Periodic Reporting for period 1 - CONNECT (From Supramolecular to Covalent Boron Clusters Membrane Carriers)
Periodo di rendicontazione: 2023-06-01 al 2025-05-31
Overview of the Research Project
The transport of small biomolecules and molecular drugs such as proteins, nucleic acids, and synthetic nanomaterials within living cells has remained a significant challenge in life sciences. At the same time, due to its capacity to enable targeting of molecules to specific locations or organelles within the cell, research on intracellular transport for imaging and drug delivery is receiving special attention. However, direct delivery of exogenous cargo into the cytosol requires overcoming the plasma membrane, which serves as a protective barrier separating the cell's internal environment from the outside. To transport molecules across membranes, synthetic carriers have been designed and, to date, they have all been based on the amphiphilic dogma. As a consequence of their amphiphilicity, current synthetic transporters tend to aggregate, alone or with other (bio)molecules, limiting their aqueous solubility and stability. Thus, this project abandoned the typical amphiphilic model of membrane transport and proposed the use of superchaotropic boron clusters for the transport of a broad range of hydrophilic chemical substances across lipid membranes and inside cells.
This transport is enabled by the superchaotropic properties of the boron cluster, which disrupt the hydrogen bonding network of water more effectively than standard chaotropic ions in the Hofmeister series. These carriers offer distinct advantages over their amphiphilic counterparts, including high water solubility, preventing aggregation, and great stability. Currently, these carriers operate through weak supramolecular interactions, which facilitate the release of the cargo within the cell. However, the exact mechanism and specificity of this transport, as well as the cargo capacity and potential for co-transport of other substances, remain unclear. To address this knowledge gap, the project aimed to covalently attach a cargo molecule to the dodecaborate cluster, ensuring exclusive transport of the desired cargo.
Therefore, the primary goal of the project was to explore, for the first time, the covalent modification of model biomolecules (e.g. peptides, fluorophores) with boron superchaotropic clusters to develop a new range of self-transported molecules. One of the potential synthetic routes that was explored was the formation of a triazole group using Copper(I)-catalyzed Azide-Alkyne Cycloaddition (CuAAC) reaction to link the cargo and the dodecaborate cluster. To achieve this, we developed a synthetic route to functionalize the dodecaborate cluster starting with the B–H functionalization of the commercial compound salt to obtain B–OH (hydroxylated cluster). Then, we proceed to halogenate (Br) the dodecaborate cluster, which involves the exchange of B-H bonds to yield B–Br, as the brominated cluster has previously demonstrated a good balance between carrier activity and toxicity. Finally, we introduce an alkyne moiety through a nucleophilic substitution reaction to yield the final alkyne compound, which will enable the covalent bonding of the cargo via a CuAAC reaction. This study demonstrates the viability of functionalizing boron clusters with bioactive cargos such as 5-Carboxytetramethylrhodamine azide.
The development of these revolutionary molecules as membrane transporters will give rise to a new type of penetration molecule. The creation of a new technology for membrane transport using the covalent introduction of superchaotropic substituents into prototype peptide sequences or other biomolecules, such as fluorophores, will potentially impact the market of pharmaceutical formulation and potentially increase the competitiveness of European institutions and companies. Nevertheless, the introduction of clusters with amino acids is still in progress; we are confident (preliminary studies) that the new application of chaotropic clusters will impact the field of membrane transport by using more controllable, non-aggregating, and less toxic vehicles.
The transport of small biomolecules and molecular drugs such as proteins, nucleic acids, and synthetic nanomaterials within living cells has remained a significant challenge in life sciences. At the same time, due to its capacity to enable targeting of molecules to specific locations or organelles within the cell, research on intracellular transport for imaging and drug delivery is receiving special attention. However, direct delivery of exogenous cargo into the cytosol requires overcoming the plasma membrane, which serves as a protective barrier separating the cell's internal environment from the outside. To transport molecules across membranes, synthetic carriers have been designed and, to date, they have all been based on the amphiphilic dogma. As a consequence of their amphiphilicity, current synthetic transporters tend to aggregate, alone or with other (bio)molecules, limiting their aqueous solubility and stability. Thus, this project abandoned the typical amphiphilic model of membrane transport and proposed the use of superchaotropic boron clusters for the transport of a broad range of hydrophilic chemical substances across lipid membranes and inside cells.
