Next Article in Journal
The Influence of Stress and Binge-Patterned Alcohol Drinking on Mouse Skeletal Muscle Protein Synthesis and Degradation Pathways
Next Article in Special Issue
Generation of Rapid and High-Quality Serum by Recombinant Prothrombin Activator Ecarin (RAPClot™)
Previous Article in Journal
Alpha-Melanocyte-Stimulating Hormone Maintains Retinal Homeostasis after Ischemia/Reperfusion
Previous Article in Special Issue
Advancements in the Application of Ribosomally Synthesized and Post-Translationally Modified Peptides (RiPPs)
 
 
Search for Articles:
Title / Keyword
Author / Affiliation / Email
Journal
Article Type
 
 
Advanced Search
 
Section
Special Issue
Volume
Issue
Number
Page
 
Journals
Biomolecules
Volume 14
Issue 5
10.3390/biom14050526
biomolecules-logo
Submit to this Journal Review for this Journal Propose a Special Issue
► Article Menu

Article Menu

share Share announcement Help format_quote Cite question_answer Discuss in SciProfiles thumb_up
...
Endorse textsms
...
Comment
first_page
settings
Order Article Reprints
Font Type:
Arial Georgia Verdana
Font Size:
Aa Aa Aa
Line Spacing:
Column Width:
Background:
Article

Synthesis of the Antimicrobial Peptide Murepavadin Using Novel Coupling Agents

by
Júlia García-Gros
1,
Yolanda Cajal
2,3,
Ana Maria Marqués
4 and
Francesc Rabanal
1,*
1
Section of Organic Chemistry, Department of Inorganic and Organic Chemistry, Faculty of Chemistry, Universitat de Barcelona, 08028 Barcelona, Spain
2
Department of Pharmacy, Pharmaceutical Technology and Physical Chemistry, Faculty of Pharmacy and Food Sciences, Universitat de Barcelona, 08028 Barcelona, Spain
3
Institute of Nanoscience and Nanotechnology (IN2UB), Universitat de Barcelona, 08028 Barcelona, Spain
4
Laboratory of Microbiology, Faculty of Pharmacy and Food Sciences, Universitat de Barcelona, 08007 Barcelona, Spain
*
Author to whom correspondence should be addressed.
Biomolecules 2024, 14(5), 526; https://doi.org/10.3390/biom14050526
Submission received: 5 April 2024 / Revised: 18 April 2024 / Accepted: 23 April 2024 / Published: 27 April 2024
(This article belongs to the Special Issue Development, Characterization, and Application of Bioactive Peptides, Diagnostic Biomarkers, and Pharmaceutical Proteins)
Browse Figures
Versions Notes

Abstract

:
The problem of antimicrobial resistance is becoming a daunting challenge for human society and healthcare systems around the world. Hence, there is a constant need to develop new antibiotics to fight resistant bacteria, among other important social and economic measures. In this regard, murepavadin is a cyclic antibacterial peptide in development. The synthesis of murepavadin was undertaken in order to optimize the preparative protocol and scale-up, in particular, the use of new activation reagents. In our hands, classical approaches using carbodiimide/hydroxybenzotriazole rendered low yields. The use of novel carbodiimide and reagents based on OxymaPure® and Oxy-B is discussed together with the proper use of chromatographic conditions for the adequate characterization of peptide crudes. Higher yields and purities were obtained. Finally, the antimicrobial activity of different synthetic batches was tested in three Pseudomonas aeruginosa strains, including highly resistant ones. All murepavadin batches yielded the same highly active MIC values and proved that the chiral integrity of the molecule was preserved throughout the whole synthetic procedure.

