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Synthesis and Biological Activity of Thymosin #4-Anionic Boron Cluster Conjugates Krzysztof Fink, Kamil Kobak, Monika Kasztura, Janusz Boraty#ski, and Tomasz Marek Goszczy#ski Bioconjugate Chem., Just Accepted Manuscript • DOI: 10.1021/acs.bioconjchem.8b00646 • Publication Date (Web): 26 Oct 2018 Downloaded from http://pubs.acs.org on October 27, 2018

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Bioconjugate Chemistry

Synthesis and Biological Activity of Thymosin β4-Anionic Boron Cluster Conjugates Krzysztof Fink†, Kamil Kobak‡, Monika Kasztura‡, Janusz Boratyński† and Tomasz M. Goszczyński†* † Laboratory

of Biomedical Chemistry, Department of Experimental Oncology, Hirszfeld Institute of Immunology and Experimental Therapy PAS, 12 Rudolf Weigl St., 53-114 Wrocław, Poland ‡ Laboratory for Applied Research on Cardiovascular System, Department of Heart Diseases, Wrocław Medical University, 5 Rudolf Weigl St., 50-981 Wrocław, Poland Abstract Anionic boron clusters are man-made, inorganic compounds with potential applications in therapeutic peptides modification to improve their biological activity and pharmacokinetics, e.g., by enabling complexation with serum albumin. However, the conjugation of anionic boron clusters and peptides remains poorly understood. Here, we report a solid-state, thermal reaction to selectively conjugate carboxylic groups in the peptide thymosin β4 (Tβ4) with cyclic oxonium derivatives of anionic boron clusters (dodecaborate anion [B12H12]2- and cobalt bis(1,2dicarbollide), [COSAN]- [3,3՛-Co(1,2-C2B9H11)2]-). Modification of the carboxylic groups retains the negative charge at the modification site and leads to the formation of ester bonds. The ester bonds in the conjugates undergo hydrolysis at different rates depending on the site of the modification. We obtained conjugates with dramatically different stabilities (τ1/2 from 3-836 h (Tβ4-[B12H12]2- conjugates) and 9-1329 h (Tβ4-[COSAN]- conjugates)) while retaining or improving the prosurvival activity of Tβ4 towards cardiomyocytes (H9C2 cell line).

Anionic boron clusters are man-made polyhedral boranes with a delocalized negative charge.1 These species have unique properties, such as spherical or ellipsoid geometries, high chemical, thermal and biological stability, the ability to form dihydrogen bonds, and high susceptibility to derivatization.2-5 Anionic boron clusters are amphiphilic because of the hydridic hydrogens on their surface and their delocalized negative charge.6 They lack hydrophobic and hydrophilic domains in their structure and are thus called intrinsic amphiphiles to distinguish them from classical amphiphiles.7-9 Anionic boron clusters are also characterized as superchaotropic ions.10 Because of these properties, anionic boron clusters are relatively soluble in water11, 12 but can also interact with hydrophobic surfaces such as lipid membranes,13-17 hydrophobic cavities of proteins,18, 19 including serum albumin,20, 21 and macrocyclic hosts such as cyclodextrins16, 22-24 and calixarenes.25 Additionally, anionic boron clusters readily cross biological membranes and accumulate inside cells, including cell nuclei,26, 27 while showing low toxicity in in vitro and in vivo studies.17, 28

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The properties of anionic boron clusters can be used in many areas of peptide modifications to develop 1) peptide analogs with high affinities for albumin, 2) cell-penetrating peptides, 3) selfassembled or supramolecular vehicles for drug delivery, and 4) peptides with antimicrobial and antitumor properties.29 However, the chemistry of the conjugation of anionic boron clusters and peptides remains unexplored. The main strategy for coupling anionic boron clusters with peptides involves the synthesis of anionic boron cluster derivatives containing linkers with carboxylic groups and conjugating them to the ε-amine group of lysine via amide bonds.30-32 This strategy requires complex, multistep routes for the functionalization of the anionic boron clusters. Thus, synthetic strategies that are simpler and allow modifications of the peptides at the side chains of various amino acid residues are needed. Cyclic oxonium derivatives of anionic boron clusters offer a convenient way to functionalize anionic boron clusters and allow facile conjugation to various functional groups.33, 34 The ringopening reactions of oxonium derivatives of cyclic ethers and polyhedral boron hydrides with nitrogen, oxygen, and sulfur nucleophiles are well recognized and widely used in the bioorganic boron chemistry of low-molecular-weight compounds.33-37 However, to be effective, these reactions must be conducted in anhydrous organic solvents. The necessity of conducting these reactions in anhydrous organic solutions is a major obstacle to the modification of biological macromolecules such as proteins and peptides, which can undergo denaturation and lose their biological activity in such environments. Problems with the solubility of biological macromolecules can also occur. Therefore, applications of this type of boron cluster donor for the modification of biological macromolecules are very limited. We have proposed a solid-state thermal reaction38, 39 for the conjugation of proteins to anionic boron clusters.40, 41 In this reaction, the protein/peptide dissolved in an aqueous buffer is mixed with the 1,4-dioxane-based oxonium derivative of a boron cluster dissolved in DMSO. The mixture is immediately frozen in liquid nitrogen, freeze dried and heated. The solid state protects proteins against the denaturing effect of high temperatures and preserves their biological activity.40, 42 This approach offers an efficient, facile, and straightforward synthetic route for the modification of peptides and proteins with anionic boron clusters. In this study, the cyclic oxonium derivatives of anionic boron clusters and a solid-state thermal reaction were for the first time used to modify the carboxylic groups of peptides. Derivatization of the carboxylic groups enables the preservation of the negative charge at a modification site, as the anionic center of the carboxylate is replaced by an anionic boron cluster with a delocalized negative charge. Furthermore, reactions at the carboxylic groups enable the attachment of anionic boron clusters to the peptide through ester bonds. Ester bonds are labile because they undergo hydrolysis in aqueous solutions and by esterases. For conjugation, we selected thymosin β4 (Tβ4), a therapeutic peptide with pleiotropic activity resulting in enhanced healing and regeneration of injured tissues43, e.g., of the heart after myocardial infarction.44, 45 Tβ4 promotes cell survival, angiogenesis and progenitor cell differentiation and reduces inflammation. Tβ4 has three regions responsible for its activity: the Nterminal tetrapeptide Ac-SDKP, the central actin-binding domain 17-LKKTET-22, and the Cterminal tetrapeptide AGES.43, 46, 47 The pleiotropic activity of Tβ4 enables the study of the 2 ACS Paragon Plus Environment

