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Published in final edited form as: Bioorg Med Chem Lett. 2015 Mar 12;25(21):4961–4969. doi: 10.1016/j.bmcl.2015.03.012

Small cyclic agonists of iron regulatory hormone hepcidin

Kristine Chua a, Eileen Fung a,†, Ewa D Micewicz b, Tomas Ganz a, Elizabeta Nemeth a, Piotr Ruchala c,d,*
PMCID: PMC4567957  NIHMSID: NIHMS671818  PMID: 25813158

Abstract

Minihepcidins are in vitro and in vivo active mimetics of iron-regulatory hormone hepcidin. They contain various unusual amino acids including: N-substituted, β-homo-, and D-amino acids with their combination depending on particular minihepcidin. In the current study, we sought to limit the use of unusual/more expensive amino acids derivatives by peptide cyclisation. Novel cyclic mimetics of hepcidin were synthesized and tested in vitro and showed activity at low nanomolar concentration. Nonetheless, the most active cyclic compound (mHS17) is approximately ten times less active than the parental minihepcidin PR73. Collectively, our findings suggest that cyclisation is viable approach in the synthesis of hepcidin mimetics.

Keywords: Minihepcidins, Peptides, S-Alkylation of peptides, Iron, Cyclisation

Hepcidin (Figure 1A), a 25 amino acids long peptide hormone, is a key regulator of iron homeostasis in verterbrates1. Its function is mediated through the membrane receptor/iron exporter, ferroportin (Fpn)2. Hepcidin binds to Fpn which in turn causes the internalization of Fpn and its subsequent proteasomal degradation. In consequence, diminished levels of surface Fpn on duodenal enterocytes and hepatic and splenic macrophages restrict systemic iron availability, by decreasing both the absorption of dietary iron in duodenum and the release of recycled iron from macrophages3.

Figure 1.

Figure 1

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Comparison of structures of (A) human hepcidin, (B) minihepcidin PR73, (C) inactive disulfide-bond cyclized analog cycloDTHFPI(CIF-CONHCH2CH2S-), and (D) bioactive disulfide-bond cyclized analog cyclo(CDTHFPIC)IF-CONH2.

Hepcidin, ferroportin and related pathways are currently under scrutiny as novel target(s) in the search for new therapeutics for iron disorders, with several leading compounds in advanced stage of development4–6. Hepcidin itself has unfavorable pharmacological properties (t1/2<2.5min)7 and, because of its 4 disulfide bonds, it is notoriously difficult to synthesize8. We developed minihepcidins, rationally designed peptide-based hepcidin mimetics, with potent in vitro and in vivo bioactivity9;10. Minihepcidins are currently under commercial development by Merganser Biotech LLC. Data available to date indicate that minihepcidins could be useful in stand-alone and combination-therapy-regimen(s) for β-thalassemia and polycythemia vera as well as in the treatment of iron-dependent bacterial infections11–14.

Most active minihepcidins contain both natural and unusual amino acids, including N-substituted- and β-homo- amino acids, with the unusual amino acids conferring resistance to proteolysis15–17. The C-terminus of minihepcidins contains a conjugated lipid moiety (C16) that increases plasma half-life and stabilizes the analog(s)18–21. Lipidation may also anchor the peptide in the hydrophobic environment of lipid rafts, increasing its local concentration, and bioactivity21–25. Notably, lipidated retro-inverso minihepcidins were also synthesized and showed potent bioactivity9.

