Next Article in Journal
Effects of l-Carnitine in Patients with Autism Spectrum Disorders: Review of Clinical Studies
Next Article in Special Issue
Convergent Synthesis of Thioether Containing Peptides
Previous Article in Journal
Evidence for Effective Inhibitory Actions on Hyperpolarization-Activated Cation Current Caused by Ganoderma Triterpenoids, the Main Active Constitutents of Ganoderma Spores
Previous Article in Special Issue
Conformation and Dynamics of the Cyclic Lipopeptide Viscosinamide at the Water-Lipid Interface
 
 
Search for Articles:
Title / Keyword
Author / Affiliation / Email
Journal
Article Type
 
 
Advanced Search
 
Section
Special Issue
Volume
Issue
Number
Page
 
Journals
Molecules
Volume 24
Issue 23
10.3390/molecules24234261
molecules-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

Solid-Phase Insertion of N-mercaptoalkylglycine Residues into Peptides

by
Spyridon Mourtas
1,2,
Dimitrios Gatos
1 and
Kleomenis Barlos
1,3,*
1
Department of Chemistry, University of Patras, 26510 Rio Patras, Greece
2
Institute of Chemical Engineering Sciences of the Foundation for Research and Technology Hellas (FORTH/IEC-HT), 26504 Rio Patras, Greece
3
CBL-Patras, Patras Industrial Area, Block 1, 25018 Patras, Greece
*
Author to whom correspondence should be addressed.
Molecules 2019, 24(23), 4261; https://doi.org/10.3390/molecules24234261
Submission received: 1 November 2019 / Revised: 18 November 2019 / Accepted: 20 November 2019 / Published: 22 November 2019
(This article belongs to the Special Issue Cyclic Peptide Analogues and Non-peptide Mimetics)
Browse Figures
Versions Notes

Abstract

:
N-mercaptoalkylglycine residues were inserted into peptides by reacting N-free amino groups of peptides, which were initially synthesized on 2-chlorotrityl resin (Cltr) using the Fmoc/tBu method, with bromoacetic acid and subsequent nucleophilic replacement of the bromide by reacting with S-4-methoxytrityl- (Mmt)/S-trityl- (Trt) protected aminothiols. The synthesized thiols containing peptide–peptoid hybrids were cleaved from the resin, either protected by treatment with dichloromethane (DCM)/trifluoroethanol (TFE)/acetic acid (AcOH) (7:2:1), or deprotected (fully or partially) by treatment with trifluoroacetic acid (TFA) solution using triethylsilane (TES) as a scavenger.

Graphical Abstract

1. Introduction

Peptides as pharmaceutical compounds suffer from unfavorable pharmacokinetics such as slow uptake into cells and rapid proteolytic cleavage, among other factors. N-alkylation of peptides can overcome these limitations, thus, structures such as N-alkylated/methylated peptides [1,2] and N-substituted glycine residues (peptoids) [3,4] are of great importance. Oligomers of N-substituted glycine residues (peptoids) differ from peptides in that the side chains are connected to amide nitrogen atoms rather than carbon atoms (Figure 1). They are classified as unnatural molecules that confer a high degree of resistance to proteolytic degradation, while the absence of any backbone hydrogen bonding means that peptoids exhibit a high degree of conformational flexibility [5,6], while they also present a broad variety of biological activities [7,8,9,10,11,12,13,14,15,16]. They also present bio-nanotechnological and bio-medicinal interest as novel therapeutics [17,18,19,20], while peptide–peptoid hybrids (peptomers) are also interesting structures which mimic biologically active peptides [21,22,23]. All of the aforementioned points have contributed to peptoids increasingly being investigated in the field of medicinal chemistry as an option to replace peptides in the development of new therapeutics [24].
The solid-phase synthesis (SPS) of peptoids was a major breakthrough because it greatly increased the synthetic efficiency, synthesis yields, and available side chain diversity, thereby dramatically reducing time and costs [3]. Since then, several methods have been used for the solid-phase synthesis (SPS) of peptoids [25,26,27,28] including the use of 2-chlorotrityl resin (Cltr) [29,30].
The thiol group is a structural element which is contained in important biologically active compounds, like peptides, proteins, coenzymes (acetyl coenzyme A), and vitamins (biotin). Examples of FDA-approved drugs include the enzyme inhibitors captopril (which is approved as an Angiotensin-Converting Enzyme (ACE) inhibitor for the treatment of hypertension) [31,32] and thiorphan (the active metabolite of the antidiarrheal racecadotril (acetorphan), which prevents the degradation of endogenous enkephalins by acting as an enkephalinase inhibitor) [33], tiopronin (another approved drug against heavy metal poisoning, liver diseases, or cystenuria), N-acetylcysteine (used to treat paracetamol (acetaminophen) overdose and to loosen thick mucus in individuals with cystic fibrosis or chronic obstructive pulmonary disease), and amifostine (a cytoprotective adjuvant used in cancer chemotherapy and radiotherapy) [34], among others.
It is therefore of great interest to develop methods for the solid-phase synthesis (SPS) of thiol-containing peptide–peptoid hybrids in order to mimic cysteine-like structures. For this reason, we prepared S-protected aminothiols, which were linear or derived from naturally occurring aminoacids, and applied using solid-phase peptide synthesis (SPPS), which is the most common method of peptide production [35] by applying common methods of peptoid synthesis.
In general, peptoid submonomers are synthesized using a two-step procedure. The first step is an acylation step with bromoacetic acid, using N,N′-diisopropylcarbodiimide (DIC) as the condensing agent, while the second step is nucleophilic displacement using a monosubstituted amine, resulting in the peptoid submonomer B (Figure 1).
By using S-4-methoxytrityl- (Mmt)/S-trityl- (Trt) protected aminothiols, we envisioned the incorporation of the peptoid submonomer into peptide–peptoid hybrids like molecule C (Figure 1). Solid-phase peptide synthesis (SPPS) using Cltr and fluorenylmethoxycarbonyl (Fmoc)/tert-butyl (tBu) was the selected method, which is now the most commonly used methodology for the preparation of peptides [35]. The use of the S-Mmt and S-Trt protecting groups is of high importance, since S-Mmt derivatives can be removed selectively in the presence of tBu-type protecting groups, thereby enabling selective derivatization via alkylation, pegylation, labeling of the liberated thiol, or synthesis of cystine analogues of peptide–peptoid hybrids after oxidation of the liberated thiols, as examples. Thus, besides the general method for the preparation of the hybrid C, we also provide selected examples for the applicability of our method in the synthesis of the protected or deprotected (fully or partially) thiol-containing peptide–peptoid hybrids, and the preparation of inter-disulfide bridged peptide–peptoid structures based on the oxidation of different thiol groups of peptoid submonomers that are present on the peptide chain.

