e)-alkene and ethylene isosteres substantially alter the ... · 2 (84 ml), and triethylamine (8.4...

Supporting Information for

(E)-Alkene and Ethylene Isosteres Substantially Alter the

Hydrogen-Bonding Network in Class II MHC

Aq/Glycopeptide Complexes and Affect T-Cell Recognition

Ida E. Andersson,a Tsvetelina Batsalova,b Sabrina Haag,b Balik Dzhambazov,b Rikard

Holmdahl,b Jan Kihlberga,c,* and Anna Linusson,a,*

a Department of Chemistry, Umeå University, SE-901 87 Umeå, Sweden. b Medical Inflammation Research,

Department of Medical Biochemistry and Biophysics, Karolinska Institute, SE-171 77 Stockholm, Sweden. c

AstraZeneca R&D Mölndal, SE-431 83 Mölndal, Sweden.

Table of Contents

Synthetic experimental procedures........................................................................... S2-S18

Biological assays ...........................................................................................................S19

Molecular dynamics simulations ....................................................................................S20

NMR spectra.......................................................................................................... S21-S45

Aq binding curves for triplicates.....................................................................................S46

References .....................................................................................................................S47

RMSD for Aq/glycopeptide complexes vs simulation time .............................................S48

Backbone dihedral angle for 1, 31, 33, and 35 vs simulation time...................................S50

Backbone dihedral angle for 1, 32, 34, and 36 vs simulation time...................................S51

RMSD for Ile260-Phe263 sequences vs simulation time ....................................................S52

Phe dihedral angles vs simulation time...........................................................................S53

RMSD of the α1 helix of Aq vs simulation time .............................................................S54

RMSD of the β1 helix of Aq vs simulation time ............................................................S55

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Experimental Section

General Methods. Aldehyde 12 was prepared in three steps from (S)-methyl-3-hydroxy-propanoate; protection with TBDPSCl and imidazole,1 reduction of the ester to the primary alcohol followed by oxidation to aldehyde 12.2 Compounds 17,3 26,4 (5R)-Nα-(Fluoren-9-ylmethoxycarbonyl)-Nε-benzyloxycarbonyl-5-O-(2,3,4,6-tetra-O-acetyl-β-D-galactopyranosyl)-5-hydroxy-L-lysine3,5 and glycopeptide 333 were synthesized as described in the literature. n-Butyllithium was titrated with diphenyl acetic acid as indicator.6

All reactions were carried out in an inert atmosphere with dry solvents under anhydrous conditions, unless otherwise stated. CH2Cl2 and MeCN were distilled from calcium hydride, whereas THF was distilled from potassium. DMF was distilled under reduced pressure and then dried over 3 Å molecular sieves. MeOH was also dried over 3 Å molecular sieves. After workup, organic solutions were dried over Na2SO4 before being concentrated under reduced pressure. TLC analysis was performed on silica gel 60 F254 (Merck) with detection by UV light and staining with alkaline aqueous KMnO4 followed by heating. Flash column chromatography was performed on silica gel (Matrex, 60 Å, 35-70 µm, Grace Amicon).

Optical rotations were measured with a Perking-Elmer model 343 polarimeter at 20 °C. 1H and 13C NMR spectra of the isostere building blocks and their intermediates were recorded at 298 K on a Bruker DRX-400 spectrometer at 400 MHz and 100 MHz, respectively. Calibration was performed using the residual peak of the solvent as internal standard [CDCl3 (CHCl3 δH 7.26 ppm, CDCl3 δC 77.0 ppm) or CD3OD (CD2HOD δH 3.31 ppm, CD3OD δC 49.0 ppm)]. First-order chemical shifts and coupling constants were obtained from one-dimensional spectra; carbon and proton resonances were assigned from COSY, NOESY and HETCOR experiments. Signals that could not be assigned are not reported. The 1H NMR spectra of compounds 2, 3, 5, 6 and 7 all contained broad, minor peaks that are not reported. These peaks have previously been shown to be caused by the existence of rotamers about the amide bond in the Fmoc urethane.7 Spectra of glycopeptides 31, 32, 34, 35 and 36 were recorded at 298 K on a Bruker Avance spectrometer at 500 MHz in H2O/D2O (9:1) with H2O (δH 4.76) as internal standard. COSY, TOCSY, 1H-13C-HSQC, and ROESY experiments were used for assignment of signals and determination of chemical shifts. HRMS data were recorded with fast atom bombardment (FAB+) or electron impact (ES+) ionization.

Analytical reversed-phase HPLC was performed on a Beckman System Gold HPLC equipped with a Supelco Discovery® Bio Wide Pore C18 column (250 × 4.6 mm, 5 µm) using a flow-rate of 1.5 mL/min and detection at 214 nm. Preparative reversed-phase HPLC was performed on a Supelco Discovery® Bio Wide Pore C18 column (250 × 21.2 mm, 5 µm) using the same eluent as for the analytical HPLC, a flow-rate of 11 mL/min, and detection at 214 nm.

N-Trifluoroacetyl-(S)-isoleucinol (9). (S)-Isoleucinol (1.93 g, 16.5 mmol) was dissolved in

CH2Cl2 (84 mL), and triethylamine (8.4 mL, 60.6 mmol) was added to the stirred solution at room temperature. The solution was cooled to 0 °C, trifluoroacetic anhydride (3.4 mL, 24.1 mmol) was slowly added over 30 min, and after complete addition, the reaction was stirred for 6 h. The solution was concentrated under reduced pressure at low temperature and the

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crude product was dissolved in EtOAc and washed with NaHCO3 (aq., sat) followed by HCl (0.1 M, aq.) and brine. The organic layer was dried and concentrated under reduced pressure to afford alcohol 9 (3.51 g, 100%) as a slightly yellow solid, which was used without further purification. [α]D

20 –21.3 (c 1.0, CHCl3); 1H NMR (CDCl3): δ 6.76 (d, J = 7.3 Hz, 1H, NH), 3.88-3.80 (m, 1H, NCH), 3.75 (t, J = 4.1 Hz, 2H, CH2OH), 2.53 (br s, 1H, OH), 1.78-1.67 (m, 1H, CHCH3), 1.53-1.42 (m, 1H, CH2CH3), 1.21-1.09 (m, 1H, CH2CH3), 0.95 (d, J = 6.9 Hz, 3H, CHCH3), 0.91 (t, J = 7.4 Hz, 3H, CH2CH3); 13C NMR (CDCl3): δ 157.6 (q, JC-F = 37 Hz, CO), 115.9 (q, JC-F = 288 Hz, CF3), 61.9 (CH2OH), 56.0 (CH), 35.2 (CHCH3), 25.4 (CH2CH3), 15.3 (CHCH3), 11.0 (CH2CH3). HRMS (FAB+) calcd for C8H15F3NO2 (M+H)+ 214.1055, found 214.1056. N-((1S,2S)-1-Bromomethyl-2-methyl-butyl)-trifluoroacetamide (10).

