Solid Phase Peptide Synthesis (SPPS) and Solid Phase. Fragment Coupling (SPFC) Mediated by Isonitriles

Similar documents
Supporting Information

Supporting Information

p-toluenesulfonic Acid-Mediated 1,3-Dipolar Cycloaddition of

Zinc Chloride Promoted Formal Oxidative Coupling of Aromatic Aldehydes and Isocyanides to α- Ketoamides

Development of a Cell-penetrating Peptide that Exhibits Responsive. Changes in its Secondary Structure in the Cellular Environment

Eur. J. Org. Chem WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim, 2009 ISSN X SUPPORTING INFORMATION

An efficient methodology to introduce o-(aminomethyl) phenyl-boronic acids into peptides: alkylation of secondary amines

Supporting Information

Supporting Information for

Supporting Materials. Experimental Section. internal standard TMS (0 ppm). The peak patterns are indicated as follows: s, singlet; d,

Supporting Information. Recyclable hypervalent-iodine-mediated solid-phase peptide

Nitro-Grela-type complexes containing iodides. robust and selective catalysts for olefin metathesis

ph Switchable and Fluorescent Ratiometric Squarylium Indocyanine Dyes as Extremely Alkaline Sensors

Electronic Supplementary Information

Supporting Information

Masatoshi Shibuya,Takahisa Sato, Masaki Tomizawa, and Yoshiharu Iwabuchi* Department of Organic Chemistry, Graduate School of Pharmaceutical Sciences,

Supporting Information. Efficient copper-catalyzed Michael addition of acrylic derivatives with primary alcohols in the presence of base

Manganese powder promoted highly efficient and selective synthesis of fullerene mono- and biscycloadducts at room temperature

Preparation of Stable Aziridinium Ions and Their Ring Openings

Supplementary Material

ELECTRONIC SUPPLEMENTARY INFORMATION

Facile Cu(II) mediated conjugation of thioesters and thioacids to peptides and proteins under mild conditions

Acyl Radical Reactions in Fullerene Chemistry: Direct Acylation of. [60]Fullerene through an Efficient Decatungstate-Photomediated Approach.

Novel D-erythro N-Octanoyl Sphingosine Analogs As Chemo- and Endocrine. Resistant Breast Cancer Therapeutics

Triptycene-Based Small Molecules Modulate (CAG) (CTG) Repeat Junctions

SUPPORTING INFORMATION TO: Oligonucleotide cyclization: The thiol-maleimide reaction revisited. Albert Sánchez, Enrique Pedroso, Anna Grandas*

Supporting Information. for. Synthesis of dye/fluorescent functionalized. dendrons based on cyclotriphosphazene

Supplementary Notes. HTS compatible FRET-based conformational sensors clarify membrane receptor activation

Electronic Supplementary Information

Supporting Information

Supporting Information. First synthetic entry to the trimer stage of 5,6-dihydroxyindole polymerization: orthoalkynylaniline-based

Preparation of Fluorinated Tetrahydropyrans and Piperidines using a New Nucleophilic Fluorination Reagent DMPU/HF

Regioective Halogenation of 2-Substituted-1,2,3-Triazole via sp 2 C-H Activation

Enabling N-to-C Ser/Thr Ligation for Convergent Protein Synthesis via Combining Chemical Ligation Approaches. Supplementary Information

Supporting Information. Radical fluorination powered expedient synthesis of 3 fluorobicyclo[1.1.1]pentan 1 amine

Schwartz s reagent-mediated regiospecific synthesis of 2,3-disubstituted indoles from isatins

Efficient Metal-Free Pathway to Vinyl Thioesters with Calcium Carbide as the Acetylene Source

An Unusual Glycosylation Product from a Partially Protected Fucosyl Donor. under Silver Triflate activation conditions. Supporting information

SUPPLEMENTARY INFORMATION

All chemicals were obtained from Aldrich, Acros, Fisher, or Fluka and were used without

Thiol-Activated gem-dithiols: A New Class of Controllable. Hydrogen Sulfide (H 2 S) Donors

SUPPORTING INFORMATION. Transition metal-promoted synthesis of 2-aryl/heteroaryl-thioquinazoline: C-S

Supplementary Information

SUPPLEMENTAL FIGURE 1 Structures and IC50 values of compounds 13 32

Synthesis of Human GLP-1 (7-36) by Chemoselective α-ketoacid Hydroxylamine Peptide Ligation of Unprotected Fragments

Rameshwar Prasad Pandit and Yong Rok Lee * School of Chemical Engineering, Yeungnam University, Gyeongsan , Korea

Supporting Information

Stereoselective Aza-Darzens Reactions of Tert- Butanesulfinimines: Convenient Access to Chiral Aziridines

Supporting Information

Microwave heating in peptide side chain modification via sulfhydryl reaction

Synthetic chemistry-led creation of a difluorinated biaryl ether non-nucleoside reverse transcriptase inhibitor

Supporting Information

Development of a near-infrared fluorescent probe for monitoring hydrazine in serum and living cells

Small Scale Preparative Isolation of Corticosteroid Degradation Products Using Mass-Based Fraction Collection Application

Preparation, isolation and characterization of N α -Fmoc-peptide isocyanates: Solution synthesis of oligo-α-peptidyl ureas