2. Results and Discussion

Peptoids are usually synthesized using the submonomer synthetic approach consisting of two steps. The first is an acylation step of the available amine group by bromoacetic acid and diisopropylcarbodiimide (DIC) (which acts as an activator of the bromoacetic acid carboxylic group), whilst the second involves nucleophilic displacement using a monosubstituted amine. This procedure results in peptoid structure B (Figure 1). It is of high importance that no racemization was observed by the application of the submonomer method for peptoid synthesis [36,37,38].
By using S-4-methoxytrityl- (Mmt)/S-trityl- (Trt) protected aminothiols, we envisioned the solid-phase synthesis of peptoid–peptide hybrids like molecule C (Figure 1). For this, we first synthesized S-Mmt- and S-Trt-protected derivatives of cysteamine or aminothiols derived from optically active aminoacids [39] by their reaction with trityl- or 4-methoxytrityl chloride in the presence of N,N-diisopropylethylamine (DIPEA) as a base (Scheme 1). The derived S-Mmt/Trt-protected aminothiols were applied in the synthesis of thiol-containing peptide–peptoid hybrids (Figure 1) using solid phase peptide synthesis (SPS) and fluorenylmethoxycarbonyl (Fmoc)/tert-butyl (tBu) methodology and 2-chlorotrityl resin (Cltr), which is now the most commonly used methodology for the production of peptides [38].
For this, resin-bound peptide 1 was assembled on 2-chlorotrityl resin [40] (Scheme 2) using N-Fmoc-amino acids and 1-hydroxy benzotriazole (HOBt)/DIC for their activation. Then, bromoacetic acid was coupled on the N-terminal function of 1 using DIC in N-methyl-2-pyrrolidone (NMP). The obtained bromoacetyl resin-bound peptide 2 was further reacted with the aminothiol 3, which was protected at its thiol function with the acid-sensitive Trt and Mmt groups. In all cases, the nucleophilic replacement of the bromine atom in 2 with the aminothiol 3 was completed within 12 h at room temperature, resulting in the thiol-containing resin-bound peptide 4.
At this step of the proposed synthetic procedure, two options existed. In the first option, acylation of 4 with acetic anhydride (Ac2O), acyl (Ac) chlorides, and aroyl (Ar) chlorides in NMP/DIPEA gave 5 within 2 h at room temperature. Treatment of 5 with dichloromethane (DCM)/trifluoroethanol (TFE)/acetic acid (AcOH) (7:2:1) for 15 min at room temperature gave the acylated mercaptoalkyl peptoid 6. By applying this method, the peptoids 9–12 (Figure 2) were prepared and analyzed by LC-MS (Table 1). The second option included the effective acylation by the coupling reaction of 4 with acids such as bromoacetic acid in NMP using DIC as the condensing agent, thereby enabling the insertion of another peptoid submonomer in the peptide chain or the continuation of peptide synthesis. In the latter case, resin-bound aminothiol containing peptide 4 was chain-elongated by using DIC/HOBt or 2-(1H-Benzotriazole-1-yl)-1,1,3,3-tetramethylaminium hexafluorophosphate (HBTU)/DIPEA/HOBt [41,42], but only in the case of using S-protected cysteamine as the mercaptoalkyl moiety (R1=H). In contrast, the coupling of Fmoc-amino acids with the more steric-hindered aminothiol containing peptide 4 could not be completed under these conditions, regardless of the use of excess amino acid or excess activation reagent. At the end of the synthesis, treatment of 7 with DCM/TFE/AcOH (7:2:1) gave the peptide–peptoid hybrid 8. By applying this method, the peptoids 13–16 (Figure 2) were prepared and analyzed by LC-MS (Table 1). High purities (>88%) were seen using LC-MS analysis for all synthesized derivatives (9–16), as can be seen by the summarized results in Table 1.
Besides bromoacetic acid, coupling with the resin-bound peptide 1 and subsequent replacement of the halogen was also performed using the m- and p-chloromethylbenzoic acid. In one example of this approach, H-Glu(tBu)-2-Cltr resin reacted with p-chloromethylbenzoic acid in the presence of DIC. The derived product was then coupled with Fmoc–Leucine in the presence of HOBt/DIC, and the derived hybrid was cleaved from the resin in its protected form using DCM/TFE/AcOH (7:2:1) to the hybrid 17 (Scheme 3), which was also analyzed by LC-MS (Table 1).
As an example of the HPLC analysis chromatograms of the different products 9–17, we present the synthesis of the S-Mmt-protected peptide–peptoid hybrids 10 and 17. These were obtained after treatment of the resin-bound peptide–peptoid hybrids with DCM/TFE/AcOH (7:2:1), which resulted in the cleavage of the S-Mmt-protected peptide–peptoid hybrids from the resins (Figure 3A,B). In addition, in order to investigate the effect of different acidic conditions, we treated the resin-bound hybrid 18 with 65% trifluoroacetic acid (TFA)/triethylsilane (TES) (95:5) (Scheme 4). Under these conditions, the hybrid was cleaved from the resin, while the S-Mmt and tBu groups were simultaneously removed to form hybrid 19. This was prepared in high purity (Figure 3C).
Disulfides in proteins play an important role in the maintenance of biological activity and conformational stability, thus, their formation is often a crucial final stage in peptide synthesis [43]. For this reason, we were also interested in evaluating the applicability of our method in the synthesis of thiol-bridged peptoid–peptide hybrids. As an example, we synthesized the resin-bound peptide-peptoid hybrid 20 (Scheme 5), which contained two S-Mmt-protected peptoid submonomers and tBu-protected amino acids. This was treated with 1.1% TFA in DCM/TES (95:5) for 5 min at room temperature, thereby enabling the cleavage of the hybrid from the resin and selectively removing the S-Mmt groups, while the tBu-groups remained unaffected.
Via this procedure, the partially tBu-protected hybrid 21 was obtained and further oxidized using dimethyl sulfoxide (DMSO), which is known as a mild oxidant. The progress of the oxidation was followed by LC-MS (Figure 4). As can be seen, the treatment of the resin 20 with 1.1% TFA in DCM/TES (95:5) gave the S-free (tBu-protected) hybrid 21. HPLC analysis of the crude hybrid showed the existence of Mmt-H (identified by ESI-MS), which was the product of the reaction of TES with Mmt cations formed during cleavage/deprotection. Hybrid 21 was further oxidized to the tBu-protected peptide–peptoid hybrid 22. Under these mild conditions, oxidation was completed in 48 h without the formation of any by-products, which was shown by LC-MS analysis (Figure 4B). Moreover, by following the HPLC analysis, we were able to observe the conversion of 21 to 22. Thus, the oxidation of 21 was completed at about 50% in 12 h (Figure 4A), while after 48 h, 21 was completely oxidized to 22 under the applied conditions (Figure 4B). Both products were identified by MS analysis (see Figure 4C for 22).
In order to investigate the selective deprotection of the S-Mmt over S-Trt protecting groups, we studied the gradual removal of the protecting groups of the S-Mmt/S-Trt type. For this reason we prepared the resin-bound hybrid 23 and initially treated it with 1.1% TFA in DCM/TES (95:5) for 5 min at room temperature (Scheme 6).
Under these conditions, the fully S-Mmt/S-Trt-protected hybrid 14 was synthesized in high selectivity, as shown by HPLC analysis (Figure 5A). In addition, the resin-bound hybrid 23 was also treated with 1.1% TFA in DCM/TES (95:5). Under these conditions, the direct cleavage of the hybrid from the resin and removal of the S-Mmt groups enabled the synthesis of the S-free/S-Trt-protected hybrid 24 (Figure 5B). Finally, the resin-bound hybrid 23 was treated with 65% TFA in DCM/TES (95:5), which resulted in the cleavage of the hybrid from the resin and the simultaneous removal of both S-Mmt/S-Trt groups to form the fully unprotected hybrid 25 (Figure 5C).

