Introduction

Transmissible spongiform encephalopathies are fatal neurodegenerative diseases of the central nervous system characterized by loss of motor control, dementia, and paralysis (Cohen and Prusiner 1998; Weissman 1999; Caughey 2000). They include scrapie of sheep and goats, bovine spongiform encephalopathy of cattle, and several human diseases, such as Creutzfeldt–Jakob disease, Gerstmann–Straussler–Scheinker syndrome, and fatal familial insomnia (Will et al. 1996; Prusiner 1998; Bons et al. 1999). The “protein-only” hypothesis formulated by Prusiner and co-workers (Cohen and Prusiner 1998; Prusiner 1998) considers that the infectious form of the prion (PrPSc from scrapie) has an amino acid sequence identical to a normal host protein (PrPC) and infection of organism with PrPSc would result in the conversion of PrPC into a conformational isomer of itself resulting in PrPSc aggregation in the brain. It has been proposed that a predominantly alpha-helical structure in the PrPC converts into beta-sheet rich PrPSc, especially in the region 90–145 (Cohen and Prusiner 1998; Pan et al. 1993; Huang et al. 1996; Jackson et al. 1999; Supattapone et al. 1999; Kaneko et al. 2000; Forloni et al. 1993). Recently, Legname et al. (2004) showed that a recombinant mouse PrP(89–230) with the P101L mutation folded in amyloid fibrils “in vitro” induced prion-like disease in transgenic mouse over expressing identical PrP. In addition, methods of “protein misfolding cyclic amplification” succeed in generation of infectious PrPSc “in vitro” (Castilla et al. 2005). The protein-only theory gained great support with the finding that PrP knockout mice do not propagate prion and are completely resistant to prion infection (Bueler et al. 1992). Even the mechanism of prion conversion remains unknown, most models predict an autocatalytic process (Cohen and Prusiner 1998; Weissman 1999; Prusiner 1998; Jarrett and Lansbury 1993; Come et al. 1993). However, to gain more knowledge about the structure of prions and the mechanism of their transmission, additional studies and new tools are necessary to unravel remaining uncertainties. One such a tool could be synthetic prion protein with precisely positioned fluorescent labels allowing testing of PrPSc structure by fluorescent method. However, prions posses more than 200 amino acid residues, which strongly disfavor straightforward peptide synthesis. It requires syntheses of shorter fragments, which are finally linked together. This “small building blocks approach” would also simplify the synthesis of prion protein with any modification, including fluorescent amino acid labels. The Boc/Bzl strategy previously provided prion polypeptides of up to 112 amino acids with some point mutations (Ball et al. 2001), i.e. the biggest prion chemically synthesized was the bovine PrP(90–200) with one more Lys residue.

In this work, our interest was focused on syntheses of building blocks of the C-domain of mouse prion protein MoPrP(93–231) by Fmoc/tBu strategy. Thioesters of corresponding peptide segments could play a role of the building blocks for native chemical ligation method for the total chemical synthesis of proteins (Dawson and Kent 2000; Milton et al. 1992). We intended to check this approach firstly in the synthesis of the building blocks from the human prion-derived peptide HuPrP106–126 (Fig. 1) as a model pattern, because its sequence was found to be a difficult one when using the classical Fmoc synthetic strategy (Jobling et al. 1999), which provided 9% yield only (Šebestik et al. 2006; Table 2, method A). We optimized the syntheses of various sequences with aim to find the best synthetic route for corresponding thioesters suitable for a final ligation steps. Finally, we applied these results in the syntheses of peptide thioesters from the sequence of the mouse prion domain 93–231 (MoPrP93–231) (Fig. 2).

Fig. 1
figure 1

Sequence of human prion derived peptide H-HuPrP(106–126)-OH

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Fig. 2
figure 2

Sequence of mouse prion domain MoPrP(93–231)

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Materials and methods

General methodologies

The solvents DCM, DMF, cleaving reagent TFA and other chemicals were purchased from Sigma-Aldrich–Fluka corporations (Prague, Czech Republic), the HPLC solvent ACN was from Scharlau, Germany, the 2-chlorotrityl chloride resin (substitution 1.2 mmol/g), Wang polystyrene resin (substitution 0.8 mmol/g), 4-sulfamylbutyryl AM resin (0.69 mmol/g), DIC, HOBt and other reagents for amino acid couplings were purchased from Merck-Novabiochem, Czech Republic and protected amino acids from Iris Biotech GMBH, Germany. The indole-3-carboxaldehyde-polystyrene resin (1.03 mmol/g; Šebestik et al. 2004) was prepared in our laboratory. Peptides were synthesized by Fmoc/tBu method (Fields and Noble 1990) either manually in plastic syringes (5–10 ml) with Teflon filter, or by automatic solid-phase synthesizer ABI 433A (Applied Biosystems, Foster City, CA, USA). During manual synthesis, progress of coupling in peptide synthesis was monitored by Kaiser (Kaiser et al. 1970; Sarin et al. 1981), chloranil (Vojkovský 1995) or bromophenol blue tests (Krchňák et al. 1988a, b). Fmoc deprotection was monitored by precipitation of dibenzofulvene in water. An automated synthesizer used the FastMoc 0.1 mmol program (SynthAssist™ version 3.1) with a double coupling: 10 eq excess of protected amino acids and HBTU coupling reagent and 20 eq excess of DIEA were used. Molecular weights of the compounds prepared were determined by mass spectrometry using either an ESI technique (Agilent 5975B MSD) from Agilent Technologies, USA, or MALDI-TOF spectra, recorded on mass spectrometer Lasermat (Finnigan, USA) with Lasermat 2000 program. For semipreparative HPLC, a Spectra Physics SP8800 pump with a TSP Chrom Jet SP4290 integrator and a Spectra 100 UV detector were used with a Vydac RP-18, 25 × 1 cm, 10 μm column (Separations Group, Hesperia, CA, USA) of flow rate 3 mL/min and detection at 220 nm using a 0–100% gradient of ACN in 0.05% aqueous TFA over 120 min unless stated otherwise. For analytical HPLC, Agilent 1200 instrument with quaternary pump, thermostat, diode array detector and reverse phase C18 columns were used with a flow rate of 1 mL/min, detection at 220 nm. HPLC columns and gradients are enlisted in Table 1. When necessary, the NMR spectra were recorded on a Bruker Avance I™ 400 MHz spectrometer (Bruker BioSpin GmbH, Rheinstetten, Germany). Products were dried in a vacuum drying box (Salvis AG, Emmenbrűcke, Lucern, Switzerland) at room temperature for 16 h.