Triphenylphosphine (3.95 g, 15.1 mmol) was added to alcohol 9 (3.02 g, 14.2 mmol) dissolved in acetonitrile (35 mL). The solution was cooled to 0 °C, and tetrabromomethane (5.00 g, 15.1 mmol) dissolved in acetonitrile (14 mL) was slowly added via a cannula. The solution was stirred for 50 h while allowed to attain room temperature, and was then concentrated under reduced pressure to approximately 10 mL. CH2Cl2 was added and the solution was then washed with H2O. The phases were separated and the aqueous phase was extracted with CH2Cl2. The combined organic layers were washed with brine, dried and concentrated. The residue was purified by flash chromatography (n-heptane/EtOAc 30:1→5:1) to afford bromide 10 (3.35 g, 85%) as a white solid. [α]D

20 –42.9 (c 1.0, CHCl3); 1H NMR (CDCl3): δ 6.38 (br s, 1H, NH), 4.01-3.94 (m, 1H, NCH), 3.60 (d, J = 3.9 Hz, 2H, CH2Br), 1.81-1.70 (m, 1H, CHCH3), 1.57-1.46 (m, 1H, CH2CH3), 1.26-1.11 (m, 1H, CH2CH3), 0.97 (d, J = 6.8 Hz, 3H, CHCH3), 0.92 (t, J = 7.4 Hz, 3H, CH2CH3); 13C NMR (CDCl3): δ 157.4 (q, JC-F = 37 Hz, CO), 115.8 (q, JC-F = 288 Hz, CF3), 53.9 (CH), 36.5 (CHCH3), 35.3 (CH2Br), 25.0 (CH2CH3), 15.0 (CHCH3), 10.8 (CH2CH3); HRMS (EI+) calcd for C8H14BrF3NO (M+H)+ 276.0211, found 276.0204.

[(3S,2S)-3-Methyl-2-(trifluoroacetamido)-pentyl]-triphenyl-phosphonium bromide (11). Bromide 10 (2.78 g, 10.1 mmol) and triphenylphosphine (10.6 g, 40.2 mmol) were refluxed in toluene (35 mL) for 22 h. The resulting slurry was allowed to cool to room temperature and was then concentrated under reduced pressure to approximately 5 mL. Et2O (40 mL) was added and the suspension was sonicated followed by siphoning off the solvent supernatant after the particles had settled. This procedure was repeated with 4×40 mL Et2O followed by drying in vacuo overnight to afford 11 (5.27 g, 97%) as a white solid. [α]D

20 –27.8 (c 1.0, CHCl3); 1H NMR (CDCl3): δ 9.80 (d, J = 9.1 Hz, 1H, NH), 7.92-7.85 (m, 6H, Ph), 7.82-7.77 (m, 3H, Ph), 7.71-7.65 (m, 6H, Ph), 5.69-5.58 (m, 1H, CH2P), 4.42-4.30 (m, 1H, NCH), 2.93-2.84 (m, 1H, CH2P), 2.04-1.93 (m, 1H, CHCH3), 1.49-1.37 (m, 1H, CH2CH3), 1.18-1.06 (m, 1H, CH2CH3), 0.95 (d, J = 6.7 Hz, 3H, CHCH3), 0.78 (t, J = 7.4 Hz, 3H, CH2CH3); 13C NMR (CDCl3): δ 157.2 (q, JC-F = 38 Hz, CO), 135.2 (d, JC-P = 3.0 Hz, Ph), 133.9 (d, JC-P = 10 Hz, Ph), 130.4 (d, JC-P = 13 Hz, Ph), 117.5 (d, JC-P = 86 Hz, Ph), 115.3 (q, JC-F = 288 Hz, CF3), 48.6 (d, JC-P = 5.3 Hz, NCH), 40.5 (d, JC-P = 12 Hz, CHCH3), 25.3 (CH2CH3), 24.0 (d, JC-P = 51 Hz, CH2P), 15.3 (CHCH3), 11.0 (CH2CH3); HRMS (FAB+) calcd for C26H28F3NOP (M+) 458.1861, found 458.1862.

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N-[(E)-(1S,1S’,4R)-1-sec-Butyl-5-(tert-butyl-diphenyl-silanyloxy)-4-methyl-pent-2-enyl]-trifluoroacetamide (13). Phosphonium salt 11 (317 mg, 0.588 mmol) was suspended in THF (5 mL) and sonicated for 30 min. The suspension was cooled to –78 °C and n-BuLi (1.55 M in hexanes, 770 µL, 5.88 mmol) was slowly added whereupon the solution turned bright yellow. The reaction was allowed to reach room temperature during 15 min. All of the phosphonium salt was dissolved and the bright orange solution was again cooled to –78 °C. After stirring for 30 min, aldehyde 12 (192 mg, 0.587 mmol) dissolved in THF (2 mL) was added dropwise via a cannula. The reaction was allowed to reach 0 °C over a period of 2.5 h, followed by addition of Et2O (10 mL) and 0.1 M HCl saturated with NH4Cl (10 mL). The organic layer was separated and the aqueous phase was extracted with Et2O. The combined organic layers were washed with brine, dried and concentrated under reduced pressure. The residue was purified by flash chromatography (n-heptane/EtOAc 1:0→20:1) to afford (E)-alkene 13 (259 mg, 87%) as a white solid. [α]D

20 –14.8 (c 1.0, CHCl3); 1H NMR (CDCl3): δ 7.67-7.62 (m, 4H, Ph), 7.46-7.35 (m, 6H, Ph), 6.12 (d, J = 8.4 Hz, 1H, NH), 5.64 (ddd, J = 15.7, 7.1, and 0.9 Hz, 1H, NCHCH=CH), 5.37 (ddd, J = 15.7, 7.1, and 0.9 Hz, 1H, NCHCH=CH), 4.42-4.35 (m, 1H, NCH), 3.56-3.48 (m, 2H, CH2OSi), 2.45-2.34 (m, 1H, CHCH2OSi), 1.68-1.57 (m, 1H, NCHCHCH3), 1.50-1.39 (m, 1H, CH2CH3), 1.16-1.05 (m, 1H, CH2CH3), 1.05 (s, 9H, C(CH3)3), 1.01 (d, J = 6.9 Hz, 3H, CH3CHCH2O), 0.91 (t, J = 7.4 Hz, 3H, CH2CH3), 0.88 (d, J = 6.8 Hz, 3H, NCHCHCH3); 13C NMR (CDCl3): δ 156.2 (q, JC-F = 37 Hz, CO), 137.1 (NCHCH=CH), 135.6, 133.7 (split), 129.6, 127.6, 125.5 (NCHCH=CH), 115.9 (q, JC-F = 288 Hz, CF3), 68.3 (CH2O), 56.1 (NCH), 39.2 (CHCH2O), 38.6 (NCHCHCH3), 26.8 (C(CH3)3), 25.3 (CH2CH3), 19.3 (C(CH3)3), 16.5 (CH3CHCH3O), 14.8 (NCHCHCH3), 11.5 (CH2CH3). HRMS (FAB+) calcd for C28H39F3NO2Si (M+H)+ 506.2702, found 506.2700. N-((1S,1S’,4R)-1-sec-Butyl-5-hydroxy-4-methyl-pent-2-enyl)-trifluoroacetamide (14).

Alkene 13 (151 mg, 0.300 mmol) was dissolved in THF (2 mL) and treated with tetrabutylammonium fluoride (1 M solution in THF, 1.2 mL, 1.2 mmol) for 3 h at room temperature. The solvent was evaporated under reduced pressure and the residue was purified by flash chromatography (n-heptane/EtOAc 4:1→3:1) to give alcohol 14 (75 mg, 94%) as a white amorphous solid. [α]D

20 –24.2 (c 1.0, CHCl3); 1H NMR (CDCl3): δ 6.36 (d, J = 7.0 Hz, 1H, NH), 5.54 (ddd, J = 15.7, 7.2, and 0.9 Hz, 1H, NCHCH=CH), 5.42 (ddd, J = 15.7, 6.9, and 0.8 Hz, 1H, NCHCH=CH), 4.39-4.32 (m, 1H, NCH), 3.54-3.47 (m, 1H, CH2OH), 3.46-3.39 (m, 1H, CH2OH), 2.43-2.32 (m, 1H, CHCH2OH), 1.70-1.58 (m, 2H, NCHCHCH3, OH), 1.51-1.40 (m, 1H, CH2CH3), 1.17-1.06 (m, 1H, CH2CH3), 1.00 (d, J = 6.8 Hz, 3H, CH3CHCH2OH), 0.92 (t, J = 7.4 Hz, 3H, CH2CH3), 0.90 (d, J = 6.8 Hz, 3H, NCHCHCH3); 13C NMR (CDCl3): δ 156.5 (q, JC-F = 37 Hz, CO), 136.4 (NCHCH=CH), 126.8 (NCHCH=CH), 115.9 (q, JC-F = 288 Hz, CF3), 67.1 (CH2O), 56.3 (NCH), 39.3 (CHCH2O), 38.4 (NCHCHCH3), 25.4 (CH2CH3), 16.1 (CH3CHCH3O), 14.9 (NCHCHCH3), 11.4 (CH2CH3). HRMS (FAB+) calcd for C12H21F3NO2 (M+H)+ 268.1524, found 268.1527.