This transport is enabled by the superchaotropic properties of the boron cluster, which disrupt the hydrogen bonding network of water more effectively than standard chaotropic ions in the Hofmeister series. These carriers offer distinct advantages over their amphiphilic counterparts, including high water solubility, preventing aggregation, and great stability. Currently, these carriers operate through weak supramolecular interactions, which facilitate the release of the cargo within the cell. However, the exact mechanism and specificity of this transport, as well as the cargo capacity and potential for co-transport of other substances, remain unclear. To address this knowledge gap, the project aimed to covalently attach a cargo molecule to the dodecaborate cluster, ensuring exclusive transport of the desired cargo.
Therefore, the primary goal of the project was to explore, for the first time, the covalent modification of model biomolecules (e.g. peptides, fluorophores) with boron superchaotropic clusters to develop a new range of self-transported molecules. One of the potential synthetic routes that was explored was the formation of a triazole group using Copper(I)-catalyzed Azide-Alkyne Cycloaddition (CuAAC) reaction to link the cargo and the dodecaborate cluster. To achieve this, we developed a synthetic route to functionalize the dodecaborate cluster starting with the B–H functionalization of the commercial compound salt to obtain B–OH (hydroxylated cluster). Then, we proceed to halogenate (Br) the dodecaborate cluster, which involves the exchange of B-H bonds to yield B–Br, as the brominated cluster has previously demonstrated a good balance between carrier activity and toxicity. Finally, we introduce an alkyne moiety through a nucleophilic substitution reaction to yield the final alkyne compound, which will enable the covalent bonding of the cargo via a CuAAC reaction. This study demonstrates the viability of functionalizing boron clusters with bioactive cargos such as 5-Carboxytetramethylrhodamine azide.
The development of these revolutionary molecules as membrane transporters will give rise to a new type of penetration molecule. The creation of a new technology for membrane transport using the covalent introduction of superchaotropic substituents into prototype peptide sequences or other biomolecules, such as fluorophores, will potentially impact the market of pharmaceutical formulation and potentially increase the competitiveness of European institutions and companies. Nevertheless, the introduction of clusters with amino acids is still in progress; we are confident (preliminary studies) that the new application of chaotropic clusters will impact the field of membrane transport by using more controllable, non-aggregating, and less toxic vehicles.
Our main scientific objective was to synthesize functionalized dodecaborate clusters [B12H12]2- with prototype biomolecules (e.g. peptides, fluorophores). To accomplish this, we design, develop, and optimize different synthetic protocols to covalently introduce superchaotropic substituents into amino acids or different fluorophores. Given the unique reactivity of boron clusters, we adapted synthetic routes described in the literature to efficiently attach the desired organic molecule to the closo-dodecaborate anion. Thus, we modified the commercial dodecaborate cluster Na2[B12H12] to allow covalent attachment of a cargo. For this purpose, we explored two different synthetic routes. The two proposed routes were: (a) formation of an ester link and (b) formation of an alkyne moiety to use CuAAC reaction. On one hand, for the ester linker, we designed a synthetic route to achieve an ester bond between the cluster and the cargo. This ester linkage is particularly interesting because it is a reversible covalent bond; inside the cell, it is easily cleaved by enzymes like esterases, releasing the cargo within the cell. On the other hand, for the CuAAC reaction of the alkyne-equipped cluster, we used a cargo with an azide moiety. After experimentation, the most efficient approach was the functionalization with an alkyne moiety. Then, we covalently attached 5-Carboxytetramethylrhodamine azide (TAMRA-azide) and Lysine-azide via Cu-catalyzed Azide-Alkyne cycloaddition reaction to the modified cluster.
It was successfully synthesized the hydroxylated cluster (B-OH) Na2[B12H11OH] from the commercially available Na2[B12H12] by optimizing the addition rate of sulfuric acid and reaction time to minimize byproducts (dehydroxylated and/or starting material). The synthesis of Na2[B12Br11OH] was achieved, with Na2[B12HBr10OH] as a minor byproduct. Furthermore, it was performed a nucleophilic substitution reaction to attach an alkyne to the -OH group of the cluster, yielding as the final product Na2[B12Br11OC3H3] with some remaining starting material Na2[B12Br11OH], purification of this material was not trivial but using a Sephadex LH-20 column with an eluent of 80/20 MeOH/ACN it was achieved a yield of 40.38% with a mass of 0.209g. Finally, we successfully synthesized a triazole linkage between the cluster and TAMRA-azide as well as Lysine-azide using a Copper(I)-catalyzed Azide-Alkyne Cycloaddition (CuAAC) Click Reaction. The formation of our final product was confirmed by 1H NMR and IR spectroscopy, although further systematic methods for purification needed to be optimized.
