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Design and Synthesis of a
1
8-Cyclodextrin Oligomer: A New Platform with
Potential Application as a Dendrimeric Multicarrier
DOI: 10.1002/chem.20130121
BARGE A.; CAPORASO M.; CRAVOTTO G.; MARTINA K.; TOSCO P.; AIME S.; CARRERA
C.; GIANOLIO E.; PARIANI G.; CORPILLO D.,CHEMISTRY-A EUROPEAN JOURNAL (2013)
19: 12086-12092
The definitive version is available at:
La versione definitiva è disponibile alla URL:
Design and Synthesis of a
1
8-Cyclodextrin Oligomer: A New Platform with
Potential Application as a Dendrimeric Multicarrier
Alessandro Barge,[a] Marina Caporaso,[a] Giancarlo Cravotto,*[a] Katia Martina,[a] Paolo Tosco,[a] Silvio Aime,[b] Carla Carrera,[b] Eliana Gianolio,[b] Giorgio Pariani,[b] and Davide Corpillo[c]
[a] Dr. A. Barge, M. Caporaso, Prof. G. Cravotto, Dr. K. Martina, Dr. P. Tosco Department of Drug Science and Technology
University of Turin, Via Pietro Giuria 9, 10125 Torino (Italy) Fax: (+39) 011-6707687
E-mail: giancarlo.cravotto@unito.it
[b] Prof. S. Aime, Dr. C. Carrera, Dr. E. Gianolio, Dr. G. Pariani Department of Chemistry IFM and
Molecular & Preclinical Imaging Centers
University of Turin, Via Nizza 52, 10126 Torino (Italy) [c] Dr. D. Corpillo
ABLE Biosciences, Bioindustry Park Silvano Fumero S.p.A. Via Ribes 5, 10010 Colleretto Giacosa (Italy)
Abstract : We report the synthesis and characterization of a water-soluble, star-shaped macromolecular platform consisting of eight -cyclodextrin (- CD) units anchored to the narrower rim of a -CD core through bis(triazo- lyl)alkyl spacers. The efficient synthetic protocol is based on the microwave (MW)-promoted Cu-catalyzed 1,3-di-polar cycloaddition of CD monoazides to CD monoacetylenes. The ligand- hosting capability of the construct has been assessed by relaxometric titration and nuclear magnetic relaxation dispersion (NMRD) profiling, which showed it to be good, and this was supported by molecular dynamics simulations. To demonstrate the feasibility of obtaining supramolecular structures with high hosting ability, we designed a dimeric platform, formed by joining two nonamers through the -CD cores through a bis(lithocholic acid) linker. With a view to the potential biological applications, cytotoxicity and extent of binding to human serum albumin were assessed. The properties of this dendrimeric multicarrier make it suitable for pharmaceutical and diagnostic purposes, ranging from targeted drug delivery to molecular imaging
Keywords: click chemistry, cyclodextrin oligomers, Gd-based contrast agents, imaging agents, microwave chemistry. Introduction
The search for new polyvalent and dendritic compounds with a well-defined chemical architecture is a hot topic in drug delivery and theranostic applications. Although significant progress has been achieved, the development of alternative template molecules as efficient carriers is still very much a work in progress.[1] An ideal vehicle requires specific structural
features: first of all, a highly functionalized surface, which facilitates selective chemical and biological interaction; secondly, a propensity for selforder and assembly; and thirdly, predictable and reproducible pharmacokinetics (PK) and pharmacodynamics (PD), which ideally require size monodispersity and well-established loading ratio.[2] Precise knowledge of
the loading stoichiometry is mandatory when such carriers are employed for the delivery of multiple therapeutic agents in a single formulation, or for the combination of imaging and drug therapy to monitor effects in real time. Functionalized liposomes[3] and nanoparticles[4] that allow molecular recognition of the target tissue have been widely used for active or triggered delivery of drugs.[5] Unfortunately, the key design parameters that govern the performance of nanocarriers and liposomes (size, surface properties, modulus, and shape) suffer from a lack of preparative reproducibility.[6] Since particle size and surface chemistry properties strongly affect in vivo distribution, metabolism, and drug release,[7] PK/PD reproducibility represents a major issue. Further requirements for the new generation of carriers are very low toxicity, compatible immunogenicity, and easy excretion.[8]