3. Materials and Methods

3.1. Materials

All chemicals were purchased from Sigma-Aldrich OM, Athens, Greece, except 2-chlorotrityl polystyrene resin (Cltr) and Fmoc-protected amino acids, which were gifted from CBL Patras S.A. (Industrial area of Patras, Building block 1, GR-25018, Patras, Greece). All chemicals were used without further purification.

3.2. Analytical Methods

Thin layer chromatography (TLC) was performed on precoated silica gel 60 F254 plates (Merck, Darmstadt, Germany) and spot detection was carried out by UV light or by charring with a ninhydrin solution. HPLC analysis was performed on a Waters 600E multisolvent delivery system (Milford, MA, USA), combined with Waters 991 photodiode array detector, using the following methods: (A) column: Nucleosil C8, 7 μm, 125 × 4 mm; flow rate: 1 mL/min; gradient: from 50 to 100% acetonitrile in water within 30 min; (B): column: Zorbax SB-C18, 3.5 μm; 2.1 × 30 nm; gradient: from 50% to 100% acetonitrile in water within 15 min, 100% acetonitrile within 15 min or from 20% to 100% acetonitrile in water within 30 min or from 20% to 100% acetonitrile in water within 30 min; flow rate: 0.4 mL/min; (C): Lichrospher RP-8 (4 × 150 mm, 5 μm); gradient: from 20% to 100% acetonitrile in water within 30 min; flow rate: 1 mL/min. All chromatograms were detected at 265 nm. ES-MS spectra were recorded on a Micromass Platform L.C. (Manchester, UK) at 30 V.

3.3. Synthetic Procedures

3.3.1. General Protocol for the Synthesis of S-trityl (Trt) and S-4-methoxytrityl (Mmt) Aminothiols

Cysteamine or aminothiols derived from optically active amino acids (1 mol) [34] were dissolved in the highest possible concentration in dichloromethane (DCM), and diisopropyletheylamine (DIPEA) (2.5 mol) was added. A 1 M solution (with respect to the aminothiol) of trityl chloride (1.2 mol) or 4-methoxytrityl chloride (1.2 mol) in DCM was prepared. This was added dropwise to the stirred aminothiol solution. The completion of the reaction was checked by TLC. The DCM was removed by rotary evaporation and diethylether (DEE) was added to the residual oil. The precipitated solid was filtered, washed with DEE, and dried in vacuo.