The characterization of the resulting synthesized products was performed using mass spectrometry (ESI-MS; MW min 50Da in negative/positive mode), nuclear magnetic resonance (NMR 500/300 MHz; different deuterated solvents; 1H, 11B, 13C, HSQC, TOCSY), and infrared spectroscopy (IR). The NMR spectrum of the hydroxylated cluster Na2[B12Br11OH] displayed a 1:5:5:1 pattern in organic solvents and water, consistent as reported in the literature. The MS was checked as well, given a peak at [M]2- ≈ 89.98 m/z, corresponding to the product. The next compound was the halogen-substituted cluster Na2[B12Br11OH], which induces a shift in the NMR spectrum from a 1:5:5:1 pattern to a 1:10:1 pattern. Additionally, the MS spectrum showed a fragment with the characteristic bromine isotope pattern at [M]2- ≈ 512 m/z, which corresponds to the product. It was also verified by IR that the vibrations (OH, B-H, B-OH) were according to the material synthesized. Furthermore, for the alkyne substitution, [B12Br11(OC3H3)]2-, the 1H NMR spectrum showed two key signals: a doublet at 4.70 ppm integrating for two protons, corresponding to the internal protons of the alkyne, and a triplet at 2.50 ppm integrating for one proton, corresponding to the terminal alkyne proton. Both signals have the same coupling constant (J4HH = 2.46 Hz). The TOCSY experiment showed that the two signals correspond to the same spin system. The 13C signals are assigned with the aid of the HSQC experiment. In addition, the characterization was through MS, giving a peak at [M]2- ≈ 531.87 m/z, corresponding to the final product. For the final product, the attachment of the TAMRA-azide to the cluster, the negative mass spectrum displayed two principal peaks: one at [M]2- ≈ 786.96 m/z, corresponding to the [B12Br11O(C31H32N6O4)]2- unit, and another at [M]2- ≈ 747.40 m/z, corresponding to the [B12HBr10O(C31H32N6O4)]2- unit. The characterization of the triazole group formation was confirmed using 1H and TOCSY, as well the comparison of IR spectra between the reaction crude, alkyne, and azide confirmed the progress of the reaction as the azide signal disappeared.
Summarized key achievements:
• It was successfully synthesized Na2[B12H11OH] from the commercially available Na2[B12H12] by optimizing the addition rate of sulfuric acid and reaction time to minimize the dihydroxylated byproduct. No signals from the starting material were observed in the 11B NMR spectrum. However, the large number of salts formed during the neutralization step prevented the calculation of the product mass and yield by weight.
• The synthesis of Na2[B12Br11OH], with Na2[B12HBr10OH] as a minor byproduct, in a 62.09% yield corresponding to 1.63 g of product was achieved.
• The esterification of Na2[B12Br11OH] with Fmoc-Glu(OAll)-OH using a standard procedure was attempted, but the reaction did not proceed as expected. The low nucleophilicity of Na2[B12Br11OH] hinders its ability to attack a chloride acid, requiring a strong base like NaH to deprotonate the hydroxyl group and generate a nucleophilic alkoxide. This is similar to the SN2 reaction, where NaH is used to deprotonate a leaving group to facilitate nucleophilic substitution. Therefore, the standard esterification methodology proved to be ineffective in this case.
• It was performed a nucleophilic substitution reaction to attach an alkyne to the -OH group of the cluster, yielding the final product Na2[B12Br11OC3H3] with some remaining starting material Na2[B12Br11OH].
• It was also possible to covalently attach a fluorophore, TAMRA-azide, to the compound through CuAAC reaction. Using TAMRA-azide as a fluorescent marker in the boron cluster could allow an effective tracking of the product.
It was successfully synthesized the hydroxylated cluster (B-OH) Na2[B12H11OH] from the commercially available Na2[B12H12] by optimizing the addition rate of sulfuric acid and reaction time to minimize byproducts (dehydroxylated and/or starting material). The synthesis of Na2[B12Br11OH] was achieved, with Na2[B12HBr10OH] as a minor byproduct. Furthermore, it was performed a nucleophilic substitution reaction to attach an alkyne to the -OH group of the cluster, yielding as the final product Na2[B12Br11OC3H3] with some remaining starting material Na2[B12Br11OH], purification of this material was not trivial but using a Sephadex LH-20 column with an eluent of 80/20 MeOH/ACN it was achieved a yield of 40.38% with a mass of 0.209g. Finally, we successfully synthesized a triazole linkage between the cluster and TAMRA-azide as well as Lysine-azide using a Copper(I)-catalyzed Azide-Alkyne Cycloaddition (CuAAC) Click Reaction. The formation of our final product was confirmed by 1H NMR and IR spectroscopy, although further systematic methods for purification needed to be optimized.