Supporting information

Supplementary Materials Contents

Allenylphosphine oxides as simple scaffolds for. phosphinoylindoles and phosphinoylisocoumarins

Toxins 2016, 8, 222; doi: /toxins

Supporting Information

Catalytic decarboxylative alkylation of β-keto acids with sulfonamides via the cleavage of carbon nitrogen and carbon carbon bonds

Supporting Information

Copyright Wiley-VCH Verlag GmbH, D Weinheim, Angew. Chem

Automated synthesis of backbone protected peptides

Supporting Information for. Boronic Acid Functionalized Aza-Bodipy (azabdpba) based Fluorescence Optodes for the. analysis of Glucose in Whole Blood

Supporting Information. for. Access to pyrrolo-pyridines by gold-catalyzed. hydroarylation of pyrroles tethered to terminal alkynes

Improved Carbonylation of Heterocyclic Chlorides and Challenging Aryl Bromides

Synthesis of cationic porphyrin modified amino. acids

Supplementary Information. Intramolecular 5-exo, 7-endo-dig Transition Metal-Free Cyclization

Supporting Information

An Orthogonal Array Optimization of Lipid-like Nanoparticles for. mrna Delivery in Vivo

Electronic Supplementary Information

# Supplementary Material (ESI) for Chemical Communications # This journal is The Royal Society of Chemistry 2005

Cu-Catalyzed Direct C6-Arylation of Indoles

Inhibition of glyoxalase I: the first low-nanomolar tight-binding inhibitors. Swati S. More ξ and Robert Vince*

Supporting Information

Lewis acid-catalyzed regioselective synthesis of chiral α-fluoroalkyl amines via asymmetric addition of silyl dienolates to fluorinated sulfinylimines

Reaction of difluorocarbene with acetylene ethers generates novel fluorinated 5- and 7-membered carbacycles.

Electronic Supplementary Information. Quinine/Selectfluor Combination Induced Asymmetric Semipinacol Rearrangement of

Supporting Information. Nitrodibenzofuran: a One- and Two-Photon Sensitive Protecting Group that is Superior to

Selective Functionalization of Antimycin A Through an N- Transacylation Reaction. Arnaud Chevalier, Yanmin Zhang, Omar M. Khdour and Sidney M.

mm C3a. 1 mm C3a Time (s) C5a. C3a. Blank. 10 mm Time (s) Time (s)

The oxazoline 6 was prepared according to a literature procedure 2 but on a 30g scale. The 1 H NMR is identical to what was reported.

Pyridazine N-Oxides as Precursors of Metallocarbenes: Rhodium-Catalyzed Transannulation with Pyrroles. Supporting Information

A pillar[2]arene[3]hydroquinone which can self-assemble to a molecular zipper in the solid state

Supporting Information. for. Synthesis of 2,1-benzisoxazole-3(1H)-ones by basemediated. photochemical N O bond-forming

Thermal shift binding experiments were carried out using Thermofluor 384 ELS system. Protein

Chiral Squaramide Derivatives are Excellent Hydrogen Bond Donor Catalysts. Jeremiah P. Malerich, Koji Hagihara, and Viresh H.

Design, synthesis and evaluation of ph-dependent hydrolysable emetine analogs as treatment for prostate cancer.

Ruthenium-Catalyzed C H Oxygenation on Aryl Weinreb Amides

A Stable Evans Blue Derived Exendin-4 peptide for Type 2 Diabetes Treatment

Supporting Information

Fluorescent probes for detecting monoamine oxidase activity and cell imaging

Direct ortho-c H Functionalization of Aromatic Alcohols Masked by Acetone Oxime Ether via exo-palladacycle

Supplementary Material (ESI) for Chemical Communications This journal is (c) The Royal Society of Chemistry 2008

Supporting Information. Copyright Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim, 2007

Study of On-Resin Convergent Synthesis of N-Linked. Glycopeptides Containing a Large High Mannose N- Linked Oligosaccharide

Supporting Information Synthesis of 2-Aminobenzonitriles through Nitrosation Reaction and Sequential Iron(III)-Catalyzed C C Bond Cleavage of 2-Arylin

Supporting Information. Copyright Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim, 2007

National Defense Academy, Hashirimizu, Yokosuka, , Japan

Transcription:

Solid Phase Peptide Synthesis (SPPS) and Solid Phase Fragment Coupling (SPFC) Mediated by Isonitriles Ting Wang a and Samuel J. Danishefsky a,b,* alaboratory for Bioorganic Chemistry, Sloan- Kettering Institute for Cancer Research, 1275 York Avenue, ew York, Y 165 bdepartment of Chemistry, Columbia University, avemeyer all, 3 Broadway, ew York, Y 127 SUPPRTIG IFRMATI General Information. All commercial materials (Aldrich, Alfa Aesar, ovabiochem) were used without further purification. All solvents were reagent grade or PLC grade (Fisher, Aldrich). Anhydrous TF, C2Cl2 were passed through column of alumina and used without further drying. 1 MR spectra and 13 C MR spectra were recorded on a Bruker Advance DRX- 6 Mz at ambient temperature unless otherwise stated. Chemical shifts were reported in parts per million relative to residual solvent CDCl3 ( 1, 7.26 ppm, 13 C, 77. ppm). Multiplicities were reported as follows: s= singlet, d= doublet, t= triplet, m= multiplet, app.= apparent, br s= broad singlet. All 13 C MR spectra were recorded with complete proton decoupling. Low- resolution mass spectral analyses were performed with a JEL JMS- DX- 33- F mass spectrometer or Waters Micromass ZQ mass spectrometer. All reactions were carried out in oven- dried glassware under an argon or nitrogen atmosphere unless otherwise noted. Analytical TLC were performed on E. Merck silica gel 6 F254 plates and visualized by UV fluorescence quenching and CAM staining. Flash column