3.3.2. Solid-Phase Peptide Assembly, General Protocol

Solid-phase peptide synthesis was carried out manually in plastic syringes equipped with porous polypropylene frits at room temperature using the fluorenylmethoxycarbonyl (Fmoc)/tert-butyl (tBu) strategy [44].

Pre-Activation of Fmoc-Amino Acids

Fmoc-amino acid (4.5 mmol) and 1-hydroxybenzotriazole hydrate (HOBt) (5.8 mmol) were dissolved in N-methyl-2-pyrrolidone (NMP) (4.5 mL) and cooled to 5 °C. Then, diisopropylcarbodiimide (DIC) (5.0 mmol) was added and the mixture was stirred for 15 min at 5 °C.

Coupling

The solution of the pre-activated amino acid was added to the resin-bound amino-acid or peptide or peptide–peptoid hybrid (5.0 g, loading 0.3 mmol/g) and shaken for 3 h at room temperature. A sample was taken to check the reaction completion by Kaiser test. In case of incomplete coupling (positive Kaiser test), recoupling was performed with a fresh solution of activated amino acid (6 mmol).

Fmoc-Group Removal and Fmoc-Removal Test

The resin-bound Fmoc-protected amino acids, peptides, or hybrids were treated twice with 25% piperidine in NMP (25 mL each) for 30 min at room temperature. To check the completion of the Fmoc-removal, 25% piperidine in NMP (20 μL) was added to a resin probe (approximately 2 mg) and the mixture was heated for 5 min at 100 °C. From the resulting solution, 10 μL was spotted on a TLC plate and checked under a UV lamp for UV-absorbing material. Alternatively, the solution was injected on HPLC and the Fmoc-material produced and released into solution was quantified at 265 nm. If the Fmoc-removal test remained positive (violet spot under UV) the piperidine treatment was prolonged or repeated until a negative test was achieved. The resin was filtered and washed with NMP (×5), DCM (×3), and DEE (×2), and dried in vacuo; otherwise, it was used directly in the next step.

3.3.3. General Procedure for the Solid-Phase Synthesis of Bromoacylated Peptides

Haloacid (3-fold molar excess over the resin bound peptide) was dissolved in NMP. The solution was cooled on ice and then DIC (equivalent to 3 moles) was added. The reaction mixture was added to the resin-bound peptide and allowed to react for 1–3 h at 25 °C. The resin was filtered and washed with NMP (×5), DCM (×3), and DEE (×2), and dried in vacuo; otherwise, it was used directly in the next step.

3.3.4. General Procedure for Peptoid Formation

A solution of the S-Mmt and S-Trt aminothiols (2-fold molar excess over the resin bound peptide) in NMP and DIPEA (equivalent to 2.2 moles) was added and the mixture was left to react for 2–12 h at 25 °C. The reaction could be easily followed by TLC and HPLC analysis. Thus 2–3 mg of resin samples were taken and the protected peptides were cleaved from the resin using a 15 min treatment with DCM/trifluoroethanol (TFE)/acetic acid (AcOH) (7:2:1). The resins were then applied to TLC and HPLC analysis. After completion of the synthesis, the resins were extensively washed with NMP (×5), DCM (×3), and DEE (×2), and then dried in vacuo; otherwise, they were used directly in the next step.
The obtained resins were either reacted with acetic anhydride (3 mol), or acyl chlorides (3 mol), or aroyl chlorides (3 mol) in NMP and DIPEA (6 mol) for 1–2 h at 25 °C, or subjected to SPS/SPPS, as previously described. The peptoid formation was easily followed by TLC and HPLC analysis. Thus, 2–3 mg of resin samples were taken and the protected peptides were cleaved from the resin using a 15 min treatment with DCM/TFE/AcOH (7:2:1) and were applied to TLC and HPLC analysis. After completion of the synthesis, the resin was extensively washed with NMP (×5), DCM (×3), and DEE (×2), and then dried in vacuo.

3.3.5. General Procedure for the Cleavage of the Peptide–Peptoid Hybrids from the Resin

Resin (0.5 g) was treated with 5 mL of a mixture of DCM/TFE/AcOH (7:2:1) for 15 min at 25 °C, and washed with DCM (3 × 5 mL). The combined filtrates were concentrated by rotary evaporation. The fully protected crude peptides (S-Mmt/S-Trt/tBu-protected) were precipitated by the addition of DEE, filtered, washed with DEE, and dried in vacuo. Treatment with 1.1% trifluoroacetic acid (TFA) in DCM/triethylsilane (TES) (95:5) gave the S-free (S-Trt/tBu-protected) hybrids (by removal of the S-Mmt protecting group), while treatment with 65% TFA in DCM/TES (95:5) gave the completely unprotected peptides (by removal of the S-Mmt/S-Trt/tBu protecting groups).

3.3.6. General Procedure for the Oxidation of Mercaptoalkyl Peptoid Side Chains

100 mg of resin was treated with 1 mL of 1.1% TFA in DCM/TES (95:5) for 10 min at 25 °C. This treatment resulted in the cleavage of the protected peptide–peptoid hybrid from the resin and the simultaneous removal of the S-Mmt groups. In the case of the S-Trt protecting groups, a solution of 65% TFA in DCM/TES (95:5) for 1 h at 25 °C was necessary for the complete removal of the S-Trt and tBu-type protecting groups. The resin was filtered off and 0.2 mL methanol (MeOH) was added to the filtrates. The mixture was left for 5 min at room temperature and then evaporated. Dimethylsulfoxide (DMSO)/water (H2O) (7:3) was added to the oily residue and the mixture was left at room temperature for 48 h. The progress of the oxidation was followed by HPLC analysis.