1. Introduction

Peptides are a class of chemical compounds that offer immense potential as therapeutic tools. These biomolecules are highly selective and effective, as they can bind to specific cell surface receptors and trigger intracellular effects while being relatively safe and well tolerated. Consequently, there is a rising interest in therapeutic peptides, and nowadays, more than eighty peptide drugs have already been approved worldwide [1,2,3]. Since the introduction of solid-phase peptide synthesis (SPPS) by Bruce Merrifield in 1963 [4], this field has experienced a boost in research and development [5]. However, scale-up and production are still complex issues during pharmaceutical development. In fact, small chemical optimization may imply great improvements in yields and purities due to the repetitive nature of the peptide assembly process [3].
In recent decades, a vast arsenal of coupling reagents has been developed for peptide bond formation [6,7,8], including aminium salts of benzotriazoles, such as 2-(1H-benzotriazole-1-yl)-1,1,3,3-tetramethyluronium hexafluorophosphate (HBTU) [9]; phosphonium salts, such as (Benzotriazol-1-yloxy)tris(dimethylamino)phosphonium hexafluorophosphate (BOP) or (Benzotriazol-1-yloxy)tripyrrolidinophosphonium hexafluorophosphate (PyBOP) [10], and carbodiimides, among others. Carbodiimides, in fact, are still the compounds of choice because of their lower cost and good performance in non-hindered peptide bond formation in comparison with more complex reagents.
For solution-phase peptide synthesis, N-ethyl-N′-(3-(dimethylamino)propyl)carbodiimide (EDC) is commonly used because its urea is highly soluble in aqueous solutions. On the other hand, N,N′-diisopropylcarbodiimide (DIC) is preferred in SPPS because the generated urea byproduct is soluble in organic solvents, while the urea of dicylohexylcarbodiimide (DCC) is prone to precipitation. It is well known, though, that carbodiimides can induce a certain degree of epimerization on the alpha-C of the activated acid group [6]. For this reason, additives, such as 1-hydroxybenzotriazole (HOBt) or 1-hydroxy-7-azabenzotriazole (HOAt) [11], are commonly used. These reagents reduce the high reactivity of O-acylisourea by forming intermediate active esters and inhibit side-reactions, such as racemization, through oxazolones or the formation of N-acylureas [12].
Unfortunately, additives, such as HOBt, can induce a certain premature cleavage of the peptide chains, especially when using acid-labile trityl-based resins, because of their acidic nature [13]. Moreover, HOBt and HOAt are classified in the Class I category according to the U.S. Department of Transportation (DOT) and United Nations (UN) guidelines due to their explosive character, and hence, their use is restricted [14]. In this sense, ethylcyano(hydroxyimino)acetate (OxymaPure, first described in 1973 by Itoh [15]) was introduced and was shown to perform better than HOBt in terms of yield and epimerization, and more importantly, it also showed better thermal stability [16]. Later, other oxyma-based compounds were developed, including the COMU reagent ((1-cyano-2-ethoxy-2-oxoethylideneaminooxy)-dimethyl-morpholino-carbeniumhexafluorophosphate [17]) or (Z)-ethyl 2-cyano-3-hydroxyacrylate potassium salt (K-Oxyma) [18], among others (Figure 1). The latter is the potassium salt of OxymaPure, a more appropriate choice when dealing with highly acid-labile resins due to the lack of the acid N-hydroxy proton (Figure 1) [18].
However, the combination of DIC with OxymaPure in dimethylformamide (DMF) at 20 °C was demonstrated to lead to the formation of oxadiazole and the harmful volatile HCN [19]. In order to avoid this drawback, the reaction of OxymaPure with carbodiimides was studied. It was found that different sterically hindered N,N′-ditertbutylcarbodiimide (DTBC) containing tertiary carbon groups did not produce either oxadiazole or HCN but showed poor reactivity. Also, the primary substituted carbodiimide EDC did not evolve into the formation of these undesirable products but showed nonproductive consumption. In this sense, a hybrid carbodiimide-based reagent, N,N′-tertbutylethylcarbodiimide (TBEC), which ensures an HCN-free reaction, was finally found to be the best reagent [20,21,22].
The problem of antimicrobial resistance is becoming a daunting challenge for human society and healthcare systems around the world. Hence, there is a constant need to develop new antibiotics to fight resistant bacteria, among other important social and economic measures. Our group has been involved in the preparation of cyclic peptide antibiotics to improve their therapeutic window and study their mechanisms of action [5,23,24,25,26,27,28,29]. The purpose of this study is to evaluate and test novel coupling reagents to optimize the synthesis of murepavadin, a novel antimicrobial peptide in clinical development, which is highly active against Pseudomonas aeruginosa, an infectious bacterial agent considered a serious threat to human health [30].
Murepavadin (Figure 2), previously known as POL7080, is an antimicrobial peptidomimetic first-in-class of the Outer Membrane Protein Targeting Antibiotics (OMPTA), developed initially by Polyphor Ltd. (and now in the portfolio of Basilea Pharmaceutica) [31]. It is a fully synthetic non-branched cyclic peptide composed of 14 amino acids with a sequence related to that of the membranolytic host-defense peptide protegrin-I (PG-I), which contains a β-hairpin conformation stabilized by a dPro-Pro turn dipeptide [32,33]. This cyclic peptide binds to the lipopolysaccharide transport protein D (LptD), an outer membrane (OM) protein involved in the lipopolysaccharide (LPS) biogenesis in Gram-negative bacteria. It inhibits the LPS transport function of LptD, altering the OM and finally causing cell death [34,35]. Murepavadin exhibits a specific and potent bactericidal activity in vitro and in vivo against Pseudomonas aeruginosa [36,37,38]. Unfortunately, the phase III trials of intravenous murepavadin in hospitalized patients with pneumonia showed an unexpectedly high incidence of acute kidney injury. Currently, a clinical phase I trial evaluating an inhaled form of the antibiotic is being evaluated for the treatment of respiratory diseases, particularly cystic fibrosis and non-cystic fibrosis bronchiectasis [39,40].

2. Materials and Methods

2.1. Chemicals

2-Chlorotrityl chloride resin (2-CTC), trityl chloride resin (Cl-Trt), N-fluorenylmethoxycarbonyl (Fmoc)-protected amino acids, trifluoroacetic acid (TFA), and N-hydroxybenzotriazole (HOBt) were purchased from Fluorochem (Hadfield, UK) and Iris Biotech GmbH (Marktredwitz, Germany). N,N′-diisopropylcarbodiimide was from Thermo Fisher Scientific (Waltham, MA, USA). TBEC and Oxyma-B were given by Luxembourg Bio Technologies. Acetonitrile (ACN) HPLC gradient grade was purchased from Labkem (Barcelona, Spain). All chemicals were of the highest available purity. The solvents used were HPLC grade, except for the water, which was doubly distilled and deionized (Milli-Q system, Millipore Corp., Burlington, MA, USA).

2.2. Synthesis of K-Oxy-B

Oxy-B (1 g, 5.4 mmol) was dissolved in 40 mL of H2O. Then, 5.4 mL of KOH 1 M was slowly added until pH 7 was reached. The mixture was lyophilized, and 1.177 g of a purple/brownish solid (yield of 97%) was obtained. MS analysis: m/z = [M]− = 184. HPLC analysis: purity of >99%, with relative absorption max. at 235 nm, using a Kinetex® (Phenomenex, Torrance, CA, USA) reversed-phase column (4.6 × 250 mm) of a 5 μm particle diameter and a pore size of 100 Å, with 5–95% gradient of 0.036% TFA/ACN and 0.045% TFA/H2O over 30 min, flow = 1 mL·min−1 (tR = 8.401 min).

2.3. Peptide Synthesis

Manual solid-phase peptide synthesis was performed following a Fmoc/tBu protection strategy in polypropylene syringes fitted with a polyethylene disc, which was attached to a vacuum system for the rapid removal of solvents and any excess of reagents. 2-CTC or Cl-Trt resins (1.67 mmol·g−1, 250 mg) were washed with dichloromethane (DCM), DMF, and DCM (2 × 4 mL each). After resin conditioning, 2 eq of Fmoc-Dab(Boc)-OH was added along with 4 eq of DIPEA and the minimum quantity of DCM. The first coupling was left to react for 3 h at room temperature. After that, the resin was washed with DMF and DCM, capped with MeOH (0.8 mL·g−1resin) for 20 min, and washed again with DCM. The loading of the first amino acid residue was assessed by Fmoc quantification. The peptide sequence was assembled by the addition of the Fmoc-protected amino acid (3 eq), coupling reagent (3 eq), additive (3 eq), and the minimum quantity of DMF for 1 h. All couplings yielded ≥99%, as assessed by the Kaiser test. The Fmoc removal was achieved by successive treatments with 20% piperidine/DMF (1 × 1 min, 2 × 10 min). Cleavage of the peptide from the resin was carried out by two successive treatments with hexafluoroisopropanol (HFIP)/DCM (1:4, v/v, 3 mL·g−1 resin) for 1 h. The crude protected peptide was obtained after solvent evaporation under reduced pressure. For cyclization, the resulting solid was dissolved in DMF (2.5 mg·mL−1), and 3 eq HATU, 3 eq HOBt, and 6 eq DIPEA were added. The reaction mixture was stirred, monitored by a ninhydrin test, and after 18 h, the DMF was removed under high vacuum. To remove any excess of coupling agents, the crude product was dissolved in DCM and extracted with ACN/H2O (1:9, v/v). The protected peptide was isolated after solvent evaporation. The crude was treated with trifluoroacetic acid (TFA)/triisopropylsilane (TIPS)/H2O (95:3:2, v/v/v) during 90 min with stirring. Then, the reaction mixture was precipitated in ice-cold diethyl ether (70 mL), centrifuged, and washed two more times with anhydrous Et2O. The lyophilized peptide was dissolved in ACN/H2O (1:1, v/v, 100 mg·mL−1), purified by semipreparative HPLC, and characterized by analytical HPLC and ESI mass spectrometry.