Table 1 Parameters used in HPLC analysis of peptides and peptide thioesters 2–65
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Glycine trithioorthoester hydrochloride 1

The compound 1 was prepared according to Brask et al. (2003). Briefly, to a mixture of dry DCM (80 mL) and AlMe3 (2 M heptane solution, 52 mL), EtSH (23.5 mL) was added drop wise under argon at 0°C. To this mixture, the solution of H-Gly-OtBu acetate (2.08 g; 11 mmol) in DCM (15 mL) was slowly added. The reaction mixture was left stirred over night and then poured to ice (300 mL). The reaction flask was washed with additional DCM (120 mL) and the combined organic phases were separated and washed with 1% HCl (4 × 200 mL). The aqueous solution was alkalized with solid NaOH (10.5 g) to pH 11 and then washed with DCM (3 × 200 mL), as well. The combined DCM solutions were finally dried with Na2SO4, separated from drying agent and evaporated to dryness. The oily residue was dissolved in 1% HCl (30 mL), the excess of HCl removed in vacuum at 0°C and the solution was lyophilized with a yield 0.8 g (27%) of the white hygroscopic solid 1. 1H-NMR (400 MHz, dmso-d6): δ 8.35 (bs, 3H, NH3 +), δ 3.22 (s, 2H, α-CH2), δ 2.71 (q, 6H, 3 J = 7.5 Hz, CH2), δ 1.20 (t, 9H, 3 J = 7.5 Hz, CH3).

The syntheses of peptides 2–5 and peptide thiosters 6–15 of HuPrP(106–126), (Fig. 1) using methods A–E (Tables 1, 2; Fig. 3)

Method A

Synthesis on Wang polystyrene resin (200 mg; substitution of the Fmoc-Gly was 0.26 mmol/g of the resin 0.052 mmol) was performed by the Fmoc/Boc strategy, using the 4 eq. excess of corresponding Fmoc-amino acids (0.21 mmol) and coupling reagents DIC (33 μL; 0.21 mmol) and HOBt (28 mg; 0.21 mmol) in DMF (2 mL), followed by deprotection with our standard mixture M: TFA (90%)–TIS (2.5%)–H2O (2.5%)–EDT (5%), 4 mL.

Table 2 Syntheses of the difficult sequence H-HuPrP(106–126)-OH and related subsequences
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Fig. 3
figure 3

The scheme of investigated syntheses of the peptide thioesters. Method B starts from resin (R1). Method C is using the resin (R3). In the method D, the resin (R5) serves as a starting compound. The most effective method E for short peptides begins with the 2-chlorotrityl chloride resin (R7)

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Obtained peptides

H-[Sec 120 ]HuPrP(120–126)-OH—H-Sec-Val-Val-Gly-Gly-Leu-Gly-OH (2)—Yield 26 mg (65%). HPLC retention time (RT; Ecli25) was 8.0 min (95%). For C25H45N7O8Se (651.25) found ESI-MS, m/z: 673.2 (M + Na+).

H-[Sec 115 ]HuPrP(115–126)-OH—H-Sec-Ala-Ala-Ala-Gly-Ala-Val-Val-Gly-Gly-Leu-Gly-OH (3)—Yield 5.8 mg (10%). HPLC RT (Ecli25) was 11.4 min (96%). For C39H68N12O13Se (992.42) found MALDI-MS, m/z: 1,015.2 (M + Na+), 1,031.2 (M + K+).

H-[Cys 120 ]HuPrP(120–126)-OH—H-Cys-Val-Val-Gly-Gly-Leu-Gly-OH (4)—Yield 6.0 mg (16%). HPLC RT (Ecli25) was 8.5 min (94%). For C25H45N7O8S (603.31) found ESI-MS, m/z: 604.2 (M + H+), 626.3 (M + Na+).

H-[Cys 115 ]HuPrP(115–126)-OH—H-Cys-Ala-Ala-Ala-Gly-Ala-Val-Val-Gly-Gly-Leu-Gly-OH (5)—Yield 7.2 mg (13%). HPLC RT (Ecli25) was 13.8 min (97%). For C39H68N12O13S (944.47) found ESI-MS, m/z: 945.3 (M + H+), 967.5 (M + Na+).

Method B

Synthesis on Wang polystyrene resin was carried out by method A. However, the Boc-Lys(Boc)-OH was coupled as the last amino acid. The cleavage of the peptides from the resin and their simultaneous thiolation were carried out by Hilvert’s method (Sewing and Hilvert 2001) with a mixture of AlMe3 (1 mmol; 500 μL 2 M AlMe3 in heptane), EtSH (3 mmol; 222 μL) in dry DCM (5 mL) under inert conditions of argon, followed by final deprotection with our standard mixture M.

Obtained peptides

H-[Asi 108 ]HuPrP(106–114)-SEt—H-Lys-Thr-Asi-Met-Lys-His-Met-Ala-Gly-SEt (6)—Yield 7.0 mg (9%). HPLC RT (Ecli25) was 8.0 min (95%). For C43H73N13O11S3 (1,043.47) found ESI-MS, m/z: 1,044.1 (M + H+).

H-[ΔThr 107 , Asi 108 ]HuPrP(106–119)-SEt—H-Lys-Asi-Met-Lys-His-Met-Ala-Gly-Ala-Ala-Ala-Ala-Gly-SEt (7)—Yield 10.0 mg (11%). HPLC RT (Ecli25) was 7.9 min (98%). For C53H89N17O14S3 (1,283.59) found MALDI-MS, m/z: 1,283.4 (M+), 1,305.4 (M + Na+), 1,321.5 (M + K+).

Method C

The trithioorthoester masking of thioester function on the C-terminal Gly residue was applied (Brask et al. 2003). An indole-3-carboxaldehyde-polystyrene resin (100 mg; substitution 1.06 mmol/g of the resin) with backbone acid labile linker was loaded with compound 1 (0.42 mmol; 111 mg) by the procedure described by Šebestik et al. (2004) with a yield 112 mg (the substitution 0.45 mmol/g was determined by elemental analysis as a content of sulfur). The peptide chain was elongated using coupling procedure described in Method A, with the Boc-Lys(Boc)-OH coupled as the last amino acid. The cleavage of peptide thioesters from the resin was performed by a TFA-thioanisole-DCM (2:1:20, 3 mL) mixture and the final deprotection with the standard mixture M (3 mL).

Obtained peptides

H-HuPrP(106–114)-SEt—H-Lys-Thr-Asn-Met-Lys-His-Met-Ala-Gly-SEt (8)—Yield 9.2 mg (12%). HPLC RT (Ecli25) was 9.6 min (98%). For C43H76N14O11S3 (1,060.50) found MALDI-MS, m/z: 1,060.5 (M+).