The characterization of the resulting synthesized products was performed using mass spectrometry (ESI-MS; MW min 50Da in negative/positive mode), nuclear magnetic resonance (NMR 500/300 MHz; different deuterated solvents; 1H, 11B, 13C, HSQC, TOCSY), and infrared spectroscopy (IR). The NMR spectrum of the hydroxylated cluster Na2[B12Br11OH] displayed a 1:5:5:1 pattern in organic solvents and water, consistent as reported in the literature. The MS was checked as well, given a peak at [M]2- ≈ 89.98 m/z, corresponding to the product. The next compound was the halogen-substituted cluster Na2[B12Br11OH], which induces a shift in the NMR spectrum from a 1:5:5:1 pattern to a 1:10:1 pattern. Additionally, the MS spectrum showed a fragment with the characteristic bromine isotope pattern at [M]2- ≈ 512 m/z, which corresponds to the product. It was also verified by IR that the vibrations (OH, B-H, B-OH) were according to the material synthesized. Furthermore, for the alkyne substitution, [B12Br11(OC3H3)]2-, the 1H NMR spectrum showed two key signals: a doublet at 4.70 ppm integrating for two protons, corresponding to the internal protons of the alkyne, and a triplet at 2.50 ppm integrating for one proton, corresponding to the terminal alkyne proton. Both signals have the same coupling constant (J4HH = 2.46 Hz). The TOCSY experiment showed that the two signals correspond to the same spin system. The 13C signals are assigned with the aid of the HSQC experiment. In addition, the characterization was through MS, giving a peak at [M]2- ≈ 531.87 m/z, corresponding to the final product. For the final product, the attachment of the TAMRA-azide to the cluster, the negative mass spectrum displayed two principal peaks: one at [M]2- ≈ 786.96 m/z, corresponding to the [B12Br11O(C31H32N6O4)]2- unit, and another at [M]2- ≈ 747.40 m/z, corresponding to the [B12HBr10O(C31H32N6O4)]2- unit. The characterization of the triazole group formation was confirmed using 1H and TOCSY, as well the comparison of IR spectra between the reaction crude, alkyne, and azide confirmed the progress of the reaction as the azide signal disappeared.
Summarized key achievements:
• It was successfully synthesized Na2[B12H11OH] from the commercially available Na2[B12H12] by optimizing the addition rate of sulfuric acid and reaction time to minimize the dihydroxylated byproduct. No signals from the starting material were observed in the 11B NMR spectrum. However, the large number of salts formed during the neutralization step prevented the calculation of the product mass and yield by weight.
• The synthesis of Na2[B12Br11OH], with Na2[B12HBr10OH] as a minor byproduct, in a 62.09% yield corresponding to 1.63 g of product was achieved.
• The esterification of Na2[B12Br11OH] with Fmoc-Glu(OAll)-OH using a standard procedure was attempted, but the reaction did not proceed as expected. The low nucleophilicity of Na2[B12Br11OH] hinders its ability to attack a chloride acid, requiring a strong base like NaH to deprotonate the hydroxyl group and generate a nucleophilic alkoxide. This is similar to the SN2 reaction, where NaH is used to deprotonate a leaving group to facilitate nucleophilic substitution. Therefore, the standard esterification methodology proved to be ineffective in this case.
• It was performed a nucleophilic substitution reaction to attach an alkyne to the -OH group of the cluster, yielding the final product Na2[B12Br11OC3H3] with some remaining starting material Na2[B12Br11OH].
• It was also possible to covalently attach a fluorophore, TAMRA-azide, to the compound through CuAAC reaction. Using TAMRA-azide as a fluorescent marker in the boron cluster could allow an effective tracking of the product.
The scientific efforts of this project (CONNECT) were primarily concerned with building a completely new platform for boron clusters as membrane transporters. While the current trend in membrane translocation of hydrophilic substances is to use amphiphilic molecules, the proposed introduction of superchaotropic boron clusters to improve the membrane transport efficiency of amphiphilic bioactive peptides and other molecules is revolutionary, and it could lead to a new generation of self-transported chaotropic biomolecules. Although the study is still in progress, continued efforts are aimed at optimizing the synthesis and conducting in vitro evaluations. In this effort, we were able to develop methods to facilitate the synthesis and characterization of superchaotropic clusters supplemented with other relevant biomolecules, such as fluorescent dyes (e.g. TAMRA-azide). Furthermore, the project has generated valuable knowledge that is fostering new opportunities for collaboration and joint funding applications. For instance, one of the collaborating researchers was recently awarded an MSCA fellowship in 2024 for a project entitled Chaotropic Delivery of Fully Anionic Oligonucleotides (BOOST–MSCA-2024-EF), which emerged from a novel research direction inspired by the current work.