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individual biological activities of the peptide in various in vitro and in vivo models. Furthermore, Tβ4 has 12 carboxylic groups distributed along its amino acid sequence, which allowed us to determine the influence of the modification site on the stability and activity of Tβ4. Herein, we present peptide-boron cluster conjugates linked via ester bonds with various stabilities. We conjugated the peptide Tβ4 (Fig. 1A) to the 1,4-dioxane-based, zwitterionic, oxonium derivative of dodecaborate anion [B12H11O(CH2CH2)2O]- (Tβ4-[B12H12]2- conjugates) or cobalt bis(1,2-dicarbollide)

[3,3՛-Co(8-O(CH2CH2)2O-1,2-C2B9H10)(1՛,2՛-C2B9H11)]

(Tβ4-[COSAN]-

conjugates) (Fig. 1B and C), which are the two most studied icosahedral anionic boron clusters. The anionic boron clusters were attached via an ether linker to the carboxylic groups of Tβ4 using a solid-state thermal reaction. The ester bonds of the conjugates had various stabilities depending on the amino acid that was modified: the conjugates in which an aspartic acid residue was modified had short half-lives of several hours, and the conjugates in which a glutamic acid residue was modified had long half-lives of several hundred hours. The conjugates were more stable at lower pH values; the half-lives of the conjugates decreased in order of pH: 6.4 > 7.2 > 8.0. Furthermore, native Tβ4 was recovered upon hydrolysis of the conjugate. Finally, the conjugates retained or improved the prosurvival activity of native Tβ4 towards cardiomyocytes.

Table 1. Identification of synthesis products of Tβ4-[B12H12]2- and Tβ4-[COSAN]- conjugates. Mpeptide/conjugate (Da)

Mpeptide/conjugate (Da)

calc./found

calc./found

4963.45/4963.38

-

Tβ4

5192.38/5193.27

228.93/229.89

Tβ4-[B12H12]2singly substituted

5421.30/5423.40

457.85/460.02

Tβ4-[B12H12]2doubly substituted

4963.45/4963.38

-

Tβ4

5374.30/5374.29

410.85/410.91

Tβ4-[COSAN]singly substituted

Peptide/conjugate

First, we optimized the reaction conditions to obtain the highest yields of the singly substituted conjugates. The reactions were conducted using buffers of various pH and ionic strength values and different concentrations of DMSO (Table S1). The pH range was from 5 to 9 to ensure that the carboxylic groups in the peptide are deprotonated and can act as nucleophiles and that the amine groups in the peptide are protonated and cannot act as nucleophiles. The products of the reactions 3 ACS Paragon Plus Environment


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were separated by RP-HPLC (Fig. S1) and identified using LC-MS (Fig. 1D and E, Table 1). The postreaction solutions contained unmodified Tβ4, singly substituted conjugates, and overmodified conjugates. We obtained the highest yields of the singly substituted conjugates when the substrates were dissolved in 20 mM MOPS (3-(N-morpholino)propanesulfonic acid) at pH 6.3 or 7.5 with 20% DMSO (Table S1). Because MOPS has a higher buffer capacity at pH 7.5, we used MOPS pH 7.5 for further syntheses. The reaction between the zwitterionic cyclic oxonium derivatives and Tβ4 did not occur when the reaction mixtures were left in solution and not freeze dried (Table S1, Fig. S1). This observation is in agreement with our previous observations40 and shows that the presence of water molecules, which can act as nucleophiles, is detrimental to the conjugation reaction. Therefore, the reaction in the solid-state is beneficial for providing an environment that supports the conjugation reaction while preserving the structure and biological activity of the peptides.

Fig. 1. The amino acid sequence of thymosin β4 (Tβ4) with carboxylic groups in the peptide marked in red (A). Structures of anionic boron clusters used for conjugation with Tβ4 (B). Scheme for synthesis of Tβ4-boron cluster conjugates (C). Tβ4 dissolved in 20 mM MOPS was mixed with 4 ACS Paragon Plus Environment