Cyclodextrins (CDs) are natural cyclic oligosaccharides that constitute exceptional building blocks for dendrimeric architectures, as they are essentially non-immunogenic and non-toxic in animals and humans. CDs can accommodate various organic molecules within their truncated cone-shaped hydrophobic cavity, generating host–guest supramolecular species in aqueous solution. This property has been exploited in pharmaceutical applications, and CDs have been incorporated into polymers or nanoparticles[9] to bind, stabilize, solubilize and/or reduce the toxicity of drugs, oli-gonucleotides, and proteins. The synthetic design of CD-based oligomers such as trimers[10] and tetramers[11] has al-ready been described; however, there are very few examples of larger constructs.[12]. The assembly of large CD constructs requires the use of reactions capable of efficiently linking two or more CD units. It is well established that the
micro-wave (MW)-promoted Cu-catalyzed 1,3-dipolar cycloaddi-tion (CuAAC) between CD monoazides and monoacetylene moieties, which results in the formation of a triazole bridge, is the most efficient way of modifying the CD surface.[13] Using the aforementioned procedure, we have previously published the preparation of water-soluble CD homo- and heterodimers and trimers of -, -, and -CD as host molecules.[10b, 14]
The aim of this work was the design, preparation, and characterization of a new water-soluble oligocyclodextrin heterononamer for use as a dendrimeric multicarrier. The loading ratio of this nanostructure was first predicted in silico through molecular dynamics (MD) simulations combined with solvent-accessible surface (SAS) calculations, then measured through the use of a paramagnetic Gd complex. The possibility of assembling multiple nonamers into supramolecular structures with high hosting ability was explored by preparing a dimeric platform through a bis(litho-cholic acid) linker. Adducts between Gd complexes and the dendrimeric platforms were studied by relaxometric titration to assess their potential as contrast agents for MRI diagnostics; finally, their cytotoxicity and binding affinity towards albumin were evaluated.
Results and Discussion
The designed platform consists of eight -CD units anch-ored to a -CD core through bis(triazolyl)alkyl spacers to give a star-shaped molecule (Figure 1). The difference in cavity size between and -CDs allows for selective complexation. For instance, the eight -CD units could host a drug or a contrast agent, while the --CD could form a non-covalent adduct with a vector unit capable of guiding the whole platform to the target site. Alternatively, the -CD cavity could be exploited to form supramolecular adducts between multiple CD dendrimers through a linker, thus multiplying the number of -CD cavities available for the inclusion of drugs or diagnostic agents.
Figure 1. Three-dimensional model of g1b8-cyclodextrin-nonamer 4.
Chemistry: The first step in the synthetic pathway (Scheme 1) was the selective monosubstitution of -CD to afford the 6-monoazido--CD 1.[15] This was followed by selective per-substitution of the primary -OH groups on the - CD to give the 6-perazido--CD 3.[16] Two MW-assisted click reactions with a linear bis(alkyne), carried out in succession, generated the 18-nonamer 4 (Figure 1). In the first CuAAC, the use of an excess of 1,8-nonadiyne prevented the formation of the -CD dimer and allowed the 6-heptynyltriazolyl--CD 2 to be isolated in good yield. The promoting effect of MW irradiation on this reaction was striking both in terms of reaction time and yield. Derivative 2 was precipitated due to its poor solubility in water, washed with water, and used without further purification. The higher water solubility of 4, compared to its precursors, facilitated its purification after the second CuAAC. Nonamer 4 was further purged of residual copper salts and other impurities by preparative HPLC and the product was obtained at a purity greater than 90 % (by analytical HPLC-ELSD). With a view to possible scale-up, an alternative, faster purification could be accomplished through the addition of ethylenediaminetetraacetic acid disodium salt dehydrate (Na2H2EDTA) to the reaction mixture, followed by ultrafiltration through a 3 kDa cut-off membrane (purity 72–75 % by analytical HPLC-ELSD). The final product did not contain impurities, but contained less-substituted analogues, from which one or two -CD units were missing.
Scheme 1. Synthetic route leading to the CD-nonamer 4.
