Peptide N-glycanase (PNGase), the enzyme responsible for the deglycosylation of N-linked glycoproteins, has an active site related to that of cysteine proteases. Chitiobiose was equipped with electrophilic traps often used in cysteine protease inhibitors, and the resulting compounds were evaluated as PNGase inhibitors. We found that the electrophilic trap of the inhibitor has a great influence on the potency of the compounds with the chloromethyl ketone inhibitor being the first potent C-glycoside-based PNGase inhibitor.

Horst, Danielle,

Marel, Gijsbert A. van der,

Overkleeft, Herman S.,

Wiertz, Emmanuel J.H.J.,

Witte, Martin D.,

University of Groningen Digital Archive

Synthesis and Biological Evaluation of a Chitobiose-Based Peptide N-Glycanase Inhibitor Library

Witte, Martin D.

Horst, Danielle

Wiertz, Emmanuel J.H.J.

Marel, Gijsbert A. van der

Overkleeft, Herman S.

University of Groningen

   University of GroningenSynthesis and Biological Evaluation of a Chitobiose-Based Peptide N-Glycanase InhibitorLibraryWitte, Martin D.; Horst, Danielle; Wiertz, Emmanuel J.H.J.; Marel, Gijsbert A. van der;Overkleeft, Herman S.Published in:The Journal of Organic ChemistryDOI:10.1021/jo801906sIMPORTANT NOTE: You are advised to consult the publisher's version (publisher's PDF) if you wish to cite fromit. Please check the document version below.Document VersionPublisher's PDF, also known as Version of recordPublication date:2009Link to publication in University of Groningen/UMCG research databaseCitation for published version (APA):Witte, M. D., Horst, D., Wiertz, E. J. H. J., Marel, G. A. V. D., & Overkleeft, H. S. (2009). Synthesis andBiological Evaluation of a Chitobiose-Based Peptide N-Glycanase Inhibitor Library. The Journal of OrganicChemistry, 74(2), 605-616. https://doi.org/10.1021/jo801906sCopyrightOther than for strictly personal use, it is not permitted to download or to forward/distribute the text or part of it without the consent of theauthor(s) and/or copyright holder(s), unless the work is under an open content license (like Creative Commons).The publication may also be distributed here under the terms of Article 25fa of the Dutch Copyright Act, indicated by the “Taverne” license.More information can be found on the University of Groningen website: https://www.rug.nl/library/open-access/self-archiving-pure/taverne-amendment.Take-down policyIf you believe that this document breaches copyright please contact us providing details, and we will remove access to the work immediatelyand investigate your claim.Downloaded from the University of Groningen/UMCG research database (Pure): http://www.rug.nl/research/portal. For technical reasons thenumber of authors shown on this cover page is limited to 10 maximum.Supporting Information  Synthesis and biological evaluation of a chitobiose based peptide N-glycanase inhibitor library  Martin D. Wittea, Daniëlle Horstb, Emmanuel J. H. J. Wiertzb, Gijs A. van der Marela* and Herman S. Overkleefta*.  aLeiden Institute of Chemistry, Leiden University, P.O. Box 9502, 2300 RA Leiden, The Netherlands. Email: marel_g@ chem.leidenuniv.nl, h.s.overkleeft@chem.leidenuniv.nl bDepartment of Medical Microbiology, Center of Infectious Diseases, Leiden University Medical Center, P.O. Box 9600, 2300 RC Leiden, The Netherlands.  Table of contents General Procedures S4Synthesis of 5 S5Synthesis of 7 S6Synthesis of 26 S6Synthesis of 39 S7Synthesis of 49 S8Competition assay S9In vivo inhibition of human PNGase  S9Copy of 1H-NMR of 5 S11S1 Copy of 13C-NMR of 5 S12Copy of 1H-NMR of 7 S13Copy of 13C-NMR of 7 S14Copy of 1H-NMR of 10 S15Copy of 13C-NMR of 10 S16Copy of LC/MS spectrum of 10 S17Copy of 1H-NMR of 11 S18Copy of 13C-NMR of 11 S19Copy of LC/MS spectrum of 11 S20Copy of 1H-NMR of 12 S21Copy of 13C-NMR of 12 S22Copy of LC/MS spectrum of 12 S23Copy of 1H-NMR of 13 S24Copy of 13C-NMR of 13 S25Copy of LC/MS spectrum of 13 S26Copy of 1H-NMR of 14 S27Copy of 13C-NMR of 14 S28Copy of LC/MS spectrum of 14 S29Copy of 1H-NMR of 17 S30Copy of 13C-NMR of 17 S31Copy of LC/MS spectrum of 17 S32Copy of 1H-NMR of 18 S33Copy of 13C-NMR of 18 S34S2 Copy of LC/MS spectrum of 18 S35Copy of 1H-NMR of 19 S36Copy of 13C-NMR of 19 S37Copy of LC/MS spectrum of 19 S38Copy of 1H-NMR of 20 S39Copy of 13C-NMR of 20 S40Copy of LC/MS spectrum of 20 S41Copy of 1H-NMR of 21 S42Copy of 13C-NMR of 21 S43Copy of LC/MS spectrum of 21 S44Copy of 1H-NMR of 24 S45Copy of 13C-NMR of 24 S46Copy of LC/MS spectrum of 24 S47Copy of 1H-NMR of 25 S48Copy of 13C-NMR of 25 S49Copy of LC/MS spectrum of 25 S50       S3 General Procedures:  All reagents were commercial grade and were used as received unless stated otherwise. TTBP was synthesized as described. Diethyl ether (Et2O), ethyl acetate (EtOAc), light petroleum ether (PE) and toluene (Tol) were purchased from Riedel-de Haën. Acetonitrile (MeCN), dichloroethane, dichloromethane (CH2Cl2), N,N-dimethylformamide (DMF), methanol (MeOH), pyridine (pyr), tetrahydrofuran (THF) were obtained from Biosolve. THF was distilled over LiAlH4 prior to use. Before use CH2Cl2 was refluxed over CaH2 for 2h and distilled. Trace of water were removed by refluxing n-Butanol over sodium for 2h followed by distilling and storing over 3Å MS. Trifluoromethanesulfonic anhydride was distilled from P2O5. Molecular sieves 3Å were flame dried before use. All reactions were performed under an inert atmosphere of Argon unless stated otherwise. Solvents used for flash chromatography were of pro analysi quality. Flash chromatography was performed on Screening Devices silica gel 60 (0.04 – 0.063 mm). TLC-analysis was conducted on DC-alufolien (Merck, Kieselgel60, F254) with detection by UV-absorption (254 nm) were applicable and by spraying with 20% sulfuric acid in ethanol followed by charring at ~150°C or by spraying with a solution of (NH4)6Mo7O24·H2O (25 g/l) and (NH4)4Ce(SO4)4·2H2O (10g/l) in 10% sulfuric acid in water followed by charring at ~150°C. 1H and 13C NMR spectra were recorded on a 400 400/100 MHz, 500/125 MHz and a 600/150 MHz spectrometer. Chemical shifts (δ) are given in ppm relative to the chloroform residual solvent peak or tetramethylsilane as internal standard. Coupling constants are given in Hz. All given 13C spectra are proton decoupled. High resolution mass spectra were recorded by direct injection (2 µL of a 2 µM solution in water/acetonitrile; 50/50; v/v and 0.1% formic acid) on a mass S4 spectrometer equipped with an electrospray ion source in positive mode (source voltage 3.5 kV, sheath gas flow 10, capillary temperature 250 °C) with resolution R = 60000 at m/z 400 (mass range m/z = 150-2000) and dioctylpthalate (m/z = 391.28428) as a “lock mass”. The high resolution mass spectrometer was calibrated prior to measurements with a calibration mixture (Thermo Finnigan). LC-MS analysis was performed on a HPLC system with a standard C18 (4.6 mmD × 250 mmL, 5μ particle size) column (detection at 200-600 nm) in combination with buffers A: H2O, B: MeCN and C: 0.5% aq. TFA and coupled to a mass spectrometer with ESI. For RP-HPLC purifications an automated HPLC system equipped with a C18 (5μm 250×10 mm) column was used. The applied buffers were A: 0.1% TFA in H2O, B: MeCN.  