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a 1,4-dioxane-based oxonium derivative of boron clusters ([3,3՛-Co(8-O(CH2CH2)2O-1,2C2B9H10)(1՛,2՛-C2B9H11)] or (Bu4N)[B12H11O(CH2CH2)2O]) dissolved in DMSO (final concentration of DMSO in reaction mixture, 20% v/v), freeze dried, and heated at 80°C for 15 min. Mass spectra of singly substituted Tβ4-[B12H12]2- (D) and Tβ4-[COSAN]- (E) conjugates. The singly substituted conjugates were separated according to the site of modification in the amino acid sequence of Tβ4 using RP-HPLC. Tβ4 has 12 carboxylic groups: 3 in side chains of aspartic acid residues (D, β-carboxylic group), 8 in side chains of glutamic acid residues (E, γ-carboxylic group) and 1 at the C-terminus of the peptide (α-carboxylic group) (Fig. 1A). Separation by HPLC allowed us to obtain 10 fractions of singly substituted Tβ4-[B12H12]2- conjugates and 8 fractions of Tβ4-[COSAN]- conjugates (Fig. S2). Next, we digested the conjugates in each fraction using trypsin and identified which amino acid residues were modified using ESI-MS/MS (Table S2, S3, and S4). We confirmed that the boron clusters were selectively attached to carboxylic groups in the peptide. Some of the fractions contained mixtures of the conjugates modified at different sites of the amino acid sequence of Tβ4 because of incomplete separation of the conjugates. For further experiments, we excluded those mixtures and selected fractions containing the conjugates modified at single sites of amino acid sequence of Tβ4. An exception was fractions containing a mixture of two conjugates, one with a modified E42 residue and one with a modified C-terminal carboxylic group: Tβ4-[B12H12]2-(E42/S43) and Tβ4-[COSAN]-(E42/S43). These fractions were also selected for further research to determine the stability of conjugates with modified C-terminal α-carboxylic groups and the influence of a modification in the C-terminal region of Tβ4 on the biological activity of the peptide. Ultimately, we obtained 9 Tβ4-[B12H12]2- conjugates and 5 Tβ4-[COSAN]conjugates for further studies; the names of the individual conjugates include the site of modification. The ester bonds in the conjugates undergo hydrolysis, leading to restoration of native Tβ4 (Fig. 1A, Fig. S3, Table S5). Thus, to determine the stability of Tβ4-[B12H12]2- and Tβ4-[COSAN]conjugates, we incubated them in 0.01 M sodium phosphate with 0.15 M NaCl at 3 different pH values (6.4, 7.2, and 8.0). The content of unmodified Tβ4 released during hydrolysis of the ester bond in the conjugates was monitored using LC-MS (Fig. 2B and C and Fig. S4). The peak areas of the extracted ion chromatograms for Tβ4 and the conjugates were calculated, and the ratios of unmodified Tβ4 to the sum of unmodified Tβ4 and the conjugates were determined. Because Tβ4 is susceptible to oxidation at M6 residue, forming Tβ4 sulfoxide (Tβ4-SO), we also included this derivative of Tβ4 in our calculations (Table S6). The stability of the conjugates was highly dependent on the modified amino acid residue. The conjugates with modified β-carboxylic groups (D) had short half-lives of several hours, and those with modified γ-carboxylic groups (E) had long half-lives of several hundred hours (over 1300 h for Tβ4-[COSAN]-(E37) at pH 7.2 (Fig. 2B and C, Table S7 and S8)). The mixtures of the conjugates (E42/S43) with modified γ- and α-carboxylic groups had half-lives shorter than those with only modified γ-carboxylic groups, which suggests that the attachment of the boron cluster to the C-terminal α-carboxylic group is less stable than that to the γ-carboxylic group of E residues.



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These results show that we can obtain conjugates with dramatically different stabilities depending on which amino acid residue is modified. Tβ4-[B12H12]2- and Tβ4-[COSAN]- conjugates were more stable in acidic environments (Table S7 and S8). Each studied conjugate had its longest half-life when incubated at pH 6.4 and its shortest half-life when incubated at pH 8.0. We observed the same trend for the conjugates modified with both boron clusters. Because the influence of anionic boron clusters on biological systems is not well established, determining the influence of modifications on the toxicity and activity of Tβ4 is of great interest. To assess the toxicity of the conjugates, we incubated rat cardiomyocytes (H9C2 cell line) in normoxic conditions for 48 h in the presence of Tβ4 or the conjugates and measured the viability of cardiomyocytes using the MTS test. The viabilities of the cardiomyocytes incubated in the presence of Tβ4 or the conjugates were similar to that of the control (Fig. S6), which indicates that the conjugates are not toxic toward these cells at the tested concentration (0.2 µM). For studies on the influence of the modification on the activity of Tβ4, we selected the conjugates with [COSAN]- because this boron cluster interacts with various proteins18, 20 and can thus influence interactions between Tβ4 and its binding partners. Tβ4 supports survival of cardiomyocytes in hypoxic conditions after myocardial infarction.48, 49 To assess the activity of the conjugates, we incubated rat cardiomyocytes (H9C2 cell line) under hypoxic conditions for 48 h in the presence of Tβ4 or Tβ4-[COSAN]- conjugates and measured viability using the MTS test. The viabilities of the cardiomyocytes incubated in the presence of Tβ4 or the conjugates were significantly higher than that of the control (Fig. 2D). In addition, the viabilities of the cardiomyocytes incubated in the presence of the conjugates were similar to those incubated with Tβ4, which indicates that the conjugates retained the activity of the native peptide after modification.