Nonamer 4 was characterized by MALDI-TOF MS, 1H NMR, 2D-COSY NMR, 2D-1H–13C HMQC NMR, and 2D-1H–13C HMBC NMR spectroscopies (Figures S1–S4, Supporting Information). All spectroscopic data were consistent with the proposed structure. In silico simulations: The behavior of 4 in aqueous solution was simulated in silico by molecular dynamics (MD). In order to determine whether pH might influence the conformational profile, five different oligomer models were generated, characterized by 0, 25, 50, 75, and 100 % of the 1,2,3-triazole rings in the protonated form, respectively; protonated nitrogen atoms were symmetrically distributed across the 16 rings. To estimate the SAS of the CDs in the oligomer relative to their monomeric -CD and -CD counterparts, an open-source software was developed, Open-CDSurf,[17] which is freely available for download. Open-CDSurf analyzes MD trajectories, identifying all CDs present in each trajectory frame, and filling their internal cavity with spheres (Videos S1–S5, Supporting Information); subsequently, it computes the SAS of each CD cavity by using the EDTSurf algorithm.[18] For each of the simulated protonation states, the SAS of each -CD and the SAS of the -CD were plotted as a function of time (Figure S5, Supporting Information), and compared to those of the reference -CD and -CD monomers. Time averages of SAS values were computed along the trajectory, and are plotted in Figure 2 asthe average SAS of - and -CDs in the oligomer relative to the average SAS of the respective monomeric CD. The SAS values of the -CDs display almost no dependence on the protonation state until half of the triazole moieties are non-protonated; under such conditions, about 60 % of the accessibility of a monomeric -CD is retained. The relative accessibility of -CDs reaches a maximum of 85 % when the degree of protonation reaches 75 %, and then decreases once more to 64 % when all of the triazole moieties are protonated.
Figure 2. Average SAS of - (▪) and -CDs (□) in the oligomer as a function of the degree of protonation, relative to their reference monomeric counterparts
This behaviour can be attributed to the role of electrostatic repulsion between charged centres, which up to a certain degree appears to favour access to the -CD cavities. The same trend roughly applies to the -CD, accessibility to which is also maximized at 75 % protonation. Interestingly, the accessibility of the -CD in the nonamer is consistently more than 1.5-fold greater than that of the CD monomer; this means that the eight -CD arms contribute to holding open the entrance to the core
-CD. Since the pKa of 1-methyl-1,2,3-triazole is 1.25,
[19] only a small fraction of the triazole moieties in the oligomer are
expected to be protonated at physiological pH, the range in which average CD accessibility is most stable and predictable. Generation of supramolecular adducts: To demonstrate the feasibility of obtaining supramolecular structures with high hosting ability, we designed the dimeric platform 6 (Figure 3), which was formed by joining the -CD cores of two nanomers through a bis(lithocholic acid) linker 5.[20] For this purpose, 1,2-bis(2-aminoethoxy)ethane was conjugated with two units of lithocholic acid through amide bonds. The synthesis involved a twofold coupling reaction of activated lithocholic acid with the diamino derivative (Scheme 2).[21] The length of the linker was carefully chosen to grant each dendrimer suitable mobility, while preventing them from collapsing onto one another. It is known that lithocholic acid shows stronger interactions with -CDs than -CDs.[22, 23] This was also confirmed by 1H NMR experiments (Figure S6, Supporting Information), which showed that lithocholic acid has a higher affinity constant for -CD (3.85•104 and 5.28•102 for - and
-CD, respectively). The supramolecular adduct 6 was obtained by simply mixing a solution of 4 in water with a solution of 5 in THF. After removal of the solvents, this adduct was dissolved in water.
Figure 3. Schematic representation of the supramolecular adduct 6.
To confirm the interaction between 4 and 5, an NMR titration was performed. Increasing amounts of 4 were added to a solution of 5 in deuterated methanol/water. 1H NMR spectra recorded after each addition showed a characteristic shift and line broadening of the signals of the methyl protons of 5 (Figure S7, Supporting Information) owing to the interaction between 4 and 5. In fact, upon interaction with the CD dendrimer, the lithocholic acid residue increased its reorientational correlation time, resulting in a reduction of the transversal relaxation time and an increase in line width.
Scheme 2. Synthetic route leading to the lithocholic acid dimer 5 (DCC =N,N-dicyclohexylcarbodiimide; NHS = N-hydroxysuccinimide).