The discovery of new transport mechanisms using boron clusters, such as modified chaotropic amino acid residues or chaotropic modified fluorophores, has the potential to be exploited in pharmaceutical applications, such as topical medication delivery or improved biomolecule monitoring in cell biology. Although various tests are currently being developed to confirm their prospective uses, preliminary results are promising. Thus, the project's implementation will raise societal awareness of the importance of science in developing better delivery methods to target and treat diseases, which is consistent with the Horizon Europe Cluster 1 goal (Health) of generating new knowledge to prevent, diagnose, treat, and cure diseases.
Our results were highly interdisciplinary and of interest to the chemistry, nanotechnology, and pharmaceutical communities, as evidenced by our dissemination work during this action, which included various conferences (national and international) and oral presentations to various scientific communities and the general public. During the dissemination activities, the various audiences expressed interest in this new form of organic/inorganic cluster compounds for transporting molecules across membranes and acknowledged the need to develop new transport mechanisms. We also believe that this knowledge will contribute significantly to Horizon Europe's new Key Enabling Technologies, which have been highlighted as a crucial asset for the EU's development. Furthermore, future publications based on the research findings will be of interest in improving the membrane transport efficiency of amphiphilic bioactive peptides using superchaotropic boron clusters and other fluorophores as trackers in cells.
Additionally, the development of these revolutionary compounds as membrane transporters will result in a new class of zwitterionic hydrophilic penetration molecules. The development of a new membrane transport technology that involves the covalent introduction of superchaotropic substituents into prototype peptide sequences or other biomolecules such as fluorophores, has the potential to impact the pharmaceutical formulation market and increase the competitiveness of European institutions and companies. Nonetheless, while the introduction of amino acid clusters is still ongoing, we are convinced (based on preliminary studies) that the new application of chaotropic clusters will have an influence on membrane transport by using more controlled, non-aggregating, and less hazardous vehicles. Furthermore, these activities will raise the visibility of the institutions involved and strengthen their industry links, potentially attracting support from additional funding programs and stakeholders such as the Ignicia Program (Galician Innovation Agency), La Caixa Foundation (LaCaixa Research), or the EIC Pathfinder Initiative.
The discovery of new transport mechanisms using boron clusters, such as modified chaotropic amino acid residues or chaotropic modified fluorophores, has the potential to be exploited in pharmaceutical applications, such as topical medication delivery or improved biomolecule monitoring in cell biology. Although various tests are currently being developed to confirm their prospective uses, preliminary results are promising. Thus, the project's implementation will raise societal awareness of the importance of science in developing better delivery methods to target and treat diseases, which is consistent with the Horizon Europe Cluster 1 goal (Health) of generating new knowledge to prevent, diagnose, treat, and cure diseases.
Our results were highly interdisciplinary and of interest to the chemistry, nanotechnology, and pharmaceutical communities, as evidenced by our dissemination work during this action, which included various conferences (national and international) and oral presentations to various scientific communities and the general public. During the dissemination activities, the various audiences expressed interest in this new form of organic/inorganic cluster compounds for transporting molecules across membranes and acknowledged the need to develop new transport mechanisms. We also believe that this knowledge will contribute significantly to Horizon Europe's new Key Enabling Technologies, which have been highlighted as a crucial asset for the EU's development. Furthermore, future publications based on the research findings will be of interest in improving the membrane transport efficiency of amphiphilic bioactive peptides using superchaotropic boron clusters and other fluorophores as trackers in cells.
Additionally, the development of these revolutionary compounds as membrane transporters will result in a new class of zwitterionic hydrophilic penetration molecules. The development of a new membrane transport technology that involves the covalent introduction of superchaotropic substituents into prototype peptide sequences or other biomolecules such as fluorophores, has the potential to impact the pharmaceutical formulation market and increase the competitiveness of European institutions and companies. Nonetheless, while the introduction of amino acid clusters is still ongoing, we are convinced (based on preliminary studies) that the new application of chaotropic clusters will have an influence on membrane transport by using more controlled, non-aggregating, and less hazardous vehicles. Furthermore, these activities will raise the visibility of the institutions involved and strengthen their industry links, potentially attracting support from additional funding programs and stakeholders such as the Ignicia Program (Galician Innovation Agency), La Caixa Foundation (LaCaixa Research), or the EIC Pathfinder Initiative.