4. Conclusions

Peptoids are very important peptide-mimicking compounds, since they can resist proteolytic degradation, thereby retaining a high degree of conformational flexibility. Thiols are very important chemical groups found in drugs and peptides with very important biological activities. Thus, we synthesized thiol-containing peptide–peptoid hybrids using standard methods of solid-phase synthesis (SPS). For this, suitably protected aminothiols (S-4-methoxytrityl (Mmt)/S-trityl (Trt)), linear and derived from amino acids, were used, and peptide synthesis was performed on 2-chlorotrityl resin (Cltr) using the fluorenylmethoxycarbonyl (Fmoc)/tert-butyl (tBu) strategy. This approach allowed for the selective deprotection of the side groups, giving many possibilities regarding peptide synthesis, including (a) the preparation of peptide–peptoid hybrids with selected thiol-free peptoid moieties (which have the ability to act as new nucleophiles) and (b) the synthesis of peptide–peptoid hybrids with inter- and intra-thiol-containing bridges.

Author Contributions

Conceptualization, K.B.; K.B., D.G. and S.M. designed the synthetic methodologies; S.M. performed the experiments; K.B. and S.M. wrote the manuscript. All authors approved the final version of the manuscript.

Funding

This research received no external funding.

Acknowledgments

The authors acknowledge C.B.L. Patras S.A., Patras, Greece, for financial support.

Conflicts of Interest

The authors declare no conflict of interest.