H-HuPrP(106–119)-SEt—H-Lys-Thr-Asn-Met-Lys-His-Met-Ala-Gly-Ala-Ala-Ala-Ala-Gly-SEt (9)—Yield 9.4 mg (10%). HPLC RT (Ecli25) was 11.4 min (95%). For C57H99N19O16S3 (1,401.67) found MALDI-MS, m/z: 1,402.7 (M + H+), 1,424.8 (M + Na+).

Method D

Synthesis on 4-sulfamylbutyryl AM resin (150 mg) loaded with Fmoc-Gly-OH (0.18 mmol/g) (Backes and Ellman 1999) used the coupling procedure described in Method A with the Boc-Lys(Boc)-OH coupled as the last amino acid. After extensive washing with freshly dried THF, a solution of TMS-CHN2 (15 mmol; 7.5 mL of 2 M solution in hexane) in dry THF (8 mL) was added to a swollen resin. After gentle stirring for 2 h, the resin was washed with dry THF (3 × 10 mL) and DCM (4 × 10 mL) and was dried under vacuum for 6 h. The peptide segments were cleaved from the resin and thiolated with Mpa-OnBu (18 μL; 0.11 mmol) in DMF (2 mL) at 55°C for 3–7 days. The protecting groups were finally removed by the standard mixture M (3 mL).

Obtained peptides

H-HuPrP(106–114)-Mpa-OnBu—H-Lys-Thr-Asn-Met-Lys-His-Met-Ala-Gly-Mpa-OnBu (10)—Yield 2.6 mg (6%). HPLC RT (Ecli25) was 10.0 min (95%). For C48H84N14O13S3 (1,160.55) found MALDI-MS, m/z: 1,183.6 (M + Na+).

H-HuPrP(106–119)-Mpa-OnBu—H-Lys-Thr-Asn-Met-Lys-His-Met-Ala-Gly-Ala-Ala-Ala-Ala-Gly-Mpa-OnBu (11)—Yield 1.1 mg (2%). HPLC RT (Ecli25) was 10.4 min (97%). For C62H107N19O18S3 (1,501.72) found MALDI-MS, m/z: 1,524.8 (M + Na+).

Method E

Synthesis on 2-chlorotrityl chloride resin (250 mg, the substitution 0.27 mmol/g of the resin) used the coupling procedure described in the method A, with the Boc-Lys(Boc)-OH coupled as the last amino acid. The protected peptides were cleaved from the resin by HFIP (1 mL) in DCM (3 mL), 5 min, and then treated with an excess of EtSH (97 μL; 1.35 mmol) in the presence of the DIC (12 μL; 75 μmol) and DMAP (0.82 mg; 6.75 μmol) mixture in DCM (3 mL). The resulting thioesters were finally deprotected with the standard reagent M (4 mL).

Obtained peptides

H-HuPrP(106–114)-SEt—H-Lys-Thr-Asn-Met-Lys-His-Met-Ala-Gly-SEt (12)—Yield 30.7 mg (30%). HPLC RT (Ecli25) was 9.6 min (98%). For C43H76N14O11S3 (1,060.50) found MALDI-MS, m/z: 1,060.5 (M+).

H-HuPrP(106–119)-SEt—H-Lys-Thr-Asn-Met-Lys-His-Met-Ala-Gly-Ala-Ala-Ala-Ala-Gly-SEt (13)—Yield 7.5 mg (6%). HPLC RT (Ecli25) was 11.5 min (96%). For C57H99N19O16S3 (1,401.67) found MALDI-MS, m/z: 1,402.7 (M + H+), 1,424.8 (M + Na+).

H-[Cys 115 ] HuPrP(115–119)-SEt—H-Cys-Ala-Ala-Ala-Gly-SEt (14)—Yield 20 mg (54%). HPLC RT (Ecli25) was 6.2 min (95%). For C16H29N5O5S2 (435.16) found ESI-MS, m/z: 458.1 (M + Na+), 436.2 (M + H+).

H-[Thz 115 ]HuPrP(115–119)-SEt—H-Thz-Ala-Ala-Ala-Gly-SEt (15)—Yield 19.7 mg (52%). HPLC RT (Ecli25) was 6.7 min (95%). For C17H29N5O5S2 (447.16) found ESI-MS, m/z: 470.2 (M + Na+).

Syntheses of peptide segments 16–39 of the mouse prion domain MoPrP (93–231, Table 3)

The peptide segments 16–39 from the mouse prion domain 93–231 (Table 3) were assembled on automated solid phase synthesizer as specified in the General methodologies (in some cases a combination with manually driven approach was chosen) using 2-chlorotritylchloride resin (200–400 mesh, 0.65 mmol/g). Fully protected peptides were cleaved from the 2-chlorotrityl resin by HFIP (1 mL) in DCM (3 mL) to obtain protected substrates for peptide thioesters preparation. The small portions of these protected peptides (mostly 50 mg) were fully deprotected by the standard mixture M: (TFA (90%)–TIS (2.5%)–H2O (2.5%)–EDT (5%), and characterized with HPLC/MS as described in General methodologies.

Table 3 Availability of sequences from Mouse Prion Protein MoPrP(93–231)
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H-MoPrP(93–104)-OH—H-Gly-Thr-His-Asn-Gln-Trp-Asn-Lys-Pro-Ser-Lys-Pro-OH (16)—150 mg Fmoc-Pro-O-ClTrt-resin (0.37 mmol g−1). HFIP treatment yielded 292 mg of the protected peptide. By deprotection of the sample with M and HPLC purification, the 7.5 mg of 16 was prepared. HPLC retention time (RT) (Poro15a) was 3.69 min (crude 77%; after HPLC purification 98%). For C61H92N20O18 (1,392.69) found MALDI-MS, m/z: 1,393.6 (M + H+), 1,415.7 (M + Na+).

H-[Thz 105 ]MoPrP(105–113)-OH—H-Thz-Thr-Asn-Leu-Lys-His-Val-Ala-Gly-OH (17)—150 mg Fmoc-Gly-O-ClTrt-resin (0.59 mmol g−1). HFIP treatment yielded 420 mg of the protected peptide that after deprotection of the sample with M and purification afforded peptide 17 (5.5 mg). HPLC RT (Poro15b) was 3.92 min (crude 62%; after HPLC purification 98%). For C40H67N13O12S (953.48) found ESI-MS, m/z: 954.4 (M + H+), 477.7 (M + 2 H+/2).

H-[Thz 114 ]MoPrP(114–118)-OH—H-Thz-Ala-Ala-Ala-Gly-OH (18)—150 mg Fmoc-Gly-O-ClTrt-resin (0.59 mmol g−1). After treatment with HFIP, the protected peptide (300 mg) was obtained. Deprotection of the sample with M and purification yielded peptide 18 (4.5 mg) with HPLC RT (PoroIso) 0.5 min (crude 84%; after HPLC purification >99%). For C15H25N5O6S (403.15) found ESI-MS, m/z: 404.1 (M + H+), 426.1 (M + Na+).