OOF +K-OOF Potassium fluoroacetate (5) Ethyl fluoroacetate, was synthesized as described in literature.1 Ethyl fluoroacetate (0.107 g, 1 mmol) was dissolved in absolute EtOH, cooled to 0°C and potassium hydroxide (56.1 mg, 1 mmol) was added. The reaction was stirred overnight after which petroleum ether was added. The solution was cooled to -20°C and filtered giving title compound 5 (66%, 77 mg, 0.66 mmol) as a white solid. 1H-NMR (400 MHz, D2O) δ 4.75 (d, J = 48.3, 2H). 13C-NMR (101 MHz, D2O) δ ppm 79.5 (d, J = 176). FT-IR: vmax(neat)/cm-1 3381, 1606, 1423, 1398, 1337, 1009. Mp: 189°C (start of decomposition).   S5   OOBr HOOOO  2-(2,6-dimethylbenzoyloxy)acetic acid (7) Tert-butyl bromoacetate (0.437 ml, 3 mmol), dimethylbenzoic acid (0.496 g, 3.3 mmol) and potassium fluoride (0.261 g, 4.5 mmol) were dissolved in DMF. After overnight stirring, Et2O was added after which the solution was extracted with NaHCO3 (sat. aq.). The organic layer was dried, concentrated in vacuo and subsequently dissolved in TFA/H2O (95/5). The solution was concentrated after 1h, coevaporated with toluene to remove residual traces of TFA and applied to silica gel chromatography (0→10% EA/Tol) giving title compound 7 (81%, 0.506 g, 2.43 mmol) as a white solid. 1H-NMR (400 MHz, CDCl3) δ ppm 7.22 (dd, J = 15.2 Hz, 7.6 Hz, 1H), 7.05 (d, J = 7.6 Hz), 4.89 (s, 2H), 2.37 (s, 6H). 13C-NMR (101 MHz, CDCl3) δ ppm 173.7, 169.1, 135.7, 132.1, 129.9, 127.7, 60.4, 19.8. FT-IR: vmax(neat)/cm-1 2955.7, 1720.7, 1597.4, 1462.9, 1424.3, 1306.1, 1281.6, 1240.1, 1165.5, 1113.7, 1082.7, 1029.1, 1003.3. Mp: 128.7-130.4°C.    OOOBnONPhthPh OBnOHOBnONPhth  3-C-(3,6-di-O-benzyl-2-deoxy-2-phthalimido-β-D-glucopyranosyl) propene (26)  Known 3-C-(3-O-benzyl-4,6-O-benzylidene-2-deoxy-2-phthalimido-β-D-glucopyranosyl)-1-propene2 (0.512 g, 1 mmol) was coevaporated with toluene (3×) before being dissolved in freshly distilled CH2Cl2. Activated molsieves (4 Å) and triethylsilane (0.533 mL, 3.3 mmol) were added. The mixture was stirred at room temperature for 15 min, cooled to -78°C followed by the addition of S6 trifluoromethanesulfonic acid (0.265 mL, 3 mmol). TLC-analysis showed complete conversion after 45 min after which the reaction was quenched with MeOH/Et3N, extracted with NaHCO3 and concentrated. Silica gel purification (0→30% EA/PE) afforded acceptor 26 (70%, 0.359 g, 0.70 mmol) as an oil. 1H-NMR (400 MHz, CDCl3) δ ppm 7.80-7.75 (m, 1H), 7.70-7.63 (m, 3H), 7.38-7.23 (m, 5H), 7.05-6.98 (m, 2H), 6.95-6.90 (m, 3H), 5.72 (dt, J = 16.9, 16.8, 7.0 Hz, 1H), 4.89 (d, J = 17.2 Hz, 1H), 4.83 (d, J = 10.2 Hz, 1H), 4.76 (d, J = 12.2 Hz, 1H), 4.64 (d, J = 12.1 Hz, 1H), 4.58 (d, J = 12.1 Hz, 1H), 4.52 (d, J = 12.2 Hz, 1H), 4.30-4.18 (m, 2H), 4.08 (t, J = 10.0, 10.0 Hz, 1H), 3.82-3.71 (m, 3H), 3.61 (td, J = 9.1, 4.4, 4.4 Hz, 1H), 3.04 (s, 1H), 2.27-2.13 (m, 2H). 13C-NMR (101 MHz, CDCl3) δ ppm 168.1, 167.8, 138.2, 137.7, 133.8, 133.7, 133.3, 131.5, 131.4, 128.4, 128.0, 127.70, 127.65, 127.2, 123.2, 123.1, 116.9, 79.6, 77.6, 74.4, 74.2, 73.6, 70.6, 55.3, 36.9. FT-IR: vmax(neat)/cm-1 3473.9, 2869.8, 1774.6, 1709.7, 1383.9, 1201.4, 1076.3, 913.3. ESI/MS: m/z = 514.13 (M+H)+. HRMS: (M + Na+) calc. for C31H31NO6 536.20436, found 536.20397.  OOO OHBnONPhthPh OOO OBnONPhthPhCCl3NH  Imidate (39) 3-O-benzyl-4,6-O-benzylidene-2-deoxy-2-phthalimido-β-D-glucopyranose3 (2.88 g, 5.9 mmol) was dissolved in anhydrous CH2Cl2. K2CO3 (3.017 g, 21.83 mmol) and trichloroacetonitrile (2.97 mL, 29.5 mmol) were added. After overnight stirring, the mixture was filtered, concentrated in vacuo and purified by silicagel column chromatography. 1H-NMR (400 MHz, CDCl3) δ ppm 8.58 (s, 1H), 7.78-7.66 (m, 4H), S7 7.55-7.51 (m, 2H), 7.44-7.37 (m, 3H), 7.03-6.99 (m, 2H), 6.95-6.86 (m, 3H), 6.50 (d, J = 8.3 Hz, 1H), 5.64 (s, 1H), 4.82 (d, J = 12.4 Hz, 1H), 4.60-4.45 (m, 4H), 3.94-3.80 (m, 3H). 13C-NMR (101 MHz, CDCl3) δ ppm 167.4, 160.8, 137.7, 137.1, 134.0, 131.4, 129.1, 128.3, 128.0, 127.4, 126.1, 123.4, 101.4, 94.3, 82.6, 74.4, 74.2, 68.5, 66.9, 54.7. FT-IR: vmax(neat)/cm-1 1776.4, 1714.1, 1677.8, 1453.7, 1383.8, 1294.2, 1112.9, 1053.4, 1023.1. The imidate was hydrolyzed during mass-spectrometry. HRMS: (M-imidate + Na+) calc. for C31H31NO6 510.15232, found 510.15212. ONHAcOHOHOBnOONHAcBnOBnOHNOOOEtOONHAcOAcOAcOAcOONHAcAcOAcOHNOOOEtO   (2S,3S)-3-N-(O-(2-acetamido-3,4,6-tri-O-acetyl-2-deoxy-β-D-glucopyranosyl)-(1→4)-2-acetamido-3,6-di-O-acetyl-2-deoxy-β-D-glucopyranosylcarbamoyl) oxirane-2-carboxylic acid ethyl ester (49) Known epoxide2 (166 mg, 0.199 mmol) was dissolved in EtOH/Dioxane (2/1 v/v, 5 mL). Pd(OH)2 on charcoal (25 mg) was added followed by stirring under H2 atm for 1h. The solution was filtered, concentrated, redissolved in pyridine (5 mL) and cooled to 0°C before being treated with Ac2O (3 mL). The mixture was stirred overnight, concentrated, redissolved in CH2Cl2, washed with H2O, dried (Na2SO4) and concentrated in vacuo. RP-HPLC (linear gradient 22 → 32% B in 10.00 min) gave title compound 49 (26%, 39.69 mg, 51.2 μmol) as a white solid.  1H-NMR (400 MHz, CDCl3/MeOD) δ ppm 5.27 (dd, J = 10.2, 9.5 Hz, 1H), 5.09 (dd, J = 10.5, 8.4 Hz, 1H), 5.07-4.98 (m, 2H), 4.67 (d, J = 8.3 Hz, 1H), 4.43 (dd, J = 12.1, 1.4 Hz, 1H), 4.42 (dd, J = 12.4, 4.4 Hz, 1H), 4.28 (m, 2H), 4.07 (dd, J = 11.9, 4.7 Hz, 1H), 4.05-3.98 (m, 2H), 3.78-3.69 (m, 4H), 3.62 (d, J = 1.7 S8 Hz, 1H), 3.48 (d, J = 1.7 Hz, 1H), 2.14 (s, 3H), 2.09 (s, 3H), 2.07 (s, 3H), 2.02 (s, 6H), 1.94 (s, 3H), 1.91 (s, 3H), 1.32 (t, J = 7.1, 7.1 Hz, 3H). 13C-NMR (101 MHz, CDCl3/MeOD) δ ppm 172.3, 171.6, 171.2, 170.7, 170.6, 170.5, 169.6, 167.0, 166.6, 100.2, 78.2, 75.2, 74.2, 72.6, 71.9, 71.2, 68.1, 62.14, 62.09, 61.5, 54.4, 52.9, 52.4, 51.8, 22.11, 22.08, 20.3, 20.2, 20.1, 20.04, 19.97, 13.5. FT-IR: vmax(neat)/cm-1 3288.1, 1738.9, 1665.5, 1541.9, 1435.1, 1372.0, 1228.2, 1046.1, 902.1, 600.8. LC/MS: Rt 4.700 min; linear gradient 10→90% B in 13.5 min; ESI/MS: m/z = 776.5 (M+H)+.  HRMS: (M + H+) calc. for C32H45N3O19 776.27200, found 776.27207.  Competition assay To 9 μL of a solution of yeast PNGase (11.1 ng/μL) in PBS (pH = 7.2, 20 mM sodium phosphate, 150 mM NaCl, BSA (1 μg/μL) was added inhibitor (1 μL) and Bodipy-VAD-Fmk (1μL , 0.5 μM final concentration). The resulting solution was incubated at 37°C for 1h, after which it was quenched by the addition of 4 × SDS-PAGE sample buffer (4 μL) and boiling for 5 min. The proteins were separated on a 10% SDS-PAGE gel and subsequently the fluorescence was measured in the wet gel slabs by using the CY3/Tamra settings (λex 532 λem560) on a Typhoon Variable Mode Imager (Amersham Biosciences). The remaining fluorescence was quantified with ImageQuant followed by analysis with Graphpad Prism.    