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Application of the Logic of Cysteine-Free Native Chemical Ligation to the Synthesis of Human Parathyroid Hormone (hPTH)

Shiying Shang,§ Zhongping Tan,§ and Samuel J. Danishefsky§¶║

§Bio-Organic Chemistry Laboratory, Molecular Pharmacology and Chemistry Program, Memorial

Sloan-Kettering Cancer Center, 1275 York Avenue, New York, NY 10065; ¶Department of Chemistry,

Columbia University, New York, NY 10027

║ To whom reprint requests should be addressed.

E-mail: s-danishefsky@ski.mskcc.org

Content Material and Methods S2 Solid Phase Peptide Synthesis S2 Preparation and Characterization of Peptide Precursors for Ligation S3 Preparation and Characterization of Ligation and Desulfurization Products S5 Circular Dichroism Spectra for the synthetic hPTH S9 Reference S9

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Materials and Methods All commercial materials (Aldrich, Fluka, Nova) were used without further purification. All solvents were reagent grade or HPLC grade (Fisher). Anhydrous THF, diethyl ether, CH2Cl2, toluene, and benzene were obtained from a dry solvent system (passed through column of alumina) and used without further drying. All reactions were performed under an atmosphere of pre-purified dry Ar(g). NMR spectra (1H and 13C) were recorded on a Bruker Advance II 600 MHz or Bruker Advance DRX-500 MHz, referenced to TMS or residual solvent. Low-resolution mass spectral analyses were performed with a JOEL JMS-DX-303-HF mass spectrometer or Waters Micromass ZQ mass spectrometer. Analytical TLC was performed on E. Merck silica gel 60 F254 plates and flash column chromatography was performed on E. Merck silica gel 60 (40–63 mm). Yields refer to chromatographically pure compounds. HPLC: All separations involved a mobile phase of 0.05% TFA (v/v) in water (solvent A)/0.04% TFA in acetonitrile (solvent B). LCMS analyses were performed using a Waters 2695 Separations Module and a Waters 996 Photodiode Array Detector equipped with Varian Microsorb 100-5, C18 150x2.0mm and Varian Microsorb 300-5, C4 250x2.0mm columns at a flow rate of 0.2 mL/min. UPLC-MS analyses were performed using a Waters AcquityTM Ultra Preformance LC system equipped with Acquity UPLC® BEH C18, 1.7µl, 2.1 x 100 mm, Acquity UPLC® BEH C8, 1.7µl, 2.1 x 100 mm, Acquity UPLC® BEH 300 C4, 1.7µl, 2.1 x 100 mm columns at a flow rate of 0.3 mL/min. Preparative separations were performed using a Ranin HPLC solvent delivery system equipped with a Rainin UV-1 detector and Varian Dynamax using Varian Microsorb 100-5, C18 250x21.4mm and Varian Microsorb 300-5, C4 250x21.4mm columns at a flow rate of 16.0 mL/min. Solid Phase Peptide Synthesis Automated peptide synthesis was performed on an Applied Biosystems Pioneer continuous flow peptide synthesizer. Peptides were synthesized under standard automated Fmoc protocols. The deblock mixture was a mixture of 100/5/5 of DMF/piperidine/DBU. The following Fmoc amino acids from NovaBiochem were employed: Fmoc-Ala-OH, Fmoc-Arg(Pbf)-OH, Fmoc-Asn(Trt)-OH, Fmoc-Asp(OtBu)-OH, Boc-Thz-OH, Fmoc-Glu(OtBu)-OH, Fmoc-Gln(Trt)-OH, Fmoc-Gly-OH, Fmoc-His(Trt)-OH, Fmoc-Ile-OH, Fmoc-Leu-OH, Fmoc-Lys(Boc)-OH, Fmoc-Met-OH, Fmoc-Phe-OH, Fmoc-Pro-OH, Fmoc-Ser(tBu)-OH, Fmoc-Thr(tBu)-OH, Fmoc-Val-OH.

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Preparation and Characterization of Peptide Precursors for Ligation Peptide Thiophenyl Esters 1 The fully protected peptidyl acid was prepared by SPPS using the general procedure described above. After cleavage, 156.4 mg crude peptide was obtained (68% yield). The fully protected peptidyl acid (71.7 mg, 15.8 µM, 1.1 equiv) and HCl⋅H-Trp-SPh (4.8 mg, 14.4 µM, 1.0 equiv) in CHCl3/TFE (v/v = 3/1, 620 µL) was cooled to –10 °C. HOOBt (2.6 mg, 15.8 µM, 1.1 equiv) and EDCI (2.8 µL, 15.8 µM, 1.1 equiv) were added. The reaction mixture was stirred at room temperature for 3 h. The solvent was then blown off under a gentle N2 stream and TFA/H2O/TIS (95:2.5:2.5) was added. After deprotection for 45 min, TFA was blown off and the oily residue was triturated with diethyl ether. The precipitate was pelleted and the ether was subsequently decanted. The resulting solid was purified by HPLC to give 11.5 mg 1, 28% yield.