2.4. Minimum Inhibitory Concentration Determination

Minimum inhibitory concentrations (MICs) of the different murepavadin batches were determined by following the Clinical and Laboratory Standards Institute (CLSI) guidelines. Bacteria used in this study were standard isolates from the American Type Culture Collection (ATCC) and German Collection of Microorganisms (DSM) (P. aeruginosa ATCC 27853, P. aeruginosa DSM 24600, and DSM 25716). Sterile microtiter plates (96 wells of 100 μL) were filled with 50 μL of cation-adjusted Mueller–Hinton broth (MHB) culture medium. Serial 2-fold dilutions of the peptides were arranged in rows ranging from 8 to 0.016 μg·mL−1. The last two columns were used as positive and negative controls, respectively. The bacterial suspensions prepared with an optical density at 600 nm (OD600) of 0.2 were diluted 100-fold, and 50 μL of the resulting suspension was added to each well (excluding the negative control). This resulted in a final concentration of approximately 5 × 105 CFU·mL−1. The plates were incubated at 37 °C for 18–20 h, and the MICs were determined. The MIC was defined as the lowest concentration of the antimicrobial agent at which the visible growth of bacteria was prevented. Each determination was carried out in triplicate. To be considered acceptable, the three MIC results have to differ in only one well, and the result is always given as the higher of the three.

3. Results and Discussion

3.1. Murepavadin Synthesis

The synthesis of murepavadin is not described in detail in the literature. The preparation of closely related peptides (i.e., POL7001, L27-11, and L26-19) followed a general Fmoc/tBu scheme of protection, and cyclization was performed between residues Pro11 (C-terminal end) and Thr10 (N-terminal, Figure 29) [38,41]. A different approach has been described using a native chemical ligation (NCL)/desulfurization methodology. In this approach, the cyclization site was the Dab-Ala amide bond, as an Ala amino acid is needed for this strategy. Cyclization took place in 30 min and reached a yield of 70% [42].
In our case, we decided to cyclize through the potentially less hindered peptide bond to reduce the risk of epimerization and facilitate amide formation. We avoided β-branched amino acids (Ile, Thr(tBu)) and, in general, other tBu- or Boc- side chain protected amino acids. The chosen bond for cyclization was the amide between Dab and Ala, obtaining yields between 89 and 96%, as described below. Hence, the first amino acid to be attached to the resin was the corresponding Fmoc-Dab(Boc)-OH (Dab1, as shown in Figure 2).
Overall, the synthesis of murepavadin was undertaken by SPPS following a Fmoc/tBu scheme of protection on the trityl-type of resins using several acylating agents, as indicated in Table 1 [38]. After the assembly of the linear protected peptide sequence on resin, a mild acidolysis using HFIP/DCM (1:4) was performed in order to obtain the partially protected peptide. The cyclization of the lineal crude was achieved with 3 eq O-(7-azabenzotriazol-1-yl)-1,1,3,3-tetramethyl-uronium hexafluorophosphate (HATU), 3 eq HOBt, and 6 eq N,N-diisopropylethylamine (DIPEA). After cyclization, the crude protected peptide was treated with an acidolysis reagent composed of TFA/TIPS/H2O (95:3:2, v/v/v) to obtain the totally deprotected cyclic peptide (Scheme 1). The purity of the fully deprotected peptide was assessed by HPLC by the integration of peaks at 220 nm.
Two different trityl-based solid supports (2-CTC and Cl-Trt) were evaluated (Figure 3). In the conditions used, we found that the synthesis performed using 2-CTC resin (Table 1, synthesis A) yielded higher loading in comparison to Cl-Trt (Table 1, synthesis C). Dealing with highly functionalized resins can sometimes be disadvantageous, especially when treating with difficult peptide sequences, as they can lead to uncomplete acylation and make re-coupling of the amino acids necessary [43,44]. However, in the present case, the assembly was quite straightforward, and only a few re-couplings were needed in some of the syntheses performed. According to the results, 2-CTC seems to be a better option than Cl-Trt in terms of loading and overall yield (Table 1 and Table 2). Therefore, 2-CTC was the resin of choice in this study.
When HOBt was used as the additive in carbodiimide (DIC) acylations, a low resin detachment yield was observed in both cases (entries A and C, Table 1). This result could be expected according to the literature since the use of HOBt has been described to cause premature peptide cleavage during assembly on 2-CTC resin due to its acidic character [13]. Similarly, OxymaPure (Luxembourg Bio Technologies, Ness Ziona, Israel) has also been reported to cause the same problem of peptide losses. For this reason, we decided to perform the synthesis using K-Oxyma (a conjugated base of OxymaPure) instead of HOBt or OxymaPure [18]. The resin detachment yield increased from 46 to 91% on the 2-CTC resin (entry B).
We then decided to test the novel protocol involving TBEC. Originally, the TBEC protocol was developed to avoid the formation of oxadiazole and HCN due to the reaction of DIC and OxymaPure [21,22]. In order to avoid losses during the assembly, we used TBEC in combination with K-Oxyma and the newly described additives Oxy-B and its conjugate base K-Oxy-B [18,21,45,46]. According to the literature, the use of K-Oxyma prevents premature peptide detachment from 2-chlorotrityl resin. Our results show that both Oxy-B and K-Oxy-B additives performed satisfactorily from this point of view. All syntheses rendered comparable detachment yields, ranging from 89 to 93% (entries D to F), and much better than the HOBt-based assemblies and similar to DIC/K-Oxyma. This fact could be attributed to the lower acidic character of Oxy-B (pKa 8.20) in comparison with the higher acidity of HOBt and OxymaPure (the pKa for both is 4.60) [20]. Finally, it can be seen that both Oxy-B and K-Oxy-B performed similarly well. Therefore, there is no apparent need to prepare the corresponding conjugate base of Oxy-B.
In all syntheses, after the acidolysis of the peptidyl resins, the linear protected peptide was cyclized. Macrolactamization was performed in solution using HATU, HOBt, and DIPEA (3:3:6 mole ratio) according to the literature [38]. The cyclization yields ranged from 89 to 96%. It is worth mentioning that this reaction was monitored by ninhydrin since both the linear and cyclic protected peptides did not elute by HPLC using ACN/H2O mixtures.
Total deprotection was achieved by treatment with TFA/TIPS/H2O (95:3:2, v/v/v%) to obtain the cyclic deprotected peptide. After precipitation in anhydrous diethyl ether and isolation of the crude peptides, the yields ranged from 83 to 91%.
The assessment of the purity of the different batches was not a straightforward task. As mentioned before, murepavadin contains a dPro-Pro moiety within the sequence. It is well known that proline residues may lead to conformational equilibria due to the possibility of significant populations of both the cis and trans conformers [47,48]. When assessing purity, we sometimes found out that two peaks with the same m/z ratio were obtained by HPLC-MS (Figure 4d,g). However, if the samples (previously lyophilized) were pre-heated to 50 °C for 30 min or the chromatography was performed using an oven at 50 °C, the coalescence of peaks took place into a single major peak (Figure 4e,f,h). In all cases, the purity was determined by the integration of peaks at 220 nm corresponding to a m/z ratio of a 777.9 mass unit, which corresponds to the [M+2H+]+2 ion, as determined by ESI-MS.
The final overall crude yields ranged from 70 to 80% in all cases except those involving the use of HOBt (30–40%). Crude purities went from 58 to 67%. Hence, the use of K-Oxyma, Oxy-B, and K-Oxy-B yielded similar good results. The highest purity (67%) was obtained for the combination of TBEC/Oxy-B in the 2-CTC resin, as shown in Table 1 and Figure 4.
After purification by semipreparative HPLC, all the murepavadin batches were obtained with a purity higher than 99%. As seen in Table 2, global yields of synthesis A and C were clearly affected by premature loss during the peptide assembly due to the acidic nature of HOBt. In comparison, the syntheses performed using K-Oxyma, Oxy-B, and K-Oxy-B rendered much higher yields (Table 2 and Syntheses B, D, E, and F). As expected, the higher yields and purities obtained for the crudes were reflected in the global yield after purification. Oxy-B is as effective as its conjugate base; hence, there is no need to prepare the potassium salt of Oxy-B (K-Oxy-B) in clear contrast to the pair OxymaPure/K-Oxyma described in the literature. In our hands, the best yield for the total synthesis of murepavadin in the 2-CTC resin was obtained for the acylating agents TBEC/Oxy-B.