In vivo inhibition of human PNGase Control and US11-expressing U373 cells (1.5*106 cells per lane) were collected, washed with PBS and resuspended in Met-/Cys- medium (2 mL) containing 10% FCS and the S9 inhibitors ZL3VS (1.25 μM), ZVAD-Fmk, 2, 49 (30 μM and 100 μM respectively) and NGT (50 μM) as indicated. The cells were incubated for 45 min at 37°C, pelleted by centrifugation and resuspended in 250 µL Met-/Cys- medium, again in the presence of the inhibitors. [35S]-labeled Cys/Met (5 μL, 200 μCi/mL) was added and the cells were incubated for 30 minutes at 37°C. The cells were washed with PBS and subsequently  lysed in 1 ml cold NP-40 buffer (0.5% NP-40, 50 mM Tris HCl pH 7.5, 5 mM MgCl2, 10 µM leupeptin, and 1 mM 4-(2-aminoethyl)benzenesulfonyl fluoride) for 45 min at 4°C. The lysed cells were centrifuged (16.000 g, 15 min) at 4°C in order to obtain post-nuclear lysates. These lysates were precleared twice by the addition of Protein G- and Protein A-sepharose beads (7.5 μL, GE Healthcare), normal rabbit serum (1.5 μL) and normal mouse serum (1.5 μL), followed by incubation for 60 min at 4°C. Next, the class I heavy chains were immunoprecipitated. Specific antibodies (mAb HC10), Protein G- and Protein A-sepharose beads were added to the lysates and samples were incubated o/n at 4°C. Isolated immune complexes were washed thrice with NET buffer containing 0.1% SDS and denatured in reducing sample buffer (50 μL). The samples were separated by SDS-PAGE and visualized by phosphoimaging.  References (1) Pelter, A.; Kvicala, J. Tetrahedron Lett. 1997, 38, 4877-4880. (2) Witte, M. D.; Descals, C. V.; De Lavoir, S. V. P.; Florea, B. I.; Van der Marel, G. A.; Overkleeft, H. S. Org. Biomol. Chem. 2007, 5, 3690-3697. (3) Chiara, J. L.; García, Á.; Cristóbal-Lumbroso, G. J. Org. Chem. 2005, 70, 4142-4150  S10F-OOK+5ppm (f1)0.01.02.03.04.05.06.07.08.0 S11F-OOK+5ppm (f1)050100150200 S12OOOHO7ppm (f1)0.05.010.0 S13OOOHO7ppm (f1)050100150200 S14ONHAcOHOHOHOONHAcHOHOHN10FOppm (f1)0.01.02.03.04.05.06.0 S15ONHAcOHOHOHOONHAcHOHOHN10FOppm (f1)050100150200 S16RT: 0.00 - 14.600 1 2 3 4 5 6 7 8 9 10 11 12 13 14Tim e (m in)020406080100Relative Abundance0200000400000600000uAU14.2412.301.280.693.65 10.3710.03 11.869.208.737.777.393.09 6.582.78 6.034.981.450.611.310.9012.4012.531.440.02 2.90 12.991.64 3.06 13.860.71 2.03 3.61 4.15 9.938.394.72 6.54 11.259.247.76 11.9410.625.33 7.29NL:7.37E5Spectrum Maximum nm=200.0-600.0  PDA mdw656NL:1.63E6Base Peak F: + p ESI Ful l  ms [ 160.00-2000.00]  MS mdw656Instrument Method: C:\Xcalibur\methods\General(TFA)\0020%B10_C=TFA_20ul_+pf 15min.methProcessing Method:Vial: A:23Injection Volume (µl):   20.00Sample Weight:    0.00m dw656 #68 RT: 1.33 AV: 1 NL: 1.19E6F: + p ESI Full m s  [ 160.00-2000.00]200 400 600 800 1000 1200 1400 1600 1800 2000m /z010000020000030000040000050000060000070000080000090000010000001100000Intensity483.93966.801449.93235.87281.00 1933.471208.53 1691.47391.07 984.00650.67 1497.601235.27500.80 1772.40720.87 1111.13902.53ONHAcOHOHOHOONHAcHOHOHN10FOS17ONHAcOHOHOHOONHAcHOHOHN11OOOEtOppm (f1)1.02.03.04.05.0 S18ONHAcOHOHOHOONHAcHOHOHN11OOOEtOppm (f1)050100150200 S19ONHAcOHOHOHOONHAcHOHOHN11OOOEtOS20ONHAcOHOHOHOONHAcHOHOHN12OOOppm (f1)1.02.03.04.05.06.07.0 S21ONHAcOHOHOHOONHAcHOHOHN12OOOppm (t1)50100150 S22ONHAcOHOHOHOONHAcHOHOHN12OOOS23ONHAcOHOHOHOONHAcHOHOHN13OHNOEtOppm (f1)1.02.03.04.05.0 S24ONHAcOHOHOHOONHAcHOHOHN13OHNOEtOppm (f1)50100150 S25ONHAcOHOHOHOONHAcHOHOHN13OHNOEtOS26ONHAcOHOHOHOONHAcHOHOHN14OHNOEtOppm (f1)0.01.02.03.04.05.0 S27ONHAcOHOHOHOONHAcHOHOHN14OHNOEtOppm (f1)050100150200 S28ONHAcOHOHOHOONHAcHOHOHN14OHNOEtOS29ONHAcOOOBnOONHAcBnOBnOHN17ONOEtOOPhppm (f1)0.01.02.03.04.05.06.07.08.0 S30ONHAcOOOBnOONHAcBnOBnOHN17ONOEtOOPhppm (t1)50100150 S31ONHAcOOOBnOONHAcBnOBnOHN17ONOEtOOPhS32ONHAcOOOBnOONHAcBnOBnOHN18ONOEtOOPhppm (t1)0.01.02.03.04.05.06.07.08.0 S33ONHAcOOOBnOONHAcBnOBnOHN18ONOEtOOPhppm (t1)050100150 S34ONHAcOOOBnOONHAcBnOBnOHN18ONOEtOOPhS35ONHAcOHOHOHOONHAcHOHOHN19ONOEtOOppm (f1)0.01.02.03.04.05.06.07.08.0 S36ONHAcOHOHOHOONHAcHOHOHN19ONOEtOOppm (f1)050100150200 S37ONHAcOHOHOHOONHAcHOHOHN19ONOEtOOS38ONHAcOHOHOHOONHAcHOHOHN20ONOEtOOppm (f1)0.01.02.03.04.05.06.07.08.0 S39ONHAcOHOHOHOONHAcHOHOHN20ONOEtOOppm (f1)050100150 S40ONHAcOHOHOHOONHAcHOHOHN20ONOEtOOS41ONHAcOOOBnOONHAcBnOBnOHN21OHOPhppm (f1)0.01.02.03.04.05.06.07.08.0 S42ONHAcOOOBnOONHAcBnOBnOHN21OHOPhppm (f1)050100150200 S43ONHAcOOOBnOONHAcBnOBnOHN21OHOPhS44ONHAcOHOHOHOONHAcHOHOHN24OOSOOppm (f1)2.002.503.003.504.004.505.00 S45ONHAcOHOHOHOONHAcHOHOHN24OOSOOppm (f1)050100150200 S46RT: 0.00 - 14.600 1 2 3 4 5 6 7 8 9 10 11 12 13 14Tim e (m in)020406080100Relative Abundance0200000400000600000uAU14.2712.292.770.68 11.833.70 11.389.35 10.158.097.80 8.846.614.511.51 2.330.612.798.02 8.189.51 10.73 14.0612.107.875.91 12.00 13.834.79 7.076.734.960.88 4.630.51 1.50 4.011.93 12.40 13.78NL:7.30E5Spectrum Maximum nm=200.0-600.0  PDA mdw707NL:1.99E6Base Peak F: + p ESI Ful l  ms [ 160.00-2000.00]  MS mdw707Instrument Method: C:\Xcalibur\methods\General(TFA)\0020%B00_C=TFA_20ul_+pf 15min.methProcessing Method:Vial: C:25Injection Volume (µl):   20.00Sample Weight:    0.00m dw707 #143 RT: 2.77 AV: 1 NL: 1.84E6F: + p ESI Full m s  [ 160.00-2000.00]200 400 600 800 1000 1200 1400 1600 1800 2000m /z020000040000060000080000010000001200000140000016000001800000Intensity560.001118.931678.601398.201151.071958.001701.80591.93357.00 1514.401312.53282.53 838.80 1057.00716.47 970.20497.53ONHAcOHOHOHOONHAcHOHOHN24OOSOOS47ONHAcOHOHOHOONHAcHOHOHN25SOppm (f1)0.01.02.03.04.05.06.07.0 S48ONHAcOHOHOHOONHAcHOHOHN25SOppm (f1)050100150200 S49RT: 0.00 - 14.600 1 2 3 4 5 6 7 8 9 10 11 12 13 14Tim e (m in)020406080100Relative Abundance0100000020000003000000uAU6.496.550.8214.3012.1511.5910.909.18 10.057.83 8.596.836.305.044.763.470.68 2.211.526.555.53 5.974.974.37 14.174.14 13.207.20 7.573.850.14 7.74 11.089.828.30 13.0711.623.162.070.59 2.11NL:3.93E6Spectrum Maximum nm=200.0-600.0  PDA mdw702NL:4.70E6Base Peak F: + p ESI Ful l  ms [ 160.00-2000.00]  MS mdw702Instrument Method: C:\Xcalibur\methods\General(TFA)\0050%B00_C=TFA_20ul_+pf 15min.methProcessing Method:Vial: C:24Injection Volume (µl):   20.00Sample Weight:    0.00m dw702 #339 RT: 6.53 AV: 1 NL: 4.15E6F: + p ESI Full m s  [ 160.00-2000.00]200 400 600 800 1000 1200 1400 1600 1800 2000m /z05000001000000150000020000002500000300000035000004000000Intensity573.931146.731719.871433.93371.13 1913.00618.80 1208.87944.67 1332.60204.00 1752.601487.60879.80 1627.20695.73 1069.00ONHAcOHOHOHOONHAcHOHOHN25SOS50