H-[Thz 119 ]MoPrP(119–131)-OH—H-Thz-Val-Val-Gly-Gly-Leu-Gly-Gly-Tyr-Met-Leu-Gly-Ser-OH (19) –120 mg Fmoc-Ser(OtBu)-O-ClTrt-resin (0.40 mmol g−1). HFIP treatment yielded the protected peptide (262 mg). After the deprotection of the sample with M and purification the peptide 19 (12.5 mg) was obtained. HPLC RT (Poro15c) was 5.58 min (crude 82%; after HPLC purification 99%). For C53H85N13O16S2 (1,223.57) found MALDI-MS, m/z: 1,246.4 (M + Na+), 1,262.4 (M + K+).

H-[Thz 132 ]MoPrP(132–141)-OH—H-Thz-Met-Ser-Arg-Pro-Met-Ile-His-Phe-Gly-OH (20)—150 mg Fmoc-Gly-O-ClTrt-resin (0.59 mmol g−1). After the peptide-resin treatment with HFIP, the protected peptide (312 mg) was obtained. The sample was deprotected by M and purified with a yield of peptide 20 (6.7 mg). HPLC RT (Poro15c) was 4.63 min (crude 69%; after HPLC purification 97%). For C51H79N15O12S3 (1,189.52) found MALDI-MS, m/z: 1,190.3 (M + H+).

H-[Thz 142 ]MoPrP(142–152)-OH—H-Thz-Asp-Trp-Glu-Asp-Arg-Tyr-Tyr-Arg-Glu-Asn-OH (21)—150 mg Fmoc-Asn(Trt)-O-ClTrt-resin (0.52 mmol g−1). HFIP treatment afforded 297 mg of protected peptide. After the sample deprotection by M and purification the 12.4 mg of peptide 21 was obtained. HPLC RT (Poro15c) was 3.66 min (crude 61%; after HPLC purification 98%). For C67H89N19O23S (1,559.61) found MALDI-MS, m/z: 1,560.6 (M + H+).

H-[Thz 153 ]MoPrP(153–164)-OH—H-Thz-Tyr-Arg-Tyr-Pro-Asn-Gln-Val-Tyr-Tyr-Arg-Pro-OH (22)—150 mg Fmoc-Pro-O-ClTrt-resin (0.37 mmol g−1). After treatment of the peptide-resin with HFIP, the protected peptide (241 mg) was obtained. Deprotection by M and purification of its sample afforded 17.5 mg of 22. HPLC RT (Poro15d) was 4.11 min (crude 67%; after HPLC purification 95%). For C76H104N20O19S (1,632.75) found MALDI-MS, m/z: 1,633.7 (M + H+).

H-[Thz 165 ]MoPrP(165–177)-OH—H-Thz-Asp-Gln-Tyr-Ser-Asn-Gln-Asn-Asn-Phe-Val-His-Asp-OH (23)—150 mg Fmoc-Asp(OtBu)-O-ClTrt-resin (0.32 mmol g−1). HFIP treatment yielded 114 mg of the protected peptide. After deprotection of the sample by M and purification the 7.8 mg of 23 was obtained. HPLC RT (Poro15c) was 3.14 min (crude 81%; after HPLC purification 99%). For C66H90N20O25S (1,594.61) found MALDI-MS, m/z: 1,595.6 (M + H+), 1,617.6 (M + Na+), 1,633.7 (M + K+).

H-[Thz 178 ]MoPrP(178–194)-OH—H-Thz-Val-Asn-Ile-Thr-Ile-Lys-Gln-His-Thr-Val-Thr-Thr-Thr-Thr-Lys-Gly-OH (24)—First, the Fmoc-MoPrP180–194-O-ClTrt-Resin was assembled by automated peptide synthesis on 150 mg Fmoc-Gly-O-ClTrt-resin (0.60 mmol g−1). Then, remaining amino acids were coupled by manual Fmoc/tBu synthesis with monitoring of coupling and Fmoc removal in each step. After HFIP treatment, the sample of the protected peptide was deprotected by M to yield peptide 24 HPLC RT (Poro15c) was 4.46 min (crude 12%). For C79H137N23O26S (1,855.98) found MALDI-MS, m/z: 1,857.1 (M + H+), 1,879.1 (M + Na+), 1,895.1 (M + K+).

H-[Thz 178 ,ΔAsn 180 ]MoPrP(178–194)-OH—H-Thz-Val-Ile-Thr-Ile-Lys-Gln-His-Thr-Val-Thr-Thr-Thr-Thr-Lys-Gly-OH (25)—By-product of synthesis of the peptide 24 was isolated and characterized with Asn180 residue missing. HPLC RT (Poro15c) was 4.20 min (crude 32%). For C75H131N21O24S (1,741.94) found MALDI-MS, m/z: 1,743.0 (M + H+), 1,765.0 (M + Na+), 1,781.0 (M + K+).

H-[Thz 178 ]MoPrP(178–194)-OH—H-Thz-Val-Asn-Ile-Thr-Ile-Lys-Gln-His-Thr-Val-Thr-Thr-Thr-Thr-Lys-Gly-OH (26)—To increase the yield of protected peptide corresponding to sequence 24, the synthesis was carried out with pseudoproline dipeptides: Fmoc-Ile-Thr(ψMe,Mepro)-OH and Fmoc-Val-Thr(ψMe,Mepro)-OH. The peptide Fmoc-[Thr(ψMe,Mepro)182,189]MoPrP180–194-OClTrt-resin was assembled by automated peptide synthesis on 150 mg Fmoc-Gly-O-ClTrt-resin (0.60 mmol g−1). The remaining four amino acids were coupled manually with a monitoring of the coupling and Nα-deprotection after each step. HFIP treatment yielded 622 mg of protected peptide. The sample was deprotected by M and free peptide 26 purified. HPLC RT (Poro15c) was 4.45 min (crude 58%). For C79H137N23O26S (1,855.98) found MALDI-MS, m/z: 1,857.2 (M + H+), 1,879.2 (M + Na+), 1,895.2 (M + K+).

H-[Thz 195 ]MoPrP(195–212)-OH—H-Thz-Asn-Phe-Thr-Glu-Thr-Asp-Val-Lys-Met-Met-Glu-Arg-Val-Val-Glu-Gln-Met-OH (27)—Synthesis was carried analogously to 24, Fmoc-MoPrP198–212-OClTrt-resin was built automatically and the remaining part by manual synthesis. However, desired product was not found in HPLC of crude mixture.