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Fig. 2. Schematic diagram of hydrolysis of the ester bond in Tβ4-boron cluster conjugates (A). Hydrolysis

of

Tβ4-[COSAN]-

conjugates

produces

native

Tβ4

and

[3,3՛-Co(8-

(OCH2CH2OCH2CH2OH)-1,2-C2B9H10)(1՛,2՛-C2B9H11)]-; hydrolysis of Tβ4-[B12H12]2- conjugates produces native Tβ4 and [B12H11OCH2CH2OCH2CH2OH]2-. Tβ4 content (%) during incubation of Tβ4-[B12H12]2- (B) or Tβ4-[COSAN]- (C) conjugates in 0.01 M sodium phosphate, pH 7.2, with 0.15 M NaCl at 37°C. The legends show the modification site and half-life (τ1/2) of each conjugate. Viabilities of rat cardiomyocytes (H9C2 cell line) incubated for 48 h under hypoxic conditions (D) or under hypoxic conditions with the addition of 100 µM deferoxamine (DFO) or 100 µM hydrogen 7 ACS Paragon Plus Environment


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peroxide (H2O2) (E) in the presence of Tβ4 and Tβ4-[COSAN]- conjugates. The concentration of Tβ4 and Tβ4-[COSAN]- conjugates was 0.2 µM. The viability of cardiomyocytes was measured (mean ± S.D., n = 3) and compared to the control, which was set as 100% (*, P < 0.05; **, P < 0.01; vs. control (ANOVA)). Only conjugates that significantly increased cardiomyocyte viability compared to the control are shown. For clarity, the names of the conjugates are abbreviated in the figure captions of B, C, D and E by omitting Tβ4-[B12H12]2- or Tβ4-[COSAN]-, and indicating only the modification sites. We also incubated cardiomyocytes under hypoxic conditions with the addition of deferoxamine (DFO) or hydrogen peroxide (H2O2). DFO is a chelator of iron and was used to simulate iron deficiency; H2O2 was used to simulate oxidative stress, which is present at the site of inflammation. Under hypoxic conditions with the addition of DFO, the viability of cardiomyocytes incubated in the presence of Tβ4 were not significantly different than that of the control (Fig. 2E). However, we observed a significant increase of the viabilities of the cardiomyocytes incubated in the presence of Tβ4-[COSAN]-(E21), Tβ4-[COSAN]-(E32) and Tβ4-[COSAN]-(E42/S43) conjugates. Similar observations were made for hypoxic conditions with the addition of H2O2: the presence of Tβ4 did not significantly affect the viability of the cardiomyocytes, whereas Tβ4-[COSAN]-(E37) and Tβ4[COSAN]-(E42/S43) conjugates significantly increased the viability of the cardiomyocytes (Fig. 2E). These results show that modification with [COSAN]- can increase the biological activity of Tβ4. For both conditions, conjugates modified at C-terminus of the peptide (Tβ4-[COSAN](E42/S43)) showed improved activity. The C-terminal tetrapeptide of Tβ4, AGES, is responsible for the beneficial activity of Tβ4 in the hypoxic heart after myocardial infarction.47 Thus, modifications in this region may have the greatest influence on the activity of Tβ4 in the biological model used in this study. Herein, we describe the first example of the application of the cyclic ether ring opening of the cyclic oxonium derivative of boron clusters for the modification of carboxylate groups (a weak nucleophile) in a peptide, which allows the preservation of the anionic character of the modification site. Previously, carboxylate groups of low-molecular-weight compounds or inorganic nanomaterials have been used to attach anionic boron clusters.50, 51 Notably, in our studies, the reaction conditions were optimized to selectively modify carboxylate groups in the peptide despite the presence of stronger nucleophiles, such as the amine groups of lysines. Because anionic boron clusters have a broad range of potential applications as modifying entities for peptides, the development of novel methods for modifying various amino acid residues may facilitate and accelerate the application of anionic boron clusters in this area. Reactions with carboxylate groups lead to the formation of ester bonds with dramatically different stabilities depending on which amino acid residue is modified. The conjugates with modified aspartic acid residues are less stable than the conjugates with modified glutamic acid residues. Attachment of boron clusters to peptides via ester bonds that can be hydrolyzed in aqueous solutions and by esterases provides more flexibility for tuning the pharmacological properties of the conjugates. The unique properties of anionic boron clusters can be used to adjust parameters critical for the biological activity of therapeutic peptides at both molecular and systemic levels. Attachment of 8 ACS Paragon Plus Environment

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[COSAN]- to tumor-selective peptide [F7,P34]-NPY did not affect the affinity but improved the selectivity of the peptide for its receptor.30 We observed differences between Tβ4 and Tβ4[COSAN]- conjugates regarding their biological activity in our in vitro studies of cardiomyocytes incubated under hypoxic conditions with the addition of DFO or H2O2. Tβ4 has a pleiotropic activity that results from interactions with various binding partners and has three domains that are responsible for its activity. Therefore, we might expect that conjugates with different modification sites will differentially affect Tβ4 activity in various in vitro models. Furthermore, [COSAN]- has a high affinity for serum albumin20 and can serve as an anchor to albumin for therapeutic peptides, which may influence the pharmacokinetic profile of Tβ4 by extending its half-life in the circulation. Because Tβ4-[COSAN]- conjugates can have dramatically different hydrolysis rates (leading to restoration of native Tβ4), we can prepare Tβ4-[COSAN]- conjugates with the desired therapeutic activity in vivo. Nonetheless, further studies are needed to establish the effectiveness of this approach. Anionic boron clusters have been conjugated to biomolecules for several decades. First, they were simply used as a source of boron for antitumor boron neutron capture therapy (BNCT).52-55 Currently, the main focus of this field is on the exploitation of the extraordinary properties of anionic boron clusters in the development of new low-molecular-weight drugs.56, 57 Due to the properties of anionic boron clusters, they are of great interest in the development of analogs of therapeutic peptides with improved pharmacological profiles, which may become an important area of application of anionic boron clusters in medicine in the near future. ASSOCIATED CONTENT Supporting Information The Supporting Information is available free of charge on the ACS Publications website. Detailed experimental procedures, Tables S1 – S8 and Figures S1 – S6 (PDF)

AUTHOR INFORMATION Corresponding author E-mail: [email protected] ORCID Tomasz M. Goszczyński: 0000-0002-9995-3260 Krzysztof Fink: 0000-0002-1238-9653 Notes The authors declare no competing financial interests.