(2R,5S,6S)-2,6-Dimethyl-5-(trifluoroacetylamino)-oct-3-enoic acid (15). A solution of CrO3 (114 mg, 1.14 mmol) in aqueous 4.4 M H2SO4 (1.1 mL) was added to alcohol 14 (102 mg, 0.382 mmol) dissolved in acetone (8 mL) at 0 °C. After 25 min, the ice bath was removed followed by stirring for a further 15 min. iPrOH (0.8 mL) was added and the pH was adjusted

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to 4 by addition of NaHCO3 (aq., sat). The aqueous phase was extracted with Et2O and the combined organic layers were dried and concentrated under reduced pressure. Purification by flash chromatography (column packed with n-heptane/EtOAc 4:1 + 1% TEA, product eluted with n-heptane/EtOAc 4:1 + 1% AcOH → 2:1+ 1% AcOH) afforded carboxylic acid 15 (80 mg, 75%) and ketone 16 (15 mg, 15%).

15: A white solid; [α]D20 –53.0 (c 1.0, CHCl3); 1H NMR (CDCl3): δ 6.25 (d, J = 7.6 Hz, 1H,

NH), 5.75 (ddd, J = 15.7, 7.5, and 1.1 Hz, 1H, NCHCHCH), 5.51 (ddd, J = 15.7, 7.2, and 0.9 Hz, 1H, NCHCHCH), 4.43-4.36 (m, 1H, NCH), 3.24-3.14 (m, 1H, CHCO2H), 1.70-1.59 (m, 1H, NCHCHCH3), 1.51-1.40 (m, 1H, CH2CH3), 1.30 (d, J = 7.1 Hz, 3H, CH3CHCO2H), 1.18-1.06 (m, 1H, CH2CH3), 0.92 (t, J = 7.4 Hz, 3H, CH2CH3), 0.89 (d, J = 6.7 Hz, 3H, NCHCHCH3); 13C NMR (CDCl3): δ 179.6 (CO2H), 156.4 (q, JC-F = 37 Hz, CO), 132.0 (NCHCH=CH), 128.2 (NCHCH=CH), 115.9 (q, JC-F = 288 Hz, CF3), 55.9 (NCH), 42.2 (CHCO2H), 38.5 (NCHCHCH3), 25.3 (CH2CH3), 16.8 (CH3CHCO2H), 14.9 (CH2CH3), 11.3 (NCHCHCH3). HRMS (FAB+) calcd for C12H19F3NO3 (M+H)+ 282.1317, found 282.1317.

16: A white solid; [α]D20 –54.4 (c 1.0, CHCl3); 1H NMR (CDCl3): δ 6.66 (dd, J = 15.8 and

6.3 Hz, 1H, NCHCH=CH), 6.44 (d, J = 7.3 Hz, 1H, NH), 6.18 (dd, J = 15.8 and 1.0 Hz, 1H, NCHCH=CH), 4.62-4.54 (m, 1H, NCH), 2.27 (s, 3H, COCH3), 1.81-1.70 (m, 1H, CHCH3), 1.55-1.43 (m, 1H, CH2CH3), 1.26-1.11 (m, 1H, CH2CH3), 0.98-0.92 (m, 6H, CHCH3 and CH2CH3); 13C NMR (CDCl3): δ 197.3 (COCH3), 156.8 (q, JC-F = 37 Hz, COCF3), 141.9 (NCHCH=CH), 131.4 (NCHCH=CH), 115.8 (q, JC-F = 288 Hz, CF3), 55.2 (NCH), 38.5 (CHCH3), 28.0 (COCH3), 25.3 (CH2CH3), 15.2 and 11.3 (CHCH3 and CH2CH3). HRMS (FAB+) calcd for C11H17F3NO2 (M+H)+ 252.1211, found 252.1216.

(2R,5S,6S)-5-(9H-Fluoren-9-ylmethoxycarbonylamino)-2,6-dimethyl-oct-3-enoic acid (2). Trifluoroacetamide 15 (69 mg, 0.244 mmol) was dissolved in MeOH (4.5 mL) and K2CO3 (10% aq., 1.8 mL) was added followed by stirring for 3 days at room temperature. A second addition of K2CO3 (10% aq., 1.8 mL) was added followed by stirring for 24 h. The solvent was removed under reduced pressure and the residue was dissolved in Na2CO3 (10% aq., 3.5 mL) and MeCN (3.5 mL) followed by addition of N-(9-fluorenylmethoxycarbonyloxy)succinimide (87 mg, 0.258 mmol). After stirring for 7 h, the MeCN was evaporated under reduced pressure. CHCl3 was added and the aqueous phase was acidified to pH 2 by addition of HCl (10% aq.) at 0 °C. The organic layer was separated and the aqueous phase was extracted with CHCl3. The combined organic layers were washed with brine, dried and concentrated under reduced pressure. Purification by flash chromatography (n-heptane/EtOAc 6:1 + 0.5% AcOH) gave the Fmoc-protected building block 2 (88 mg, 89%) as a white solid. [α]D

20 –11.7 (c 1.0, CHCl3); 1H NMR (CD3OD): δ 7.78 (d, J = 7.5 Hz, 2H, Fmoc-arom), 7.65 (d, J = 7.2 Hz, 2H, Fmoc-arom), 7.41-7.35 (m, 2H, Fmoc-arom), 7.33-7.27 (m, 2H, Fmoc-arom), 7.16 (d, J = 8.8 Hz, 1H, NH), 5.67 (dd, J = 15.6 and 7.6 Hz, 1H, NCHCH=CH), 5.51 (dd, J = 15.6 and 7.0 Hz, 1H, NCHCH=CH), 4.40-4.30 (m, 2H, Fmoc-CH2), 4.20 (t, J = 6.6 Hz, 1H, Fmoc-CH), 3.97-3.88 (m, 1H, NCH), 3.15-3.06 (m, 1H, CHCO2H), 1.55-1.42 (m, 2H, NCHCHCH3 and CH2CH3), 1.23 (d, J = 6.9 Hz, 3H, CH3CHCO2H), 1.19-1.04 (m, 1H, CH2CH3), 0.90 (t, J = 7.1 Hz, 3H, CH2CH3), 0.85 (d, J = 6.5 Hz, 3H, NCHCHCH3); 13C NMR (CD3OD): δ 178.4 (CO2H), 158.3 (NCO, split), 145.4 (split), 142.6, 132.2 (CH2CH=CH), 131.5 (NCHCH=CH), 128.7, 128.1 (split), 126.2, 120.9,

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67.5 (Fmoc-CH2), 58.7 (NCH, split), 48.6 (Fmoc-CH), 43.8 (CHCO2H), 40.4 (NCHCHCH3), 26.6 (CH2CH3), 17.7 (CH3CHCO2H), 15.7 (NCHCHCH3), 11.7 (CH2CH3); HRMS (FAB+) calcd for C25H30NO4 (M+H)+ 408.2175, found 408.2175.