H-[Thz 195 ,ΔThr 198 ,ΔVal 202 ]MoPrP(195–212)-OH—H-Thz-Asn-Phe-Glu-Thr-Asp-Lys-Met-Met-Glu-Arg-Val-Val-Glu-Gln-Met-OH (28)—By-product of 27. HPLC RT (Poro15c) was 6.81 min (crude 31%). For C82H132N22O28S4 (2,000.85) found MALDI-MS, m/z: 2,002.1 (M + H+).

H-[Thz 195 ,ΔAsn 196 ]MoPrP(195–212)-OH—H-Thz-Phe-Thr-Glu-Thr-Asp-Val-Lys-Met-Met-Glu-Arg-Val-Val-Glu-Gln-Met-OH (29)—By-product of 27 HPLC RT (Poro15c) was 7.24 min (crude 27%). For C87H142N22O29S4 (2,086.92) found MALDI-MS, m/z: 2,087.7 (M + H+).

H-[Thz 195 ]MoPrP(195–212)-OH—H-Thz-Asn-Phe-Thr-Glu-Thr-Asp-Val-Lys-Met-Met-Glu-Arg-Val-Val-Glu-Gln-Met-OH (30)—Automatically prepared Fmoc-[Thr(ψMe,Mepro)200]MoPrP198–212-OClTrt-Resin was extended by manual synthesis. However, the synthesis did not provide desired peptide according to HPLC (Poro 25a).

H-[Thz 195 ,ΔGlu-Thr 198−199 ]MoPrP(195–212)-OH—H-Thz-Asn-Phe-Thr-Asp-Val-Lys-Met-Met-Glu-Arg-Val-Val-Glu-Gln-Met-OH (31)—By-product of 30. HPLC RT (Poro25a) was 8.47 min (crude 7.1%). For C82H134N22O26S4 (1,970.87) found MALDI-MS, m/z: 1,971.6 (M + H+). Sequence was determined by Edman degradation.

H-[Thz 195 ,ΔThr-Glu-Thr 198−200 ]MoPrP(195–212)-OH—H-Thz-Asn-Phe-Asp-Val-Lys-Met-Met-Glu-Arg-Val-Val-Glu-Gln-Met-OH (32)—By-product of 30. HPLC RT (Poro25a) was 8.63 min (crude 8.4%). For C78H127N21O24S4 (1,869.82), found MALDI-MS, m/z: 1,870.5 (M + H+). Sequence was determined by Edman degradation.

Compound 33. By-product of 30. HPLC RT (Poro25a) was 8.87 min (Crude 43%). Found MALDI-MS, m/z: 1,009.3.

H-[Thz 195 ]MoPrP(195–212)-OH—H-Thz-Asn-Phe-Thr-Glu-Thr-Asp-Val-Lys-Met-Met-Glu-Arg-Val-Val-Glu-Gln-Met-OH (34)—120 mg Fmoc-Met-O-ClTrt-resin (0.42 mmol g−1). Automatically built Fmoc-MoPrP201–212-OClTrt-resin was extended by manual synthesis, which used Fmoc-Glu(OtBu)-Thr(ψMe,Mepro)-OH in the first coupling of this extension to the peptide sequence desired. HFIP treatment resulted in protected peptide (189 mg). Its sample was deprotected by M to yield peptide 34. HPLC RT (Poro25b) was 15.7 min (crude 41%). For C91H148N24O31S4 (2,200.96) found MALDI-MS, m/z: 2,204.2 (M + 3H+).

Intermediates

H-MoPrP(201–212)-OH—H-Asp-Val-Lys-Met-Met-Glu-Arg-Val-Val-Glu-Gln-Met-OH (35)—HPLC RT (Poro25b) was 12.1 min (59%). For C61H107N17O20S3 (1,493.70) found MALDI-MS, m/z: 1,494.70 (M + H+), 1,516.7 (M + Na+).

H-MoPrP(198–212)-OH—H-Thr-Glu-Thr-Asp-Val-Lys-Met-Met-Glu-Arg-Val-Val-Glu-Gln-Met-OH (36)—HPLC RT (Poro25b) was 13.1 min (44%). For C74H128N20O27S3 (1,824.84) found MALDI-MS, m/z: 1,825.8 (M + H+), 1,847.8 (M + Na+).

H-MoPrP(213–231)-OH—H-Cys-Val-Thr-Gln-Tyr-Gln-Lys-Glu-Ser-Gln-Ala-Tyr-Tyr-Asp-Gly-Arg-Arg-Ser-Ser-OH (37)—150 mg Fmoc-Ser(tBu)-O-ClTrt-resin (0.32 mmol g−1). After direct treatment of the assembled protected peptide on the resin with M and HPLC purification the peptide 37 was obtained (25 mg). HPLC RT (Poro18) was 9.15 min (crude 10%; after HPLC purification 98%). For C95H145N29O34S (2,268.02) found MALDI-MS, m/z: 2,269.0 (M + H+), 2,291.0 (M + Na+), 2,307.0 (M + K+).

H-[Asi 226 ]MoPrP(213–231)-OH—H-Cys-Val-Thr-Gln-Tyr-Gln-Lys-Glu-Ser-Gln-Ala-Tyr-Tyr-Asi-Gly-Arg-Arg-Ser-Ser-OH (38)—By-product of 37. HPLC RT (Poro18) was 9.0 min (crude 63%; after HPLC purification 98%). For C95H143N29O33S (2,251.40) found MALDI-MS, m/z: 2,251.1 (M+).

H-MoPrP(213–231)-OH—H-Cys-Val-Thr-Gln-Tyr-Gln-Lys-Glu-Ser-Gln-Ala-Tyr-Tyr-Asp-Gly-Arg-Arg-Ser-Ser-OH (39)—With aim to increase the yield of the peptide 37, the Fmoc-Asp(OtBu)-(Dmb)Gly-OH was used in automated peptide synthesis. HPLC RT (Poro18) was 9.15 min (crude 68%; after HPLC purification 98%). For C95H145N29O34S (2,268.02) found MALDI-MS, m/z: 2,269.0 (M + H+), 2,291.0 (M + Na+), 2,307.0 (M + K+).

Synthesis of thioesters 40–65 from MoPrP(93–231) sequence

Method Ea

Protected peptide (2 mg) was added to EtSH (20 eq), DIC (1.1 eq), DMAP (0.1 eq) in DCM 50 μl. After 2 h, reaction mixture was evaporated to dryness and analyzed by HPLC with subsequent MS analyzes. If necessary, the reaction time was extended up to 24 h. Deprotection of resulting thioesters was carried out with the reagent M.

Obtained thioesters

H-[Asp(S-Et) 96,99 ]MoPrP(93–104)-S-Et—H-Gly-Thr-His-Asp(S-Et)-Gln-Trp-Asp(S-Et)-Lys-Pro-Ser-Lys-Pro-S-Et (40)—HPLC RT (Poro25b) was 11.1 min (60%). For C67H102N18O17S3 (1,526.68) found MALDI-MS, m/z: 1,527.6 (M + H+), 1,549.7 (M + Na+), 1,565.8 (M + K+).