3.2. Antimicrobial Activity Determination of the Different Murepavadin Batches

As mentioned before, murepavadin exhibits potent activity against P. aeruginosa by targeting the periplasmatic β-jellyroll domain of the LptD protein of this bacteria. It is well known that drug–protein interactions are stereospecific, and studies have shown that murepavadin exerts its antipseudomonal activity by means of a mechanism of action involving a chiral receptor [49,50]. In this sense, we wanted to confirm the chiral integrity of the murepavadin purified batches by assessing their in vitro antibacterial activity. Antimicrobial activity was determined in terms of minimal inhibitory concentrations (MICs) by the broth dilution method and following the CLSI guidelines.
All murepavadin batches were tested against three different P. aeruginosa strains. Two of them (DSM 24600 [51] and DSM 25716 [52]) were resistant to ß-lactam antibiotics. P. aeruginosa DSM 24600 produces extended-spectrum ß-lactamase (ESBL) and is carbapenem-resistant (Genotype blaVIM-1). P. aeruginosa DSM 25716 is resistant to carbapenems (i.e., imipenem) as it expresses a VIM ß-lactamase but it does not produce extended-spectrum ß-lactamases (ESBL).
As seen in Table 3, this cyclic peptide exerts a potent antipseudomonal activity, with P. aeruginosa DSM 24600 being the most susceptible strain. The MIC values are in the range of those found in the literature (0.008–0.25 μg·mL−1) [36,38]. No differences were observed between the different batches, which indicates that the chiral integrity of the molecule was preserved in the different syntheses of murepavadin.

4. Conclusions

The preparation of murepavadin, a complex macrocyclic peptide antibiotic, was successfully achieved. Its assembly was accomplished using different resins and coupling reagents. Regarding the resin, 2-CTC was preferred over Cl-Trt, as the loading obtained with this solid support was higher, and the peptide assembly was quite straightforward. When using HOBt in combination with the standard carbodiimide DIC for peptide assembly, low detachment yields were obtained (41–46%), indicating premature cleavage of the peptide chains, probably due to the acidic nature of this additive. The cleavage yield was improved (91%) when the conjugate base of OxymaPure, K-Oxyma, was used with DIC. Also, different oxyma-based additives were used in combination with a relatively new carbodiimide, TBEC, and proved to be as effective as the system DIC/K-Oxyma in terms of cleavage yield (89–93%). The use of K-Oxyma/DIC, Oxy-B/TBEC, and K-Oxy-B/TBEC yielded similar good yields and purities. Oxy-B is as effective as its conjugate base; hence, there is no need to prepare the potassium salt of Oxy-B (K-Oxy-B) in clear contrast to the pair OxymaPure/K-Oxyma. Altogether, the best yield for the total synthesis of murepavadin was obtained using the 2-CTC resin and the acylating agents TBEC/Oxy-B. Chromatographic elution by HPLC at a high temperature (50 °C or pre-heating samples at 50 °C) was necessary for the correct assessment of murepavadin crude purity.
Finally, the antimicrobial activity of the different synthetic batches was tested in three P. aeruginosa strains, including highly resistant varieties that consisted of two carbapenem-resistant strains and an ESBL producer. All murepavadin batches yielded the same MIC values, which demonstrates that the chiral integrity of the molecule was preserved throughout the whole synthetic procedure.