(2R,5S,6S)-5-(9H-Fluoren-9-ylmethoxycarbonylamino)-2,6-dimethyl-octanoic acid (3). Pd/C (10%, 5 mg) was added to alkene 2 (56 mg, 0.138 mmol) dissolved in MeOH (2 mL). After six vacuum/H2 cycles, the mixture was stirred under one atmosphere pressure of H2 for 1 h 40 min at room temperature, then filtered through a pad of Celite and concentrated under reduced pressure. Purification by flash chromatography (n-heptane/EtOAc 5:1 + 0.5% AcOH) gave the saturated analogue 3 (43 mg, 77%) as a white solid. [α]D

20 –2.6 (c 0.5, CHCl3); 1H NMR (CDCl3): δ 7.76 (d, J = 7.6 Hz, 2H, Fmoc-arom), 7.59 (d, J = 7.3 Hz, 2H, Fmoc-arom), 7.42-7.36 (m, 2H, Fmoc-arom), 7.34-7.28 (m, 2H, Fmoc-arom), 4.57-4.51 (m, 1H, NH), 4.44 (d, J = 6.7 Hz, 2H, Fmoc-CH2), 4.22 (t, J = 6.6 Hz, 1H, Fmoc-CH), 3.58-3.48 (m, 1H, NCH), 2.54-2.44 (m, 1H, CHCO2H), 1.72-1.61 (m, 1H, CH2CHCO2H), 1.61-1.37 (m, 3H, CH2CHCO2H, NCHCH2 and NCHCHCH3), 1.35-1.23 (m, 2H, NCHCH2 and CH2CH3), 1.18 (d, J = 7.0 Hz, 3H, CH3CHCO2H), 1.15-1.00 (m, 1H, CH2CH3), 0.90 (t, J = 7.5 Hz, 3H, CH2CH3), 0.86 (d, J = 6.7 Hz, 3H, NCHCHCH3); 13C NMR (CDCl3): δ 180.6 (CO2H), 156.5 (NCO), 144.0, 141.3, 127.6, 127.0, 125.0, 119.9, 66.3 (Fmoc-CH2), 55.3 (NCH), 47.4 (Fmoc-CH), 38.9 (split, CHCO2H), 30.1 (CH2CHCO2H), 28.9 (NCHCH2), 25.0 (CH2CH3), 16.7 (CH3CHCO2H), 15.2 (NCHCHCH3), 11.7 (CH2CH3); HRMS (FAB+) calcd for C25H32NO4 (M+H)+ 410.2331, found 410.2332. N-((1S)-5-Hydroxy-1-methyl-pentyl)-trifluoroacetamide (19). Pd/C (10%, 51 mg) was

added to the mixture of alkenes 17 (747 mg, 1.66 mmol, E/Z 1:2) dissolved in MeOH (15 mL). After six vacuum/H2 cycles, the mixture was stirred under one atmosphere pressure of H2 for 3 h at room temperature, and was then filtered through a pad of Celite and concentrated under reduced pressure. The crude saturated analogue 18 was dissolved in THF (5 mL) and treated with tetrabutylammonium fluoride (1 M solution in THF, 6.65 mL, 6.65 mmol) for 2 h at room temperature. The solvent was evaporated under reduced pressure and the residue was purified by flash chromatography (n-heptane/EtOAc 3:1→2:1) to give alcohol 19 (253 mg, 71%) as a white solid. [α]D

20 –11.1 (c 0.5, CHCl3); 1H NMR (CDCl3): δ 6.46 (br s, 1H, NH), 4.07-3.95 (m, 1H, CH), 3.66-3.59 (m, 2H, CH2OH), 1.93-1.78 (m, 1H, OH), 1.64-1.48 (m, 4H, CHCH2 and CH2CH2OH), 1.45-1.35 (m, 2H, CHCH2CH2), 1.21 (d, J = 6.7 Hz, 3H, CH3); 13C NMR (CDCl3): δ 156.7 (q, JC-F = 37 Hz, CO), 115.9 (q, JC-F = 288 Hz, CF3), 62.4 (CH2OH), 46.5 (CH), 35.9 (CHCH2), 32.1 (CH2CH2OH), 22.1 (CHCH2CH2), 20.3 (CH3); HRMS (FAB+) calcd for C8H15F3NO2 (M+H)+ 214.1055, found 214.1053.

(5S)-5-(Trifluoro-acetylamino)-hexanoic acid (20). A solution of CrO3 (156 mg, 1.56 mmol) in aqueous 4.4 M H2SO4 (1.6 mL) was added to alcohol 19 (111 mg, 0.522 mmol) dissolved in acetone (10 mL) at 0 °C. After 5 min, the icebath was removed followed by stirring for a further 40 min. iPrOH (0.8 mL) was added and the pH was adjusted to 3 by addition of NaHCO3 (aq., sat). The aqueous phase was extracted with Et2O, and the combined organic layers were dried and concentrated under reduced pressure. Purification by flash chromatography (n-heptane/EtOAc 2:1 + 1% AcOH) afforded carboxylic acid 20 (104 mg, 88%) as a white solid. [α]D

20 –13.0 (c 1.0, CHCl3); 1H NMR (CDCl3): δ 6.30 (br s, 1H, NH), 4.09-3.97 (m, 1H, CH), 2.43-2.38 (m, 2H, CH2CO2H), 1.72-1.62 (m, 2H, CHCH2CH2), 1.62-

S6

1.54 (m, 2H, CHCH2), 1.24 (d, J = 6.7 Hz, 3H, CH3); 13C NMR (CDCl3): δ 178.9 (CO2H), 156.7 (q, JC-F = 37 Hz, NCO), 115.8 (q, JC-F = 288 Hz, CF3), 46.3 (CH), 35.4 (CHCH2), 33.2 (CH2CO2H), 20.7 (CHCH2CH2), 20.2 (CH3); HRMS (FAB+) calcd for C8H13F3NO3 (M+H)+ 228.0848, found 228.0853.

(5S)-5-(9H-Fluoren-9-ylmethoxycarbonylamino)-hexanoic acid (5). Trifluoroacetamide 20 (84 mg, 0.357 mmol) dissolved in MeOH (6.5 mL) was treated with K2CO3 (10% aq., 2.6 mL) for 46 h at room temperature. The solvent was removed under reduced pressure and the residue was dissolved in 10% aqueous Na2CO3/MeCN (1:1, 10 mL) followed by addition of N-(9-fluorenylmethoxycarbonyloxy)succinimide (126 mg, 0.374 mmol). After stirring for 8 h, the MeCN was evaporated under reduced pressure, CHCl3 was added and the aqueous phase was acidified to pH 2 by addition of HCl (10% aq.) at 0 °C. The organic layer was separated and the aqueous phase was extracted with CHCl3. The combined organic layers were washed with brine, dried and concentrated under reduced pressure. Purification by flash chromatography (n-heptane/EtOAc 6:1 + 0.5% AcOH → 6:1 + 0.5% AcOH) afforded the Fmoc-protected building block 5 (116 mg, 92%) as a white solid. [α]D

20 –7.9 (c 0.5, CHCl3); 1H NMR (CDCl3): δ 7.78 (d, J = 7.4 Hz, 2H, Fmoc-arom), 7.64 (d, J = 7.4 Hz, 2H, Fmoc-arom), 7.41-7.35 (m, 2H, Fmoc-arom), 7.33-7.27 (m, 2H, Fmoc-arom), 6.9 (d, J = 7.8 Hz, 1H, NH), 4.42-4.35 (m, 1H, Fmoc-CH2), 4.35-4.27 (m, 1H, Fmoc-CH2), 4.23-4.16 (m, 1H, Fmoc-CH), 3.67-3.55 (m, 1H, NCH), 2.32-2.25 (m, 2H, CH2CO2H), 1.69-1.52 (m, 2H, CH2CH2CO2H), 1.51-1.41 (m, 2H, NCHCH2), 1.11 (d, J = 6.5 Hz, 3H, CH3); 13C NMR (CDCl3): δ 177.5 (CO2H), 158.4 (NCO), 145.4 (split), 142.6, 128.7, 128.1, 126.2 (split), 120.9, 67.4 (Fmoc-CH2), 48.6 (Fmoc-CH), 47.9 (NCH), 37.1 (NCHCH2), 34.7 (CH2CO2H), 22.7 (NCHCH2CH2), 21.4 (CH3); HRMS (FAB+) calcd for C21H24NO4 (M+H)+ 354.1705, found 354.1705.

