 3.59-3.54 ( m, 1H, OCH2 ) , 3.48-3.44 ( m, 1H, CH2 ) , 1.82-1.76 ( m, 1H, CH2 ) , 1.71-1.65 ( m, 1H ; CH2 ) , 1.60-1.44 ( m, 4H ; CH2 ) . 13C NMR ( 125.7 MHz, CDCl3, 25A°C ) i?¤ 170.26, 135.38, 128.53, 128.35, 128.33, 98.85, 70.88, 70.57, 70.55, 70.53, 70.50, 70.46, 68.61, 66.57, 66.41, 62.12, 30.49, 25.36, 19.41. HRMS i?›M+Nai??+ calcd 449.2117 found 449.2142.

Compound Ten: A moved solution of 5 ( 4.98 g, 11.3 mmol, 1 combining weight. ) and pyridinium methylbenzene sulfonate ( 0.95 g, 3.78 mmol, 0.33 combining weight ) in ethyl alcohol was heated overnight at 45A°C. The dissolver was removed under decreased force per unit area and the oily residue purified by column chromatography ( AcOEt ) . Vaporization of the solvent gives compound 6 as colorless oil ( 3.2 g ; 83 % ) . Spectroscopic informations are indistinguishable to those antecedently reported in the literature.

Compound Ten: para-chloro methylbenzene sulfonate ( 1.5 g, 7.87 mmol, 1.3 combining weight ) was added at 0A°C in one part to a moved solution of compound 6 ( 2g, 5.84 mmol ) in pyridine ( 6 milliliter ) . The solution was stirred at this temperature for an extra 2 hours. Water ( 10 milliliter ) and diethyl ather ( 40 milliliter ) were so added to the solution. The organic bed is collected and the aqueous bed is extracted twice with diethyl quintessence ( 2 i‚? 20 milliliter ) . The combined organic bed are so washed with seawater ( 20 milliliter ) and H2O ( 10 milliliter ) and so dried over Na sulphate. Vaporization of the dissolver leaves a residue, which was purified by column chromatography ( AcOEt ) . Different fractions are collected and the vaporization of the solvent gives compound 7 ( 2.32 g, 80 % ) as colorless oil. Additional information 1H NMR ( 500 MHz, CDCl3, 25A°C ) i?¤ 7.77 ( vitamin D, 3J ( H, H ) = 8.3 Hz, 2H ; Ar ) , 7.33-7.29 ( m, 7H ; Ar ) , 5.16 ( s, 2H ; OCH2Bn ) , 4.17 ( s, 2H ; OCH2COO ) , 4.13 ( T, 3J ( H, H ) = 4.9 Hz, 2H ; OCH2 ) , 3.71-3.54 ( m, 14H, OCH2 ) , 2.42 ( s, 3H ; CH3 ) .

Synthesis of cryptophanol-A and of the linker. Derivative X has been prepared from cryptophanol X and a PEG molecule aimed at increasing meagerly the solubility of the concluding molecule in H2O and leting its fond regard to the protein ( olo-transferrin ) . Herein, cryptophanol X whose synthesis has been independently reported by Darzac et Al. and Spence et Al. was prepared in a individual measure from cryptophane-A.[ I ]This new scheme allows the rapid synthesis for the mono-hydroxyled cryptophane and avoids the usage of the multi-step synthesis described antecedently in the literature. Thus a remotion of a individual methyl group has been achieved by utilizing iodotrimethylsilane in CH2Cl2 and provides cryptophanol in a 37 % output. During the purification process the unreacted cryptophane-A was recovered, which can be reused for subsequent reactions. This scheme affords an alternate manner for fixing this of import compound X, which has been used to construct up a big scope of different compounds.[ two ]The output and the easiness of purification are strongly dependent of the quality of the extremely reactive iodo-trimethylsilane used for the reaction and a monitoring of the reaction is normally necessary ( the clip of the reaction every bit good as the sum of the reactant are strongly dependent of the quality of the iodotrimethylsilane used ) . It is notable that efforts to execute the reaction with iodo-trimethylsilane prepared in situ from chlorotrimethylsilane and Na iodide in a mixture of methylene chloride and acetonitrile failed to accomplish the mono-deprotection. Similarly efforts to utilize other reactants such as Li diphenylphosphide in THF or Na isopropylthiolate in DMF failed to accomplish the demethylation reaction.