English

Peptide N-glycanase (PNGase), the enzyme responsible for the deglycosylation of N-linked glycoproteins, has an active site related to that of cysteine proteases. Chitiobiose was equipped with electrophilic traps often used in cysteine protease inhibitors, and the resulting compounds were evaluated as PNGase inhibitors. We found that the electrophilic trap of the inhibitor has a great influence on the potency of the compounds with the chloromethyl ketone inhibitor being the first potent C-glycoside-based PNGase inhibitor

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 University of Groningen
Synthesis and Biological Evaluation of a Chitobiose-Based Peptide N-Glycanase Inhibitor
Library
Witte, Martin; Horst, Danielle; Wiertz, Emmanuel J.H.J.; Marel, Gijsbert A. van der;
Overkleeft, Herman S.
Published in:
The Journal of Organic Chemistry
DOI:
10.1021/jo801906s
IMPORTANT NOTE: You are advised to consult the publisher's version (publisher's PDF) if you wish to cite from
it. Please check the document version below.
Document Version
Publisher's PDF, also known as Version of record
Publication date:
2009
Link to publication in University of Groningen/UMCG research database
Citation for published version (APA):
Witte, M. D., Horst, D., Wiertz, E. J. H. J., Marel, G. A. V. D., & Overkleeft, H. S. (2009). Synthesis and
Biological Evaluation of a Chitobiose-Based Peptide N-Glycanase Inhibitor Library. The Journal of Organic
Chemistry, 74(2), 605-616. DOI: 10.1021/jo801906s
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Download date: 10-02-2018
Supporting Information 
 
Synthesis and biological evaluation of a chitobiose based peptide N-glycanase 
inhibitor library 
 
Martin D. Wittea, Daniëlle Horstb, Emmanuel J. H. J. Wiertzb, Gijs A. van der Marela* 
and Herman S. Overkleefta*. 
 
aLeiden Institute of Chemistry, Leiden University, P.O. Box 9502, 2300 RA Leiden, The 
Netherlands. Email: marel_g@ chem.leidenuniv.nl, h.s.overkleeft@chem.leidenuniv.nl 
bDepartment of Medical Microbiology, Center of Infectious Diseases, Leiden University 
Medical Center, P.O. Box 9600, 2300 RC Leiden, The Netherlands. 
 
Table of contents 
Copy of 1H-NMR of 26 S4
Copy of 13C-NMR of 26 S5
Copy of 1H-NMR of 29 S6
Copy of 13C-NMR of 29 S7
Copy of 1H-NMR of 30 S8
Copy of 13C-NMR of 30 S9
Copy of LC/MS spectrum of 30 S10
Copy of 1H-NMR of 31 S11
Copy of 13C-NMR of 31 S12
S1
Copy of LC/MS spectrum of 31 S13
Copy of 1H-NMR of 32 S14
Copy of 13C-NMR of 32 S15
Copy of LC/MS spectrum of 32 S16
Copy of 1H-NMR of 33 S17
Copy of 13C-NMR of 33 S18
Copy of LC/MS spectrum of 33 S19
Copy of 1H-NMR of 36 S20
Copy of 13C-NMR of 36 S21
Copy of 1H-NMR of 37 S22
Copy of 13C-NMR of 37 S23
Copy of 1H-NMR of 38 S24
Copy of 13C-NMR of 38 S25
Copy of LC/MS spectrum of 38 S26
Copy of 1H-NMR of 39 S27
Copy of 13C-NMR of 39 S28
Copy of 1H-NMR of 40 S29
Copy of 13C-NMR of 40 S30
Copy of 1H-NMR of 41 S31
Copy of 13C-NMR of 41 S32
Copy of LC/MS spectrum of 41 S33
Copy of 1H-NMR of 42 S34
Copy of 13C-NMR of 42 S35
S2
Copy of LC/MS spectrum of 42 S36
Copy of 1H-NMR of 43 S37
Copy of 13C-NMR of 43 S38
Copy of LC/MS spectrum of 43 S39
Copy of 1H-NMR of 44 S40
Copy of 13C-NMR of 44 S41
Copy of LC/MS spectrum of 44 S42
Copy of 1H-NMR of 45 S43
Copy of 13C-NMR of 45 S44
Copy of LC/MS spectrum of 45 S45
Copy of 1H-NMR of 47 S46
Copy of 13C-NMR of 47 S47
Copy of LC/MS spectrum of 47 S48
Copy of 1H-NMR of 48 S49
Copy of 13C-NMR of 48 S50
Copy of LC/MS spectrum of 48 S51
Copy of 1H-NMR of 49 S52
Copy of 13C-NMR of 49 S53
Copy of LC/MS spectrum of 49 S54
 
S3
OBnOHO
NPhth
BnO
26
ppm (f1)
0.01.02.03.04.05.06.07.08.0 S4
OBnOHO
NPhth
BnO
26
ppm (f1)
050100150200 S5
ONPhth
O
AcO
AcO
AcO
O
NPhth
BnO
BnO
29
ppm (f1)
1.02.03.04.05.06.07.08.0 S6
ONPhth
O
AcO
AcO
AcO
O
NPhth
BnO
BnO
29
ppm (f1)
50100150 S7
ONHAc
O
AcO
AcO
AcO
O
NHAc
BnO
BnO
30
ppm (f1)
2.03.04.05.06.07.08.0 S8
ONHAc
O
AcO
AcO
AcO
O
NHAc
BnO
BnO
30
ppm (f1)
50100150 S9
RT: 0.00 - 14.60
0 1 2 3 4 5 6 7 8 9 10 11 12 13 14
Tim e (m in)
0
20
40
60
80
100
R
e
l
a
t
i
v
e
 
A
b
u
n
d
a
n
c
e
0
200000
400000
600000
800000
u
A
U
14.27
13.93
11.247.96
0.84
0.65
8.494.07
6.244.953.381.02 2.13
7.99
8.08
6.73 7.224.53 8.35 9.00 9.362.97 3.482.061.561.21 11.250.43 12.404.67 10.93 13.425.61 6.59 13.76
NL:
8.33E5
Spectrum 
Maximum 
nm=200.0-600.0 
 PDA mdw630
NL:
1.39E7
Base Peak F: + p 
ESI Ful l  ms [ 
160.00-2000.00] 
 MS mdw630
Instrument Method: C:\Xcalibur\methods\General(TFA)\1090%B10_C=TFA_20ul_+pf 15min.meth
Processing Method:
Vial: C:21
Injection Volume (µl):   10.00
Sample Weight:    0.00
m dw630 #414 RT: 7.97 AV: 1 NL: 1.14E7
F: + p ESI Full m s  [ 160.00-2000.00]
200 400 600 800 1000 1200 1400 1600 1800 2000
m /z
0
1000000
2000000
3000000
4000000
5000000
6000000
7000000
8000000
9000000
10000000
11000000
I
n
t
e
n
s
i
t
y
755.00
1508.80
799.87
329.87 1553.33209.80 825.87 1884.671078.40 1645.27427.00 926.73
O
NHAc
O
AcO
AcO
AcO
O
NHAc
BnO
BnO
30
S10
ONHAc
O
AcO
AcO
AcO
O
NHAc
AcO
AcO
31
ppm (f1)
2.03.04.05.06.0 S11
ONHAc
O
AcO
AcO
AcO
O
NHAc
AcO
AcO
31
ppm (f1)
050100150 S12
RT: 0.00 - 14.60
0 1 2 3 4 5 6 7 8 9 10 11 12 13 14
Tim e (m in)
0
20
40
60
80
100
R
e
l
a
t
i
v
e
 