Supplementary Materials

The following supporting information can be downloaded at: https://www.mdpi.com/article/10.3390/biom14050526/s1, Table S1: Quantities obtained in each step of the synthesis of murepavadin for the different batches; Figure S1: HPLC and MS analysis of K-Oxy-B; Figures S2–S7: HPLC and MS analysis of murepavadin batches A to F.

Author Contributions

Conceptualization, F.R. and Y.C.; investigation, J.G.-G. and A.M.M.; writing—original draft preparation, J.G.-G. and F.R.; writing—review and editing, F.R., J.G.-G., A.M.M., and Y.C.; funding acquisition, F.R. All authors have read and agreed to the published version of the manuscript.

Funding

This research was funded by the AEI of the Ministry of Science, Innovation and Universities [European project Muryxin, JPI-AMR-2022-066 DRUID call, PCI2023-143362, and PID2021-124342OB-I00]. J.G-G discloses support for the execution of her doctoral thesis from the Agència de Gestió d’Ajuts Universitaris i de Recerca (FISDU 00155, Generalitat de Catalunya) and Grup de Recerca de la Generalitat de Catalunya (2021 SGR 00288).

Institutional Review Board Statement

Not applicable.

Informed Consent Statement

Not applicable.

Data Availability Statement

Dara are contained within the article and Supplementary Materials.

Acknowledgments

We want to thank Josep Martí (Section of Organic Chemistry, UB) for his invaluable support and advice with the ESI-MS analyses. We also acknowledge the generous gift of TBEC and Oxy-B from Luxembourg Biotechnologies.

Conflicts of Interest

The authors declare no conflicts of interest.