The PEG map used for the fond regard of the biosensor molecule to the protein has been prepared in three stairss from a mono-protected PEG derivative X antecedently described in the literature.[ three ]An activated acid map has been introduced at one appendage in order to let the matching reaction with the protein and a tosyl group has been introduced at the other appendage to let the matching reaction between the phenol mediety of the cryptophane and the PEG molecule. First the debut of the carboxylic map was achieved by responding the PEG X with bromomethylacetic acid in THF in presence of strong base. Second the protection of the acerb derivative with benzyl bromide was carried out to let the purification by column chromatography of compound X with 75 % output. Deprotection of the tetrahydropyranyl mediety was so achieved with pyridinium p-toluene sulfonate in Ethanol to give rise to intensify X with 83 % after purification.[ four ]In bend the free intoxicant map was used to present a tosyl group to supply derivative Ten with 80 % output.[ V ]

& lt ; Scheme 1 & gt ;

Synthesis of cryptophane-PEG-succinimide. The efficient matching reaction between cryptophanol-A and derivative Ten has been carried out in DMF in presence of caesium carbonate and gave rise to monofunctionalized cryptophane Ten with a 69 % . The formation of the coveted merchandise can be easy detected by 1H NMR spectrometry. Indeed the presence in the 1H NMR spectrum of two diastereotopic protons located at 3.9 ppm appears characteristic of the matching reaction since this signal bases for the two protons of the PEG derivative straight attached to the cryptophane molecule. The deprotection of the carboxylic acid was obtained by hydrogenation at atmospheric force per unit area in presence of a Pd/C accelerator in a mixture of methylene chloride and ethyl alcohol. A filtration of the solution after the reaction allows the obtainment of the clean derivative Ten with 89 % output. The activation of the acid map was eventually achieved with Nhydroxysuucinimide in the presence of dicyclohexylcarbodiimide. After an appropriate intervention the cryptophane-PEG-succinimide was used without farther purification for the matching reaction with the protein.

& lt ; Scheme 2 & gt ;

B. Preparation of the biosensors

Biosensor B1. The human beta globulin and Bovin Serum Albumin were purchased from Sigma Aldrich. Each protein was incubated with the activated cryptophanes during one hr in Phosphate Buffer Saline ( PBS ) medium pH 7.4 at room temperature. The ratio of cryptophanes versus protein used for NMR experiments was 5:1 and 2:1 for fluorescence imagination. The unreacted dye was removed from the protein solution utilizing a gel Sephadex G25 medium chromatography. The elution was made with PBS, pH 7.4.

Rhodamine Green -transferrin. Rhodamine green succinimide ester was purchased from Molecular Probe. The protein was incubated with the fluorescent dye in hydrogen carbonate medium pH=8,4 during 1h, at room temperature under agitation. The ratio of 2 was used. The free dye was removed from the protein solution utilizing a Sephadex G25 medium gel chromatography. The elution was made with PBS. The labeling efficiency was controlled utilizing an soaking up spectrometer.

c. Characterization of the concepts

The folding of the labelled proteins was controlled with 1H NMR on a 700 MHz Bruker spectrometer equipped with a HCN cryoprobe. Solutions at 1 millimeter in 500 AµL H2O: D2O 90:10 were analyzed in 5 millimeter tubings.

d. Cells

Cell civilization. Human erythroleukaemic K562 cells were purchased from ATCC and grown in IMDM medium ( Sigma-Aldrich ) supplemented with 10 % heat-inactivated foetal bovine serum, 1 % of l-glutamine, 100 U/ml penicillin and 100 g/ml streptomycin. The cells were maintained in exponential growing in a 5 % CO2 brooder at 37A°C. Prior to the experiment, the cells were washed tree times in PBS and feasible cells were counted utilizing trypan blew exclusion. The chosen sum of cells is re-suspended to a concluding volume of 1,5 L.

Fluorescent labeling. Cells were incubated with 200 nanometers of fluorescent investigation during 1h. The cells were washed 3 times with PBS at 4A°C in order to barricade the membrane moral force. Then the cells were fixed on the glass substrate utilizing PFA 5 % during 30 proceedingss at 4A°C. The observation was made utilizing an upside-down microscope.

Preparation of the samples. 120 1000000s of cells were washed three times with PBS. For the pronase sample, the cells were treated utilizing a 2 mg/mL solution of pronase during 30 proceedingss at 37A°C. ( a voucher ) Then the cells were washed 3 times with PBS and incubated with 5 AµM of the biosensor during 1h at 37A°C. At the terminal, the cells were washed 3 times at 4A°C in order to barricade plasma membrane kineticss, and re-suspended in a concluding volume of 1,5mL.

e. 129Xe NMR experiments

Laser-polarized Xe. Xenon 86 % -enriched in isotope 129 was purchased from CornetNet, France. The hyperpolarized gas was prepared via the spin-exchange method, utilizing our batch setup antecedently described. [ ref EPJD, PINS ] . The sum of gas produced with this set-up utilizing a Ti: sapphire optical maser, on the order of 1 milliliter ( in ~10 proceedingss ) , was sufficient to make full the NMR tubing with a force per unit area of ~1 saloon and an mean polarisation of 40 % . The transportation of Xe between the cold finger designed to divide Xe from N after optical pumping and the NMR tubing of involvement was made through a vacuity line in the fringe field of the NMR magnet in order to continue polarisation.