Peptide 1: Chemical Formula: C124H193N35O35S3, Expected Mass: 2828.36, [M+2H]2+ m/z = 1415.18, [M+3H]3+ m/z = 943.79. Thioleucine-containing Peptide Alkyl Thioester 2 The peptide resin from the Fmoc SPPS (6.49 µmol, 1.0 equiv) was mixed with Boc-Leu(SSMe)-OH (2.0 mg, 6.49 µmol, 1.0 equiv), HATU (7.6 mg, 19.5 µmol, 3.0 equiv) and DIEA (6.8 µL, 39.0 µmol, 6.0 equiv) in DMF (200 µL) and stirred at room temperature for 10 min. The resin was washed with DMF, DCM and MeOH several times and dried under vacuum. The dried resin was cleaved by treatment with AcOH/TFE/DCM (1:1:8) for 2 × 1 hour to yield the fully protected peptidyl acids. The above crude peptidyl acid (6.49 µM, 1.0 equiv) and HCl⋅H-Gly-3-thiopropionic acid ethyl ester (7.79 µM, 1.2 equiv) in CHCl3/TFE (v/v = 3/1, 435 µL) was cooled to –10 °C. HOOBt (6.49 µM, 1.0 equiv) and EDCI (6.49 µM, 1.0 equiv) were added. The reaction mixture was stirred at room temperature for 3.5 h. The solvent was then blown off under a gentle N2 stream and TFA/H2O/TIS (95:2.5:2.5) was added. After deprotection for 20 min, TFA was blown off and the oily residue was triturated with diethyl ether. The precipitate was pelleted and the ether was subsequently decanted. The resulting solid was purified by HPLC to give 3.7 mg 2, 30% yield (calculated based on the resin weight).

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Peptide 2: Chemical Formula: C85H142N24O21S3, Expected Mass: 1930.99, [M+2H]2+ m/z = 966.50. Peptide Thiophenyl Esters 3 The fully protected peptidyl acid was prepared by SPPS using the general procedure described above. After cleavage, 45.5 mg crude peptide was obtained (23% yield). The fully protected peptidyl acid (45.5 mg, 11.3 µM, 1.1 equiv) and HCl⋅H-Leu-SPh (2.7 mg, 10.3 µM, 1.0 equiv) in CHCl3/TFE (v/v = 3/1, 440 µL) was cooled to –10 °C. HOOBt (1.8 mg, 11.3 µM, 1.1 equiv) and EDCI (2.0 µL, 11.3 µM, 1.1 equiv) were added. The reaction mixture was stirred at room temperature for 3 h. The solvent was then blown off under a gentle N2 stream and TFA/H2O/TIS (95:2.5:2.5) was added. After deprotection for 45 min, TFA was blown off and the oily residue was triturated with diethyl ether. The precipitate was pelleted and the ether was subsequently decanted. The resulting solid was purified by HPLC to give 7.2 mg 3, 28% yield.

Peptide 3: Chemical Formula: C105H172N34O30S2, Expected Mass: 2453.24, [M+2H]2+ m/z = 1227.62, [M+3H]3+ m/z = 818.75. Thiovaline-containing Peptide 4 The peptide resin from the Fmoc SPPS (6.64 µmol, 1.0 equiv) was mixed with Boc-Val(SSMe)-OH (2.0 mg, 6.64 µmol, 1.0 equiv), HATU (7.6 mg, 19.9 µmol, 3.0 equiv) and DIEA (6.9 µL, 39.8 µmol, 6.0

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equiv) in DMF (200 µL) and stirred at room temperature for 10 min. The resin was washed with DMF, DCM and MeOH several times and dried under vacuum. The peptide was cleaved and deprotected by treatment with TFA/H2O/TIS (95:2.5:2.5) for 1 h 10 min. TFA was then blown off and the oily residue was triturated with diethyl ether. The precipitate was pelleted and the ether was subsequently decanted. The resulting solid was purified by HPLC to give 8.9 mg 2, 49% yield (calculated based on the resin weight).

Peptide 3: Chemical Formula: C114H193N33O42S2, Expected Mass: 2760.34, [M+2H]2+ m/z = 1381.17, [M+3H]3+ m/z = 921.11. Preparation and Characterization of Ligation and Desulfurization Products Ligated Peptide 9 The synthesis of 9 was carried out under kinetically controlled ligation conditions. Peptide 1 (6.1 mg, 2.2 µmol, 1.1 equiv) and peptide 2 (3.7 mg, 1.9 µmol, 1.0 equiv) were dissolved in ligation buffer (600 µL, 6 M Gdn⋅HCl, 100 mM Na2HPO4, 50 mM TCEP, pH 7.5). The reaction mixture was stirred at room temperature for 9 h. The reactions were monitored by LC-MS and purified directly by HPLC to give 5.2 mg ligated peptide 9, 59% yield. As estimated by LC-MS analysis, the ratio between the cyclization product of 2 and the ligation product 9 is 1:10.