References

  1. Lau, J.L.; Dunn, M.K. Therapeutic Peptides: Historical Perspectives, Current Development Trends, and Future Directions. Bioorg. Med. Chem. 2018, 26, 2700–2707. [Google Scholar] [CrossRef] [PubMed]
  2. Al Musaimi, O.; Al Shaer, D.; Albericio, F.; de la Torre, B.G. 2020 FDA TIDES (Peptides and Oligonucleotides) Harvest. Pharmaceuticals 2021, 14, 145. [Google Scholar] [CrossRef] [PubMed]
  3. Wang, L.; Wang, N.; Zhang, W.; Cheng, X.; Yan, Z.; Shao, G.; Wang, X.; Wang, R.; Fu, C. Therapeutic Peptides: Current Applications and Future Directions. Signal. Transduct. Target. Ther. 2022, 7, 48. [Google Scholar] [CrossRef] [PubMed]
  4. Merrifield, R.B. Solid Phase Peptide Synthesis. I. The Synthesis of a Tetrapeptide. J. Am. Chem. Soc. 1963, 85, 2149–2154. [Google Scholar] [CrossRef]
  5. Segovia, R.; Díaz-Lobo, M.; Cajal, Y.; Vilaseca, M.; Rabanal, F. Linker-Free Synthesis of Antimicrobial Peptides Using a Novel Cleavage Reagent: Characterisation of the Molecular and Ionic Composition by NanoESI-HR MS. Pharmaceutics 2023, 15, 1310. [Google Scholar] [CrossRef]
  6. El-Faham, A.; Albericio, F. Peptide Coupling Reagents, More than a Letter Soup. Chem. Rev. 2011, 111, 6557–6602. [Google Scholar] [CrossRef] [PubMed]
  7. Al-Warhi, T.I.; Al-Hazimi, H.M.A.; El-Faham, A. Recent Development in Peptide Coupling Reagents. J. Saudi Chem. Soc. 2012, 16, 97–116. [Google Scholar] [CrossRef]
  8. Albericio, F.; El-Faham, A. Choosing the Right Coupling Reagent for Peptides: A Twenty-Five-Year Journey. Org. Process Res. Dev. 2018, 22, 760–772. [Google Scholar] [CrossRef]
  9. Carpino, L.A.; Imazumi, H.; El-Faham, A.; Ferrer, F.J.; Zhang, C.; Lee, Y.; Foxman, B.M.; Henklein, P.; Hanay, C.; Wenschuh, H.; et al. The Uronium/Guanidinium Peptide Coupling Reagents: Finally the True Uronium Salts. Angew. Chem. Int. Ed. 2002, 41, 441–445. [Google Scholar] [CrossRef]
  10. Coste, J.; Lenguyen, D.; Castro, B. PyBOP®: A New Peptide Coupling Reagent Devoid of Toxic by-Product. Tetrahedron. Lett. 1990, 31, 205–208. [Google Scholar] [CrossRef]
  11. Carpino, L.A.; El-Faham, A.; Minorb, C.A.; Albericio, F. Advantageous Applications of Azabenzotriazole (Triazolopyridine)-Based Coupling Reagents to Solid-Phase Peptide Synthesis. J. Chem. Soc. Chem. Commun. 1994, 201–203. [Google Scholar] [CrossRef]
  12. Carpino, L.A. 1-Hydroxy-7-Azabenzotriazole. An Efficient Peptide Coupling Additive. J. Chem. Soc. Chem. Commun. 1970, 103, 858. [Google Scholar] [CrossRef]
  13. Spengler, J.; Fernandez-Llamazares, A.I.; Albericio, F. Use of an Internal Reference for the Quantitative HPLC-UV Analysis of Solid-Phase Reactions: A Case Study of 2-Chlorotrityl Chloride Resin. ACS Comb. Sci. 2013, 15, 229–234. [Google Scholar] [CrossRef]
  14. Wehrstedt, K.D.; Wandrey, P.A.; Heitkamp, D. Explosive Properties of 1-Hydroxybenzotriazoles. J. Hazard. Mater. 2005, 126, 1–7. [Google Scholar] [CrossRef] [PubMed]
  15. Itoh, M. Racemization Suppression by the Use of Ethyl 2-Hydroximino-2-Cyanoacetate and Its Amide. Bull. Chem. Soc. Jpn. 1973, 46, 2219–2221. [Google Scholar] [CrossRef]
  16. Subirós-Funosas, R.; Prohens, R.; Barbas, R.; El-Faham, A.; Albericio, F. Oxyma: An Efficient Additive for Peptide Synthesis to Replace the Benzotriazole-Based HOBt and HOAt with a Lower Risk of Explosion. Chem.—A Eur. J. 2009, 15, 9394–9403. [Google Scholar] [CrossRef]
  17. El-Faham, A.; Albericio, F. COMU: A Third Generation of Uronium-Type Coupling Reagents. J. Pept. Sci. 2010, 16, 6–9. [Google Scholar] [CrossRef] [PubMed]
  18. Cherkupally, P.; Acosta, G.A.; Nieto-Rodriguez, L.; Spengler, J.; Rodriguez, H.; Khattab, S.N.; El-Faham, A.; Shamis, M.; Luxembourg, Y.; Prohens, R.; et al. K-Oxyma: A Strong Acylation-Promoting, 2-Ctc Resin-Friendly Coupling Additive. Eur. J. Org. Chem. 2013, 28, 6372–6378. [Google Scholar] [CrossRef]
  19. McFarland, A.D.; Buser, J.Y.; Embry, M.C.; Held, C.B.; Kolis, S.P. Generation of Hydrogen Cyanide from the Reaction of Oxyma (Ethyl Cyano(Hydroxyimino)Acetate) and DIC (Diisopropylcarbodiimide). Org. Process. Res. Dev. 2019, 23, 2099–2105. [Google Scholar] [CrossRef]
  20. Manne, S.R.; Sharma, A.; Sazonovas, A.; El-Faham, A.; de la Torre, B.G.; Albericio, F. Understanding OxymaPure as a Peptide Coupling Additive: A Guide to New Oxyma Derivatives. ACS Omega 2022, 7, 6007–6023. [Google Scholar] [CrossRef]
  21. Manne, S.R.; Akintayo, D.C.; Luna, O.; El-Faham, A.; De La Torre, B.G.; Albericio, F. Tert-Butylethylcarbodiimide as an Efficient Substitute for Diisopropylcarbodiimide in Solid-Phase Peptide Synthesis: Understanding the Side Reaction of Carbodiimides with OxymaPure. Org. Process. Res. Dev. 2022, 26, 2894–2899. [Google Scholar] [CrossRef]
  22. Manne, S.R.; Luna, O.; Acosta, G.A.; Royo, M.; El-Faham, A.; Orosz, G.; De La Torre, B.G.; Albericio, F. Amide Formation: Choosing the Safer Carbodiimide in Combination with OxymaPure to Avoid HCN Release. Org. Lett. 2021, 23, 6900–6904. [Google Scholar] [CrossRef] [PubMed]
  23. Rabanal, F.; Grau-Campistany, A.; Vila-Farrés, X.; Gonzalez-Linares, J.; Borràs, M.; Vila, J.; Manresa, A.; Cajal, Y. A Bioinspired Peptide Scaffold with High Antibiotic Activity and Low in Vivo Toxicity. Sci. Rep. 2015, 5, 10558. [Google Scholar] [CrossRef] [PubMed]
  24. Grau-Campistany, A.; Manresa, Á.; Pujol, M.; Rabanal, F.; Cajal, Y. Tryptophan-Containing Lipopeptide Antibiotics Derived from Polymyxin B with Activity against Gram Positive and Gram Negative Bacteria. Biochim. Biophys. Acta Biomembr. 2016, 1858, 333–343. [Google Scholar] [CrossRef] [PubMed]
  25. Segovia, R.; Solé, J.; Marqués, A.M.; Cajal, Y.; Rabanal, F. Unveiling the Membrane and Cell Wall Action of Antimicrobial Cyclic Lipopeptides: Modulation of the Spectrum of Activity. Pharmaceutics 2021, 13, 2180. [Google Scholar] [CrossRef] [PubMed]
  26. Rabanal, F.; Cajal, Y. Recent Advances and Perspectives in the Design and Development of Polymyxins. Nat. Prod. Rep. 2017, 34, 886–908. [Google Scholar] [CrossRef] [PubMed]
  27. Rudilla, H.; Pérez-Guillén, I.; Rabanal, F.; Sierra, J.M.; Vinuesa, T.; Viñas, M. Novel Synthetic Polymyxins Kill Gram-Positive Bacteria. J. Antimicrob. Chemother. 2018, 73, 3385–3390. [Google Scholar] [CrossRef] [PubMed]
  28. Clausell, A.; Rabanal, F.; Garcia-Subirats, M.; Alsina, M.A.; Cajal, Y. Synthesis and Membrane Action of Polymyxin B Analogues. Luminescence 2005, 20, 117–123. [Google Scholar] [CrossRef] [PubMed]
  29. Clausell, A.; Rabanal, F.; Garcia-Subirats, M.; Alsina, M.A.; Cajal, Y. Membrane Association and Contact Formation by a Synthetic Analogue of Polymyxin B and Its Fluorescent Derivatives. J. Phys. Chem. B 2006, 110, 4465–4471. [Google Scholar] [CrossRef]
  30. Priorization of Pathogens to Guide Discovery, Research and Development of New Antibiotics for Drug-Resistant Bacterial Infections, Including Tuberculosis; World Health Organization: Geneva, Switzerland, 2017.
  31. Martin-Loeches, I.; Dale, G.E.; Torres, A. Murepavadin: A New Antibiotic Class in the Pipeline. Expert Rev. Anti Infect. Ther. 2018, 16, 259–268. [Google Scholar] [CrossRef]
  32. Shankaramma, S.C.; Athanassiou, Z.; Zerbe, O.; Moehle, K.; Mouton, C.; Bernardini, F.; Vrijbloed, J.W.; Obrecht, D.; Robinson, J.A. Macrocyclic Hairpin Mimetics of the Cationic Antimicrobial Peptide Protegrin I: A New Family of Broad-Spectrum Antibiotics. ChemBioChem 2002, 3, 1126–1133. [Google Scholar] [CrossRef]
  33. Robinson, J.A.; Shankaramma, S.C.; Jetter, P.; Kienzl, U.; Schwendener, R.A.; Vrijbloed, J.W.; Obrecht, D. Properties and Structure-Activity Studies of Cyclic β-Hairpin Peptidomimetics Based on the Cationic Antimicrobial Peptide Protegrin I. Bioorg. Med. Chem. 2005, 13, 2055–2064. [Google Scholar] [CrossRef] [PubMed]
  34. Schmidt, J.; Patora-Komisarska, K.; Moehle, K.; Obrecht, D.; Robinson, J.A. Structural Studies of β-Hairpin Peptidomimetic Antibiotics That Target LptD in Pseudomonas sp. Bioorg. Med. Chem. 2013, 21, 5806–5810. [Google Scholar] [CrossRef] [PubMed]
  35. Andolina, G.; Bencze, L.C.; Zerbe, K.; Müller, M.; Steinmann, J.; Kocherla, H.; Mondal, M.; Sobek, J.; Moehle, K.; Malojčić, G.; et al. A Peptidomimetic Antibiotic Interacts with the Periplasmic Domain of LptD from Pseudomonas aeruginosa. ACS Chem. Biol. 2018, 13, 666–675. [Google Scholar] [CrossRef]