ACKNOWLEDGEMENTS 9 ACS Paragon Plus Environment


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The authors are grateful to the National Science Centre, Poland, Grant No. 2015/19/N/NZ7/02726 and Grant No. 2016/23/D/NZ1/02611 for partial financial support. The contribution from the Statutory Fund of HIIET PAS is also gratefully acknowledged. Synthetic thymosin β4 was a kind gift from RegeneRx Biopharmaceuticals Inc. (Rockville, MD, USA).

References (1) (2) (3) (4) (5)

(6) (7) (8)

(9) (10) (11) (12) (13)

Masalles, C., Borrós, S., Viñas, C., and Teixidor, F. (2000) Are low-coordinating anions of interest as doping agents in organic conducting polymers? Adv. Mater. 12, 1199-1202. Fanfrlik, J., Lepsik, M., Horinek, D., Havlas, Z., and Hobza, P. (2006) Interaction of carboranes with biomolecules: formation of dihydrogen bonds. ChemPhysChem 7, 11001105. Juárez‐Pérez, E. J., Núñez, R., Viñas, C., Sillanpää, R., and Teixidor, F. (2010) The role of C-H∙∙∙H-B interactions in establishing rotamer configurations in metallabis(dicarbollide) systems. Eur. J. Inorg. Chem. 2010, 2385-2392. Grimes, R. N. (2016) Carboranes, Academic Press, London. Housecroft, C. E. (2011) Boron: Metallacarbaboranes. Encyclopedia of Inorganic and Bioinorganic Chemistry (Wells, S. A., Sartbaeva, A., Kuznetsov, V.L., Edwards, P.P., Nowakowski, A., Karim, M., Petering, D., Rais, D., Vilar, R., Hollingsworth, J. A., et al., Eds.), John Wiley & Sons. Brusselle, D., Bauduin, P., Girard, L., Zaulet, A., Viñas, C., Teixidor, F., Ly, I., and Diat, O. (2013) Lyotropic lamellar phase formed from monolayered θ-shaped carborane-cage amphiphiles. Angew. Chem. Int. Ed. 52, 12114-12118. Fernandez-Alvarez, R., Ďorďovič, V., Uchman, M., and Matějíček, P. (2018) Amphiphiles without head-and-tail design: nanostructures based on the self-assembly of anionic boron cluster compounds. Langmuir 34, 3541-3554. Gassin, P. M., Girard, L., Martin-Gassin, G., Brusselle, D., Jonchère, A., Diat, O., Viñas, C., Teixidor, F., and Bauduin, P. (2015) Surface activity and molecular organization of metallacarboranes at the air-water interface revealed by nonlinear optics. Langmuir 31, 2297-2303. Viñas, C., Tarrés, M., González-Cardoso, P., Farràs, P., Bauduin, P., and Teixidor, F. (2014) Surfactant behaviour of metallacarboranes. A study based on the electrolysis of water. Dalton Trans. 43, 5062-5068. Assaf, K. I., Nau, W. M. (2018) The chaotropic effect as an assembly motif in chemistry. Angew. Chem. Int. Ed. 57, 13968-13981. Plešek, J., Baše, K., Mareš, F., Hanousek, F., Štibr, B., and Heřmánek, S. (1984) Potential uses of metallocarborane sandwich anions for analysis, characterization and isolation of various cations and organic-bases. Collect. Czech. Chem. Commun. 49, 2776-2789. Farràs, P., Juárez-Pérez, E. J., Lepšík, M., Luque, R., Núñez, R., and Teixidor, F. (2012) Metallacarboranes and their interactions: theoretical insights and their applicability. Chem. Soc. Rev. 41, 3445-3463. Gabel, D., Awad, D., Schaffran, T., Radovan, D., Dărăban, D., Damian, L., Winterhalter, M., Karlsson, G., and Edwards, K. (2007) The anionic boron cluster (B12H11SH)2- as a means to trigger release of liposome contents. ChemMedChem 2, 51-53. 10 ACS Paragon Plus Environment

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(14) (15) (16) (17)

(18)

(19) (20) (21)

(22) (23)

(24) (25) (26)

(27)