chromatography was performed on E. Merck silica get 6 (4-63mm). Yields refer to chromatographically and spectroscopically pure compounds. PLC: All separations of peptides involved a mobile phase of.5 TFA (v/v) in water (solvent A)/.4 TFA in acetonitrile (solvent B). Preparative and analytical PLC separation was performed using a Rainin PXL solvent delivery system equipped with a Rainin UV- 1 detector. LC- MS chromatographic separations were performed using a Waters Acquity Ultra Performance LC system equipped with acquity UPLC BE C18 column (1.7 µm, 2.1 1. mm) at a flow rate of.3 ml/min, X-Terra TM MS C18 column (3.5 µm, 2.1 1. mm) or Varain Microsorb C18 column (2 15. mm) at a flow rate of.2 ml/min. PLC separations were performed using: X-Bridge TM Prep C18 column BDTM (5. µm, 19 15 mm) at a flow rate of 16 ml/min, Microsorb 1-5 C18 column at a flow rate of 16. ml/min.

General procedure for isonitrile mediated amidation X R 1 R 2 Fmoc To a suspension of solid support amino acid 8 (.21 mmol) in DMF (1 ml) was added Fmoc- Asn(Trt)- S 9 (.315 mmol), Bt(4.3 mg,.315 mmol) followed by t- BuC(3.56 μl,.315 mmol). The reaction mixture was shaken at ambient temperature 3-15 hours. The resin was then washed sequentially with C2Cl2/Me (1:1, v/v, 3 1 ml), DMF (3 1 ml) and Me (3 1 ml), dried by a stream of 2 and put on high vacuum overnight. The resin cleavage was then executed by treatment with TFA/TIPS/2 (9.5 ml/.25 ml/.25 ml) for 9 min. After removal of TFA by evaporation with a stream of 2, the residue was washed by cold ether, dissolved in C3C/2 (1:1, 1 ml) and lyophilized to yield peptide 1 in good purity.

2 Fmoc 2 1 C18, 1-5 TW-4-184 1:34 4.68 Range: 9.419 C18, 1-5 TW-4-184 911 (4.719) Cm (911:913) 1 483.3 5.82e6 8.5 8. 7.5 7. 6.5 6. 5.5 5. 4.5 4. 3.5 3. 2.5 2. 1.5 1. 5.e-1. 2. 2.25 2.5 2.75 3. 3.25 3.5 3.75 4. 4.25 4.5 4.75 5. 5.25 5.5 5.75 6. 3 32 34 36 38 4 42 44 46 48 5 52 54 56 58 6 62 64 66 68 7 Figure S- 1. (Left) UV trace from UPLC- MS analysis of dipeptide 1; retention time = 4.68 min, gradient 1-5 C3C/2 with.5 TFA over 6 min at a flow rate of.3 ml/min, C18 column. (Right) ESI- MS of 1. LRMS (ESI+) calcd. for C242747 [(M+) + ]: 483.2, found: 483.3.

Fmoc 2 12 C18, 3-6 TW-4-94-2 1:43 4.48 Range: 2.176e+2 C18, 3-6 TW-4-94-2 658 (4.511) Cm (656:668) 1 482.4 6.1e7 2.e+2 1.8e+2 1.6e+2 1.4e+2 1.2e+2 1.e+2 8.e+1 6.e+1 4.e+1 963.7 2.e+1. 1.5 2. 2.5 3. 3.5 4. 4.5 5. 5.5 6. 2 3 4 5 6 7 8 9 1 11 Figure S- 2. (Left) UV trace from UPLC- MS analysis of dipeptide 12; retention time = 4.48 min, gradient 3-6 C3C/2 with.5 TFA over 6 min at a flow rate of.3 ml/min, C18 column. (Right) ESI- MS of 12. LRMS (ESI+) calcd. for C263236 [(M+) + ]: 482.2, found: 482.4.

2 15 Fmoc C18, 1-5 TW-4-96 1:44 3.65 Range: 1.684e+2 C18, 1-5 TW-4-96 544 (3.73) Cm (534:548) 1 484.4 9.35e7 1.6e+2 1.5e+2 1.4e+2 1.3e+2 1.2e+2 1.1e+2 1.e+2 9.e+1 8.e+1 7.e+1 6.e+1 5.e+1 4.e+1 3.e+1 2.e+1 1.e+1. 1.5 1.75 2. 2.25 2.5 2.75 3. 3.25 3.5 3.75 4. 4.25 4.5 4.75 5. 5.25 5.5 2 3 4 5 6 7 8 9 1 11 Figure S- 3. (Left) UV trace from UPLC- MS analysis of dipeptide 15; retention time = 3.65 min, gradient 1-5 C3C/2 with.5 TFA over 6 min at a flow rate of.3 ml/min, C18 column. (Right) ESI- MS of 15. LRMS (ESI+) calcd. for C24356 [(M+) + ]: 484.2, found: 484.4.