[5-((4R,5S)-4-Methyl-2-oxo-5-phenyl-oxazolidin-3-yl)-5-oxo-pent-2-enyl]-carbamic acid tert-butyl ester (27). Triethylamine (310 µL, 2.24 mmol) was added to a solution of carboxylic acid 26 (443 mg, 2.06 mmol) in THF (14 mL) cooled to -78 °C. After stirring for 5 min, pivaloyl chloride (270 µL, 2.19 mmol) was added dropwise followed by stirring for 30 min at -78 °C and then at 0 °C for 60 min. The solution was again cooled to -78 °C and a mixture of (4R,5S)-4-Methyl-5-phenyl-2-oxazolidinone (391 mg, 2.21 mmol) and n-BuLi (1.55 M in hexanes, 1.45 mL, 2.24 mmol) in THF (8 mL) cooled to -78 °C was transferred to this solution via a cannula. The resulting mixture was stirred at -78 °C for 40 min, and then at room temperature for 2 h 40 min. NH4Cl (aq., sat., 4 mL) and brine (5 mL) were added, the phases were separated and the aqueous layer was extracted with CH2Cl2. The combined organic layers were dried over Na2SO4, filtered and concentrated. Purification by flash chromatography (n-heptane/EtOAc 3:1) gave 27 (633 mg, 82%) as a white amorphous solid.

[α]D20 +24.0 (c 0.5, CHCl3); 1H NMR (CDCl3): δ 7.45-7.35 (m, 3H, Ph), 7.32-7.28 (m, 2H,

Ph), 5.84-5.75 (m, 1H, CHCH2CO), 5.68 (d, J = 7.3 Hz, 1H, CHPh), 5.71-5.63 (m, 1H, NCH2CH), 4.79-4.71 (m, 1H, NCH), 4.59 (br s, 1H, NH), 3.80-3.70 (m, 4H, NCH2 and CH2CO), 1.45 (s, 9H, C(CH3)3), 0.90 (d, J = 6.6 Hz, 3H, NCHCH3); 13C NMR (CDCl3): δ 170.9 (COCH2), 155.7 (CO2tBu), 152.9 (CO2CH), 133.2 (CHCH2CO), 131.3, 128.8, 128.7, 125.6, 123.3 (NCH2CH), 79.4 (C(CH3)3), 79.1 (CO2CH), 54.8 (NCH), 42.2 (NCH2), 38.8

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(CH2CO), 28.4 (C(CH3)3), 14.5 (NCHCH3). HRMS (FAB+) calcd for C20H27N2O5 (M+H)+ 375.1920, found 375.1920.

(4R,5S)-3-[5-(Benzhydrylidene-amino)-pent-3-enoyl]-4-methyl-5-phenyl-oxazolidin-2-one (28). Compound 27 (622 mg, 1.66 mmol) was dissolved in CH2Cl2 (24 mL) and treated with TFA (6 mL) for 15 min at room temperature. The solution was concentrated under reduced pressure, and then redissolved in CHCl3 and concentrated. The latter procedure was repeated twice to remove residual TFA. The residue was dissolved in CH2Cl2 (10 mL) and benzophenone imine (305 µL, 1.82 mmol) was added followed by stirring for 20 h at room temperature. The solution was concentrated under reduced pressure and the residue was purified by flash chromatography (n-heptane/EtOAc 10:1 + 1% TEA → 5:1 + 1% TEA) to afford 28 (526 mg, 72%) as a slightly yellow amorphous solid. [α]D

20 +15.0 (c 1.0, CHCl3); 1H NMR (CDCl3): δ 7.65-7.60 (m, 2H, Ph), 7.49-7.27 (m, 11H, Ph), 7.21-7.16 (m, 2H, Ph), 5.90 (dt, J = 15.5 and 5.4 Hz, 1H, NCH2CH), 5.82-5.73 (m, 1H, CHCH2CO), 5.67 (d, J = 7.3 Hz, 1H, CHPh), 4.79-4.71 (m, 1H, NCH), 4.04 (d, J = 5.4 Hz, 2H, NCH2), 3.75 (d, J = 6.7 Hz, 2H, CH2CO), 0.90 (d, J = 6.6 Hz, 3H, CH3); 13C NMR (CDCl3): δ 171.2 (COCH2), 169.0 (N=C), 152.9 (CO2CH), 139.7, 136.6, 133.3, 133.0 (NCH2CH), 130.0, 128.8, 128.7 (2C), 128.5, 128.4, 128.0, 127.7, 125.6, 122.3 (CHCH2CO), 79.0 (CO2CH), 55.5 (NCH2), 54.8 (NCH), 39.1 (CH2CO), 14.5 (CH3). HRMS (FAB+) calcd for C28H27N2O3 (M+H)+ 439.2022, found 439.2017.

(4R,5S)-3-[5-(Benzhydrylidene-amino)-2-benzyl-pent-3-enoyl]-4-methyl-5-phenyl-oxazolidin-2-one (29). n-BuLi (1.55 M in hexanes, 760 µL, 1.18 mmol) was added dropwise to a solution of diisopropylamine (170 µL, 1.21 mmol) in THF cooled to 0 °C. After stirring for 30 min, the solution was cooled to -78 °C, and 28 (503 mg, 1.15 mmol) dissolved in THF (5.5 mL) was added dropwise via a cannula whereupon the solution quickly turned black. After stirring for 30 min, benzyl bromide (682 µL, 5.74 mmol) was slowly added over 20 min. The solution was stirred at -78 °C for 50 min and was then allowed to reach 0 °C during 45 min. After stirring for 70 min at 0 °C, the solution was cooled to -40 °C and NH4Cl (aq., sat., 4 mL) was added. After warming to room temperature, brine (2 mL) was added, the phases were separated and the organic layer was washed with brine. The combined aqueous layers were extracted with CH2Cl2, and the combined organic layers were then dried and concentrated under reduced pressure. Purification by flash chromatography (n-heptane/EtOAc 15:1 + 1% TEA → 5:1 + 1% TEA) afforded a mixture of diastereomers 29 (dr 93:7, total yield of diastereomers: 426 mg, 70%). 1H NMR (CDCl3): δ 7.64-7.60 (m, 2H, Ph), 7.47-7.31 (m, 9H, Ph), 7.26-7.21 (m, 6H, Ph), 7.19-7.11 (m, 3H, Ph), 5.86 (dt, J = 15.5 and 5.3 Hz, 1H, NCH2CH), 5.72 (dd, J = 15.5 and 8.5 Hz, 1H, CHCHCO), 5.59 (d, J = 7.3 Hz, 1H, CHPh), 4.98-4.90 (m, 1H, CHCO), 4.73-4.65 (m, 1H, CH3CH), 4.02-3.96 (m, 2H, NCH2), 3.18 (dd, J = 13.5 and 8.2 Hz, 1H, PhCH2), 2.88 (dd, J = 13.5 and 7.0 Hz, 1H, PhCH2), 0.66 (d, J = 6.6 Hz, 3H, CH3); 13C NMR (CDCl3): δ 173.6 (COCH), 169.1 (N=C), 152.5 (CO2CH), 139.7, 138.6, 136.5, 132.4, 133.0 (NCHCH), 129.4, 128.7, 128.6, 128.5, 128.2 (2C), 128.0, 127.8, 127.7, 126.3 (CHCH2CO), 125.6, 77.3 (CO2CH), 55.3 (NCH2), 54.8 (NCH), 47.8 (CHCO), 14.3 (CH3). HRMS (FAB+) calcd for C35H33N2O3 (M+H)+ 529.2491, found 529.2499.