H-[Thz 105 ]MoPrP(105–113)-S-Et—H-Thz-Thr-Asn-Leu-Lys-His-Val-Ala-Gly-S-Et (41)—HPLC RT (Disc25) was 9.6 min (22%). For C42H71N13O11S2 (997.48) found ESI-MS, m/z: 998.5 (M + H+), 1,020.6 (M + Na+).

H-[Thz 114 ]MoPrP(114–118)-S-Et—H-Thz-Ala-Ala-Ala-Gly-S-Et (42)—HPLC RT (Poro25b) was 7.04 min (89%). For C17H29N5O5S2 (447.16) found ESI-MS, m/z: 470.2 (M + Na+).

H-[Thz 119 ]MoPrP(119–131)-S-Et—H-Thz-Val-Val-Gly-Gly-Leu-Gly-Gly-Tyr-Met-Leu-Gly-Ser-S-Et (43)—HPLC RT (Poro40) was 8.4 min (20%). For C55H89N13O15S3 (1,267.58) found ESI-MS, m/z: 1,268.6 (M + H+), 1,290.6 (M + Na+).

H-[Thz 132 ]MoPrP(132–141)-S-Et—H-Thz-Met-Ser-Arg-Pro-Met-Ile-His-Phe-Gly-S-Et (44)—HPLC RT (Poro25b) was 16.6 min (47%). For C53H83N15O11S4 (1,233.53) found MALDI-MS, m/z: 1,235.3 (M + 2H+).

H-[Thz 142 ]MoPrP(142–152)-S-Et—H-Thz-Asp-Trp-Glu-Asp-Arg-Tyr-Tyr-Arg-Glu-Asn-S-Et (45)—HPLC RT (Poro25b) was 12.0 min (43%). For C69H93N19O22S2 (1,603.62) found MALDI-MS, m/z: 1,604.5 (M + H+).

H-[Thz 153 ,Asp(S-Et) 158 ]MoPrP(153–164)-S-Et—H-Thz-Tyr-Arg-Tyr-Pro-Asp(S-Et)-Gln-Val-Tyr-Tyr-Arg-Pro-S-Et (46)—HPLC RT (Poro25b) was 14.0 min (66%). For C80H111N19O18S3 (1,721.75) found ESI-MS, m/z: 1,783.6 (M + Na+ K+).

H-[Thz 165 ]MoPrP(165–177)-S-Et—H-Thz-Asp-Gln-Tyr-Ser-Asn-Gln-Asn-Asn-Phe-Val-His-Asp-S-Et (47)—HPLC RT (Disc25) was 7.6 min (5%). For C68H94N20O24S2 (1,638.62) found MALDI-MS, m/z: 1,639.4 (M + H+).

Method Eb

Protected peptide (2 mg) was added to PyBOP (3 eq) and DIEA (4 eq) in 50 uL of DCM. After 2 min stirring, EtSH (3 eq) was added. Work-up was according to the method Ea.

Obtained peptides

H-[Thz 178 ]MoPrP(178–194)-S-Et—H-Thz-Val-Asn-Ile-Thr-Ile-Lys-Gln-His-Thr-Val-Thr-Thr-Thr-Thr-Lys-Gly-S-Et (48)—Peptide thioester desired was not identified by HPLC analysis (Poro 25b).

H-[Thz 178 ]MoPrP(178–194)-pyrrolidine—H-Thz-Val-Asn-Ile-Thr-Ile-Lys-Gln-His-Thr-Val-Thr-Thr-Thr-Thr-Lys-Gly-pyrrolidine (49)—By product of 48. HPLC RT (Poro25b) was 9.1 min (51%). For C83H144N24O25S (1,909.05) found MALDI-MS, m/z: 1,909.8 (M+), 1,932.0 (M + Na+).

H-[Thz 195 ]MoPrP(195–212)-S-Et (50)—H-Thz-Asn-Phe-Thr-Glu-Thr-Asp-Val-Lys-Met-Met-Glu-Arg-Val-Val-Glu-Gln-Met-S-Et—Peptide thioester was not identified by HPLC analysis (Poro 25b).

H-[Thz 195 ]MoPrP(195–212)-pyrrolidine—H-Thz-Val-Asn-Ile-Thr-Ile-Lys-Gln-His-Thr-Val-Thr-Thr-Thr-Thr-Lys-Gly-pyrrolidine (51)—By product of 50. HPLC RT (Poro25b) was 16.0 min (60%). For C95H155N25O30S4 (2,254.03) found MALDI-MS, m/z: 2,252.9 (M − H+).

Method Ec

This method was modified according to von Eggelkraut-Gottanka et al. (2003) method. Corresponding peptide (2 mg) was added to PyBOP (3 eq), DIEA (5 eq) in 50 μl DCM. After 2 min stirring, p-acetamidothiophenol (3 eq) was added. After 2 h, reaction mixture was processed in accordance with the procedure Ea.

Obtained peptides

H-MoPrP(93–104)-S-Ph-NHCOCH 3 —H-Gly-Thr-His-Asn-Gln-Trp-Asn-Lys-Pro-Ser-Lys-Pro-S-Ph-NHCOCH3 (52)—HPLC RT (Poro25b) was 9.32 min (68%). For C69H99N21O18S (1,541.72) found MALDI-MS, m/z: 1,542.6 (M + H+), 1,564.7 (M + Na+), 1,580.7 (M + K+).

H-[Thz 114 ]MoPrP(114–118)-S-Ph-NHCOCH 3 —H-Thz-Ala-Ala-Ala-Gly-S-Ph-NHCOCH3 (53)—HPLC RT (Poro25b) was 8.68 min (47%). For C23H32N6O6S2 (552.18) found ESI-MS, m/z: 553.2 (M + H+).

H-[Thz 114 ]MoPrP(114–118)-pyrrolidine—H-Thz-Ala-Ala-Ala-Gly-pyrrolidine (54)—By-product of 53 synthesis. HPLC RT (Poro25b) was 5.40 min (31%). For C19H32N6O5S (456.22) found ESI-MS, m/z: 457.2 (M + H+), 479.2 (M + Na+).

H-[Thz 119 ]MoPrP(119–131)-S-Ph-NHCOCH 3 —H-Thz-Val-Val-Gly-Gly-Leu-Gly-Gly-Tyr-Met-Leu-Gly-Ser-S-Ph-NHCOCH3 (55)—HPLC RT (Poro25b) was 15.9 min (34%). For C61H92N14O16S3 (1,372.60) found MALDI-MS, m/z: 1,395.7 (M + Na+), 1,262.4 (M + K+).