Awad, D., Damian, L., Winterhalter, M., Karlsson, G., Edwards, K., and Gabel, D. (2009) Interaction of Na2B12H11SH with dimyristoyl phosphatidylcholine liposomes. Chem. Phys. Lipids 157, 78-85. Awad, D., Bartok, M., Mostaghimi, F., Schrader, I., Sudumbrekar, N., Schaffran, T., Jenne, C., Eriksson, J., Winterhalter, M., Fritz, J., et al. (2015) Halogenated dodecaborate clusters as agents to trigger release of liposomal contents. ChemPlusChem 80, 656-664. Uchman, M., Jurkiewicz, P., Cigler, P., Grüner, B., Hof, M., Procházka, K., and Matejicek, P. (2010) Interaction of fluorescently substituted metallacarboranes with cyclodextrins and phospholipid bilayers: fluorescence and light scattering study. Langmuir 26, 6268-6275. Verdiá-Báguena, C., Alcaraz, A., Aguilella, V. M., Cioran, A. M., Tachikawa, S., Nakamura, H., Teixidor, F., and Viñas, C. (2014) Amphiphilic COSAN and I2-COSAN crossing synthetic lipid membranes: planar bilayers and liposomes. Chem. Commun. 50, 6700-6703. Cigler, P., Kozisek, M., Rezácová, P., Brynda, J., Otwinowski, Z., Pokorná, J., Plesek, J., Grüner, B., Dolecková-Maresová, L., Mása, M., et al. (2005) From nonpeptide toward noncarbon protease inhibitors: metallacarboranes as specific and potent inhibitors of HIV protease. Proc. Natl. Acad. Sci. U. S. A. 102, 15394-15399. Rezácová, P., Pokorná, J., Brynda, J., Kozisek, M., Cigler, P., Lepsik, M., Fanfrlik, J., Rezác, J., Grantz Sasková, K., Sieglová, I., et al. (2009) Design of HIV protease inhibitors based on inorganic polyhedral metallacarboranes. J. Med. Chem. 52, 7132-7141. Goszczyński, T. M., Fink, K., Kowalski, K., Leśnikowski, Z. J., and Boratyński, J. (2017) Interactions of boron clusters and their derivatives with serum albumin. Sci. Rep. 7, 9800. Kuperman, M. V., Losytskyy, M. Y., Bykov, A. Y., Yarmoluk, S. M., Zhizhin, K. Y., Kuznetsov, N. T., Varzatskii, O. A., Gumienna-Kontecka, E., and Kovalska, V. B. (2017) Effective binding of perhalogenated closo-borates to serum albumins revealed by spectroscopic and ITC studies. J. Mol. Struct. 1141, 75-80. Assaf, K. I., Ural, M. S., Pan, F., Georgiev, T., Simova, S., Rissanen, K., Gabel, D., and Nau, W. M. (2015) Water structure recovery in chaotropic anion recognition: high-affinity binding of dodecaborate clusters to γ-cyclodextrin. Angew. Chem. Int. Ed. 54, 6852-6856. Eyrilmez, S. M., Bernhardt, E., Dávalos, J. Z., Lepšík, M., Hobza, P., Assaf, K. I., Nau, W. M., Holub, J., Oliva-Enrich, J. M., Fanfrlik, J., et al. (2017) Binary twinned-icosahedral [B21H18]- interacts with cyclodextrins as a precedent for its complexation with other organic motifs. Phys. Chem. Chem. Phys. 19, 11748-11752. Assaf, K. I., Suckova, O., Al Danaf, N., von Glasenapp, V., Gabel, D., and Nau, W. M. (2016) Dodecaborate-functionalized anchor dyes for cyclodextrin-based indicator displacement applications. Org. Lett. 18, 932-935. Assaf, K. I., Hennig, A., Peng, S., Guo, D. S., Gabel, D., and Nau, W. M. (2017) Hierarchical host-guest assemblies formed on dodecaborate-coated gold nanoparticles. Chem. Commun. 53, 4616-4619. Viñas, C., Fuentes, I., Garcia-Mendiola, T., Sato, S., Pita, M., Nakamura, H., Lorenzo, E., Teixidor, F., and Marques, F. (2018) Metallacarboranes on the road to anticancer therapies: cellular uptake, DNA interaction and biological evaluation of cobaltabisdicarbollide ([COSAN]-). Chem. – Eur. J., doi: 10.1002/chem.201803178 Tarrés, M., Canetta, E., Viñas, C., Teixidor, F., and Harwood, A. J. (2014) Imaging in living cells using νB-H Raman spectroscopy: monitoring COSAN uptake. Chem. Commun. 50, 3370-3372. 11 ACS Paragon Plus Environment


(28)

(29) (30) (31)

(32) (33)

(34) (35) (36)

(37)

(38) (39) (40) (41) (42)

Page 12 of 17

Zheng, Y.., Liu, W., Chen, Y., Jiang, H., Yan, H., Kosenko, I., Chekulaeva, L., Sivaev, I., Bregadze, V., and Wang, X. (2017) A highly potent antibacterial agent targeting methicillin-resistant Staphylococcus aureus based on cobalt bis(1,2-dicarbollide) alkoxy derivative. Organometallics 36, 3484-3490. Goszczyński, T. M., Fink, K., and Boratyński, J. (2018) Icosahedral boron clusters as modifying entities for biomolecules. Expert Opin. Biol. Ther. 18, 205-213. Frank, R., Ahrens, V. M., Boehnke, S., Beck-Sickinger, A. G., and Hey-Hawkins, E. (2016) Charge-compensated metallacarborane building blocks for conjugation with peptides. ChemBioChem 17, 308-317. Kimura, S., Masunaga, S., Harada, T., Kawamura, Y., Ueda, S., Okuda, K., and Nagasawa, H. (2011) Synthesis and evaluation of cyclic RGD-boron cluster conjugates to develop tumor-selective boron carriers for boron neutron capture therapy. Bioorg. Med. Chem. 19, 1721-1728. Betzel, T., Hess, T., Waser, B., Reubi, J. C., and Roesch, F. (2008) closo-borane conjugated regulatory peptides retain high biological affinity: synthesis of closo-borane conjugated Tyr(3)-octreotate derivatives for BNCT. Bioconjugate Chem. 19, 1796-1802. Sivaev, I. B., Semioshkin, A. A., Brellochs, B., Sjöberg, S., and Bregadze, V. I. (2000) Synthesis of oxonium derivatives of the dodecahydro-closo-dodecaborate anion [B12H12]2. Tetramethylene oxonium derivative of [B12H12]2- as a convenient precursor for the synthesis of functional compounds for boron neutron capture therapy. Polyhedron 19, 627632. Semioshkin, A. A., Sivaev, I. B., and Bregadze, V. I. (2008) Cyclic oxonium derivatives of polyhedral boron hydrides and their synthetic applications. Dalton Trans., 977-992. Wojtczak, B. A., Andrysiak, A., Grüner, B., and Lesnikowski, Z. J. (2008) "Chemical ligation": a versatile method for nucleoside modification with boron clusters. Chem. – Eur. J. 14, 10675-10682. Semioshkin, A., Laskova, J., Ilinova, A., Bregadze, V., and Lesnikowski, Z. J. (2011) Reactions of oxonium derivatives of [B12H12]2- with sulphur nucleophiles. Synthesis of novel B12-based mercaptanes, sulfides and nucleosides. J. Organomet. Chem. 696, 539543. Teixidor, F., Pedrajas, J., Rojo, I., Viñas, C., Kivekäs, R., Sillanpää, R., Sivaev, I., Bregadze, V., and Sjöberg, S. (2003) Chameleonic capacity of [3,3'-Co(1,2-C2B9H11)2]- in coordination. Generation of the highly uncommon S(thioether)-Na bond. Organometallics 22, 3414-3423. Boratyński, J., and Roy, R. (1998) High temperature conjugation of proteins with carbohydrates. Glycoconjugate J. 15, 131-138. Boratynski, J. (1998) Dry reaction of proteins with carbohydrates at 120 degrees C yields neoglycoconjugates. Biotechnol. Tech. 12, 707-710. Goszczyński, T. M., Kowalski, K., Leśnikowski, Z. J., and Boratyński, J. (2015) Solid state, thermal synthesis of site-specific protein-boron cluster conjugates and their physicochemical and biochemical properties. Biochim. Biophys. Acta 1850, 411-418. Kowalski, K., Goszczyński, T., Leśnikowski, Z. J., and Boratyński, J. (2015) Synthesis of lysozyme-metallacarborane conjugates and the effect of boron cluster modification on protein structure and function. ChemBioChem 16, 424-431. Kanska, U., Omar, M. S., Budzynska, R., Nevozhay, D., Jagiello, M., Opolski, A., and Boratynski, J. (2005) Antileukemic activity of glycated fibrinogen-methotrexate conjugates. Anticancer Res. 25, 2229-2234. 12 ACS Paragon Plus Environment