Due to their content of unusual amino acids, certain minihepcidins may be expensive to produce, especially in the large quantities. Additional costs result mainly from the use of β-homo-amino acids (β-homo-L-proline (βhPro) and β-homo-L-phenylalanine (βhPhe)) and the unusual amino acid, 3,3′-diphenyl-L-alanine (Dpa). Therefore we explored a different strategy that would avoid the use of the most expensive amino acid derivatives, produce active hepcidin mimetics, and decrease the cost of synthesis. We chose the peptide cyclisation approach which has a long standing history26. Interestingly, head-to-tail cyclisation of full length hepcidin with and without spacer-residue(s) was recently reported27. However these modifications resulted in inactive derivatives. In our case though, the starting point for modifications was the PR73 minihepcidin (Figure 1B) rather than the full length hormone. Specifically, we employed the previously described S-alkylation method for the bridging of peptide(s) containing two strategically placed Cys residues with either symmetrical bis-halogeno-derivatives28;29 or divinyl sulfone (DVS)30. The use of this particular cyclisation method was prompted both by low cost and wide range of various alkylating analogs available, with an additional benefit of increased rigidity of the bridge that may stabilize the active conformer(s). The availability of various cysteine homologs: (L)Cys, (D)Cys, (L)homoCys, (D)homoCys, (L)Pen, and (D)Pen provides the option of “fine tuning” of selected active derivatives. Moreover, all reactions can be carried out in solution without any protecting groups. Notably, this approach was already applied in peptide drug development31, including phage display32–42 as well as peptide-albumin43;44 and peptide-antibody drug conjugates (ADCs)45.

All linear peptides necessary for this study were synthesized by the solid phase method using CEM Liberty automatic microwave peptide synthesizer (CEM Corporation Inc., Matthews, NC), employing 9-fluorenylmethyloxycarbonyl (Fmoc) chemistry and commercially available amino acid derivatives and reagents (Chem-Impex International, Inc., Wood Dale, IL)46;47. Since minihepcidins contain the biologically important cysteine residue in position 79;10 we used S-tert-butyl protected Cys-derivative (Cys(tBu)) to avoid unwanted interference with S-alkylation of cysteines in these positions.

S-Alkylation/cyclisation of reverse-phase high-performance liquid chromatography (RP-HPLC) purified linear analogs was performed using 2 different protocols, depending on the particular compound’s structure. Generally, for S-alkylation of synthetic linear peptides which are moderately or well soluble in water, we employed the protocol described by Timmerman and co-workers28. These reactions were carried out at ambient temperature in 50 mM ammonium bicarbonate (NH4HCO3) dissolved in a mixture of acetonitrile (ACN) and water 46. In case of hydrophobic peptides (e.g. lipidated analogs) we utilized previously described 1,1,3,3-tetramethylguanidine (TMG) driven reaction of thiol(s) containing compounds with bis-halogeno-derivatives in organic solvent/low molecular weight alcohol (preferably methanol)48, that we adapted to peptides46. Since peptides are rarely well soluble in methanol, we used dimethyl sulfoxide (DMSO) as a solubilizing additive (up to 25%) and excess of TMG (final conc.=0.35%, vol/vol). In addition, we also synthesized bi- and tri-cyclic concatenated derivatives (mHS11-mHS13), in an attempt to benefit from the potential multivalency of binding to multimerized Fpn.

Cyclic crude analogs containing Cys(tBu) residue(s) (position 7) were selectively deprotected using either modified TFMSA protocol or DMSO/TFA oxidation/dimerization followed by reduction with excess of tris(2-carboxyethyl)phosphine hydrochloride (TCEP, 100 eq/30 min)46;49. Notably, an alternative S-tert-butyl deprotection protocol, utilizing 1M HBF4/thioanisole was also described50.

All synthesized cyclic analogs were purified by preparative RP-HPLC and characterized by matrix-assisted laser desorption ionization spectrometry (MALDI-MS) as well as analytical RP-HPLC51 (see Table 1). Structural details for the synthesized analogs are presented in Figures 2, 4 and 5. Notably, for hydrophobic/lipidated analogs, an alternative synthetic protocol was also described52. Generally, cyclisation leading to monocyclic mHS derivatives proceeded efficiently with 1,3-bis(bromomethyl)benzene and 2,6-bis(bromomethyl)pyridine giving superior results. On the other hand, analogs mHS8-10 were particularly difficult to obtain, regardless on used protocol giving estimated yields <5% (based on RP-HPLC purified material).

Table 1.

Analytical and in vitro activity data for mSH analogs.