17 Fmoc C18, 4-8 TW-5-72-2 1:39 3.47 Range: 1.585e+2 C18, 4-8 TW-5-72-2 692 (3.533) Cm (688:73) 1 411.3 2.14e7 1.5e+2 1.4e+2 1.3e+2 1.2e+2 1.1e+2 1.e+2 9.e+1 8.e+1 7.e+1 6.e+1 5.e+1 4.e+1 3.e+1 2.e+1 1.e+1...5 1. 1.5 2. 2.5 3. 3.5 4. 4.5 5. 2 225 25 275 3 325 35 375 4 425 45 475 5 525 55 575 6 625 65 675 7 725 75 775 8 Figure S- 4. (Left) UV trace from UPLC- MS analysis of dipeptide 17; retention time = 3.47 min, gradient 4-8 C3C/2 with.5 TFA over 6 min at a flow rate of.3 ml/min, C18 column. (Right) ESI- MS of 17. LRMS (ESI+) calcd. for C232725 [(M+) + ]: 411.2, found: 411.3.

2 SSt-Bu 2 2 Fmoc C18, 4-6 TW-3-162-3 3.4 1:41 Range: 8.272e+1 C18, 4-6 TW-3-162-3 54 (3.455) Cm (499:519) 1 741.6 4.18e7 8.e+1 7.5e+1 7.e+1 6.5e+1 6.e+1 5.5e+1 5.e+1 4.5e+1 4.e+1 3.5e+1 3.e+1 2.5e+1 2.e+1 1.5e+1 1.e+1 5. 2. 2.25 2.5 2.75 3. 3.25 3.5 3.75 4. 4.25 4.5 4.75 5. 5.25 5.5 3 35 4 45 5 55 6 65 7 75 8 85 9 95 1 15 11 115 Figure S- 5. (Left) UV trace from UPLC- MS analysis of dipeptide 2; retention time = 3.4 min, gradient 4-6 C3C/2 with.5 TFA over 6 min at a flow rate of.3 ml/min, C18 column. (Right) ESI- MS of 2. LRMS (ESI+) calcd. for C354986S2 [(M+) + ]: 741.3, found: 741.6.

General procedure for isonitrile/bt- mediated SPPS To a suspension of solid support amino acid (.2 mmol) was added piperidine/dmf solution (1:4, v/v, 2 ml). The mixture was allowed to shake for 3 min before the solution was drained away. Another portion of piperidine/dmf solution (1:4, v/v, 2 ml) was added and the mixture was allowed to shake for 7 min. After drain away the DMF solution, the resin was then washed sequentially with C2Cl2/Me (1:1, v/v, 3 1 ml), DMF (3 1 ml) and Me (3 1 ml), dried by a stream of 2. To the resulting solid support amino acid/peptide was added DMF (1 ml), Fmoc- XXX- S (.3 mmol), Bt(4.1 mg,.3 mmol) followed by t- BuC (3.4 μl,.3 mmol). The reaction mixture was shaken at ambient temperature 3-15 hours. The resin was then washed sequentially with C2Cl2/Me (1:1, v/v, 3 1 ml), DMF (3 1 ml) and Me (3 1 ml), dried by a stream of 2. The resin cleavage of peptide product was executed by treatment of with TFA/TIPS/2 (9.5 ml/.25 ml/.25 ml) for 9 min. After removal of TFA by evaporation with a stream of 2, the residue was washed by cold ether, dissolved in C3C/2 (1:1, 1 ml) and lyophilized to yield peptide, which was then purified by PLC.

21 Fmoc The product was purified by reversed- phase PLC (C18 column, gradient 38-78, C3C/2 with.5 TFA over 17 min). The fractions were collected and lyophilized to give 21 (8.63 mg, 17 µmol) as a white solid. C18, 4-8 TW-5-PLG-3 1:38 3.31 Range: 2.281e+2 C18, 4-8 TW-5-PLG-3 492 (3.347) Cm (486:499) 1 58.4 6.8e7 2.2e+2 2.e+2 1.8e+2 1.6e+2 1.4e+2 1.2e+2 1.e+2 8.e+1 6.e+1 4.e+1 2.e+1..5 1. 1.5 2. 2.5 3. 3.5 4. 4.5 5. 5.5 2 25 3 35 4 45 5 55 6 65 7 75 8 85 9 95 1 Figure S- 6. (Left) UV trace from UPLC- MS analysis of purified dipeptide 21; retention time = 3.31 min, gradient 4-8 C3C/2 with.5 TFA over 6 min at a flow rate of.3 ml/min, C18 column. (Right) ESI- MS of 21. LRMS (ESI+) calcd. for C293436 [(M+) + ]: 58.2, found: 58.4.