Page 13 of 17 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


(43) (44) (45) (46) (47)

(48) (49) (50) (51) (52) (53)

(54) (55) (56) (57)

Goldstein, A. L., and Kleinman, H. K. (2015) Advances in the basic and clinical applications of thymosin β4. Expert Opin. Biol. Ther. 15, S139-145. Rossdeutsch, A., Smart, N., and Riley, P. R. (2008) Thymosin β4 and Ac-SDKP: tools to mend a broken heart. J. Mol. Med. 86, 29-35. Smart, N., Bollini, S., Dubé, K. N., Vieira, J. M., Zhou, B., Riegler, J., Price, A. N., Lythgoe, M. F., Davidson, S., Yellon, D., et al. (2012) Myocardial regeneration: expanding the repertoire of thymosin β4 in the ischemic heart. Ann. N. Y. Acad. Sci. 1269, 92-101. Sosne, G., Qiu, P., Goldstein, A. L., and Wheater, M. (2010) Biological activities of thymosin β4 defined by active sites in short peptide sequences. FASEB J. 24, 2144-2151. Hinkel, R., Ball, H. L., DiMaio, J. M., Shrivastava, S., Thatcher, J. E., Singh, A. N., Sun, X., Faskerti, G., Olson, E. N., Kupatt, C., et al. (2015) C-terminal variable AGES domain of thymosin β4: the molecule's primary contribution in support of post-ischemic cardiac function and repair. J. Mol. Cell. Cardiol. 87, 113-125. Bock-Marquette, I., Saxena, A., White, M. D., DiMaio, J. M., and Srivastava, D. (2004) Thymosin β4 activates integrin-linked kinase and promotes cardiac cell migration, survival and cardiac repair. Nature 432, 466-472. Peng, H. M., Xu, J., Yang, X. P., Dai, X., Peterson, E. L., Carretero, O. A., and Rhaleb, N. E. (2014) Thymosin-β4 prevents cardiac rupture and improves cardiac function in mice with myocardial infarction. Am. J. Physiol.: Heart Circ. Physiol. 307, H741-H751. Farràs, P., Teixidor, F., Kivekäs, R., Sillanpää, R., Viñas, C., Grüner, B., and Cisarova, I. (2008) Metallacarboranes as building blocks for polyanionic polyarmed aryl-ether materials. Inorg. Chem. 47, 9497-9508. Cabana, L., Gonzàlez-Campo, A., Ke, X., Van Tendeloo, G., Núñez, R., and Tobias, G. (2015) Efficient chemical modification of carbon nanotubes with metallacarboranes. Chem. – Eur. J. 21, 16792-16795. Valliant, J. F., Guenther, K. J., King, A. S., Morel, P., Schaffer, P., Sogbein, O. O., and Stephenson, K. A. (2002) The medicinal chemistry of carboranes. Coord. Chem. Rev. 232, 173-230. Kimura, S., Masunaga, S., Harada, T., Kawamura, Y., Ueda, S., Okuda, K., and Nagasawa, H. (2011) Synthesis and evaluation of cyclic RGD-boron cluster conjugates to develop tumor-selective boron carriers for boron neutron capture therapy. Bioorg. Med. Chem. 19, 1721-1728. Barth, R. F., Coderre, J. A., Vicente, M. G., and Blue, T. E. (2005) Boron neutron capture therapy of cancer: current status and future prospects. Clin. Cancer Res. 11, 3987-4002. Hawthorne, M. F. (1998) New horizons for therapy based on the boron neutron capture reaction. Mol. Med. Today 4, 174-181. Leśnikowski, Z. J. (2016) Challenges and opportunities for the application of boron clusters in drug design. J. Med. Chem. 59, 7738-7758. Viñas, C. (2013) The uniqueness of boron as a novel challenging element for drugs in pharmacology, medicine and for smart biomaterials. Future Med. Chem. 5, 617-619.