Peptide Composition MW Calc/Found RT [min] EC50 [nM] TREX-hFpn-GFP cells
PR73 C86H133N21O15S 1733.19/1734.34 47.11* 4.2±0.3
mHS1 C46H69N15O9S3 1072.32/1072.85 29.47 >600
mHS2 C52H77N17O11S3 1212.47/1212.20 28.06 >600
mHS3 C50H75N15O11S4 1190.47/1190.96 29.24 >600
mHS4 C54H75N15O9S3 1174.46/1174.73 33.42 585.6±71.1
mHS5 C54H75N15O9S3 1174.46/1174.86 34.19 272.2±18.9
mHS6 C54H75N15O9S3 1174.46/1174.60 34.18 598.9±114.2
mHS7 C53H74N16O9S3 1175.45/1175.90 27.65 >600
mHS8 C58H77N15O9S3 1224.52/1225.92 30.95 >600
mHS9 C60H79N15O10S3 1266.56/1266.74 39.58 477.4±76.3
mHS10 C61H81N15O9S3 1264.58/1264.52 41.14 516.9±131.7
mHS11 C96H134N28O16S5 2096.59/2097.25 34.74 >600
mHS12 C138H193N41O23S7 3018.73/3018.75 34.00/34.89 >600 regioisomers
mHS13 C141H199N41O31S7 3188.80/3188.87 34.33 >600
mHS14 C60H85N17O13S3 1348.62/1348.85 30.44 531.7±36.1
mHS15 C60H79N15O9S3 1250.56/1250.82 36.16 54.3±2.7
mHS16 C66H89N17O13S3 1424.71/1425.16 32.47 75.1±0.3
mHS17 C86H128N18O12S3 1702.25/1702.26 58.73 44.2±6.8
mHS18 C53H69N13O7S3 1096.39/1096.45 40.94 103.8±13.0
mHS19 C59H73N13O7S3 1172.49/1172.74 45.16 112.7±10.5
mHS20 C53H69N13O7S3 1096.39/1096.61 41.01 335.3±41.2
mHS21 C52H68N12O6S3 1053.36/1053.87 44.03 268.8±29.2
mHS22 C66H92N16O11S3 1381.73/1381.51 38.32 164.5±14.7
mHS23 C64H89N15O10S3 1324.68/1324.28 40.54 92.3±19.5
mHS24 C74H107N15O9S3 1446.93/1446.57 50.68* >600
mHS25 C73H106N14O8S3 1403.91/1403.63 50.73* 369.0±35.0
mHS26 C75H112N14O10S3 1465.98/1466.43 55.52* 52.3±10.8
mHS27 C68H99N13O7S3 1306.79/1306.43 57.32* >600
mHS28 C68H99N13O7S3 1306.79/1306.90 57.72* 458.3±87.7
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Analytical RP-HPLC was performed on a Varian ProStar 210 HPLC system equipped with ProStar 325 Dual Wavelength UV-Vis detector with the wavelengths set at 220 nm and 280 nm (Varian Inc., Palo Alto, CA). Mobile phases consisted of solvent A, 0.1% TFA in water, and solvent B, 0.1% TFA in acetonitrile. Analyses of peptides were performed with an analytical reversed-phase C18 SymmetryShield™ column, 4.6×250 mm, 5 μm (Waters, Milford, MA) or (*) an analytical reversed-phase C4 XBridge™ BEH300 column, 4.6×150 mm, 3.5 μm (Waters, Milford, MA), applying linear gradient of solvent B from 0 to 100% over 100 min (flow rate: 1 ml/min).

Figure 2.

Figure 2

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General structure of synthesized analogs mHS1–mHS10.

Figure 4.

Figure 4

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Structure of concatenated multi-cyclic derivatives mHS11÷mHS13. For mHS12, examples of 2 regioisomers are depicted.

Figure 5.

Figure 5

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Structure of cyclic mHS14÷mHS28 derivatives.