(2R)-2-Benzyl-5-(9H-fluoren-9-ylmethoxycarbonylamino)-pent-3-enoic acid (6). H2O2 (30 wt. % in H2O, 0.4 mL) was added to a solution of 29 (369 mg, 0.698 mmol) in THF (7

S8

mL) and H2O (3 mL) cooled to 0 °C. LiOH (67 mg, 2.97 mmol) dissolved in H2O (2.5 mL) was added dropwise followed by stirring for 40 min at 0 °C. Na2SO3 (1 M aq., 3.6 mL) was then added followed by stirring for 10 min. Following this, CH2Cl2 was added and the aqueous layer was acidified to pH 3 by addition of 1 M HCl (aq.). The phases were separated and the aqueous layer was extracted with EtOAc. The combined organic layers were washed with brine, dried and concentrated under reduced pressure. This afforded the crude carboxylic acid 30, which was dissolved in THF (13 mL) and treated with citric acid (0.5 M aq., 3.4 mL) for 1 h 50 min at room temperature. The solution was cooled to 0 °C, and NaHCO3 (10% aq., 10 mL) was added. The THF was removed under reduced pressure and acetone (10 mL) was added followed by addition of N-(9-fluorenylmethoxycarbonyloxy)succinimide (248 mg, 0.735 mmol). After stirring for 3 h, the acetone was removed under reduced pressure and CHCl3 was added. The aqueous phase was acidified to pH 2 and the phases were separated. The aqueous layer was extracted with CHCl3, and the combined organic layers were then washed with brine, filtered and concentrated under reduced pressure. Purification by flash chromatography (n-heptane/EtOAc 3:1 + 1% AcOH) followed by preparative chiral HPLC purification using a Chiralpak AD-H column and heptane/isopropylalcohol/formic acid 80:20:0.1 as eluent (detection at 254 nm) afforded the Fmoc-protected building block 6 (196 mg, 66%, > 99% ee) as a colorless oil. [α]D

20 –33.5 (c 1.0, CHCl3); 1H NMR (CD3OD): δ 7.81 (m, 2H, Fmoc-arom), 7.64 (d, J = 7.2 Hz, 2H, Fmoc-arom), 7.41-7.35 (m, 2H, Fmoc-arom), 7.33-7.28 (m, 2H, Fmoc-arom), 7.25-7.10 (m, 6H, Ph and NH), 5.67 (dd, J = 15.6 and 8.4 Hz, 1H, CH2CH=CH), 5.50 (dt, J = 15.6 and 5.4 Hz, 1H, CH2CH=CH), 4.36-4.29 (m, 2H, Fmoc-CH2), 4.22-4.15 (m, 1H, Fmoc-CH), 3.69-3.62 (m, 2H, NCH2), 3.31-3.22 (m, 1H, CHCO2H), 3.03 (dd, J = 13.6 and 7.8 Hz, 1H, PhCH2), 2.78 (dd, J = 13.6 and 7.1 Hz, 1H, PhCH2); 13C NMR (CD3OD): δ 177.2 (CO2H), 158.7 (NCO), 145.3, 142.6, 140.2, 130.7 (CH2CH=CH), 130.3 (CH2CH=CH), 130.2, 129.3, 128.8, 128.2, 127.3, 126.2, 120.9, 67.8 (Fmoc-CH2), 52.2 (CH2CO2H), 48.5 (Fmoc-CH), 43.1 (NCH2), 39.7 (PhCH2); HRMS (FAB+) calcd for C27H26NO4 (M+H)+ 428.1862, found 428.1859.

(2R)-2-Benzyl-5-(9H-fluoren-9-ylmethoxycarbonylamino)-pentanoic acid (7). Pd/C (10%, 6 mg) was added to alkene 6 (62 mg, 0.145 mmol) dissolved in MeOH (3 mL). After six vacuum/H2 cycles, the mixture was stirred under an atmospheric pressure of H2 for 50 min at room temperature, then filtered through a pad of Celite and concentrated under reduced pressure. Purification by flash chromatography (n-heptane/EtOAc 4:1 + 0.5% AcOH) gave the saturated analogue 7 (53 mg, 84%) as a colorless oil. [α]D

20 +3.0 (c 1.0, CHCl3); 1H NMR (CD3OD): δ 7.78 (d, J = 7.5 Hz, 2H, Fmoc-arom), 7.62 (d, J = 7.5 Hz, 2H, Fmoc-arom), 7.40-7.34 (m, 2H, Fmoc-arom), 7.32-7.27 (m, 2H, Fmoc-arom), 7.25-7.10 (m, 5H, Ph), 7.04 (t, J = 5.4 Hz, 1H, NH), 4.31 (d, J = 6.9 Hz, 2H, Fmoc-CH2), 4.16 (t, J = 6.9 Hz, 1H, Fmoc-CH), 3.13-3.04 (m, 2H, NCH2), 2.90 (dd, J = 13.5 and 8.4 Hz, 1H, PhCH2), 2.73 (dd, J = 13.5 and 6.4 Hz, 1H, PhCH2), 2.68-2.59 (m, 1H, CHCO2H), 1.68-1.40 (m, 4H, NCHCH2 and NCHCH2CH2); 13C NMR (CD3OD): δ 179.3 (CO2H), 158.9 (NCO), 145.3 (split), 142.6, 140.8 (Ph), 130.0 (Ph), 129.3 (Ph), 128.7, 128.1, 127.3 (Ph), 126.2, 120.9, 67.6 (Fmoc-CH2), 48.7 (CHCO2H), 48.5 (Fmoc-CH), 41.5 (NCH2), 39.5 (PhCH2), 30.3 (NCH2CH2CH2), 28.7 (NCH2CH2); HRMS (FAB+) calcd for C27H28NO4 (M+H)+ 430.2018, found 430.2029.

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General procedure for solid-phase glycopeptide synthesis. Glycopeptides 31, 32, and 34-36 were synthesized in mechanically agitated reactors on Tentagel-S-PHB-Thr(tBu)-Fmoc resins using standard solid-phase methodology as described elsewhere with minor modifications.8 All couplings were performed in DMF. Nα-Fmoc amino acids with standard side-chain protecting groups (4 equiv) were activated with 1-hydroxybenzotriazole (HOBt, 6 equiv) and 1,3-diisopropylcarbodiimide (DIC, 3.9 equiv), and the couplings were monitored using bromophenol blue9 as indicator. (5R)-Nα-(Fluoren-9-ylmethoxycarbonyl)-Nε-benzyloxycarbonyl-5-O-(2,3,4,6-tetra-O-acetyl-β-D-galactopyranosyl)-5-hydroxy-L-lysine3,5 (1.5 equiv) and the isostere building blocks 2, 3, 5, 6 and 7 (1.5 equiv) were activated with O-(7-azabenzotriazol-1-yl)-1,1,3,3-tetramethyluronium hexafluorophosphate (HATU, 1.5 equiv) and 2,4,6-collidine (3.0 equiv) and coupled for 24 h. Fmoc deprotection after each coupling cycle was accomplished by treatment with 20% piperidine in DMF for 10 min. The glycopeptides were cleaved from the resin with trifluoroacetic acid/H2O/thioanisole/ethanedithiol (35:2:2:1) for 3 h at 40 °C with workup performed essentially as described elsewhere.8 Purification by reversed-phase HPLC was followed by deacetylation with NaOMe in MeOH (20 mM, 1 mL/mg peptide) for 2-3 h at room temperature (monitored by analytical reversed-phase HPLC). Neutralization was achieved by addition of AcOH and concentration under reduced pressure, and the residue was purified using reversed-phase HPLC and lyophilization.