  36. Sader, H.S.; Dale, G.E.; Rhomberg, P.R.; Flamm, R.K. Antimicrobial Activity of Murepavadin Tested against Clinical Isolates of Pseudomonas aeruginosa from the United States, Europe, and China. Antimicrob. Agents Chemother. 2018, 62, e00311–e00318. [Google Scholar] [CrossRef]
  37. Werneburg, M.; Zerbe, K.; Juhas, M.; Bigler, L.; Stalder, U.; Kaech, A.; Ziegler, U.; Obrecht, D.; Eberl, L.; Robinson, J.A. Inhibition of Lipopolysaccharide Transport to the Outer Membrane in Pseudomonas aeruginosa by Peptidomimetic Antibiotics. ChemBioChem 2012, 13, 1767–1775. [Google Scholar] [CrossRef]
  38. Srinivas, N.; Jetter, P.; Ueberbacher, B.J.; Werneburg, M.; Zerbe, K.; Steinmann, J.; Van Der Meijden, B.; Bernardini, F.; Lederer, A.; Dias, R.L.A.; et al. Peptidomimetic Antibiotics Target Outer-Membrane Biogenesis in Pseudomonas aeruginosa. Science 2010, 327, 1010–1013. [Google Scholar] [CrossRef]
  39. Díez-Aguilar, M.; Hernández-García, M.; Morosini, M.I.; Fluit, A.; Tunney, M.M.; Huertas, N.; Del Campo, R.; Obrecht, D.; Bernardini, F.; Ekkelenkamp, M.; et al. Murepavadin Antimicrobial Activity against and Resistance Development in Cystic Fibrosis Pseudomonas aeruginosa Isolates. J. Antimicrob. Chemother. 2021, 76, 984–992. [Google Scholar] [CrossRef]
  40. Spexis Reports Solid Safety and Pharmacokinetics Results from First-in-Human Study with Inhaled Murepavadin, a Novel Macrocycle Compound. Available online: https://www.globenewswire.com/news-release/2023/01/09/2584785/0/en/Spexis-reports-solid-safety-and-pharmacokinetics-results-from-first-in-human-study-with-inhaled-murepavadin-a-novel-macrocycle-compound.html (accessed on 16 April 2024).
  41. Obrecht, D.; Luther, A.; Bernardini, F.; Zbinden, P. Beta–Hairpin Peptidomimetics. U.S. Patent 20180044380A1 2018. [Google Scholar]
  42. Chaudhuri, D.; Ganesan, R.; Vogelaar, A.; Dughbaj, M.A.; Beringer, P.M.; Camarero, J.A. Chemical Synthesis of a Potent Antimicrobial Peptide Murepavadin Using a Tandem Native Chemical Ligation/Desulfurization Reaction. J. Org. Chem. 2021, 86, 15242–15246. [Google Scholar] [CrossRef] [PubMed]
  43. Mueller, L.K.; Baumruck, A.C.; Zhdanova, H.; Tietze, A.A. Challenges and Perspectives in Chemical Synthesis of Highly Hydrophobic Peptides. Front. Bioeng. Biotechnol. 2020, 8, 162. [Google Scholar] [CrossRef]
  44. Isidro-Llobet, A.; Kenworthy, M.N.; Mukherjee, S.; Kopach, M.E.; Wegner, K.; Gallou, F.; Smith, A.G.; Roschangar, F. Sustainability Challenges in Peptide Synthesis and Purification: From R&D to Production. J. Org. Chem. 2019, 84, 4615–4628. [Google Scholar] [CrossRef] [PubMed]
  45. Jad, Y.E.; Khattab, S.N.; De La Torre, B.G.; Govender, T.; Kruger, H.G.; El-Faham, A.; Albericio, F. Oxyma-B, an Excellent Racemization Suppressor for Peptide Synthesis. Org. Biomol. Chem. 2014, 12, 8379–8385. [Google Scholar] [CrossRef] [PubMed]
  46. Jad, Y.E.; Khattab, S.N.; de la Torre, B.G.; Govender, T.; Kruger, H.G.; El-Faham, A.; Albericio, F. EDC·HCl and Potassium Salts of Oxyma and Oxyma-B as Superior Coupling Cocktails for Peptide Synthesis. Eur. J. Org. Chem. 2015, 2015, 3116–3120. [Google Scholar] [CrossRef]
  47. O’Neal, K.D.; Chari, M.V.; Mcdonald, C.H.; Cook, R.G.; Li-Yuan, Y.-L.; Morrisett, J.D.; Shearer, W.T. Multiple Cis-Trans Conformers of the Prolactin Receptor Proline-Rich Motif (PRM) Peptide Detected by Reverse-Phase HPLC, CD and NMR Spectroscopy. Biochem. J. 1996, 315, 833–844. [Google Scholar] [CrossRef]
  48. Sui, Q.; Rabenstein, D.L. Cis/Trans Isomerization of Proline Peptide Bonds in the Backbone of Cyclic Disulfide-Bridged Peptides. Pept. Sci. 2018, 110, e24088. [Google Scholar] [CrossRef]
  49. Upert, G.; Luther, A.; Obrecht, D.; Ermert, P. Emerging Peptide Antibiotics with Therapeutic Potential. Med. Drug Discov. 2021, 9, 100078. [Google Scholar] [CrossRef]
  50. Zerbe, K.; Moehle, K.; Robinson, J.A. Protein Epitope Mimetics: From New Antibiotics to Supramolecular Synthetic Vaccines. Acc. Chem. Res. 2017, 50, 1323–1331. [Google Scholar] [CrossRef]
  51. BacDive The Bacterial Diversity Metadatabase. Pseudomonas aeruginosa DSM 24600. Available online: https://bacdive.dsmz.de/strain/12803 (accessed on 22 February 2024).
  52. BacDive The Bacterial Diversity Metadatabase. Pseudomonas aeruginosa DSM 25716. Available online: https://bacdive.dsmz.de/strain/12805 (accessed on 22 February 2024).
Biomolecules 14 00526 g001
Figure 1. Structures of HOBt and oxyma-based reagents.
Figure 1. Structures of HOBt and oxyma-based reagents.
Biomolecules 14 00526 g001
Biomolecules 14 00526 g002
Figure 2. Structure of murepavadin. The sequence consists of 14 amino acids: cyclo[Ser-dPro-Pro-Thr-Trp-Ile-Dab-Orn-dDab-Dab-Trp-Dab-Dab-Ala].
Figure 2. Structure of murepavadin. The sequence consists of 14 amino acids: cyclo[Ser-dPro-Pro-Thr-Trp-Ile-Dab-Orn-dDab-Dab-Trp-Dab-Dab-Ala].
Biomolecules 14 00526 g002
Biomolecules 14 00526 sch001
Scheme 1. Synthesis of murepavadin.
Scheme 1. Synthesis of murepavadin.
Biomolecules 14 00526 sch001
Biomolecules 14 00526 g003
Figure 3. Trityl-based polystyrene solid supports: trityl chloride resin (left), 2-chlorotrityl chloride resin (right).
Figure 3. Trityl-based polystyrene solid supports: trityl chloride resin (left), 2-chlorotrityl chloride resin (right).
Biomolecules 14 00526 g003
Biomolecules 14 00526 g004
Figure 4. Chromatograms of murepavadin crudes obtained after treating the protected peptide crude with TFA/TIPS/H2O (95:3:2, v/v/v) using different chromatographic conditions: (a) Synthesis D, HPLC 25 °C, (b) Synthesis D, HPLC column T set to 50 °C, (c) Synthesis D, sample pre-heating 50 °C, (d) Synthesis E, HPLC 25 °C, (e) Synthesis E, HPLC column T set to 50 °C, (f) Synthesis E, sample pre-heating 50 °C, (g) Synthesis F, HPLC 25 °C, (h) Synthesis F, HPLC column T set to 50 °C, (i) Synthesis F, sample pre-heating 50 °C.
Figure 4. Chromatograms of murepavadin crudes obtained after treating the protected peptide crude with TFA/TIPS/H2O (95:3:2, v/v/v) using different chromatographic conditions: (a) Synthesis D, HPLC 25 °C, (b) Synthesis D, HPLC column T set to 50 °C, (c) Synthesis D, sample pre-heating 50 °C, (d) Synthesis E, HPLC 25 °C, (e) Synthesis E, HPLC column T set to 50 °C, (f) Synthesis E, sample pre-heating 50 °C, (g) Synthesis F, HPLC 25 °C, (h) Synthesis F, HPLC column T set to 50 °C, (i) Synthesis F, sample pre-heating 50 °C.
Biomolecules 14 00526 g004
Table 1. Yields and purities for each of the syntheses performed (also see Table S1).
Table 1. Yields and purities for each of the syntheses performed (also see Table S1).
SynthesisResinLoadingCoupling AgentsResin Detachment YieldCyclization YieldAcidolysis YieldOverall Crude YieldCrude Purity a
A2-CTC0.86 mmol/g3 eq DIC
3 eq HOBt		46%96%90%40%58%
B2-CTC0.86 mmol/g3 eq DIC
3 eq K-Oxyma		91%93%83%70%64%
CCl-Trt0.45 mmol/g3 eq DIC
3 eq HOBt		41%89%83%30%67%
D2-CTC0.63 mmol/g3 eq TBEC
3 eq K-Oxyma		90%95%88%75%60%
E2-CTC0.69 mmol/g3 eq TBEC
3 eq Oxy-B		89%96%89%76%67%
F2-CTC0.70 mmol/g3 eq TBEC
3 eq K-Oxy-B		93%94%91%80%59%
a The HPLC purities were obtained by integrating the peak areas at 220 nm.
Table 2. Purification and global yields for each of the syntheses performed.
Table 2. Purification and global yields for each of the syntheses performed.
SynthesisResinCoupling AgentsPurification YieldGlobal Yield
A2-CTC3 eq DIC
3 eq HOBt		29%12%
B2-CTC3 eq DIC
3 eq K-Oxyma		40%28%
CCl-Trt3 eq DIC
3 eq HOBt		45%14%
D2-CTC3 eq TBEC
3 eq K-Oxyma		36%27%
E2-CTC3 eq TBEC
3 eq Oxy-B		39%30%
F2-CTC3 eq TBEC
3 eq K-Oxy-B		34%27%
Table 3. MIC values (μg·mL−1) for the different batches of murepavadin.
Table 3. MIC values (μg·mL−1) for the different batches of murepavadin.
ABCDEF
P. aeruginosa
ATCC 27853		0.06250.06250.06250.06250.06250.0625
P. aeruginosa
DSM 24600		0.031250.031250.031250.031250.031250.03125
P. aeruginosa
DSM 25716		0.250.250.250.250.250.25
Disclaimer/Publisher’s Note: The statements, opinions and data contained in all publications are solely those of the individual author(s) and contributor(s) and not of MDPI and/or the editor(s). MDPI and/or the editor(s) disclaim responsibility for any injury to people or property resulting from any ideas, methods, instructions or products referred to in the content.