Bicyclic mHS11 was synthesized from its linear analog: Ac-CFPRXRFCFPRXRFC-CONH2 (X=L-Cys(StBu)) using 1,3,5-tris(bromomethyl)benzene as a bridging moiety28. Tricyclic counterparts were synthesized from Ac-CFPRXRFCFPRXRFCFPRXRFC-CONH2 and either commercially available 1,2,4,5-tetrakis(bromomethyl)benzene28 or pentaerythritol tetrakis(2-bromoacetate)46;53. In this case, depending on bridging moiety structure/symmetry, single or multiple products (regioisomers) may be obtained. Single product of S-alkylation is possible if linker used in the process of S-alkylation possesses tetrahedral symmetry (Td) (e.g. pentaerythritol scaffold). As expected, cyclisation reaction with 1,2,4,5-tetrakis(bromomethyl)benzene proceeded with low efficiency, which is most likely due to the generally rigid structure of the final product(s) as well as the fact that such linker may produce theoretically 4 different regioisomers varying in Cys-linker connectivity. Analogous reaction with pentaerythritol tetrakis(2-bromoacetate) gave a single product. Subsequent selective deprotection of the side chains of X-residues (Cys(tBu)→Cys(SH)) in multicyclic analogs using trifluoromethanesulfonic acid (TFMSA) protocol produced the desired analogs mHS11-mHS13. In these harsh conditions the pentaerythritol scaffold remained intact proving that the proposed linker is relatively stable and perhaps require more attention, especially that it efficiently produced tricyclic, sterically hindered compound containing various bulky side chains in the vicinity of S-alkylation sites. Tricyclic analogs obtained via this method would be a logical extension of previously done work on therapeutic bicyclic analogs31 offering potentially even larger diversity, especially if coupled with phage display approach32–42. Notably, the pentaerythritol scaffold was used previously for combinatorial chemistry54 and in the synthesis of polymers and nanomaterials using an atom transfer radical polymerization (ATRP)55;56 but not as an S-alkylating component of peptides’ constraining scaffold(s).

All newly synthesized analogs were tested in vitro57 using a previously described Fpn degradation assay58 with results summarized in Table 1.

Our studies on cyclic analogs were prompted by observation that certain disulfide bridged derivatives of N-terminal portion of hepcidin retain moderate bioactivity59. Specifically, the disulfide –bond cyclized compound cyclo(CDTHFPIC)IF-CONH2 showed EC50=300 nM, while cycloDTHFPI(CIF-CONHCH2CH2S-) was inactive (Figures 1C and 1D respectively), proving that only cyclic analogs with certain structural features are able to bind effectively to Fpn and trigger its degradation. The need for a broader strategy was reemphasized by the inactivity of full length head to tail cyclic hepcidin analogs27. We therefore explored different available linker(s) capable of bridging intramolecular thiols in the prototypical minihepcidin peptide resulting in analogs mHS1-mHS10. The sequence and length of the peptides was chosen based on available data9;58–60. Specifically, we assumed that the hexapeptide FPRCRF, corresponding to the parental sequence FPICIF, is both necessary and sufficient to exert full bioactivity, when its conformation is “properly altered” by cyclisation constrains. This sequence was in turn flanked with Cys residues, to allow for cyclisation based on S-alkylation with bis-thiol-reactive linkers. In addition, Ile → Arg modifications were used to improve solubility and activity59. This sequence is significantly shorter than the hepcidin segment DTHFPICIF from which minihepcidins are derived. Lack of N-terminal dipeptide (DT) has limited effects on bioactivity58, therefore only remaining 3His deletion, which accounts for ~80% loss in activity when combined with the latter (e.g. lack of DTH)58, represents significant challenge and this activity loss can be compensated by proper linker design. Such assumption is plausible since substitution of 3His for fairly similar Phe or (D)His residues has limited effects on overall activity60 and S-alkylation of Cys residue with aromatic linker(s) would produce substituted phenylalanine homolog(s) (see Figure 2). Except aromatic bridging moieties we also tested 2 linear and more flexible linkers afforded by bis-reactive Michael type compound DVS and 2-chloro-N-(2-(2-chloro-acetyloamino)-ethyl)-acetamide, Notably, other thiol(s)-multireactive Michael type compounds, including aromatic derivatives, were also reported32;61.