A
b
u
n
d
a
n
c
e
0
200000
400000
600000
800000
u
A
U
14.26
13.90
0.64 0.85
8.458.145.58 7.737.114.984.674.133.601.61 1.89
5.59
7.95 7.997.857.116.735.254.62 8.29 11.72 12.43 12.912.922.41 8.503.23 9.51 11.590.45 0.90 1.39 10.87 13.56
NL:
8.31E5
Spectrum 
Maximum 
nm=200.0-600.0 
 PDA mdw636
NL:
8.87E6
Base Peak F: + p 
ESI Ful l  ms [ 
160.00-2000.00] 
 MS mdw636
Instrument Method: C:\Xcalibur\methods\General(TFA)\1090%B00_C=TFA_20ul_+pf 15min.meth
Processing Method:
Vial: C:22
Injection Volume (µl):   10.00
Sample Weight:    0.00
m dw636 #290 RT: 5.59 AV: 1 NL: 8.87E6
F: + p ESI Full m s  [ 160.00-2000.00]
200 400 600 800 1000 1200 1400 1600 1800 2000
m /z
0
1000000
2000000
3000000
4000000
5000000
6000000
7000000
8000000
I
n
t
e
n
s
i
t
y
659.00
1316.47
703.87
1358.47 1974.80329.87 1665.87720.60 845.87 1475.07614.87237.93
O
NHAc
O
AcO
AcO
AcO
O
NHAc
AcO
AcO
31
S13
ONHAc
O
AcO
AcO
AcO
O
NHAc
AcO
AcO
32
OEt
O
ppm (f1)
0.01.02.03.04.05.06.07.0 S14
ONHAc
O
AcO
AcO
AcO
O
NHAc
AcO
AcO
32
OEt
O
ppm (f1)
050100150200 S15
ONHAc
O
AcO
AcO
AcO
O
NHAc
AcO
AcO
32
OEt
O
S16
ONHAc
O
HO
HO
HO
O
NHAc
HO
HO
33
OMe
O
ppm (f1)
2.03.04.05.06.07.0 S17
ONHAc
O
HO
HO
HO
O
NHAc
HO
HO
33
OMe
O
ppm (f1)
-100-50050 S18
RT: 0.00 - 14.60
0 1 2 3 4 5 6 7 8 9 10 11 12 13 14
Tim e (m in)
0
20
40
60
80
100
R
e
l
a
t
i
v
e
 
A
b
u
n
d
a
n
c
e
0
200000
400000
600000
800000
u
A
U
7.08
14.27
12.29
7.38
11.763.67 11.369.388.04 9.908.756.846.274.770.77 1.49 1.84
7.10
7.02 14.107.39 12.049.26 11.378.43 13.769.30 12.1911.217.664.864.78 6.875.03 5.94 12.3510.923.621.50 3.231.600.34 3.750.51 13.67
13.34
NL:
9.78E5
Spectrum 
Maximum 
nm=200.0-600.0 
 PDA mdw643
NL:
1.61E6
Base Peak F: + p 
ESI Ful l  ms [ 
160.00-2000.00] 
 MS mdw643
Instrument Method: C:\Xcalibur\methods\General(TFA)\0020%B00_C=TFA_20ul_+pf 15min.meth
Processing Method:
Vial: C:23
Injection Volume (µl):   20.00
Sample Weight:    0.00
m dw643 #366 RT: 7.10 AV: 1 NL: 1.61E6
F: + p ESI Full m s  [ 160.00-2000.00]
200 400 600 800 1000 1200 1400 1600 1800 2000
m /z
0
200000
400000
600000
800000
1000000
1200000
1400000
1600000
I
n
t
e
n
s
i
t
y
507.00
1013.20
1519.13
1266.67 1350.67203.93 1772.471564.53552.00 1863.931074.07711.27305.13 391.20 1447.40973.87
O
NHAc
O
HO
HO
HO
O
NHAc
HO
HO
33
OMe
O
S19
ONHAc
O
TBSO
TBSO
TBSO
O
NHAc
TBSO
TBSO
36
ppm (f1)
0.01.02.03.04.05.06.0 S20
ONHAc
O
TBSO
TBSO
TBSO
O
NHAc
TBSO
TBSO
36
ppm (f1)
050100150200 S21
ONHAc
O
TBSO
TBSO
TBSO
O
NHAc
TBSO
TBSO
37
S
O O
ppm (f1)
0.01.02.03.04.05.06.07.0 S22
ONHAc
O
TBSO
TBSO
TBSO
O
NHAc
TBSO
TBSO
37
S
O O
ppm (f1)
050100150200 S23
ONHAc
O
HO
HO
HO
O
NHAc
HO
HO
38
S
O O
ppm (f1)
0.01.02.03.04.05.06.07.0 S24
ONHAc
O
HO
HO
HO
O
NHAc
HO
HO
38
S
O O
ppm (f1)
050100150200 S25
ONHAc
O
HO
HO
HO
O
NHAc
HO
HO
38
S
O O
S26
OOO
BnO
NPhth
O CCl3
NH
Ph
39
ppm (f1)
0.01.02.03.04.05.06.07.08.0 S27
OOO
BnO
NPhth
O CCl3
NH
Ph
39
ppm (f1)
050100150200 S28
ONPhth
O
O
O
BnO
O
NPhth
BnO
BnO
40
Ph
ppm (f1)
0.01.02.03.04.05.06.07.08.0 S29
ONPhth
O
O
O
BnO
O
NPhth
BnO
BnO
40
Ph
ppm (f1)
050100150200 S30
ONHAc
O
O
O
BnO
O
NHAc
BnO
BnO
41
OH
O
Ph
ppm (f1)
0.01.02.03.04.05.06.07.0 S31
ONHAc
O
O
O
BnO
O
NHAc
BnO
BnO
41
OH
O
Ph
ppm (f1)
050100150200 S32
ONHAc
O
O
O
BnO
O
NHAc
BnO
BnO
41
OH
O
Ph
S33
ONHAc
O
O
O
BnO
O
NHAc
BnO
BnO
42
O
O
Ph O
ppm (f1)
0.01.02.03.04.05.06.07.08.0 S34
ONHAc
O
O
O
BnO
O
NHAc
BnO
BnO
42
O
O
Ph O
ppm (f1)
050100150200 S35
RT: 0.00 - 14.60
0 1 2 3 4 5 6 7 8 9 10 11 12 13 14
Tim e (m in)
0
20
40
60
80
100
R
e
l
a
t
i
v
e
 
A
b
u
n
d
a
n
c
e
0
500000
1000000
1500000
u
A
U
10.76
14.27
13.3212.62
0.84 10.4410.045.86 7.994.11 7.355.434.643.391.56 1.98
10.81
10.96
11.119.79 12.629.90 12.94 13.47
3.07 9.652.121.790.20 3.401.25 4.43 4.91 5.89 7.956.24 6.87 9.40
NL:
1.83E6
Spectrum 
Maximum 
nm=200.0-600.0 
 PDA mdw810
NL:
1.45E7
Base Peak F: + p 
ESI Ful l  ms [ 
160.00-2000.00] 
 MS mdw810
Instrument Method: C:\Xcalibur\methods\General(TFA)\1090%B10_C=TFA_20ul_+pf 15min.meth
Processing Method:
Vial: C:28
Injection Volume (µl):   10.00
Sample Weight:    0.00
m dw810 #561 RT: 10.79 AV: 1 NL: 1.35E7
F: + p ESI Full m s  [ 160.00-2000.00]
200 400 600 800 1000 1200 1400 1600 1800 2000
m /z
0
2000000
4000000
6000000
8000000
10000000
12000000
I
n
t
e
n
s
i
t
y
971.27
1942.00
382.07 1457.201015.93 1253.07590.27314.87 1541.20 1782.20474.47 866.33
O
NHAc
O
O
O
BnO
O
NHAc
BnO
BnO
42
O
O
Ph O
S36
ONHAc
O
O
O
BnO
O
NHAc
BnO
BnO
43
O
Cl
Ph
ppm (f1)
0.01.02.03.04.05.06.07.0 S37
ONHAc
O
O
O
BnO
O
NHAc
BnO
BnO
43
O
Cl
Ph
ppm (f1)
050100150200 S38
RT: 0.00 - 14.60
0 1 2 3 4 5 6 7 8 9 10 11 12 13 14
Tim e (m in)
0
20
40
60
80
100
R
e
l
a
t
i
v
e
 