2 Fmoc 2 22 The product was purified by reversed- phase PLC (C18 column, gradient 26-56, C3C/2 with.5 TFA over 2 min). The fractions were collected and lyophilized to give 22 (12.22 mg, 14.4 µmol) as a white solid. C18, 35-65 TW-5-74 1.1e+2 1:44 2.35 Range: 1.127e+2 C18, 35-65 TW-5-74 351 (2.48) Cm (348:354) 1 85.4 2.58e7 1.e+2 9.e+1 8.e+1 7.e+1 6.e+1 5.e+1 4.e+1 3.e+1 2.e+1 425.8 1.e+1...5 1. 1.5 2. 2.5 3. 3.5 4. 4.5 5. 3 35 4 45 5 55 6 65 7 75 8 85 9 95 1 15 11 115 Figure S- 7. (Left) UV trace from UPLC- MS analysis of purified peptide 22; retention time = 2.35 min, gradient 35-65 C3C/2 with.5 TFA over 6 min at a flow rate of.3 ml/min, C18 column. (Right) ESI- MS of 22. LRMS (ESI+) calcd. for C434891 [(M+) + ]: 85.4, found: 85.4.

2 2 Fmoc 2 23 The product was purified by reversed- phase PLC (C18 column, gradient 27-57, C3C/2 with.5 TFA over 18 min). The fractions were collected and lyophilized to give 23 (14.37 mg, 13.6 µmol) as a white solid. C18, 35-65 TW-4-128 pure 1:43 2.3 Range: 1.265e+2 C18, 35-65 TW-4-128 pure 344 (2.359) Cm (34:346) 529.8 1 2.6e7 1.2e+2 1.1e+2 1.e+2 9.e+1 8.e+1 7.e+1 6.e+1 5.e+1 158.6 4.e+1 3.e+1 2.e+1 1411.3 1.e+1 1588.4...5 1. 1.5 2. 2.5 3. 3.5 4. 4.5 5. 3 4 5 6 7 8 9 1 11 12 13 14 15 16 17 Figure S- 8. (Left) UV trace from UPLC- MS analysis of purified peptide 23; retention time = 2.3 min, gradient 35-65 C3C/2 with.5 TFA over 6 min at a flow rate of.3 ml/min, C18 column. (Right) ESI- MS of 23. LRMS (ESI+) calcd. for C51681114 [(M+) + ]: 158.5, found: 158.6, 529.8 (M+2) 2+

2 24 2 2 S S 2 2 The product was purified by reversed- phase PLC (C18 column, gradient 13-24, C3C/2 with.5 TFA over 19 min). The fractions were collected and lyophilized to give 24 (23.32 mg, 21.5 µmol) as a white solid. 1 MR (6 Mz, DMS- d6, a mixture of carbamate rotamers) δ 9.21 (s, 1), 8.48 (d, J = 8.1 z, 1), 8.37 (d, J = 8. z, 1), 8.24 (dd, J = 21.7, 7.6 z, 2), 8.16 (d, J = 7.6 z, 1), 8.11 7.99 (m, 5), 7.49 7.37 (m, 2), 7.33 7.13 (m, 7), 7.13 6.98 (m, 3), 6.96 (d, J = 2.4 z, 1), 6.83 (d, J = 2.4 z, 1), 6.66 6.61 (m, 2), 4.65 4.51 (m, 2), 4.51 4.41 (m, 1), 4.34 4.16 (m, 3), 3.89 (p, J = 5.3 z, 1), 3.71 3.55 (m, 3), 3.38 (d, J = 15.9 z, 1), 3.8 (ddt, J = 19.5, 14.2, 6.2 z, 3), 2.95 (ddd, J = 17.8, 11.4, 2.8 z, 2), 2.86 2.5 (m, 5), 2.46 2.38 (m, 2), 2.16 1.95 (m, 3), 1.93 1.68 (m, 4), 1.59 1.41 (m, 3), 1.24 (d, J = 1.1 z, 1). 13 C MR (15 Mz, DMS- d6) δ 173.85, 171.74, 171.39, 171.28, 171.7, 17.97, 17.93, 17.67, 17.63, 17.62, 168.43, 168.41, 166.59, 166.57, 157.97, 157.77, 156.56, 155.81, 137.67, 13.2, 129.1, 128.2, 128., 127.53, 126.17, 118.5, 116.8, 116.7, 116.6, 114.87, 114.84, 59.73, 54.51, 53.76, 53.56, 53.2, 53.18, 52.31, 52.14, 49.48, 47.8, 41.78,

4.32, 37.24, 36.76, 36.41, 31.4, 29.12, 28.48, 28.17, 25.52, 25.38, 24.94, 24.4.; LRMS (ESI) calcd. for C46681512S2 [(M+) + ]: 186.5; found: 186.6. C18, 15-25 TW-4-dvp 3.7 1:4 Range: 6.58 C18, 15-25 TW-4-dvp 458 (3.118) Cm (454:473) 1 543.9 9.96e6 5.5 5. 4.5 4. 3.5 3. 2.5 2. 1.5 1. 5.e-1 1.5 1.75 2. 2.25 2.5 2.75 3. 3.25 3.5 3.75 4. 4.25 4.5 4.75 5. 5.25 5.5 3 35 4 45 5 55 6 65 7 75 8 85 9 95 1 15 11 115 Figure S- 9. (Left) UV trace from UPLC- MS analysis of purified dihydrovasopressin 24; retention time = 3.7 min, gradient 15-25 C3C/2 with.5 TFA over 6 min at a flow rate of.3 ml/min, C18 column. (Right) ESI- MS of 24. LRMS (ESI+) calcd. for C46681512S2 [(M+) + ]: 186.5; found: 186.6, 543.9 (M+2) 2+