As shown in Table 1 the most active analog in initial group, mHS5, was produced by bridging with 1,3-bis(bromomethyl)benzene. Interestingly, mHS7, which possesses identical geometry, resulted in inactive analog, suggesting that presence of more hydrophilic/ionizable pyridine group is strongly detrimental in this particular region of molecule. This observation is in line with results from previous mutational and SAR studies that underlined importance of hydrophobic interactions in the vicinity of 4Phe and 9Phe58;60 which are in close proximity to the bridging moiety. The role of hydrophobicity and conformational influence of the linker is additionally validated by results for mHS2 and mHS3 compounds which possess hydrophilic linkers with similar length to that of mHS4 and mHS5 analogs but are inactive. Similarly, analogs mHS4 and mHS6, that closely resemble mHS5 in terms of bridging moiety and its hydrophobicity, are approximately 2 times less active stressing importance of proper spatial arrangements of amino acids side chains for Fpn efficient binding which results from particular linker’s geometry. Based on these results we chose mHS5 and its bridging moiety, 1,3-bis(bromomethyl)benzene, for further studies.

In the next group of concatenated, multicyclic analogs mHS11-mHS13 (Figure 4) we probed whether multivalent cyclic mHS5-derivatives can exert desired bioactivity. Surprisingly all those analogs were inactive. Notably, mHS12 was tested as a mixture of regioisomers, and based on analytical RP-HPLC results there were at least 2 different regioisomers present.

Subsequently we focused on various modifications of mHS5 to determine whether its activity could be improved. Specifically, addition of the Ida-Thr dipeptide at the N-terminus portion of mHS5 is detrimental (analogs mHS5 versus mHS14 and mHS15 versus mHS16), which is unexpected since this fragment corresponds to previously deleted portion (Asp-Thr) of original minihepcidin DTHFPICIF-CONH2, and it was used based on our earlier work on linear minihepcidins10;59. This modification causes ~30–50% loss in activity and its effects may explain negative results for bicyclic mHS11 derivative, which despite being basically mHS5 dimer must have different N- and C-terminal spatial side-chains arrangements due to cyclisation-induced “crowding”, underling once again role of hydrophobic interactions provided by 4Phe and 9Phe. Even the presence of the small acetyl-amino-group in this position is undesirable as analog lacking this particular modification (mHS23) is approximately 2 times more active than AcNH-containing and otherwise identical mHS22 compound. Similarly presence of the substituents at C-terminus (e.g. amide moiety) seems to have negative effects, although they are less pronounced (mHS20 versus mHS21 and mHS24 versus mHS25). Increase of hydrophobicity in 4Phe position (4Phe→Dpa) causes a dramatic increase in potency (5×, mHS5 versus mHS15) which is analogous to the effects observed among its linear counterparts10;59. Since Dpa is considered in this study to be an “expensive” amino acid, we attempted next to develop its viable, less expensive substituent. To this end, we decided to use 3,3-diphenylpropionic- and 3,3,3-triphenylpropionic acids (Dpp and Tpp respectively), which are less expensive des-amino-Dpa homologs. However, this approach requires modification of N-terminal Cys position as both Dpp and Tpp can be only placed as “final” N-terminal residue(s), due to lack of amine group. Based on limited molecular modeling (HyperChem 8.0, Hypercube Inc., Gainesville, FL), we determined that the location and spatial arrangements of Pro residue at mHS5 are such that they permit its substitution for (L)Cys without significant structural change. As a result, we synthesized analogs mHS18 and mHS19 bearing such modifications (e.g. 5Pro→(L)Cys) which showed moderate potency (EC50≈100 nM) and were approximately 2× less potent than initial mHS15 (EC50=54.3±2.7). Notably, there was no apparent effect of hydrophobicity increase on bioactivity as Tpp containing compound (mHS19) showed similar activity to Dpp counterpart (mHS18). The change in absolute Cys configuration ((L)Cys → (D)Cys; mHS18 versus mHS20) resulted in a significant loss of bioactivity (3.2×).