A
b
u
n
d
a
n
c
e
0
200000
400000
600000
800000
u
A
U
14.26
9.85
13.9213.62
0.73
8.317.937.343.73 5.715.384.093.191.70
9.87
10.22 13.52 13.8510.509.017.37 12.8512.377.413.99 5.355.061.86 3.211.26 2.80 6.15 7.190.32
NL: 8.15E5
Spectrum 
Maximum 
nm=200.0-600.0  
PDA mdw835spot1
NL: 7.05E6
Base Peak F: + p 
ESI Ful l  ms [ 
160.00-2000.00]  
MS mdw835spot1
Instrument Method: C:\Xcalibur\methods\General(TFA)\1090%B10_C=TFA_20ul_+pf 15min.meth
Processing Method:
Vial: A:13
Injection Volume (µl):   10.00
m dw835spot1 #505 RT: 9.83 AV: 1 NL: 3.62E6
F: + p ESI Full m s [ 160.00-2000.00]
200 400 600 800 1000 1200 1400 1600 1800 2000
m /z
0
10
20
30
40
50
60
70
80
90
100
R
e
l
a
t
i
v
e
 
A
b
u
n
d
a
n
c
e
857.07
1713.87
947.20381.93 1804.87
962.00269.67 1129.27 1968.471420.201215.27752.20476.13 696.27 1680.73
O
NHAc
O
O
O
BnO
O
NHAc
BnO
BnO
43
O
Cl
Ph
S39
ONHAc
O
HO
HO
HO
O
NHAc
HO
HO
44
O
O
O
ppm (f1)
0.01.02.03.04.05.06.07.0 S40
ONHAc
O
HO
HO
HO
O
NHAc
HO
HO
44
O
O
O
ppm (f1)
050100150200 S41
RT: 0.00 - 14.60
0 1 2 3 4 5 6 7 8 9 10 11 12 13 14
Tim e (m in)
0
20
40
60
80
100
R
e
l
a
t
i
v
e
 
A
b
u
n
d
a
n
c
e
0
1000000
2000000
3000000
u
A
U
8.46
0.84
14.25
12.108.637.81 9.426.926.145.763.55 4.844.200.68 2.291.53
8.49
6.13 8.838.38 12.88 13.946.594.57 5.18 7.134.39 12.759.893.560.12 3.10 10.952.570.57 2.03
NL:
3.97E6
Spectrum 
Maximum 
nm=200.0-600.0 
 PDA mdw750
NL:
9.73E6
Base Peak F: + p 
ESI Ful l  ms [ 
160.00-2000.00] 
 MS mdw750
Instrument Method: C:\Xcalibur\methods\General(TFA)\0050%B00_C=TFA_20ul_+pf 15min.meth
Processing Method:
Vial: C:26
Injection Volume (µl):   20.00
Sample Weight:    0.00
m dw750 #439 RT: 8.47 AV: 1 NL: 8.02E6
F: + p ESI Full m s  [ 160.00-2000.00]
200 400 600 800 1000 1200 1400 1600 1800 2000
m /z
0
1000000
2000000
3000000
4000000
5000000
6000000
7000000
8000000
I
n
t
e
n
s
i
t
y
613.07
1225.07
1837.87
1531.87658.00
410.07204.07 1271.13921.73 1634.931429.53 1882.93344.20 1706.20765.27
O
NHAc
O
HO
HO
HO
O
NHAc
HO
HO
44
O
O
O
S42
ONHAc
O
HO
HO
HO
O
NHAc
HO
HO
45
Cl
O
ppm (f1)
1.02.03.04.0 S43
ONHAc
O
HO
HO
HO
O
NHAc
HO
HO
45
Cl
O
ppm (f1)
050100150200 S44
ONHAc
O
HO
HO
HO
O
NHAc
HO
HO
45
Cl
O
S45
ONHAc
O
HO
HO
HO
O
NHAc
HO
HO
47
O
P
O
Ph
Ph
O
ppm (f1)
1.02.03.04.05.06.07.08.0 S46
ONHAc
O
HO
HO
HO
O
NHAc
HO
HO
47
O
P
O
Ph
Ph
O
ppm (f1)
050100150200 S47
RT: 0.00 - 14.60
0 1 2 3 4 5 6 7 8 9 10 11 12 13 14
Tim e (m in)
0
20
40
60
80
100
R
e
l
a
t
i
v
e
 
A
b
u
n
d
a
n
c
e
0
500000
1000000
u
A
U
7.89
14.28
8.52 12.1810.849.919.296.805.854.51 5.543.723.360.67 2.151.78
7.92
8.59
7.78
13.8313.2812.194.023.67 4.382.56 5.210.39 5.811.86 6.391.28 8.687.65 10.908.93 9.87
NL:
1.40E6
Spectrum 
Maximum 
nm=200.0-600.0 
 PDA mdw816
NL:
9.46E6
Base Peak F: + p 
ESI Ful l  ms [ 
160.00-2000.00] 
 MS mdw816
Phone:
Instrument Method: C:\Xcalibur\methods\General(TFA)\0050%B10_C=TFA_20ul_+pf 15min.meth
Processing Method:
Vial:  A:6
m dw816 #411 RT: 7.92 AV: 1 NL: 9.46E6
F: + p ESI Full m s [ 160.00-2000.00]
200 400 600 800 1000 1200 1400 1600 1800 2000
m /z
0
10
20
30
40
50
60
70
80
90
100
R
e
l
a
t
i
v
e
 
A
b
u
n
d
a
n
c
e
681.07
1360.93
1701.40726.00 1815.131407.73 1598.47478.00 1936.20198.80 1041.07 1282.20343.80 833.20 914.47
O
NHAc
O
HO
HO
HO
O
NHAc
HO
HO
47
O
P
O
Ph
Ph
O
S48
ONHAc
O
HO
HO
HO
O
NHAc
HO
HO
48
S
O
ppm (t1)
0.01.02.03.04.05.06.07.0
Et3N HOAc
Et3N HOAc
S49
ONHAc
O
HO
HO
HO
O
NHAc
HO
HO
48
S
O
ppm (t1)
050100150200 S50
RT: 0.00 - 14.60
0 1 2 3 4 5 6 7 8 9 10 11 12 13 14
Tim e (m in)
0
20
40
60
80
100
R
e
l
a
t
i
v
e
 
A
b
u
n
d
a
n
c
e
0
200000
400000
600000
800000
1000000
u
A
U
4.52
14.27
13.71
6.544.38 5.383.410.64 1.07 1.99
1.42
4.54
0.02 12.87 13.3711.8710.8710.575.47 9.376.29 6.79 8.047.754.36 8.553.990.36 3.011.29 2.42
NL:
1.11E6
Spectrum 
Maximum 
nm=200.0-600.0  
PDA 
mdw871_H2O
NL:
8.73E6
Base Peak F: + p 
ESI Ful l  ms [ 
160.00-2000.00]  
MS mdw871_H2O
Sample Name:
Study:
Client:
Laboratory:
Company:
m dw871_H2O #232 RT: 4.54 AV: 1 NL: 2.81E6
F: + p ESI Full m s  [ 160.00-2000.00]
200 400 600 800 1000 1200 1400 1600 1800 2000
m /z
0
500000
1000000
1500000
2000000
2500000
I
n
t
e
n
s
i
t
y
573.00
1144.80
1716.40617.87 1189.93175.67 1439.60 1800.47725.20391.27 878.13 1296.80947.40447.80 1670.73 1943.871102.27
O
NHAc
O
HO
HO
HO
O
NHAc
HO
HO
48
S
O
S51
OAcOAcO
AcO O
OAcO
AcO
NHAc NHAc
H
N
O
O
OEt
O
49
ppm (f1)
1.02.03.04.05.0 S52
OAcOAcO
AcO O
OAcO
AcO
NHAc NHAc
H
N
O
O
OEt
O
49
ppm (f1)
050100150200 S53
ONHAc
O
AcO
AcO
AcO
O
NHAc
AcO
AcO
H
N
49
O
O
OEt
O
S54