2 2 S 24 2 S 2 2 2 2 S 24 2 S 2 2

2 25 2 2 S S 2 2 Vasopressin (25) To a solution of dihydrovasopressin 24 (1 mg, 9.26 µmol) in 2 (1 ml) was added a few drops of Ac. The p of the solution was adjusted to about 7 with 4. The solution was then aerated for about 6 h, at which time UPLC show the completion of the reaction. The crude vasopressin was obtained by lyophilization. The product was purified by reversed- phase PLC (C18 column, gradient 14-24, C3C/2 with.5 TFA over 18 min). The fractions were collected and lyophilized to give vasopressin 25 (7.1 mg, 6.536 µmol) as a white solid. 1 MR (6 Mz, DMS-d6) δ 9.28 (s, 1), 8.7 (d, J = 8. z, 1), 8.41 8.36 (m, 1), 8.34 8.28 (m, 3), 8.25 8.13 (m, 3), 7.96 (dt, J = 12.5, 6.5 z, 2), 7.57 (q, J = 6.6, 5.7 z, 1), 7.47 7.41 (m, 1), 7.4 7.18 (m, 7), 7.19 7.9 (m, 1), 6.96 (dd, J = 24.4, 5.3 z, 3), 6.92 6.81 (m, 3), 6.67 6.55 (m, 2), 4.71 (p, J = 7.1 z, 1), 4.65 4.54 (m, ), 4.52 (q, J = 6.8 z, 1), 4.4 (q, J = 8.3 z, 1), 4.35 4.1 (m, 3), 4.2 (q, J = 9.6, 7.8 z, 1), 3.92 (dt, J = 1.2, 5.3 z, 1), 3.71 3.47 (m, 4), 3.47 3.39 (m, 1), 3.2 3.6 (m, 4), 3.1 (td, J = 13.5, 6.7 z, 1), 2.92 (dd, J = 14., 1.1 z, 1), 2.87 2.76 (m, 1), 2.59 (t, J = 8.3 z, 3), 2.21 2.6 (m, 2), 2.7 1.7 (m, 5), 1.65 1.43 (m, 3), 1.31 1.2 (m, 1).; 13 C MR

(15 Mz, DMS- d6, a mixture of carbamate rotamers) δ 173.98, 171.82, 171.56, 171.47, 171.16, 17.85, 17.73, 17.69, 167.64, 166.62, 158.1, 157.9, 157.7, 157.5, 156.57, 155.91, 137.54, 129.9, 129.9, 128.19, 127.23, 126.39, 115.2, 59.89, 55.75, 55.22, 53.95, 52.36, 51.83, 51.25, 49.75, 46.78, 41.84, 4.98, 4.66, 4.33, 36.62, 35.86, 31.35, 29.6, 28.3, 26.3, 24.99, 24.42, 1.14.; LRMS (ESI) calcd. for C46661512S2 [(M+) + ]: 184.4; found: 184.7 The identity of synthetic vasopressin and authentic vasopressin (American Peptide Company) was confirmed by co- injection in UPLC. C18, 15-25 TW-4-AVP-co 6. 2.83 1:43 Range: 6.29 C18, 15-25 TW-4-syn-AVP 426 (2.92) Cm (42:431) 542.8 1 9.87e6 4. 2.. 1.25 1.5 1.75 2. 2.25 2.5 2.75 3. 3.25 3.5 3.75 4. 4.25 4.5 4.75 5. 5.25 TW-4-Aut-AVP 2.8 Range: 9.413 6. 4. 2.. 1.25 1.5 1.75 2. 2.25 2.5 2.75 3. 3.25 3.5 3.75 4. 4.25 4.5 4.75 5. 5.25 TW-4-syn-AVP 2.86 Range: 4.239 3. 2. 1. 1.25 1.5 1.75 2. 2.25 2.5 2.75 3. 3.25 3.5 3.75 4. 4.25 4.5 4.75 5. 5.25 2 3 4 5 6 7 8 9 1 11 12 13 14 Figure S- 1. (Left) UV trace from UPLC- MS analysis of purified vasopressin 25, authentic sample and co- injection; retention time = 2.83 min, gradient 15-25 C3C/2 with.5 TFA over 6 min at a flow rate of.3 ml/min, C18 column. (Right) ESI- MS of 25. LRMS (ESI+) calcd. for C46661512S2 [(M+) + ]: 184.4; found: 184.7, 542.8 (M+2) 2+

2 2 S S 2 2 25 2 Vasopressin Authentic Sample Vasopressin Synthetic Sample

2 2 S S 2 2 25 2 Vasopressin Authentic Sample Vasopressin Synthetic Sample

Single amino acid derived thioacid was synthesized following the procedure documented in ref. 12, 19. Peptide- derived thioacid was synthesized following the procedure documented in ref. 11, 13. 2 S Fmoc 2 38 The product was purified by reversed- phase PLC (C18 column, gradient 31-61, C3C/2 with.5 TFA over 17 min). C18, 3-6 TW-4-158 1.5e+2 1:4 3.49 Range: 1.56e+2 C18, 3-6 TW-4-158 52 (3.564) Cm (516:532) 1 711.6 1.53e7 1.e+2 9.5e+1 9.e+1 8.5e+1 8.e+1 7.5e+1 7.e+1 6.5e+1 6.e+1 5.5e+1 5.e+1 4.5e+1 4.e+1 3.5e+1 3.e+1 2.5e+1 2.e+1 356.3 1.5e+1 1.e+1 5...5 1. 1.5 2. 2.5 3. 3.5 4. 4.5 5. 5.5 2 25 3 35 4 45 5 55 6 65 7 75 8 85 9 95 1 Figure S- 11. (Left) UV trace from UPLC- MS analysis of purified 38; retention time = 3.49 min, gradient 3-6 C3C/2 with.5 TFA over 6 min at a flow rate of.3 ml/min, C18 column. (Right) ESI- MS of 38. LRMS (ESI+) calcd. for C344787S [(M+) + ]: 711.3, found: 711.6.