Glycyl-L-isoleucylψ[(E)-CH=CH]-L-alanyl-glycyl-L-phenylalanyl-5-O-(β-D-galactopyranosyl)-5-hydroxy-L-lysylglycyl-L-glutam-1-yl-L-glutaminylglycyl-L-prolyl-L-lysylglycyl-L-glutam-1-yl-L-threonine (31). Synthesis was performed with building block 2 on the solid phase resin (50 µmol) according to the general procedure described above. This afforded the trifluoroacetate salt of 31 (20.4 mg, 20% yield based on the amount of resin used) as a white amorphous solid after lyophilization. MS (MALDI-TOF) calcd 1636.81 [M+H]+, found 1636.85. 1H NMR data are given in Table S1.

Glycyl-L-isoleucylψ[CH2CH2]-L-alanyl-glycyl-L-phenylalanyl-5-O-(β-D-galactopyranosyl)-5-hydroxy-L-lysylglycyl-L-glutam-1-yl-L-glutaminylglycyl-L-prolyl-L-lysylglycyl-L-glutam-1-yl-L-threonine (32). Synthesis was performed with building block 3 on the solid phase resin (50 µmol) according to the general procedure described above. This afforded the trifluoroacetate salt of 32 (15.1 mg, 15% yield based on the amount of resin used) as a white amorphous solid. MS (MALDI-TOF) calcd 1638.82 [M+H]+, found 1638.84. 1H NMR data are given in Table S2.

Glycyl-L-isoleucyl-L-alanylψ[CH2CH2]glycyl-L-phenylalanyl-5-O-(β-D-galactopyranosyl)-5-hydroxy-L-lysylglycyl-L-glutam-1-yl-L-glutaminylglycyl-L-prolyl-L-lysylglycyl-L-glutam-1-yl-L-threonine (34). Synthesis was performed with building block 5 on the solid phase resin (50 µmol) according to the general procedure described above. This afforded the trifluoroacetate salt of 34 (16.6 mg, 16% yield based on the amount of resin used) as a white amorphous solid after lyophilization. MS (MALDI-TOF) calcd 1638.82 for [M+H]+, found 1638.82. 1H NMR data are given in Table S3.

Glycyl-L-isoleucyl-L-alanyl-glycylψ[(E)-CH=CH]-L-phenylalanyl-5-O-(β-D-galactopyranosyl)-5-hydroxy-L-lysylglycyl-L-glutam-1-yl-L-glutaminylglycyl-L-prolyl-L-lysylglycyl-L-glutam-1-yl-L-threonine (35). Synthesis was performed with building block 6

S10

on the solid phase resin (50 µmol) according to the general procedure described above. This afforded the trifluoroacetate salt of 35 (13.7 mg, 13.7% yield based on the amount of resin used) as a white amorphous solid after lyophilization. MS (MALDI-TOF) calcd 1636.81 [M+H]+, found 1636.83. 1H NMR data are given in Table S4.

Glycyl-L-isoleucyl-L-alanyl-glycylψ[CH2CH2]-L-phenylalanyl-5-O-(β-D-galactopyranosyl)-5-hydroxy-L-lysylglycyl-L-glutam-1-yl-L-glutaminylglycyl-L-prolyl-L-lysylglycyl-L-glutam-1-yl-L-threonine (36). Synthesis was performed with building block 7 attached to the solid phase resin (50 µmol) according to the general procedure described above. This afforded the trifluoroacetate salt of 36 (16.5 mg, 16.5% yield based on the amount of resin used) as a white amorphous solid after lyophilization. MS (MALDI-TOF) calcd 1638.82 [M+H]+, found 1638.88. 1H NMR data are given in Table S5.

Table S1. 1H NMR chemical shifts for CII259-273 with Ile260ψ[(E)-CH=CH]Ala261 (31).a

Residue NH Hα Hβ Hγ Others

Gly259 3.78b Ile260ψ[(E)-CH=CH] 8.28 4.23 1.55 1.39, 1.06,

0.81 (CH3) 0.83 (Hδ), 5.59b (CH=CH)

Ala261 3.16 1.17 Gly262 8.01 3.83b Phe263 8.01 4.62 3.07, 3.01 7.22 (Hδ), 7.33 (Hε), 7.30 (Hζ) Hyl264 8.41 4.26 2.00, 1.74 1.58b 4.00 (Hδ), 3.16 and 2.97 (Hε), 7.61 (εNH2), c Gly265 8.00 3.89b Glu266 8.19 4.34 2.08, 1.91 2.43b Gln267 8.48 4.36 2.11, 1.96 2.35b 7.48 and 6.82 (δNH2) Gly268 8.27 4.12, 3.98 Pro269 4.40 2.26, 1.90 1.99b 3.58b (Hδ) Lys270 8.44 4.28 1.83, 1.75 1.44b 1.66b (Hδ), 2.97b (Hε), 7.48 (εNH2) Gly271 8.34 3.94b Glu272 8.20 4.45 2.15, 1.97 2.46b Thr273 8.08 4.32 4.32 1.16

a Measured at 500 MHz and 298 K in water containing 10% D2O with H2O (δH 4.76 ppm) as internal standard. The glycopeptide existed in two forms in solution due to cis/trans isomerization of the Gly268-Pro269 amide bond. Shifts are reported for the major trans-isomer (displayed NOEs between Gly268 Hα and Pro269 Hδ

,δ’), while the minor cis-isomer (< 7%) could not be completely assigned due to

spectral overlap. b Degeneracy has been assumed. c Chemical shifts for the galactose moiety: δ 4.42 (H1), 3.91 (H4), 3.76 (H6), 3.68 (H5), 3.63 (H3), and 3.52 (H2).

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Table S2. 1H NMR chemical shifts for CII259-273 with Ile260ψ[CH2CH2]Ala261 (32).a


Gly259 3.76b Ile260ψ[CH2CH2] 8.09 3.66 1.47 1.36, 1.04,

0.81 (CH3) 0.82 (Hδ), 1.38b (CH2CH2), 1.41b (CH2CH2)

Ala261 2.36 1.05 Gly262 8.13 3.84b Phe263 8.02 4.63 3.08, 3.01 7.22 (Hδ), 7.33 (Hε), 7.30 (Hζ) Hyl264 8.41 4.26 2.00, 1.74 1.59b 4.01 (Hδ), 3.17 and 2.98 (Hε), 7.61 (εNH2), c Gly265 7.99 3.89b Glu266 8.18 4.35 2.09, 1.92 2.43b Gln267 8.48 4.36 2.11, 1.97 2.35b 7.48 and 6.82 (δNH2) Gly268 8.27 4.12, 3.98 Pro269 4.39 2.25, 1.91 1.99b 3.58b (Hδ) Lys270 8.44 4.28 1.83, 1.75 1.43b 1.66b (Hδ), 2.98b (Hε), 7.49 (εNH2) Gly271 8.34 3.94b Glu272 8.19 4.46 2.14, 1.97 2.46b Thr273 8.13 4.35 4.35 1.17