Assessment of ligand binding affinity and loading ratio: The carrier abilities of CD nonamer 4 and bis(CD-nonamer) adduct 6 were assessed by loading their respective -CD cavities with an appropriate paramagnetic metal complex. For this purpose, a Gd complex was functionalized with an adamantyl moiety, which is well-known to have high affinity toward the -CD cavity (7, Scheme 3).[24] The strengths of the interactions between 7 and dendrimers 4 and 6 were studied by relaxometric titration. Binding parameters (affinity constant KA, number of equivalent and independent binding sites n, and relaxivity of the supramolecular adduct 1) were determined by using the proton relaxation enhancement (PRE) method, which considers the relaxation enhancement due to the formation of a slow-moving macromolecular adduct. [25] The binding parameters were determined by two different experiments (Figure 4). In the first experiment, the water proton relaxation rate (r1 = 1/T1; 20 MHz and 25 °C) of a 0.1 mm solution of 7 was measured as a function of the concentration of 4 (or 6, data not shown). Analysis of the experimental data gave very similar affinity constants (nKA = 3.7•105 m-1 for 4 ; 2.2•105 m-1 for 6) and r
1b values (12.4 mm-1 s-1 for 4; 12.5 mm-1 s-1 for 6). The second experiment involved a relaxometric titration of a fixed concentration (0.05 mm) of 4 (or 6, data not shown) with 7. The water proton relaxation rate increased linearly with the concentration of 7 until a discontinuity in the slope, which indicated that the oligomer binding sites had been saturated. After this point, an additional contribution to the overall relaxation rate was observed, which could be attributed to the free Gd complex. The 7/4 ratio of 8 and 7/6 ratio of 16, which were found at this point, roughly correspond to the numbers of binding sites (n). These data indicate that the eight -CD cavities present on the surface of 4 and the sixteen cavities of the supramolecular adduct 6 are all fully loaded with 7. In the case of 4, the average affinity decreases slightly if the g-CD cavity is considered as a ninth interaction site. This was corroborated by MD simulations, which indicated the presence of a larger than expected cavity in the -CD unit, resulting in negligible interaction with the Gd complex.
Scheme 3. Synthesis of the adamantyl Gd complex 7 (DIPEA = N,N-di- isopropylethylamine).
Figure 4. a) Observed proton relaxation rate of a 0.1 mm solution of 7 in PBS as a function of increasing concentration of 4, measured at 20 MHz and 25 °C. b) Observed proton relaxation rate of a 0.05 mm solution of 4 in PBS as a function of increasing concentration of 7, measured at 20 MHz and 25 °C
Relaxometric characterization: Nuclear magnetic relaxation dispersion (NMRD) profiling, namely the measurement of the water proton relaxation rate over an extended range of magnetic field strengths (0.01– 80 MHz), is a commonly used method to gain insight into the relaxometric character of a paramagnetic
complex.[26] NMRD profiles of 7 and its adducts with 4 and 6, recorded at 25 °C and neutral pH, are shown in Figure 5.
Figure 5. 1/T1 NMRD profiles of 7 (triangle), and of the 4/7 (black square) and 6/7 (open square) adducts, recorded at 25 °C in PBS (pH 7.4). The data refer to a 1 mm concentration of the paramagnetic complex. Qualitative analysis of the experimental data indicates that the complexation of 7 with both 4 and 6 increases its relaxation rate over the entire frequency range investigated. On the other hand, it is worth noting that the classic relaxivity peak, usually centred at around 30 MHz and characteristic of GdIII chelates bound to macromolecules, is not so pronounced in this case. Quantitative analysis of the experimental data was performed by using the Solomon–Blombergen–Morgan inner–second-outer-sphere model.[27] In the case of unbound 7, the inner coordination sphere of the metal was considered as containing one water molecule (q=1) with an exchange lifetime (M) comparable to those reported for related mono-amido Gd 1,4,7,10-tetraazacyclododecane-1,4,7,10-tetraacetic acid (DOTA) derivatives.[28] Subsequently, data analysis for the 4/7 and 6/7 adducts (Table 1) was performed by using a q value of 1 and assuming that the observed relaxivities were also influenced by the protons of water molecules present in the second co-ordination sphere of the GdIII chelate at a distance of about 4 Ǻ. The M values in the 4/7 and 6/7 adducts were found to be more than three times longer than that of the free Gd complex. This effect can most probably be attributed to the hydrogen-bond network that is present among the water molecules and around the polar groups,[29] as has previously been reported for several Gd chelates/-CD supramolecular adducts. [10b, 28, 30]
In light of the parameters obtained from the fitting procedures and considering the spherical shape of the CD oligomer, it is possible to propose an orientation of the Gd complexes on the platform surface. Each molecule of complex 7 is inserted into one -CD cavity via the adamantyl moiety, and interacts with the contiguous -CD unit through the metal complex portion. This arrangement should increase the exchange lifetime (M) of the inner-sphere water molecule and allow for the presence of second-sphere water molecules. Moreover, as expected from the fact that the size of the 6/7 adduct is twice that of the 4/7 adduct, the R value of the former is almost twice as large as that of the latter. NMRD profiles of 1:1, 1:4, and 1:8 4/7 systems show quite similar relaxivity values over the entire frequency range (Figure S8, Supporting Information). This indicates that the interaction strength and structural features do not change significantly when the CD nonamer is loaded further.