Ac S 42 2 The product was purified by reversed- phase PLC (C18 column, gradient 5-25, C3C/2 with.5 TFA over 16 min). C18, 1-3 TW-4-54 5.e+1 2.54 1:46 Range: 5.277e+1 C18, 1-3 TW-4-54 382 (2.599) Cm (38:389) 1 581.3 1.39e7 4.75e+1 4.5e+1 4.25e+1 4.e+1 3.75e+1 3.5e+1 3.25e+1 3.e+1 2.75e+1 2.5e+1 2.25e+1 2.e+1 1.75e+1 1.5e+1 1.25e+1 1.e+1 7.5 5. 2.5..5 1. 1.5 2. 2.5 3. 3.5 4. 4.5 5. 5.5 2 25 3 35 4 45 5 55 6 65 7 75 8 85 9 95 1 Figure S- 12. (Left) UV trace from UPLC- MS analysis of purified 42; retention time = 2.54 min, gradient 1-3 C3C/2 with.5 TFA over 6 min at a flow rate of.3 ml/min, C18 column. (Right) ESI- MS of 42. LRMS (ESI+) calcd. for C243787S [(M+) + ]: 581.3, found: 581.3.

S Ac 2 45 The product was purified by reversed- phase PLC (C18 column, gradient 12-32, C3C/2 with.5 TFA over 18 min). C18, 13-33Ting Wang, sjd TW-4-18-2 8.e+1 38 18.84 2:13:3614-ct-212 Range: 8.121e+1 C18, 13-33 TW-4-18-2 1132 (19.36) Cm (1126:1171) 1 657.31 7.26e7 7.5e+1 7.e+1 6.5e+1 6.e+1 5.5e+1 5.e+1 4.5e+1 4.e+1 3.5e+1 3.e+1 658.32 2.5e+1 2.e+1 1.5e+1 1.e+1 659.21 5.. 17.45 6. 8. 1. 12. 14. 16. 18. 2. 22. 24. 26. 28. 3. 3 35 4 45 5 55 6 65 7 75 8 85 9 95 1 Figure S- 13. (Left) UV trace from PLC- MS analysis of purified 45; retention time = 18.84 min, gradient 13-33 C3C/2 with.5 TFA over 3 min at a flow rate of.3 ml/min, C18 column. (Right) ESI- MS of 45. LRMS (ESI+) calcd. for C34187S [(M+) + ]: 657.3, found: 657.3.

2 2 2 Fmoc 2 4 2 2 To the solid support peptide 37 (3.36 μmol) was added DMF (.3 ml), thioacid 38 (3.58 mg, 5.4 μmol), Bt (1.36 mg, 1.8 μmol) followed by t- BuC (1.14 μl, 1.8 μmol). The reaction mixture was shaken at ambient temperature 12 hours. The resin was then washed sequentially with C2Cl2/Me (1:1, v/v, 3 2 ml), DMF (3 2 ml) and Me (3 2 ml), dried by a stream of 2. To a suspension of the resulting solid support peptide 39 was added piperidine/dmf solution (1:4, v/v, 1 ml). The mixture was allowed to shake for 3 min before the solution was drained away. Another portion of piperidine/dmf solution (1:4, v/v, 1 ml) was added and the mixture was allowed to shake for 7 min. After drain away the DMF solution, the resin was then washed sequentially with C2Cl2/Me (1:1, v/v, 3 2 ml), DMF (3 2 ml) and Me (3 2 ml), dried by a stream of 2. To the resulting support peptide was added DMF (.3 ml), thioacid 38 (3.58 mg, 5.4 μmol), Bt (1.36 mg, 1.8 μmol) followed by t- BuC (1.14 μl, 1.8 μmol). The reaction mixture was shaken at ambient temperature 24 hours. The resin was then washed sequentially with C2Cl2/Me (1:1, v/v, 3 2 ml), DMF (3 2 ml) and Me (3 2 ml), dried by a stream of 2.