Dissertations of the University of Groningen

   University of GroningenSynthesis and Biological Evaluation of a Chitobiose-Based Peptide N-Glycanase InhibitorLibraryWitte, Martin D.; Horst, Danielle; Wiertz, Emmanuel J.H.J.; Marel, Gijsbert A. van der;Overkleeft, Herman S.Published in:The Journal of Organic ChemistryDOI:10.1021/jo801906sIMPORTANT NOTE: You are advised to consult the publisher's version (publisher's PDF) if you wish to cite fromit. Please check the document version below.Document VersionPublisher's PDF, also known as Version of recordPublication date:2009Link to publication in University of Groningen/UMCG research databaseCitation for published version (APA):Witte, M. D., Horst, D., Wiertz, E. J. H. J., Marel, G. A. V. D., & Overkleeft, H. S. (2009). Synthesis andBiological Evaluation of a Chitobiose-Based Peptide N-Glycanase Inhibitor Library. The Journal of OrganicChemistry, 74(2), 605-616. https://doi.org/10.1021/jo801906sCopyrightOther than for strictly personal use, it is not permitted to download or to forward/distribute the text or part of it without the consent of theauthor(s) and/or copyright holder(s), unless the work is under an open content license (like Creative Commons).Take-down policyIf you believe that this document breaches copyright please contact us providing details, and we will remove access to the work immediatelyand investigate your claim.Downloaded from the University of Groningen/UMCG research database (Pure): http://www.rug.nl/research/portal. For technical reasons thenumber of authors shown on this cover page is limited to 10 maximum.Download date: 12-11-2019Supporting Information  Synthesis and biological evaluation of a chitobiose based peptide N-glycanase inhibitor library  Martin D. Wittea, Daniëlle Horstb, Emmanuel J. H. J. Wiertzb, Gijs A. van der Marela* and Herman S. Overkleefta*.  aLeiden Institute of Chemistry, Leiden University, P.O. Box 9502, 2300 RA Leiden, The Netherlands. Email: marel_g@ chem.leidenuniv.nl, h.s.overkleeft@chem.leidenuniv.nl bDepartment of Medical Microbiology, Center of Infectious Diseases, Leiden University Medical Center, P.O. Box 9600, 2300 RC Leiden, The Netherlands.  Table of contents Copy of 1H-NMR of 26 S4Copy of 13C-NMR of 26 S5Copy of 1H-NMR of 29 S6Copy of 13C-NMR of 29 S7Copy of 1H-NMR of 30 S8Copy of 13C-NMR of 30 S9Copy of LC/MS spectrum of 30 S10Copy of 1H-NMR of 31 S11Copy of 13C-NMR of 31 S12S1Copy of LC/MS spectrum of 31 S13Copy of 1H-NMR of 32 S14Copy of 13C-NMR of 32 S15Copy of LC/MS spectrum of 32 S16Copy of 1H-NMR of 33 S17Copy of 13C-NMR of 33 S18Copy of LC/MS spectrum of 33 S19Copy of 1H-NMR of 36 S20Copy of 13C-NMR of 36 S21Copy of 1H-NMR of 37 S22Copy of 13C-NMR of 37 S23Copy of 1H-NMR of 38 S24Copy of 13C-NMR of 38 S25Copy of LC/MS spectrum of 38 S26Copy of 1H-NMR of 39 S27Copy of 13C-NMR of 39 S28Copy of 1H-NMR of 40 S29Copy of 13C-NMR of 40 S30Copy of 1H-NMR of 41 S31Copy of 13C-NMR of 41 S32Copy of LC/MS spectrum of 41 S33Copy of 1H-NMR of 42 S34Copy of 13C-NMR of 42 S35S2Copy of LC/MS spectrum of 42 S36Copy of 1H-NMR of 43 S37Copy of 13C-NMR of 43 S38Copy of LC/MS spectrum of 43 S39Copy of 1H-NMR of 44 S40Copy of 13C-NMR of 44 S41Copy of LC/MS spectrum of 44 S42Copy of 1H-NMR of 45 S43Copy of 13C-NMR of 45 S44Copy of LC/MS spectrum of 45 S45Copy of 1H-NMR of 47 S46Copy of 13C-NMR of 47 S47Copy of LC/MS spectrum of 47 S48Copy of 1H-NMR of 48 S49Copy of 13C-NMR of 48 S50Copy of LC/MS spectrum of 48 S51Copy of 1H-NMR of 49 S52Copy of 13C-NMR of 49 S53Copy of LC/MS spectrum of 49 S54 S3OBnOHONPhthBnO26ppm (f1)0.01.02.03.04.05.06.07.08.0 S4OBnOHONPhthBnO26ppm (f1)050100150200 S5ONPhthOAcOAcOAcOONPhthBnOBnO29ppm (f1)1.02.03.04.05.06.07.08.0 S6ONPhthOAcOAcOAcOONPhthBnOBnO29ppm (f1)50100150 S7ONHAcOAcOAcOAcOONHAcBnOBnO30ppm (f1)2.03.04.05.06.07.08.0 S8ONHAcOAcOAcOAcOONHAcBnOBnO30ppm (f1)50100150 S9RT: 0.00 - 14.600 1 2 3 4 5 6 7 8 9 10 11 12 13 14Tim e (m in)020406080100Relative Abundance0200000400000600000800000uAU14.2713.9311.247.960.840.658.494.076.244.953.381.02 2.137.998.086.73 7.224.53 8.35 9.00 9.362.97 3.482.061.561.21 11.250.43 12.404.67 10.93 13.425.61 6.59 13.76NL:8.33E5Spectrum Maximum nm=200.0-600.0  PDA mdw630NL:1.39E7Base Peak F: + p ESI Ful l  ms [ 160.00-2000.00]  MS mdw630Instrument Method: C:\Xcalibur\methods\General(TFA)\1090%B10_C=TFA_20ul_+pf 15min.methProcessing Method:Vial: C:21Injection Volume (µl):   10.00Sample Weight:    0.00m dw630 #414 RT: 7.97 AV: 1 NL: 1.14E7F: + p ESI Full m s  [ 160.00-2000.00]200 400 600 800 1000 1200 1400 1600 1800 2000m /z01000000200000030000004000000500000060000007000000800000090000001000000011000000Intensity755.001508.80799.87329.87 1553.33209.80 825.87 1884.671078.40 1645.27427.00 926.73ONHAcOAcOAcOAcOONHAcBnOBnO30S10ONHAcOAcOAcOAcOONHAcAcOAcO31ppm (f1)2.03.04.05.06.0 S11ONHAcOAcOAcOAcOONHAcAcOAcO31ppm (f1)050100150 S12RT: 0.00 - 14.600 1 2 3 4 5 6 7 8 9 10 11 12 13 14Tim e (m in)020406080100Relative Abundance0200000400000600000800000uAU14.2613.900.64 0.858.458.145.58 7.737.114.984.674.133.601.61 1.895.597.95 7.997.857.116.735.254.62 8.29 11.72 12.43 12.912.922.41 8.503.23 9.51 11.590.45 0.90 1.39 10.87 13.56NL:8.31E5Spectrum Maximum nm=200.0-600.0  PDA mdw636NL:8.87E6Base Peak F: + p ESI Ful l  ms [ 160.00-2000.00]  MS mdw636Instrument Method: C:\Xcalibur\methods\General(TFA)\1090%B00_C=TFA_20ul_+pf 15min.methProcessing Method:Vial: C:22Injection Volume (µl):   10.00Sample Weight:    0.00m dw636 #290 RT: 5.59 AV: 1 NL: 8.87E6F: + p ESI Full m s  [ 160.00-2000.00]200 400 600 800 1000 1200 1400 1600 1800 2000m /z010000002000000300000040000005000000600000070000008000000Intensity659.001316.47703.871358.47 1974.80329.87 1665.87720.60 845.87 1475.07614.87237.93ONHAcOAcOAcOAcOONHAcAcOAcO31S13ONHAcOAcOAcOAcOONHAcAcOAcO32OEtOppm (f1)0.01.02.03.04.05.06.07.0 S14ONHAcOAcOAcOAcOONHAcAcOAcO32OEtOppm (f1)050100150200 S15ONHAcOAcOAcOAcOONHAcAcOAcO32OEtOS16ONHAcOHOHOHOONHAcHOHO33OMeOppm (f1)2.03.04.05.06.07.0 S17ONHAcOHOHOHOONHAcHOHO33OMeOppm (f1)-100-50050 S18RT: 0.00 - 14.600 1 2 3 4 5 6 7 8 9 10 11 12 13 14Tim e (m in)020406080100Relative Abundance0200000400000600000800000uAU7.0814.2712.297.3811.763.67 11.369.388.04 9.908.756.846.274.770.77 1.49 1.847.107.02 14.107.39 12.049.26 11.378.43 13.769.30 12.1911.217.664.864.78 6.875.03 5.94 12.3510.923.621.50 3.231.600.34 3.750.51 13.6713.34NL:9.78E5Spectrum Maximum nm=200.0-600.0  PDA mdw643NL:1.61E6Base Peak F: + p ESI Ful l  ms [ 160.00-2000.00]  MS mdw643Instrument Method: C:\Xcalibur\methods\General(TFA)\0020%B00_C=TFA_20ul_+pf 15min.methProcessing Method:Vial: C:23Injection Volume (µl):   20.00Sample Weight:    0.00m dw643 #366 RT: 7.10 AV: 1 NL: 1.61E6F: + p ESI Full m s  [ 160.00-2000.00]200 400 600 800 1000 1200 1400 1600 1800 2000m /z02000004000006000008000001000000120000014000001600000Intensity507.001013.201519.131266.67 1350.67203.93 1772.471564.53552.00 1863.931074.07711.27305.13 391.20 1447.40973.87ONHAcOHOHOHOONHAcHOHO33OMeOS19ONHAcOTBSOTBSOTBSOONHAcTBSOTBSO36ppm (f1)0.01.02.03.04.05.06.0 S20ONHAcOTBSOTBSOTBSOONHAcTBSOTBSO36ppm (f1)050100150200 S21ONHAcOTBSOTBSOTBSOONHAcTBSOTBSO37SO Oppm (f1)0.01.02.03.04.05.06.07.0 S22ONHAcOTBSOTBSOTBSOONHAcTBSOTBSO37SO Oppm (f1)050100150200 S23ONHAcOHOHOHOONHAcHOHO38SO Oppm (f1)0.01.02.03.04.05.06.07.0 S24ONHAcOHOHOHOONHAcHOHO38SO Oppm (f1)050100150200 S25ONHAcOHOHOHOONHAcHOHO38SO OS26OOOBnONPhthO CCl3NHPh39ppm (f1)0.01.02.03.04.05.06.07.08.0 S27OOOBnONPhthO CCl3NHPh39ppm (f1)050100150200 S28ONPhthOOOBnOONPhthBnOBnO40Phppm (f1)0.01.02.03.04.05.06.07.08.0 S29ONPhthOOOBnOONPhthBnOBnO40Phppm (f1)050100150200 S30ONHAcOOOBnOONHAcBnOBnO41OHOPhppm (f1)0.01.02.03.04.05.06.07.0 S31ONHAcOOOBnOONHAcBnOBnO41OHOPhppm (f1)050100150200 S32ONHAcOOOBnOONHAcBnOBnO41OHOPhS33ONHAcOOOBnOONHAcBnOBnO42OOPh Oppm (f1)0.01.02.03.04.05.06.07.08.0 S34ONHAcOOOBnOONHAcBnOBnO42OOPh Oppm (f1)050100150200 S35RT: 0.00 - 14.600 1 2 3 4 5 6 7 8 9 10 11 12 13 14Tim e (m in)020406080100Relative Abundance050000010000001500000uAU10.7614.2713.3212.620.84 10.4410.045.86 7.994.11 7.355.434.643.391.56 1.9810.8110.9611.119.79 12.629.90 12.94 13.473.07 9.652.121.790.20 3.401.25 4.43 4.91 5.89 7.956.24 6.87 9.40NL:1.83E6Spectrum Maximum nm=200.0-600.0  PDA mdw810NL:1.45E7Base Peak F: + p ESI Ful l  ms [ 160.00-2000.00]  MS mdw810Instrument Method: C:\Xcalibur\methods\General(TFA)\1090%B10_C=TFA_20ul_+pf 15min.methProcessing Method:Vial: C:28Injection Volume (µl):   10.00Sample Weight:    0.00m dw810 #561 RT: 10.79 AV: 1 NL: 1.35E7F: + p ESI Full m s  [ 160.00-2000.00]200 400 600 800 1000 1200 1400 1600 1800 2000m /z020000004000000600000080000001000000012000000Intensity971.271942.00382.07 1457.201015.93 1253.07590.27314.87 1541.20 1782.20474.47 866.33ONHAcOOOBnOONHAcBnOBnO42OOPh OS36ONHAcOOOBnOONHAcBnOBnO43OClPhppm (f1)0.01.02.03.04.05.06.07.0 S37ONHAcOOOBnOONHAcBnOBnO43OClPhppm (f1)050100150200 S38RT: 0.00 - 14.600 1 2 3 4 5 6 7 8 9 10 11 12 13 14Tim e (m in)020406080100Relative Abundance0200000400000600000800000uAU14.269.8513.9213.620.738.317.937.343.73 5.715.384.093.191.709.8710.22 13.52 13.8510.509.017.37 12.8512.377.413.99 5.355.061.86 3.211.26 2.80 6.15 7.190.32NL: 8.15E5Spectrum Maximum nm=200.0-600.0  PDA mdw835spot1NL: 7.05E6Base Peak F: + p ESI Ful l  ms [ 160.00-2000.00]  MS mdw835spot1Instrument Method: C:\Xcalibur\methods\General(TFA)\1090%B10_C=TFA_20ul_+pf 15min.methProcessing Method:Vial: A:13Injection Volume (µl):   10.00m dw835spot1 #505 RT: 9.83 AV: 1 NL: 3.62E6F: + p ESI Full m s [ 160.00-2000.00]200 400 600 800 1000 1200 1400 1600 1800 2000m /z0102030405060708090100Relative Abundance857.071713.87947.20381.93 1804.87962.00269.67 1129.27 1968.471420.201215.27752.20476.13 696.27 1680.73ONHAcOOOBnOONHAcBnOBnO43OClPhS39ONHAcOHOHOHOONHAcHOHO44OOOppm (f1)0.01.02.03.04.05.06.07.0 S40ONHAcOHOHOHOONHAcHOHO44OOOppm (f1)050100150200 S41RT: 0.00 - 14.600 1 2 3 4 5 6 7 8 9 10 11 12 13 14Tim e (m in)020406080100Relative Abundance0100000020000003000000uAU8.460.8414.2512.108.637.81 9.426.926.145.763.55 4.844.200.68 2.291.538.496.13 8.838.38 12.88 13.946.594.57 5.18 7.134.39 12.759.893.560.12 3.10 10.952.570.57 2.03NL:3.97E6Spectrum Maximum nm=200.0-600.0  PDA mdw750NL:9.73E6Base Peak F: + p ESI Ful l  ms [ 160.00-2000.00]  MS mdw750Instrument Method: C:\Xcalibur\methods\General(TFA)\0050%B00_C=TFA_20ul_+pf 15min.methProcessing Method:Vial: C:26Injection Volume (µl):   20.00Sample Weight:    0.00m dw750 #439 RT: 8.47 AV: 1 NL: 8.02E6F: + p ESI Full m s  [ 160.00-2000.00]200 400 600 800 1000 1200 1400 1600 1800 2000m /z010000002000000300000040000005000000600000070000008000000Intensity613.071225.071837.871531.87658.00410.07204.07 1271.13921.73 1634.931429.53 1882.93344.20 1706.20765.27ONHAcOHOHOHOONHAcHOHO44OOOS42ONHAcOHOHOHOONHAcHOHO45ClOppm (f1)1.02.03.04.0 S43ONHAcOHOHOHOONHAcHOHO45ClOppm (f1)050100150200 S44ONHAcOHOHOHOONHAcHOHO45ClOS45ONHAcOHOHOHOONHAcHOHO47OPOPhPhOppm (f1)1.02.03.04.05.06.07.08.0 S46ONHAcOHOHOHOONHAcHOHO47OPOPhPhOppm (f1)050100150200 S47RT: 0.00 - 14.600 1 2 3 4 5 6 7 8 9 10 11 12 13 14Tim e (m in)020406080100Relative Abundance05000001000000uAU7.8914.288.52 12.1810.849.919.296.805.854.51 5.543.723.360.67 2.151.787.928.597.7813.8313.2812.194.023.67 4.382.56 5.210.39 5.811.86 6.391.28 8.687.65 10.908.93 9.87NL:1.40E6Spectrum Maximum nm=200.0-600.0  PDA mdw816NL:9.46E6Base Peak F: + p ESI Ful l  ms [ 160.00-2000.00]  MS mdw816Phone:Instrument Method: C:\Xcalibur\methods\General(TFA)\0050%B10_C=TFA_20ul_+pf 15min.methProcessing Method:Vial:  A:6m dw816 #411 RT: 7.92 AV: 1 NL: 9.46E6F: + p ESI Full m s [ 160.00-2000.00]200 400 600 800 1000 1200 1400 1600 1800 2000m /z0102030405060708090100Relative Abundance681.071360.931701.40726.00 1815.131407.73 1598.47478.00 1936.20198.80 1041.07 1282.20343.80 833.20 914.47ONHAcOHOHOHOONHAcHOHO47OPOPhPhOS48ONHAcOHOHOHOONHAcHOHO48SOppm (t1)0.01.02.03.04.05.06.07.0Et3N HOAcEt3N HOAcS49ONHAcOHOHOHOONHAcHOHO48SOppm (t1)050100150200 S50RT: 0.00 - 14.600 1 2 3 4 5 6 7 8 9 10 11 12 13 14Tim e (m in)020406080100Relative Abundance02000004000006000008000001000000uAU4.5214.2713.716.544.38 5.383.410.64 1.07 1.991.424.540.02 12.87 13.3711.8710.8710.575.47 9.376.29 6.79 8.047.754.36 8.553.990.36 3.011.29 2.42NL:1.11E6Spectrum Maximum nm=200.0-600.0  PDA mdw871_H2ONL:8.73E6Base Peak F: + p ESI Ful l  ms [ 160.00-2000.00]  MS mdw871_H2OSample Name:Study:Client:Laboratory:Company:m dw871_H2O #232 RT: 4.54 AV: 1 NL: 2.81E6F: + p ESI Full m s  [ 160.00-2000.00]200 400 600 800 1000 1200 1400 1600 1800 2000m /z05000001000000150000020000002500000Intensity573.001144.801716.40617.87 1189.93175.67 1439.60 1800.47725.20391.27 878.13 1296.80947.40447.80 1670.73 1943.871102.27ONHAcOHOHOHOONHAcHOHO48SOS51OAcOAcOAcO OOAcOAcONHAc NHAcHNOOOEtO49ppm (f1)1.02.03.04.05.0 S52OAcOAcOAcO OOAcOAcONHAc NHAcHNOOOEtO49ppm (f1)050100150200 S53ONHAcOAcOAcOAcOONHAcAcOAcOHN49OOOEtOS54

Synthesis and Biological Evaluation of a Chitobiose-Based Peptide N-Glycanase Inhibitor Library

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