H-[Thz 119 ]MoPrP(119–131)-pyrrolidine—H-Thz-Val-Val-Gly-Gly-Leu-Gly-Gly-Tyr-Met-Leu-Gly-Ser-pyrrolidine (56)—By-product of 55. HPLC RT (Poro25b) was 14.6 min (25%). For C57H92N14O15S2 (1,276.63) found MALDI-ESI-MS, m/z: 1,299.7 (M + Na+), 1,315.7 (M + K+).

H-[Thz 142 ]MoPrP(142–152)-S-Ph-NHCOCH 3 —H-Thz-Asp-Trp-Glu-Asp-Arg-Tyr-Tyr-Arg-Glu-Asn-S-Ph-NHCOCH3 (57)—HPLC RT (Poro25b) was 9.4 min (65%). For C75H96N20O23S2 (1,708.64) found MALDI-MS, m/z: 1,709.7 (M + H+).

H-[Thz 142 ]MoPrP(142–152)-pyrrolidine—H-Thz-Asp-Trp-Glu-Asp-Arg-Tyr-Tyr-Arg-Glu-Asn-pyrrolidine (58)—By-product of 57. HPLC RT (Poro25b) was 12.1 min (10%). For C71H96N20O22S (1,612.67) found MALDI-MS, m/z: 1,613.6 (M + H+).

H-[Thz 153 ]MoPrP(153–164)-S-Ph-NHCOCH 3 —H-Thz-Tyr-Arg-Tyr-Pro-Asn-Gln-Val-Tyr-Tyr-Arg-Pro-S-Ph-NHCOCH3 (59)—HPLC RT (Poro25b) was 12.5 min (56%). For C84H111N21O19S2 (1,781.78) found MALDI-MS, m/z: 1,783.1 (M + H+).

H-[Thz 165 ]MoPrP(165–177)-S-Ph-NHCOCH 3 —H-Thz-Asp-Gln-Tyr-Ser-Asn-Gln-Asn-Asn-Phe-Val-His-Asp-S-Ph-NHCOCH3 (60)—Peptide was not identified using HPLC analysis (Poro25b).

H-[Thz 165 ]MoPrP(165–177)-pyrrolidine—H-Thz-Asp-Gln-Tyr-Ser-Asn-Gln-Asn-Asn-Phe-Val-His-Asp-pyrrolidine (61)—By product of 60 synthesis. HPLC RT (Poro25b) was 9.0 min (33%). For C70H97N21O24S (1,647.67) found MALDI-MS, m/z: 1,648.5 (M + H+), 1,671.0 (M + Na+), 1,686.5 (M + K+).

H-[Thz 178 ]MoPrP(178–194)-S-Ph-NHCOCH 3 —H-Thz-Val-Asn-Ile-Thr-Ile-Lys-Gln-His-Thr-Val-Thr-Thr-Thr-Thr-Lys-Gly-S-Ph-NHCOCH3 (62)—HPLC RT (Poro25b) was 10.6 min (26%). For C87H144N24O26S2 (2,005.01) found MALDI-MS, m/z: 2,006.0 (M + H+).

H-[Thz 178 ]MoPrP(178–194)-pyrrolidine—H-Thz-Val-Asn-Ile-Thr-Ile-Lys-Gln-His-Thr-Val-Thr-Thr-Thr-Thr-Lys-Gly-pyrrolidine (63)—By product of 62 synthesis. HPLC RT (Poro25b) was 9.1 min (19%). For C83H144N24O25S (1,909.05) found MALDI-MS, m/z: 1,909.9 (M + H+).

H-[Thz 195 ]MoPrP(195–212)-S-Ph-NHCOCH 3 —H-Thz-Asn-Phe-Thr-Glu-Thr-Asp-Val-Lys-Met-Met-Glu-Arg-Val-Val-Glu-Gln-Met-S-Ph-NHCOCH3 (64)—HPLC RT (Poro25b) was 15.1 min (23%). For C99H155N25O31S5 (2,349.99) found MALDI-MS, m/z: 2,352.0 (M + 2+), 2,372.9 (M + Na+).

H-[Thz 195 ]MoPrP(195–212)-pyrrolidine—H-Thz-Asn-Phe-Thr-Glu-Thr-Asp-Val-Lys-Met-Met-Glu-Arg-Val-Val-Glu-Gln-Met-pyrrolidine (65)—By product of 64 synthesis. HPLC RT (Poro25b) was 12.9 min (18%). For C95H155N25O30S4 (2,254.03) found MALDI-MS, m/z: 2,255.0 (M + H+), 2,276.9 (M + Na+).

Results and discussion

To increase the overall yield of the HuPrP106–126 (Fig. 1), we decided to check the chemical ligation strategy for its synthesis. Therefore, this sequence was split to smaller fragments (Table 2), which were subjected to thioesters formation for their subsequent ligation (Dawson et al. 1994) and desulfurization (Wan and Danishefsky 2007) procedure. Two splitting sites in the molecule, between Gly114-Ala115 and Gly119-Ala120 residues, were designed (chosen) to avoid racemization in corresponding peptide thioesters with C-terminal Gly residue. An introduction of the retro synthetically functional group was applied on the N-terminal Ala120 residue, so that the Ala to Cys, resp. to Sec transformation was carried out (Wan and Danishefsky 2007). However, the syntheses of the segments with 12 (the items 4 and 5) and 14 (the items 8, 10, 12, 14) residues, due to low yields, were non-competitive to the divergent Fmoc approach described (Šebestik et al. 2006; Table 2, method A). Only the syntheses of selenocysteine derivative in the C-terminus (120–126, item 3), and N-terminal peptide thioester (106–114, item 13) on 2-chlorotrityl chloride resin (von Eggelkraut-Gottanka et al. 2003) were found to be competitive. The limited availability of the difficult peptide segments due to their low yields led us to design the shorter ones of the central part (115–119) using the Kent’s modular approach (Bang and Kent 2004), in which the both splitting sites were used in one synthetic scheme. Therefore, the middle part of the peptide was prepared according to adapted version of von Eggelkraut-Gottanka et al. (2003) with a protection of the N-terminal Cys as thiazolidine-4-carboxylic acid. This method provided two peptides (items 15 and 16) in very competitive yields to divergent peptide synthesis (item 2). From the yields of the items 3, 11, and 14, the synthesis of N-terminal peptide thioester (item 3) could be viewed as the yield-limiting step for the synthesis of the above peptide.

In general, our study on preparation of the peptide thioester segments for the HuPrP106–126 synthesis by chemical ligation have found the adopted version of von Eggelkraut-Gottanka et al. (2003) strategy as the method of choice because of the presence of the C-terminal Gly residue in HuPrP106–126.