NMR experiments. Each acquisition of an NMR spectrum was preceded by vigorous agitating ( to unnaturally present the hyperpolarized gas into solution ) and a wait clip of ~10 s to enable the riddance of bubbles. The experiments were run on a 500 MHz Avance II Bruker spectrometer equipped with a 129Xe/1H micro-imaging probehead accepting tubings with a maximum outer diameter of 8 millimeter. Selective excitement was performed utilizing a 1 % abbreviated Gaussian pulsation of 500 Aµs. Processing


In this article we have demonstrated the proof-of-concept of the 129Xe NMR-based biosensing attack on a existent in vitro system. The presence of paramagnetic Fe3+ ions on the biosensor itself and in the K562 cells did non hinder the observation of xenon caged in cryptophanes grafted on this biosensor. It is deserving observing that the 129Xe NMR spectra were obtained in one shooting by repeated selective excitements in the spectral part 55-95 ppm. At the concentrations of protein used ( ~5 AµM ) the ascertained xenon relaxation clip remains long, which enables to to the full utilize the reservoir of polarisation constituted by dissolved Xe. Furthermore, given the big reservoir of gaseous hyperpolarized Xe on top on the solution, if required we could hold benefited from extra spectra to increase the signal-to-noise ratio merely by agitating the NMR tubing between two acquisition series.

In the presence of K562 cells showing many transferrin receptors, the beta globulin biosensor is endocyted, as proved by fluorescence microscopy experiments. The laser-polarized 129Xe NMR spectra of the biosensor B1 entirely and in the presence of the cells are mostly different. However the deficiency of specificity of this biosensor for the beta globulin receptors is undeniable. We have shown that it is due to the presence of the strong hydrophobic character of the cryptophane. Without taking to protein unfolding, it tends to pull the biosensor to the cell membrane.

This shows that the current attacks taking at rendering soluble the cryptophanes in H2O through debut of a hydrophilic ligand far from the pit can hold some drawbacks, as was already observed with a cryptophane biosensor designed to acknowledge a complementary RNA strand in solution. [ ref ChemPhysChem2007 ] . The amphiphilicity created by the presence of the cage-molecule and the aliphatic spacer on one side and the oligonucleotide on the other side led to the formation of self-organized systems of the micelle or cyst type. A solution would be to put hydrophilic groups closer to the aromatic rings of the cryptophanes, nevertheless paying attending to keeping the xenon in-out exchange. [ ref cryptophan-6-ol ]


Figure 1. Structure of the biosensor B1 and laser-polarized 129Xe NMR spectrum of the baronial gas in this biosensor in PBS. In the frame, a rapid climb of the 55-95 ppm frequence scope, matching to the Xe @ cryptophane part, is displayed.

Figure 2. Fluorescence images of K562 cells incubated by the B2 biosensor, without ( left ) and with ( right ) old pronase intervention.

Figure 3. 129Xe NMR spectra obtained by selective excitement in the xenon @ cryptophane. Upper left: with K562 cells after incubation with the biosensor during 1h at 37A°C ; upper right: spectrum of the corresponding supernatant ; lower left: spectrum with K562 treated with pronase and so incubated with the biosensor during 1h at 37A°C ; lower right: spectrum of the corresponding supernatant.

Figure 4. Fluorescence images of K562 cells incubated by the B3 biosensor and 129Xe NMR spectrum obtained by selective excitement in the xenon @ cryptophane spectral part.


Scheme 1. Synthesis of cryptophanol-A from cryptophane-A and synthesis of the linker.

Scheme 2. Synthesis of cryptophane-PEG-succinimide.


Table 1. Outline of the protocols used in our batch process, designed toaˆ¦

Table 2. Relative countries of the extremums of xenon caged in cryptophaneaˆ¦

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refx. Despite a xenon polarisation non to the full consistent, quantitative appraisal of the xenon NMR signals has been rendered possible through standardization of the integral of the extremum of xenon free in solution, sing that the same sum of xenon was each clip introduced in the NMR tubing ( checked by burdening of the tubings before and after the experiment ) .

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