References

  1. Chatterjee, J.; Rechenmacher, F.; Kessler, H. N-Methylation of Peptides and Proteins: An Important Element for Modulating Biological Functions. Angew. Chem. Int. Ed. 2013, 52, 254–269. [Google Scholar] [CrossRef] [PubMed]
  2. Di Gioia, M.L.; Leggio, A.; Malagrinò, F.; Romio, E.; Siciliano, C.; Liguori, A. N-Methylated α-Amino Acids and Peptides: Synthesis and Biological Activity. Mini Rev. Med. Chem. 2016, 16, 683–690. [Google Scholar] [CrossRef] [PubMed]
  3. Zuckermann, R.N.; Kerr, J.M.; Kent, S.B.H.; Moos, W.H. Efficient method for the preparation of peptoids oligo(N-substituted glycines) by submonomer solid-phase synthesis. J. Am. Chem. Soc. 1992, 114, 10646–10647. [Google Scholar] [CrossRef]
  4. Simon, R.J.; Kania, R.S.; Zuckermann, R.N.; Huebner, V.D.; Jewell, D.A.; Banville, S.; Ng, S.; Wang, L.; Rosenberg, S.; Marlowe, C.K. Peptoids: A modular approach to drug discovery. Proc. Natl. Acad. Sci. USA 1992, 89, 9367–9371. [Google Scholar] [CrossRef]
  5. Miller, S.M.; Simon, R.J.; Ng, S.; Zuckermann, R.N.; Kerr, J.M.; Moos, W.H. Comparison of the proteolytic susceptibilities of homologous l-amino acid, d-amino acid, and N-substituted glycine peptide and peptoid oligomers. Drug Dev. Res. 1995, 35, 20–32. [Google Scholar] [CrossRef]
  6. Fowler, S.A.; Blackwell, H.E. Structure-function relationships in peptoids: Recent advances toward deciphering the structural requirements for biological function. Org. Biomol. Chem. 2009, 7, 1508–1524. [Google Scholar] [CrossRef]
  7. Goodman, M.; Bhumralkar, M.; Jefferson, E.A.; Kwak, J.; Locardi, E. Collagen mimetics. Biopolymers 1998, 47, 127–142. [Google Scholar] [CrossRef]
  8. Mas-Moruno, C.; Cruz, L.J.; Mora, P.; Francesch, A.; Messeguer, A.; Pérez-Paya, E.; Albericio, F. Smallest peptoids with antiproliferative activity on human neoplastic cells. J. Med. Chem. 2007, 50, 2443–2449. [Google Scholar] [CrossRef]
  9. Czyzewski, A.M.; McCaig, L.M.; Dohm, M.T.; Broering, L.A.; Yao, L.J.; Brown, N.J.; Didwania, M.K.; Lin, J.S.; Lewis, J.F.; Veldhuizen, R.; et al. Effective in vivo treatment of acute lung injury with helical, amphipathic peptoid mimics of pulmonary surfactant proteins. Sci. Rep. 2018, 8, 6795. [Google Scholar] [CrossRef]
  10. Young, S.C. A Systematic Review of Antiamyloidogenic and Metal-Chelating Peptoids: Two Structural Motifs for the Treatment of Alzheimer’s Disease. Molecules 2018, 23, 296. [Google Scholar] [CrossRef]
  11. Song, D.; Park, H.; Lee, S.H.; Kim, M.J.; Kim, E.J.; Lim, K.M. PAL-12, a new anti-aging hexa-peptoid, inhibits UVB-induced photoaging in human dermal fibroblasts and 3D reconstructed human full skin model, Keraskin-FT™. Arch. Derm. Res. 2017, 309, 697–707. [Google Scholar] [CrossRef] [PubMed]
  12. Gomes Von Borowski, R.; Gnoatto, S.C.B.; Macedo, A.J.; Gillet, R. Promising Antibiofilm Activity of Peptidomimetics. Front. Microbiol. 2018, 9, 2157. [Google Scholar] [CrossRef] [PubMed]
  13. Bhowmik, A.; Basu, M.; Ghosh, M.K. Design, synthesis and use of peptoids in the diagnosis and treatment of cancer. Front. Biosci. (Elite Ed) 2017, 9, 101–108. [Google Scholar] [PubMed]
  14. Mándity, I.M.; Fülöp, F. An overview of peptide and peptoid foldamers in medicinal chemistry. Expert Opin. Drug Discov. 2015, 10, 1163–1177. [Google Scholar] [CrossRef] [PubMed]
  15. Dohm, M.T.; Kapoor, R.; Barron, A.E. Peptoids: Bio-inspired polymers as potential pharmaceuticals. Curr. Pharm. Des. 2011, 17, 2732–2747. [Google Scholar] [CrossRef] [PubMed]
  16. Zuckermann, R.N.; Kodadek, T. Peptoids as potential therapeutics. Curr. Opin. Mol. 2009, 11, 299–307. [Google Scholar]
  17. Sun, J.; Zucherman, R.N. Peptoid Polymers: A Highly Designable Bioinspired Material. Acs Nano 2013, 7, 4715–4732. [Google Scholar] [CrossRef]
  18. Luo, Y.; Song, Y.; Wang, M.; Jian, T.; Ding, S.; Mu, P.; Liao, Z.; Shi, Q.; Cai, X.; Jin, H.; et al. Bioinspired Peptoid Nanotubes for Targeted Tumor Cell Imaging and Chemo-Photodynamic Therapy. Small 2019, 15, e1902485. [Google Scholar] [CrossRef]
  19. Battigelli, A. Design and preparation of organic nanomaterials using self-assembled peptoids. Adv. Sci. 2019, 110, e23265. [Google Scholar] [CrossRef]
  20. Sun, J.; Li, Z. Peptoid applications in biomedicine and nanotechnology (Chapter 7). In Peptide Applications in Biomedicine, Biotechnology and Bioengineering; Koutsopoulos, S., Ed.; Woodhead: Duxford, UK, 2018; pp. 183–213. [Google Scholar]
  21. Schneider, J.A.; Craven, T.W.; Kasper, A.C.; Yun, C.; Haugbro, M.; Briggs, E.M.; Svetlov, V.; Nudler, E.; Knaut, H.; Bonneau, R.; et al. Design of Peptoid-peptide Macrocycles to Inhibit the β-catenin TCF Interaction in Prostate Cancer. Nat. Commun. 2018, 9, 4396. [Google Scholar] [CrossRef]
  22. Olsen, C.A.; Montero, A.; Leman, L.J.; Ghadiri, M.R. Macrocyclic Peptoid–Peptide Hybrids as Inhibitors of Class I Histone Deacetylases. Acs Med. Chem. Lett. 2012, 3, 749–753. [Google Scholar] [CrossRef] [PubMed]
  23. Stawikowski, M.J. Peptoids and peptide-peptoid hybrid biopolymers as peptidomimetics. Methods Mol. Biol. 2013, 1081, 47–60. [Google Scholar] [PubMed]
  24. Patch, J.A.; Kirshenbaum, K.; Seurynck, S.L.; Zuckermann, R.N.; Barron, A.E. The Many Roles of Peptoids in Drug Discovery. In Pseudopeptides in Drug Development; Nielsen, P.E., Ed.; Wiley-VCH: Weinheim, Germany, 2004; pp. 1–31. [Google Scholar]