Assessment of oligomer ligand binding affinity versus albumin: The relaxometric PRE procedure was used to measure the binding affinity of 7 toward human serum albumin (HSA), which is the most abundant protein present in plasma. An affinity constant of 1.9•103 m-1 with one strong binding site was determined. This affinity constant is two orders of magnitude lower than those measured for 4 and 6 CD multimers. On the basis of the measured binding affinities toward HSA and 6, it was calculated that, when the con-centrations of 7 and 6 are 1 mm and 62 mm (1/16 of 7), respectively, and considering HSA to be present in human plasma at a concentration of 0.58 mm, 68 % of 7 is bound to 6, 15 % is bound to albumin, and 17 % is free. These data indicate that, once administered in vivo, most of the paramagnetic probe should be loaded on the CD supramolecular structure.
Assessment of cytotoxicity: Finally, with a view to the potential application of these macromolecular carriers in drug delivery, cell labeling, and/or in vivo applications, a cytotoxicity test against a glioblastoma cell line (U87 MG) was performed (Figure S9, Supporting Information). Cell viability was evaluated by using the Trypan Blue exclusion test and referenced to untreated cells as a control (100 % viable). An acceptable viable cell value (> 80 %) was recovered when using a carrier concentration lower than 0.1 mm, which would in turn enable the transport of drug (or diagnostic) molecules at a concentration of 0.8–1.6 mm in the case of 4 and 6, respectively. An inherent challenge with CD platforms for in vivo host–guest applications is the competition from binding of the included ligands to plasma proteins.
Conclusion
A new 18-CD platform has been designed and synthesized. It fulfils the main requirements for use as a multi-carrier, namely water solubility, surface accessibility, and hosting capability. Exploiting the different hosting abilities of - and -CDs, we have shown that it is possible to assemble supramolecular adducts by joining multiple platforms through appropriate linkers. These findings may open up access to a wide range of potential pharmaceutical and diagnostic applications.
Experimental Section
Chemistry: Commercially available reagents and solvents were used without further purification. Native CDs were kindly provided by Wacker Chemie Italia srl (Italy). Reactions were monitored by TLC on Merck 60 F254 (0.25 mm) plates. Spot detection was carried out by staining with 5% H2SO4 in ethanol. IR spectra were recorded on a Shimadzu FTIR 8001 spectrophotometer. NMR spectra were recorded at 258C on a
Bruker Avance 300 spectrometer operating at 7 T; chemical shifts () are given in ppm, coupling constants (J) in Hz. MALDI-TOF mass spectra were recorded on a Bruker Ultraflex TOF mass spectrometer. MW-promoted reactions were carried out in a professional oven (MicroSYNTHMilestone, Italy). The reaction temperature was monitored by a fibreoptic thermometer directly inserted into the reaction vessel. HPLC analyses were carried out at 258C with a flow rate of 1 mLmin-1 on a Waters XBridge BEH 130 column (4.6/100, 5 mm) by using an evaporative light scatter detector (ELSD), with H2O/trifluoroacetic acid (TFA) 0.1% v/v (phase A) and MeOH/TFA 0.1% v/v (phase B) as mobile phases. HPLC method (time (min), %B): 0, 10; 1.68, 10; 8.32, 35; 13.30, 35; 23.28, 100; 29.00, 100. The injection volume was 10 mL. Separations were carried out at 25°C with a flow rate of 20 mLmin1 by using a Waters XBridge BEH 130 column (19/100, 5 mm) with H2O/TFA 0.1% v/v (phase A) and MeOH/TFA 0.1% v/v (phase B) as mobile phases. HPLC method (time (min), %B): 0, 20; 7.10, 20; 8.51, 30; 15.60, 30; 17.02, 40; 23.00, 40.