The resin cleavage of peptide product was executed by treatment of with TFA/TIPS/2 (1.5 ml/.1 ml/.1 ml) for 9 min. After removal of TFA by evaporation with a stream of 2, the residue was washed by cold ether, dissolved in C3C/2 (1:1, 5 ml) and lyophilized to yield crude peptide. The product was then purified by reversed- phase PLC (C18 column, gradient 28-53, C3C/2 with.5 TFA over 18 min). The fractions were collected and lyophilized to give 4 (4.28 mg, 2.62 µmol) as a white solid. C18, 3-55 TW-4-192-pure 3.4e+1 1:45 2.97 Range: 3.446e+1 C18, 3-55 TW-4-192-pure 443 (3.39) Cm (433:45) 1 817.2 3.12e7 3.2e+1 3.e+1 2.8e+1 2.6e+1 2.4e+1 2.2e+1 2.e+1 1.8e+1 1.6e+1 1.4e+1 1.2e+1 1.e+1 8. 6. 189.6 4. 2. 545.4 1225.8 1633.9..5 1. 1.5 2. 2.5 3. 3.5 4. 4.5 5. 5.5 3 4 5 6 7 8 9 1 11 12 13 14 15 16 17 18 19 Figure S- 14. (Left) UV trace from UPLC- MS analysis of purified 4; retention time = 2.97 min, gradient 3-55 C3C/2 with.5 TFA over 6 min at a flow rate of.3 ml/min, C18 column. (Right) ESI- MS of 4. LRMS (ESI+) calcd. for C73114232S [(M+) + ]: 1633.8, found: 1633.9, 1225.8, 189.6, 817.2, 545.4

Ac 2 43 To the solid support peptide 41 (3.73 μmol) was added DMF (.4 ml), thioacid 42 (4.32 mg, 7.46 μmol), Bt (2.52 mg, 18.65 mmol) followed by t- BuC (2 μl, 18.65 mmol). The reaction mixture was shaken at ambient temperature 15 hours. The resin was then washed sequentially with C2Cl2/Me (1:1, v/v, 3 2 ml), DMF (3 2 ml) and Me (3 2 ml), dried by a stream of 2. The resin cleavage of peptide product was executed by treatment of with TFA/TIPS/2 (3 ml/.15 ml/.15 ml) for 9 min. After removal of TFA by evaporation with a stream of 2, the residue was washed by cold ether, dissolved in C3C/2 (1:1, 5 ml) and lyophilized to yield crude peptide. The product was then purified by reversed- phase PLC (C18 column, gradient 5-25, C3C/2 with.5 TFA over 19 min). The fractions were collected and lyophilized to give 43 (1.93 mg, 3.1 µmol) as a white solid.

C18, 5-2 TW-3-238-pure 1:42 3.46 Range: 4.162e+1 C18, 5-2 TW-3-238-pure 517 (3.517) Cm (59:523) 1 622.4 7.36e6 3.8e+1 3.6e+1 3.4e+1 3.2e+1 3.e+1 2.8e+1 2.6e+1 2.4e+1 2.2e+1 2.e+1 1.8e+1 1.6e+1 1.4e+1 1.2e+1 1.e+1 8. 6. 4. 2...5 1. 1.5 2. 2.5 3. 3.5 4. 4.5 5. 5.5 2 25 3 35 4 45 5 55 6 65 7 75 8 85 9 95 1 Figure S- 15. (Left) UV trace from UPLC- MS analysis of purified 43; retention time = 3.46 min, gradient 5-2 C3C/2 with.5 TFA over 6 min at a flow rate of.3 ml/min, C18 column. (Right) ESI- MS of 43. LRMS (ESI+) calcd. for C26499 [(M+) + ]: 622.3, found: 622.4.

2 Ac 2 44 2 To the solid support peptide 37 (3.73 μmol) was added DMF (.5 ml), thioacid 42 (4.32 mg, 7.46 μmol), Bt (2.52 mg, 18.65 mmol) followed by t- BuC (2 μl, 18.65 mmol). The reaction mixture was shaken at ambient temperature 2 hours. The resin was then washed sequentially with C2Cl2/Me (1:1, v/v, 3 2 ml), DMF (3 2 ml) and Me (3 2 ml), dried by a stream of 2. The resin cleavage of peptide product was executed by treatment of with TFA/TIPS/2 (3 ml/.15 ml/.15 ml) for 9 min. After removal of TFA by evaporation with a stream of 2, the residue was washed by cold ether, dissolved in C3C/2 (1:1, 5 ml) and lyophilized to yield crude peptide. The product was then purified by reversed- phase PLC (C18 column, gradient 8-28, C3C/2 with.5 TFA over 18 min). The fractions were collected and lyophilized to give 44 (3.2 mg, 3.6 µmol) as a white solid.

C18, 1-3 TW-4-82-pure 1:48 3.16 Range: 3.61e+1 C18, 1-3 TW-4-82-pure 471 (3.28) Cm (468:478) 524.9 1 3.18e7 3.4e+1 3.2e+1 3.e+1 2.8e+1 2.6e+1 2.4e+1 2.2e+1 2.e+1 1.8e+1 1.6e+1 1.4e+1 1.2e+1 1.e+1 8. 6. 4. 148.7 2...5 1. 1.5 2. 2.5 3. 3.5 4. 4.5 5. 5.5 3 35 4 45 5 55 6 65 7 75 8 85 9 95 1 15 11 115 Figure S- 16. (Left) UV trace from UPLC- MS analysis of purified 44; retention time = 3.16 min, gradient 1-3 C3C/2 with.5 TFA over 6 min at a flow rate of.3 ml/min, C18 column. (Right) ESI- MS of 44. LRMS (ESI+) calcd. for C4471515 [(M+) + ]: 148.5, found: 148.7, 524.9 (M+2) 2+

2024 © healthdocbox.com Privacy Policy | Terms of Service | Feedback