Generally, lipidation improves the bioactivity of cyclic mHS analogs, provided lipid moiety is conjugated via C-terminus of the molecule (mHS17 versus mHS15, mHS24 & mHS25, and mHS26 versus mHS18, mHS27 & mHS28), which agrees with observations for linear minihepcidins59. C16 moiety was introduced either using palmitic acid or iminodiacetic acid mono-n-palmityl amide (IdaNHPal) with spacers afforded by short PEG (1,8-diamino-3,6-dioxaoctane) or 6-aminohexanoic acid (Ahx) respectively. The use of short spacers is necessary for high activity of original linear minihepcidins59 and this principle was also assumed to be important for cyclic counterparts.

The most active cyclic analogs developed in this study are mHS17 (EC50=44.2±6.8 nM) and mHS26 (EC50=52.3±10.8 nM). Despite their bioactivity in the low nanomolar range they are still considerably less potent (10.5× and 12.5× respectively) than parental minihepcidin PR73 (EC50= 4.2±0.3 nM). Nonetheless, they are smaller than PR73, and considerably less expensive to produce, at least based on direct synthesis/materials costs (see Figure 6). Therefore, despite their lower potency, we decided to test whether mHS derivatives can exert desired bioactivity in vivo. We selected lipidated derivatives: mHS17 and mHS26, and “lipid-less” mHS18 as suitable candidates for animal studies62, which were carried out using previously described protocol9;10;58. The peptides were injected subcutaneously and bioactivity was tested solely at 24h post-injection time point, which roughly coincides with maximal activity for linear minihepcidin(s)9;10. Using minihepcidin PR73 as a control, we determined that analogous amounts of mHS peptides (50–200 nmoles) show no desired biological effect (Figure 3B). Since no in vivo activity was observed, we probed plasma stability63–66 of mHS26 and PR73 to determine whether the lack of activity of mHS compounds is due to their proteolytic degradation. In fact, the inactive lipidated analog mHS26 was slightly more resistant to plasma driven degradation (Figure 3C) than the highly bioactive minihepcidin PR73, suggesting that the overall potency rather than stability is the key feature determining in vivo properties/bioactivity of minihepcidins. Such assumption would certainly explain the poor performance of mHS derivatives in in vivo experiments, since animals received similar dosage (50–200 nmoles) with the potency for the most active mHS analogs being at least 10× smaller than of PR73. Cyclisation of mHS peptides certainly improves plasma stability as linear analog mHS1 was rapidly degraded in the same experimental conditions within 6 h.

Figure 6.

Figure 6

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Comparison of structures and relative production costs (materials) for analogs PR73, mHS17 and mHS26.

Figure 3.

Figure 3

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Activity of selected mHS analogs: (A) comparison of dose response curves obtained for analogs PR73 (EC50=4.2±0.3 nM), mHS17 (EC50=44.2±6.8 nM), and mHS26 (EC50=52.3±10.8 nM) in in vitro Fpn degradation assay57, (B) comparison of in vivo activity of selected mHS peptides at 24h time-point62, (C) comparison of plasma stability of PR73 and mHS2666.

In conclusion, a group of small cyclic peptide-based hepcidin agonists was synthesized and tested in vitro and principles of their SAR were established. The most active analogs, mHS17 and mHS26 are considerably cheaper to produce than prototypic, parental minihepcidin PR73, however they show no in vivo potency based on equimolar administration in a mouse model. Nonetheless, in vitro activity of selected mHS peptides is in low nanomolar range suggesting that these novel cyclic analogs may be viable leads for further development of therapeutic hepcidin agonists. Such analogs may be obtained by additional “fine tuning” of mHS properties exploiting commercially available cysteine homologs (e.g. (L)homoCys, (L)β-homoCys, (L)Pen, etc.) or change of the location of the lipidation site (use of lipid-bearing S-alkylating linkers), or both. Significant interest in pharmaceutically-relevant hepcidin agonists certainly warrants further experimentation.

Acknowledgments

This project was partially supported by funds from the Adams and Burnham endowments provided by the Dean’s Office of the David Geffen School of Medicine at UCLA (PR) and NIH grant R01 DK090554 (TG and EN).