Share and Cite

MDPI and ACS Style

García-Gros, J.; Cajal, Y.; Marqués, A.M.; Rabanal, F. Synthesis of the Antimicrobial Peptide Murepavadin Using Novel Coupling Agents. Biomolecules 2024, 14, 526. https://doi.org/10.3390/biom14050526

AMA Style

García-Gros J, Cajal Y, Marqués AM, Rabanal F. Synthesis of the Antimicrobial Peptide Murepavadin Using Novel Coupling Agents. Biomolecules. 2024; 14(5):526. https://doi.org/10.3390/biom14050526

Chicago/Turabian Style

García-Gros, Júlia, Yolanda Cajal, Ana Maria Marqués, and Francesc Rabanal. 2024. "Synthesis of the Antimicrobial Peptide Murepavadin Using Novel Coupling Agents" Biomolecules 14, no. 5: 526. https://doi.org/10.3390/biom14050526

APA Style

García-Gros, J., Cajal, Y., Marqués, A. M., & Rabanal, F. (2024). Synthesis of the Antimicrobial Peptide Murepavadin Using Novel Coupling Agents. Biomolecules, 14(5), 526. https://doi.org/10.3390/biom14050526

Note that from the first issue of 2016, this journal uses article numbers instead of page numbers. See further details here.

Article Metrics

Back to TopTop