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Peptide 9: Chemical Formula: C202H327N59O56S4, Expected Mass: 4603.34, [M+2H]2+ m/z = 2302.67, [M+3H]3+ m/z = 1535.45, [M+4H]4+ m/z = 1151.84, [M+5H]5+ m/z = 921.67. Ligated Peptide 10 and 11 Peptide 3 (2.7 mg, 1.1 µmol, 1.6 equiv) and peptide 4 (1.8 mg, 0.67 µmol, 1.0 equiv) were dissolved in ligation buffer (300 µL, 6 M Gdn⋅HCl, 100 mM Na2HPO4, 50 mM TCEP, pH 7.5). The reaction mixture was stirred at room temperature for 9 h. The reactions were monitored by LC-MS and the crude peptide 10 was deprotected directly without further purification. The Thz was converted to cysteine by addition of 0.2 M methoxylamine HCl at pH 4.0. The reaction mixture was stirred at room temperature for 5 h. The reactions were monitored by LC-MS and purified directly by HPLC to give 2.9 mg deprotected peptide 11, 86% yield.

Peptide 11: Chemical Formula: C211H357N67O72S2, Expected Mass: 5045.58, [M+3H]3+ m/z = 1682.86, [M+4H]4+ m/z = 1262.40, [M+5H]5+ m/z = 1010.12, [M+6H]7+ m/z = 841.93, [M+7H]5+ m/z = 721.81.

*: Unidentified impurity. Full purification was achieved after desulfurization. Ligated Peptide 12

Peptide 9 (1.1 mg, 0.24 µmol, 1.1 equiv) and peptide 11 (1.1 mg, 0.22 µmol, 1.0 equiv) were dissolved

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in ligation buffer (100 µL, 6 M Gdn⋅HCl, 300 mM Na2HPO4, 200 mM MPAA, 20 mM TCEP, pH 7.9). The reaction mixture was stirred at room temperature for 4 h. The reactions were monitored by LC-MS and purified directly by HPLC to give 1.3 mg ligated peptide 12, 59% yield.

Peptide 12: Chemical Formula: C408H674N126O126S5, Expected Mass: 9514.88, [M+5H]5+ m/z = 1903.98, [M+6H]6+ m/z = 1586.81, [M+7H]7+ m/z = 1360.27, [M+8H]8+ m/z = 1190.36, [M+9H]9+ m/z = 1058.21, [M+10H]10+ m/z = 952.49, [M+11H]11+ m/z = 865.99, [M+12H]12+ m/z = 793.91, [M+13H]13+ m/z = 732.91.

*: Unidentified impurity. Full purification was achieved after desulfurization. Desulfurized Peptide hPTH To a solution of the purified ligated peptide 12 (0.7 mg) in degassed CH3CN/H2O (v/v = 1:1, 0.2 ml) were added 0.2 ml of 0.5 M bond-breaker® TCEP solution (Pierce), 0.02 ml of 2-methyl-2-propanethiol and 0.2 ml of radical initiator (0.1 M in H2O). The reaction mixture was stirred at 37 °C for 2 h. The reactions were monitored by LC-MS and purified directly by HPLC to give 0.6 mg hPTH, 86%.

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Synthetic hPTH:

Human PTH: Chemical Formula: C408H674N126O126S2, Expected Mass: 9418.96, [M+5H]5+ m/z = 1884.79, [M+6H]6+ m/z = 1570.83, [M+7H]7+ m/z = 1346.57, [M+8H]8+ m/z = 1178.37, [M+9H]9+ m/z = 1047.55, [M+10H]10+ m/z = 942.90, [M+11H]11+ m/z = 857.27, [M+12H]12+ m/z = 785.91, [M+13H]13+ m/z = 725.54.

Recombinant hPTH from Bachem:

*: Unidentified impurities

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CD Spectra CD spectra were obtained on an Aviv 410 circular dichroism spectropolarimeter. Protein concentrations were determined based on the extinction coefficient, calculated according to the number of Trp residue.1 The solvent for all experiments were 1:1 CH3CN:H2O. Spectra were collected with a 1 mm pathlength cuvette at protein concentration of 14 µM and 7 µM. Reference 1. Edelhoch H. Spectroscopic determination of tryptophan and tyrosine in proteins. Biochemistry 1967;6:1948–1954.