  25. Culf, A.S.; Ouellette, R.J. Solid-Phase Synthesis of N-Substituted Glycine Oligomers (α-Peptoids) and Derivatives. Molecules 2010, 15, 5282–5335. [Google Scholar] [CrossRef] [PubMed]
  26. Seo, J.; Lee, B.-C.; Zuckermann, R.N. Peptoids: Synthesis, Characterization, and Nanostructures. Compr. Biomater. 2011, 2, 53–76. [Google Scholar]
  27. Tran, H.; Gael, S.L.; Connolly, M.D.; Zuckermann, R.N. Solid-phase submonomer synthesis of peptoid polymers and their self-assembly into highly-ordered nanosheets. J. Vis. Exp. 2011, 57, e3373. [Google Scholar] [CrossRef]
  28. Webster, A.M.; Cobb, S.L. Recent Advances in the Synthesis of Peptoid Macrocycles. Chemistry 2018, 24, 7560–7573. [Google Scholar] [CrossRef]
  29. Barlos, K.; Gatos, D.; Kallitsis, J.; Papaphotiu, G.; Sotiriu, P.; Wenqing, Y.; Schiifer, W. Darstellung geschützter peptid-fragmente unter einsatz substituierter triphenylmethyl-harze. Telrahedron Lett. 1989, 30, 3943–3946. [Google Scholar] [CrossRef]
  30. Lee, B.-C.; Zuckermann, R.N.; Dill, K.A. Folding a Nonbiological Polymer into a Compact Multihelical Structure. J. Am.Chem. Soc. 2005, 127, 10999–11009. [Google Scholar] [CrossRef]
  31. Ondetti, M.A.; Rubin, B.; Cushman, D.W. Design of specific inhibitors of angiotensin-converting enzyme: New class of orally active antihypertensive agents. Science 1977, 196, 441–444. [Google Scholar] [CrossRef]
  32. Odaka, C.; Mizuochi, T. Angiotensin-converting enzyme inhibitor captopril prevents activation-induced apoptosis by interfering with T cell activation signals. Clin. Exp. Immunol. 2000, 121, 515–522. [Google Scholar] [CrossRef]
  33. Llorens, C.; Gacel, G.; Swerts, J.P.; Perdrisot, R.; Fournié-Zaluski, M.C.; Schwartz, J.C.; Roques, B.P. Rational design of enkephalinase inhibitors: Substrate specificity of enkephalinase studied from inhibitory potency of various dipeptides. Biochem. Biophys. Res. Commun. 1980, 96, 1710–1716. [Google Scholar] [CrossRef]
  34. Kouvaris, J.R.; Kouloulias, V.E.; Vlahos, L.J. Amifostine: The first selective-target and broad-spectrum radioprotector. Oncologist 2007, 12, 738–747. [Google Scholar] [CrossRef] [PubMed]
  35. Behrendt, R.; White, P.; Offer, J. Advances in Fmoc solid-phase peptide synthesis. J. Pept. Sci. 2016, 22, 4–27. [Google Scholar] [CrossRef] [PubMed] [Green Version]
  36. Gao, Y.; Kodadek, T. Synthesis, Screening and Hit Optimization of Stereochemically Diverse Combinatorial Libraries of Peptide Tertiary Amides. Chem. Biol. 2013, 20, 360–369. [Google Scholar]
  37. Suwal, S.; Kodadek, T. Solid-phase synthesis of peptoid-like oligomers containing diverse diketopiperazine units. Org. Biomol. Chem. 2014, 12, 5831–5834. [Google Scholar] [CrossRef] [Green Version]
  38. Wender, P.A.; Mitchell, D.J.; Pattabiraman, K.; Pelkey, E.T.; Steinman, L.; Rothbard, J.B. The design, synthesis, and evaluation of molecules that enable or enhance cellular uptake: Peptoid molecular transporters. Proc. Natl. Acad. Sci. USA 2000, 97, 13003–13008. [Google Scholar] [CrossRef] [Green Version]
  39. Mourtas, S.; Katakalou, C.; Nicolettou, A.; Tzavara, C.; Gatos, D.; Barlos, K. Resin-bound aminothiols: Synthesis and application. Tetrahedron Lett. 2003, 44, 179–182. [Google Scholar] [CrossRef]
  40. Barlos, K.; Gatos, D.; Kallitsis, J.; Papaphotiu, G.; Sotiriu, P.; Wenqing, Y.; Schäfer, W. Darstellung geschützter peptid-fragmente unter einsatz substituierter triphenylmethyl-harze. Tetrahedron Lett. 1989, 30, 3943–3946. [Google Scholar] [CrossRef]
  41. El-Faham, A.; Albericio, F. Peptide Coupling Reagents, More than a Letter Soup. Chem. Rev. 2011, 111, 6557–6602. [Google Scholar] [CrossRef]
  42. Joullié, M.M.; Lassen, K.M. Evolution of amide bond formation. Arkivoc 2010, 8, 189–250. [Google Scholar]
  43. Trivedi, M.V.; Laurence, J.S.; Siahaan, T.J. The role of thiols and disulfides on protein stability. Curr. Protein Pept. Sci. 2009, 10, 614–625. [Google Scholar] [CrossRef] [PubMed] [Green Version]
  44. Amblard, M.; Fehrentz, J.-A.; Martinez, J.; Subra, G. Methods and Protocols of Modern Solid-Phase Peptide Synthesis. Mol. Biotechnol. 2006, 33, 239–254. [Google Scholar] [CrossRef]
Sample Availability: Samples of all compounds are available.
Molecules 24 04261 g001 550
Figure 1. Comparison of a peptide (A), a peptoid (B), and the newly synthesized thiol-containing peptide–peptoid structure (C).
Figure 1. Comparison of a peptide (A), a peptoid (B), and the newly synthesized thiol-containing peptide–peptoid structure (C).
Molecules 24 04261 g001
Molecules 24 04261 sch001 550
Scheme 1. Synthesis of S-Mmt/S-Trt aminothiols. R1 = H or the side chain of naturally occurring aminoacids.
Scheme 1. Synthesis of S-Mmt/S-Trt aminothiols. R1 = H or the side chain of naturally occurring aminoacids.
Molecules 24 04261 sch001
Molecules 24 04261 sch002 550
Scheme 2. Method for the synthesis of peptide–peptoid hybrids on 2-chlorotrityl resin. Pr = Mmt, Trt; R1 = H or amino acid side chain; R2 = Ac or Ar.
Scheme 2. Method for the synthesis of peptide–peptoid hybrids on 2-chlorotrityl resin. Pr = Mmt, Trt; R1 = H or amino acid side chain; R2 = Ac or Ar.
Molecules 24 04261 sch002
Molecules 24 04261 g002 550
Figure 2. The synthesized S-Mmt/S-Trt/tBu-protected peptide–peptoid hybrids.
Figure 2. The synthesized S-Mmt/S-Trt/tBu-protected peptide–peptoid hybrids.