6I-Deoxy-6I-[4-(hept-6-ynyl)-1H-1,2,3-triazolyl]--CD (2): The reaction between 1 (1.00 g, 0.862 mmol) and 1,8-nonadiyne (670 mL, 4.48 mmol) was carried out in a professional MW oven by using a pressure-resistant closed vessel equipped with a fibre-optic thermometer and a magnetic stirring bar. The reaction mixture, which also contained CuSO4 (43 mg, 0.172 mmol) and l-ascorbic acid (60.6 mg, 0.345 mmol), was dissolved in DMF (10 mL) and heated at 70°C (70 W) for 2 h. After concentration under vacuum to half of the original volume and addition of acetone (10 mL), a solid product was collected by filtration on a Hirsch funnel. The product was washed with water (3x5 mL) and used without further purification (white powder; yield 86%). Purity: 97.4% by HPLC-ELSD. Rf : 0.36 (silica; iPrOH/H2O/EtOAc/NH4OH, 5:3:1:1); IR (KBr): ˜ = 3420, 2928, 1460, 1369, 1032, 1157, 1030, 945, 756 cm-1; NMR and MS data are reported in the Supporting Information (Figures S1–S3 and respective captions).
18-CD oligomer (4): The reaction between 2 (300 mg, 0.234 mmol) and per-6-azido-per-6-deoxy--CD 3 (29.2 mg, 0.0195 mmol) was carried out in a two-necked round-bottomed flask (25 mL) equipped with a stirring bar and a fibre-optic thermometer in a professional MW oven. The reaction mixture, which also contained CuSO4 (58.8 mg, 0.234 mmol) and lascorbic acid (82.5 mg, 0.468 mmol), was suspended in a DMF/H2O mixture (1:5) and heated at 100 8C (120 W) for 3 h. The crude product was either purified by semi-preparative HPLC (pale-yellow powder; yield 22%) or by the addition of Na2H2EDTA and ultrafiltration (3 kDa disc) in a stirred cell (freeze-dried, pale-yellow powder; yield 79%). NMR and MS data are reported in the Supporting Information (Figure S4 and caption).
Bis(deoxycholamide) of 1,2-bis(2-aminoethoxy)ethane (5): N-Hydroxysuccinimide (345 mg, 3 mmol) and N,N-dicyclohexylcarbodiimide (DCC; 621 mg, 3.04 mmol) were added to a solution of lithocholic acid (1.0 g, 2.66 mmol) in THF (20 mL). The mixture was stirred for 4 h at room temperature and filtered to remove the insoluble urea. A solution prepared from 1,2-bis(2-aminoethoxy)ethane (148 mL, 1.0 mmol), triethylamine (1 mL), and water (5 mL) was added to the filtrate. After being stirred overnight, the mixture was poured into 1m aqueous HCl (25 mL). The resulting solid was collected by filtration and purified by column chromatography (silica gel; CH2Cl2/CH3OH/NH4OH, 95:5:0.3 v/v/v) to give the bis(deoxycholamide) of 1,2-bis(2-aminoethoxy)ethane (yield 51%). Rf : 0.59 (silica; CHCl3/MeOH/H2O, 6:1:0.05); 1H NMR ([D6]DMSO, 300 MHz): =0.64 (s, 6H), 0.91–2.13 (m, 68H), 3.2–3.23 (m, 4H), 3.35– 3.44 (m, overlay with water signal), 3.53 (s, 4 H), 4.49 (d, 2H), 7.84–7.88 ppm (m, 2H); MS (ESI): m/z
calcd for C54H92N2O6 : 866.32 [M+H]+; found: 866.09. 18
-Nonamer/2,2’-(ethylenedioxy)bis(ethylamine)dideoxycholamide complex (6): Nonamer 4 (50 mg, 0.00426 mmol) was dissolved in water (1 mL), and then a solution of 2,2’-(ethylenedioxy)bis(ethylamine)dideoxycholamide (1.84 mg, 0.00213 mmol) in THF (500 mL) was added. After evaporation of the THF, the solution was freeze-dried.
1-[9-(1-Adamantyl)carbonylamino-3-aza-2-oxononyl]-1,4,7,10-tetraazacyclododecane- 4,7,10-triacetate gadolinium (7): The complexation reaction was performed with GdCl3 in aqueous solution at pH 7 by the
ligand addition method.[31] An equimolar amount of aqueous GdCl3 (524 mg, 1.41 mmol) solution was slowly added to an aqueous solution of 7c (1.00 g, 1.41 mmol), maintaining the pH value at 7 with 0.1N NaOH. The mixture was stirred at room temperature until the pH remained constant. When a slight excess of the metal cation was reached, as monitored with the orange xylenol assay,[32] a small excess of ligand was added (< 0.13%). The complex was then desalted by size-exclusion chromatography on Sephadex G10 resin and freeze-dried (yield 53%).
Computational methods: Molecular simulations were carried out by using MOE,[33] AMBER 10,[34] and our in-house software OpenCDSurf.[17] Full methodological details are reported in the Supporting Information
Acknowledgements
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