Page 14 of 17

Table of Contents Graphic

Synthesis and Biological Activity of Thymosin β4-Anionic Boron Cluster Conjugates

Krzysztof Fink, Kamil Kobak, Monika Kasztura, Janusz Boratyński and Tomasz M. Goszczyński


Page 15 of 17

A

1 2 H C 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 Intens. x104 32 336 34 354 36 372 38 39 400600 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60 3

O HN

COO (β) S1


Thymosin β4 (Tβ4)

D2

COO (γ)

COO (β) D5

P4

K3

M6

E8

A7

COO (γ) P27

K25

N26

L28

E24

Q23

COO (γ)

P29 S30

E32

K31

T33

I34

E10

I9

F12

K11

E21

K14

B

=

S15 K19

T20

COO (γ)

COO (γ)

E35

E37

Q36

B

B

BORON CLUSTER

D13

COO (γ) T22

B

COO (β)

COO (γ)

Boron Clusters

K18

L17

2

B

B

B

B

B

B

B

or

B B

Co

B B

B

B

B

B

B

B

B

B B

B

B

B B

B

K16

B

B

B

B B

B

K38

Q39

A40

G41

cobalt bis(1,2-dicarbollide)

COO (γ) E42

[COSAN]-

O S43

dodecaborate

[B12H12]2-

O

= B or BH = CH

Thermal reaction in solid state

C

1,4-dioxane-based oxonium derivative of a boron cluster

O

O

...

BORON CLUSTER

O

O

...

O

∆T, solid state

...

O

O

BORON CLUSTER

O

...

Τβ4−boron cluster conjugates

Τβ4 Product identification

D

E

Τβ4-[B12H12]2-conjugate 866.465

6+ 866.465

866.298

+MS, 8.6-9.1min #(514-543)

866.797

866.132

Tβ4-[COSAN]- conjugate

Intens. x104

6+ 896.651

8

866.963

6 865.0

7+ 742.826

700

865.5

866.0

866.5

867.0

900

1000

868.0

868.5

897.150 897.317

m/z

895.5

896.0

896.5

897.0

897.5

898.0

898.5

899.0 m/z

4

5+ 1039.555

800

867.5

896.318

7+ 768.703

867.129

+MS, 10.2-10.6min #(607-629)

896.651 896.484 896.983

1100

1200

1300

5+ 1075.779

2

4+ 1299.195 m/z

0 600

700

ACS Paragon Plus Environment

800

900

1000

1100

4+ 1344.472 1200

1300

m/z


Page 16 of 17

Hydrolysis of Tβ4-boron cluster conjugates

A

O

...

O

O

O

H2O, 37°C

...

OH

...

+

O

HO

BORON CLUSTER

O

...

Tβ4-boron cluster conjugates

Intact Tβ4 B

B B

2

B

B

=

BORON CLUSTER

B

B

B

B

B

or

B B

Co

B B

B

B

B

B

B

B

B

B B

B

B

B B

B

B

B

B

B B

B

= CH

= B or BH

Stability of conjugates in aqueous solutions

C

Τβ4-[B12H12]2-conjugates D2, τ1/2=6.8h

Tβ4 content / %

D5, τ1/2=24h D13, τ1/2=3.4h E21, τ1/2=372h E24, τ1/2=418h E32, τ1/2=591h E35, τ1/2=474h

Tβ4-[COSAN]- conjugates

100

Tβ4 content / %

B

D5, τ1/2=8.8h E21, τ1/2=546h E32, τ1/2=803h

50

E37, τ1/2=1329h E42/S43, τ1/2=172h

E37, τ1/2=836h E42/S43, τ1/2=192h 50

100

150

200

0

0

100

200

300

400

t/h

t/h

Biological activity of Tβ4-[COSAN]- conjugates

E **

**

150

100

50

C

T

5

D

E

E

32

ns

** **

*

*

100

50

l

ro

43

t on

21

*

150

0

β4

E4

l ro

E3 7

0

ns

t on

C

ACS Paragon Plus Environment

4

Tβ

1 2 E2 E3

l

ro

t on

C

4

Tβ

7 2/ S4 3

**

E3

**

H2O2

E4

ns

Cell viability / %

*

DFO

200

3

200

Hypoxia

S4

Hypoxia

E4 2/

D

Cell viability / %

O

BORON CLUSTER

2/ S

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19100 20 21 22 23 24 50 25 26 27 28 29 0 0 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

ester bonds

τ = 9 - 1329 h Page 1,4-dioxane-based 17 of 17 Bioconjugate Chemistry 1/2

oxonium darivative of a boron cluster

...

B

B

B

Co

O

B

B

B

B

B

B

B

B B

B

B

B

B

B

B

B B

B

+

g

e dryin 1) freez ng 2) heati

biologically active conjugates Hypoxia

2 00

OH

Cell viability / %

O

...

DFO ns

*

H2O2

** **

ns

*

*

7

B

43

B

4

B

E3

O

B

B

Co

B

...

B B

O

O

B

1 50

ACS ... Paragon Plus Environment 50

E4 2/S

l ro

Tβ

on t C

1

E3 2 E4 2/S 43

E2

Tβ 4

0

on tr ol

Thymosin β4 (Tβ4)

1 00

C

1 2 3 4 5 6 7 8

B

B

B

B

B

O

B

B

B

B

B

B

B

B

B

O

Synthesis and Biological Activity of Thymosin Î²4 ... - ACS Publications

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