Molecules 24 04261 g002
Molecules 24 04261 sch003 550
Scheme 3. Synthesis of peptide–peptoid hybrid 17 using m-chlorobenzoic acid.
Scheme 3. Synthesis of peptide–peptoid hybrid 17 using m-chlorobenzoic acid.
Molecules 24 04261 sch003
Molecules 24 04261 sch004 550
Scheme 4. Synthesis of S-free/completely unprotected peptide–peptoid 19.
Scheme 4. Synthesis of S-free/completely unprotected peptide–peptoid 19.
Molecules 24 04261 sch004
Molecules 24 04261 g003 550
Figure 3. HPLC analysis of peptide–peptoid hybrids: 10 (A) (column: Zorbax SB-C18, 3.5 μm; 2.1 × 30 nm; gradient: from 50% to 100% acetonitrile in water within 15 min, 100% acetonitrile within 15 min; flow rate: 0.4 mL/min); 17 (B) (column: Lichrospher RP-8 (4 × 150 mm, 5 μm); gradient: from 20% to 100% acetonitrile in water within 30 min; flow rate: 1 mL/min); 19 (C) (column: Lichrospher RP-8 (4 × 150 mm, 5 μm); gradient: from 20% to 100% acetonitrile in water within 30 min; flow rate: 1 mL/min). All chromatograms were detected at 265 nm.
Figure 3. HPLC analysis of peptide–peptoid hybrids: 10 (A) (column: Zorbax SB-C18, 3.5 μm; 2.1 × 30 nm; gradient: from 50% to 100% acetonitrile in water within 15 min, 100% acetonitrile within 15 min; flow rate: 0.4 mL/min); 17 (B) (column: Lichrospher RP-8 (4 × 150 mm, 5 μm); gradient: from 20% to 100% acetonitrile in water within 30 min; flow rate: 1 mL/min); 19 (C) (column: Lichrospher RP-8 (4 × 150 mm, 5 μm); gradient: from 20% to 100% acetonitrile in water within 30 min; flow rate: 1 mL/min). All chromatograms were detected at 265 nm.
Molecules 24 04261 g003
Molecules 24 04261 sch005 550
Scheme 5. Synthesis of thiol-bridged peptoid–peptide hybrid 22.
Scheme 5. Synthesis of thiol-bridged peptoid–peptide hybrid 22.
Molecules 24 04261 sch005
Molecules 24 04261 g004 550
Figure 4. LC-MS analysis of the oxidation of crude hybrid 21 in DMSO, after 12 h (A) and 48 h (B). Column: Nucleosil C8, 7 μm, 125 × 4 mm; flow rate: 1 mL/min; gradient: from 50 to 100% acetonitrile in water within 30 min; detection at 265 nm. (C) ES-MS analysis of the oxidized dimer 22. [M + Na]+ calc: 1655.80; [M + Na]+ found: 1655.6.
Figure 4. LC-MS analysis of the oxidation of crude hybrid 21 in DMSO, after 12 h (A) and 48 h (B). Column: Nucleosil C8, 7 μm, 125 × 4 mm; flow rate: 1 mL/min; gradient: from 50 to 100% acetonitrile in water within 30 min; detection at 265 nm. (C) ES-MS analysis of the oxidized dimer 22. [M + Na]+ calc: 1655.80; [M + Na]+ found: 1655.6.
Molecules 24 04261 g004
Molecules 24 04261 sch006 550
Scheme 6. Synthesis of S-Mmt/S-Trt-protected peptide–peptoid hybrid 14, partially S-free peptide–peptoid hybrid 24, and S-free peptide–peptoid hybrid 25.
Scheme 6. Synthesis of S-Mmt/S-Trt-protected peptide–peptoid hybrid 14, partially S-free peptide–peptoid hybrid 24, and S-free peptide–peptoid hybrid 25.
Molecules 24 04261 sch006
Molecules 24 04261 g005 550
Figure 5. HPLC analysis of hybrids 14 (A), 24 (B), and 25 (C). Column: Zorbax SB-C18, 3.5 μm; 2.1 × 30 nm; gradient: (A) from 50% to 100% acetonitrile in water within 15 min, 100% acetonitrile within 15 min; (B) from 20% to 100% acetonitrile in water within 30 min; (C) from 20% to 100% acetonitrile in water within 30 min; flow rate: 0.4 mL/min; detection at 265 nm.
Figure 5. HPLC analysis of hybrids 14 (A), 24 (B), and 25 (C). Column: Zorbax SB-C18, 3.5 μm; 2.1 × 30 nm; gradient: (A) from 50% to 100% acetonitrile in water within 15 min, 100% acetonitrile within 15 min; (B) from 20% to 100% acetonitrile in water within 30 min; (C) from 20% to 100% acetonitrile in water within 30 min; flow rate: 0.4 mL/min; detection at 265 nm.
Molecules 24 04261 g005
Table
Table 1. Retention time (tR), yield of the crude product (%), HPLC purity of the crude products (%), m/z (calculated and found) of the peptide–peptoid hybrids 9–17.
Table 1. Retention time (tR), yield of the crude product (%), HPLC purity of the crude products (%), m/z (calculated and found) of the peptide–peptoid hybrids 9–17.
a/atRCrude YieldHPLC Purity cm/z
(min)(%)(%)CalculatedFound
926.7 b9291760.4 d760.6 d
1023 a94931330.63 d665.9 e
1134 b92891536.71 d768.9 a
1227.2 b8988709.33 d709.3 d
1332 b9593969.41 d969.4 d
1418.8 a93901249.52 d625.4 e
1519.3 a93891719.89 d855.0 e
1626.9 a90922365.13 d-
1728.3 b96921004.45 d1004.4 d
a Column: Zorbax SB-C18, 3.5 μm; 2.1 × 30 nm; gradient: from 50% to 100% acetonitrile in water within 15 min, 100% acetonitrile within 15 min; flow rate: 0.4 mL/min. b Column: Lichrospher RP-8, 5 μm; 4 × 150 mm; gradient: from 20% to 100% acetonitrile in water within 30 min; flow rate: 1 mL/min. c HPLC purity determined at 265 nm. d [M + H]+. e [M + 2H]+2.

Share and Cite

MDPI and ACS Style

Mourtas, S.; Gatos, D.; Barlos, K. Solid-Phase Insertion of N-mercaptoalkylglycine Residues into Peptides. Molecules 2019, 24, 4261. https://doi.org/10.3390/molecules24234261

AMA Style

Mourtas S, Gatos D, Barlos K. Solid-Phase Insertion of N-mercaptoalkylglycine Residues into Peptides. Molecules. 2019; 24(23):4261. https://doi.org/10.3390/molecules24234261

Chicago/Turabian Style

Mourtas, Spyridon, Dimitrios Gatos, and Kleomenis Barlos. 2019. "Solid-Phase Insertion of N-mercaptoalkylglycine Residues into Peptides" Molecules 24, no. 23: 4261. https://doi.org/10.3390/molecules24234261

Article Metrics

Back to TopTop