Footnotes

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    1. mHS2 - 2-Chloro-N-(2-(2-chloro-acetyloamino)-ethyl)-acetamide,

    2. (2) mHS3 - Divinylsulfone, 10 mM conc. of NH4HCO3 was used.

    3. mHS4 - 1,4-Bis(bromomethyl)benzene,

    4. mHS5 - 1,3-Bis(bromomethyl)benzene,

    5. mHS6 - 1,2-Bis(bromomethyl)benzene,

    6. mHS7 - 2,6-Bis(bromomethyl)pyridine,

    7. mHS8 - 1,8-Bis(bromomethyl)naphthalene,

    8. mHS9 - 1-(Chloromethyl)-4-[4-(chloromethyl)phenoxy]benzene,

    9. mHS10 - 1-(Bromomethyl)-3-[3-(bromomethyl)benzyl]benzene,

    10. mHS11 - 1,3,5-Tris(bromomethyl)benzene,

    11. mHS12 - 1,2,4,5-Tetrakis(bromomethyl)benzene,

    12. mHS13 - Pentaerythritol tetrakis(2-bromoacetate),

    13. mHS14-mHS16, mHS22 and mHS23 - 1,3-Bis(bromomethyl)benzene.

    Synthesis of analogs mHS17-mHS21, mHS24-mHS28.Cyclisation was carried out using modified Włostowski S-alkylation protocol48 that was adapted to peptides. Briefly, linear peptides were solubilized in anhydrous methanol (~0.5 mg/mL) containing, if necessary up 25% of DMSO. Subsequently 1 equivalent of 1,3-bis(bromomethyl)benzene was added followed by 1,1,3,3-tetramethylguanidine (final conc.=0.35%, vol/vol). Reaction mixtures were stirred 15 min at room temperature and then 90 min at 50 °C. Subsequently solutions were lyophilized, selectively deprotected (see below) and obtained crude solid residues purified by preparative RP-HPLC. Purity of obtained analogs was evaluated by MALDI-MS as well as analytical RP-HPLC.Selective deprotection of Cys(tBu).TFMSA method: Cyclic crude peptide was dissolved in the mixture of TFA:thioanisole:EDT (2750:250:75 μL, 20 mg/mL) and cooled in ice bath (10 min). Subsequently TFMSA was added (125 μL) and solution was stirred for additional 60 min at 0ºC. Then reduced peptide was precipitated by addition of ice cold diethyl ether and separated by centrifugation. DMSO/TFA oxidation method: Cyclic crude peptide was dissolved in the mixture of 5% DMSO in TFA (20 mg/mL) and agitated for 45 min at room temperature. Subsequently oxidized/dimerized peptide was precipitated by addition of ice cold diethyl ether and separated by centrifugation. Then crude residue was dissolved in 50–75% methanol in water and reduced with TCEP (100 eq/30 min). Resulting solution was freeze-dried and remaining residue purified by RP-HPLC.Synthesis of pentaerythritol tetrakis(2-bromoacetate).Pentaerythritol (0.5 g, 3.67 mmol), triethylamine (4.5 mL, 3.23 g, 32.3 mmol) and 40 mL of acetonitrile were placed in a 100 mL three-neck round bottom flask, equipped with dropping funnel, magnetic stirrer/bar and inert-gas blanket assembly (argon). An apparatus was placed in water/ice bath and solution was agitated and cooled to 0 ºC under the argon atmosphere (20 min) Subsequently, 1.92 mL of bromoacetyl bromide (4.44 g, 22 mmol) diluted in 10 mL acetonitrile was added dropwise over 30 min. The reaction mixture was stirred overnight (room temp.), then filtered and evaporated on the rotary evaporator. The resulting residue (brown/red viscous liquid) was purified using flash column chromatography on silica gel (200–425 mesh, dichloromethane/ethyl acetate (40:1)) giving 1.62 g of desired product (yield=71.2%). Small amounts of pentaerythritol tetrakis(2-bromoacetate) may be also purified using RP-HPLC and an analytical reversed-phase C4 XBridge™ column, 4.6×150 mm, 3.5 μm (Waters, Milford, MA), (RT=43.8 min).
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