Consequently, above method was employed for a scan of the mouse prion domain 93–231 (Fig. 2). The first step included the sequences assembling from the corresponding Fmoc amino acids on the 2-chlorotrityl resin as described in the method E with the exception of attachment of the last amino acid in the given sequence with Boc protection. These sequences were selected as follows: H-MoPrP(93–104)-OH, H-[Thz105]MoPrP(105–113)-OH, H-[Thz114]MoPrP(114–118)-OH, H-[Thz119]MoPrP(119–131)-OH, H-[Thz132]MoPrP(132–141)-OH, H-[Thz142]MoPrP(142–152)-OH, H-[Thz153]MoPrP(153–164)-OH, H-[Thz165]MoPrP(165–177)-OH, H-[Thz178]MoPrP(178–194)-OH, H-[Thz195]MoPrP(195–212)-OH and H-MoPrP(213–231)-OH. A usage of Thz residues introduced several mutations into prion protein: Lys105Thz, Ala114Thz, Ala119Thz, Ala132Thz, Asn142Thz, Met153Thz, Val165Thz, and Glu195Thz. Danishefsky’s reduction (Wan and Danishefsky 2007) can remove the Ala mutations (Ala114Thz, Ala119Thz, and Ala132Thz). Thus only following mutations will remain: Lys105Ala, Asn142Ala, Met153Ala, Val165Ala, and Glu195Ala. From synthetic point of view, these mutations will facilitate the protein synthesis. Since Glu195Ala is very close to Asn196, which glycosylation has no effect on biological properties of prion protein (Wollscheid et al. 2009), it can be speculated that from structural point of view the mutation cannot influence the conformation of prion protein, too. This can be confirmed by database search (GPCRDB: Swiss-Prot entry 2011) where the position 195 can contain both acidic Glu residue and basic Lys residue. Site 104 is involved in biologically active mutation: Pro104Leu where the secondary structure breaking Pro is replaced with Leu (Spielhaupter and Schaetzl 2001). Thus, we can also suggest that Lys105Ala mutation is not sensitive for overall structure changes. For sites 142, 153, and 165, there is no evidence for influence of these mutations on prion structure or function.

In the second step, the small portions of the protected peptide-resins were treated with a mixture M and cleaved peptides analyzed by HPLC/MS. HPLC yields at 220 nm were used as a criterion of the sequence availability (Table 3). The sequences shorter than 13 amino acids were available in a moderate (items 1, 2, 5–7) to excellent (items 3, 4, 8) purity when an automated peptide synthesis was employed. The moderate yields were probably caused by inappropriate sequence length: 9–11 amino acid residues (Hagenmaier 1970). The occurrence of deletion peptides in the sequences longer than 15 residues (items 9–11) demanded employment of the special dipeptides, which prevent an undesirable hydrogen bond formation during the synthesis: either an amide bond protection with Dmb group (Zahariev et al. 2005) in the H-MoPrP(213–231)-OH preparation (item 11) or pseudo-proline masking of Ser and Thr residues (White et al. 2004). The synthesis of the latter peptide without a usage of Fmoc-Asp-(Dmb)Gly-OH (Cardona et al. 2008) mostly provided corresponding aspartimide.

In the case of H-[Thz178]MoPrP(178–194)-OH, an introduction of pseudo-proline Fmoc-Ile-Thr(ψMe,Mepro)-OH at positions 181–182 and 188–189, respectively, improved the HPLC yield nearly 5 times (item 9). Since the automated synthesis of the sequence H-[Thz195]MoPrP(195–212)-OH (item 10) has failed, this segment was finally prepared manually, by continuation from its sequence 201–212, prepared on the synthesizer.

Sufficient availability of corresponding peptide segments opened a venue for syntheses of their thioesters (Table 4) with the exception of the last sequence H-MoPrP(213–231)-OH that was prepared as a free acid, because subsequent ligation was not necessary. The fully protected peptide segments were converted to thioesters either by an adopted version of von Eggelkraut-Gottanka et al. (2003) using DIC as a coupling agent (Table 4, column DIC/EtSH, Ea) or by the genuine version using PyBOP as a coupling agent and p-acetamidothiophenol as a thiolic source (Table 4, the last column, Ec).

Table 4 Conversion of protected peptides to corresponding unprotected thioesters
Full size table

The DIC coupling with poor nucleophilic EtSH require prolongated time for conversion of corresponding peptide to the peptide thioesters. During this extensive thiolic treatment a vulnerable group such as Asn and Asp(OtBu) can be converted to corresponding thioesters Asp(SEt) (Table 4, items 1 and 7; compounds 40 and 46). The longer peptides with poorer solubility did not provide corresponding thioesters at all (items 9 and 10).

On the other hand, the genuine version of von Eggelkraut-Gottanka et al. (2003) method led to desired conversion of free acid to peptide thioesters almost in every evaluated case. The exception was synthesis of H-[Thz165]MoPrP(165–177)-S-Ph(NH-Ac) (60) where only the corresponding H-[Thz165]MoPrP(165–177)-pyrrolidone (61) was obtained accompanied with an unconverted free acid. A usage of PyBOP with a poorer nucleophile—EtSH—led to pyrrolidone by-products without formation of desired peptide thioester (Table 4, items 9 and 10). In fact, the pyrrolidone derivative on C-terminus was obtained in almost all cases with PyBOP activation as either main product or by-product (Fig. 4).

Fig. 4
figure 4

Formation of by-products during PyBOP activation. Successfully prepared peptide thioesters (52, 53, 55, 57, 59, 62, 64) and obtained by-products (49, 51, 54, 56, 58, 61, 63, 65)

Full size image

The formation of H-[Thz165]MoPrP(165–177)-SEt (47) was achieved with the adopted version of von Eggelkraut-Gottanka et al. (2003) method with very poor HPLC yield (ca 5%). It is a bottleneck of the conversion of assembled peptides to thioesters. For future, we suggest the problematic peptide should be prepared on resin directly without following conversion to thioester either by Boc/Bzl approach or another method of Fmoc/tBu one. The limitation of proposed strategy is C-terminal Asp residue vulnerable to several side reactions.

Conclusions

The comparative study carried on HuPrP106–126—the difficult sequence—revealed that an assembly of fully protected peptide with subsequent conversion to thioester is the favorable way for short thioester syntheses. This strategy was evaluated on peptide thioesters derived from C-domain of mouse PrP(93–231). With the exception of H-[Thz165]MoPrP(165–177)-SEt (47), the peptide thioesters are easily available. The last mentioned sequence is accessible only in limited yield and poses a difficulty for future protein assembly by native chemical ligations. However, it may be synthesized by Boc/Bzl approach with decreased possibility for future modification of this site.