Table S3. 1H NMR chemical shifts for CII259-273 with Ala261ψ[CH2CH2]Gly262 (34).a


Gly259 3.83b Ile260 8.38 4.09 1.78 1.42, 1.14,

0.87 (CH3) 0.83 (Hδ)

Ala261ψ[CH2CH2] 8.06 3.76 1.05 1.30b ψ[CH2CH2], 1.37 and 1.47 ψ[CH2CH2] Gly262 2.18b Phe263 8.16 4.59 3.09, 2.95 7.24 (Hδ), 7.33 (Hε), 7.29 (Hζ) Hyl264 8.40 4.28 2.00, 1.75 1.60b 4.01 (Hδ), 3.17 and 2.97 (Hε), 7.61 (εNH2), c Gly265 7.98 3.88b Glu266 8.22 4.35 2.10, 1.94 2.43b Gln267 8.48 4.36 2.11, 1.96 2.34b 7.49 and 6.81 (δNH2) Gly268 8.26 4.10, 3.97 Pro269 4.38 2.25, 1.89 1.99b 3.58b (Hδ) Lys270 8.44 4.28 1.83, 1.75 1.44b 1.66b (Hδ), 2.97b (Hε), 7.49 (εNH2) Gly271 8.34 3.93b Glu272 8.20 4.59 2.14, 1.96 2.46b Thr273 8.08 4.32 4.32 1.16



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Table S4. 1H NMR chemical shifts for CII259-273 with Gly262ψ[(E)-CH=CH]Phe263 (35).a


Gly259 3.85b Ile260 8.44 4.21 1.84 1.44, 1.17,

0.91 (CH3) 0.84 (Hδ)

Ala261 8.41 4.27 1.36 Gly262ψ[(E)-CH=CH] 8.07 3.74b 5.60 ψ[(E)-CH=CH], 5.64 ψ[(E)-CH=CH] Phe263 3.41 2.93, 2.86 7.20 (Hδ), 7.30 (Hε), 7.25 (Hζ) Hyl264 8.28 4.19 2.94, 1.68 1.54b 3.98 (Hδ), 3.14 and 2.95 (Hε), 7.60 (εNH2), c Gly265 7.55 3.77, 3.84 Glu266 8.19 4.35 2.10, 1.94 2.43b Gln267 8.47 4.37 2.11, 1.97 2.35b 7.49 and 6.82 (δNH2) Gly268 8.28 4.13, 4.13 Pro269 4.39 2.26, 1.89 1.99b 3.59b (Hδ) Lys270 8.44 4.28 1.84, 1.76 1.44b 1.66b (Hδ), 2.97b (Hε), 7.49 (εNH2) Gly271 8.34 3.93b Glu272 8.20 4.46 2.14, 1.98 2.45b Thr273 8.08 4.32 4.32 1.16




Table S5. 1H NMR chemical shifts for CII259-273 with Gly262ψ[CH2CH2]Phe263 (36).a


Gly259 3.84b Ile260 8.43 4.19 1.83 1.44, 1.16,

0.91 (CH3) 0.85 (Hδ)

Ala261 8.38 4.24 1.33 Gly262ψ[CH2CH2] 7.98 3.20b 1.48b ψ[CH2CH2], 1.58b ψ[CH2CH2] Phe263 2.72 2.84, 2.72 7.20 (Hδ), 7.30 (Hε), 7.25 (Hζ) Hyl264 8.28 4.19 2.93, 1.65 1.54b 3.98 (Hδ), 3.14 and 2.95 (Hε), 7.61 (εNH2), c Gly265 7.10 3.75b Glu266 8.19 4.35 2.11, 1.95 2.44b Gln267 8.49 4.37 2.11, 1.97 2.35b 7.49 and 6.82 (δNH2) Gly268 8.28 4.14, 4.00 Pro269 4.39 2.26, 1.90 1.99b 3.59b (Hδ) Lys270 8.45 4.29 1.83, 1.75 1.43b 1.66b (Hδ), 2.97b (Hε), 7.49 (εNH2) Gly271 8.35 3.94b Glu272 8.21 4.45 2.14, 1.99 2.46b Thr273 8.08 4.32 4.32 1.16

S13




NH

OHa

NH

XF3C

O

II X = Br

21 X = PPh3 Brb

F3C

O

I

Scheme S1. Reagents and conditions: (a) CBr4, PPh3, MeCN, 0 °C → rt (70%); (b) PPh3, toluene, reflux (98%). N-(2-Bromoethyl)-trifluoroacetamide (II). Triphenylphosphine (2.83 g, 10.8 mmol) was added to alcohol I (1.67 g, 10.3 mmol) dissolved in acetonitrile (30 mL). The solution was cooled to 0 °C, and tetrabromomethane (3.63 g, 10.8 mmol) dissolved in acetonitrile (7 mL) was slowly added. The solution was stirred for 49 h while allowed to attain rt, and was then concentrated under reduced pressure to approximately 10 mL. CH2Cl2 was added and the solution was then washed with H2O. The phases were separated and the aqueous phase was extracted with CH2Cl2. The combined organic layers were washed with brine, dried and concentrated. The residue was purified by flash chromatography (n-heptane/EtOAc 30:1→3:1) to afford bromide II (1.57 g, 70%) as a white solid. 1H NMR (CDCl3): δ 7.01 (br s, 1H, NH), 3.80-3.75 (m, 2H, NCH2), 3.51 (t, J = 6.0 Hz, 2H, CH2Br); 13C NMR (CDCl3): δ 157.5 (q, JC-F = 37 Hz, CO), 115.6 (q, JC-F = 288 Hz, CF3), 41.3 (NCH2), 30.0 (CH2Br). [2-(Trifluoroacetamido)-ethyl]-triphenyl-phosphonium bromide (21). Bromide II (1.40 g, 6.36 mmol) and triphenylphosphine (6.68 g, 25.5 mmol) were refluxed in toluene (22 mL) for 18 h. The slurry was allowed to cool to rt and was then concentrated under reduced pressure to approximately 5 mL. Et2O (25 mL) was added and the suspension was sonicated followed by siphoning off the solvent supernatant after the particles had settled. This procedure was repeated with 4×40 mL Et2O followed by drying in vacuo over night to afford 21 (3.01 g, 98%) as a white solid. 1H NMR (CDCl3): δ 9.79 (m, 1H, NH), 7.84-7.74 (m, 9H, Ph), 7.73-7.66 (m, 6H, Ph), 4.01-3.92 (m, 2H, CH2P), 3.88-3.78 (m, 2H, NCH2); 13C NMR (CDCl3): δ 157.7 (q, JC-F = 38 Hz, CO), 135.4 (d, JC-P = 3.0 Hz, Ph), 133.5 (d, JC-P = 10 Hz, Ph), 130.6 (d, JC-P = 13 Hz, Ph), 117.2 (d, JC-P = 87 Hz, Ph), 115.5 (q, JC-F = 288 Hz, CF3), 33.5 (NCH), 22.6 (CH2P).

S14

HO

O

N

O

O

O

Ph

N

O

O

O

Ph

X

HO HO

OTBDPS OTBDPS

O

H

OTBDPS

O

a b

d

V X = OH

VI X = OTBDPSc

e f

III IV

VII VIII 22 Scheme S2. Reagents and conditions: (a) Et3N, t-BuCOCl, THF, -78 °C, ii) (R)-4-Benzyloxazolidin-2-one, n-BuLi, THF, -78 °C → rt (69%); (b) TiCl4, diisopropylamine, s-trioxane, CH2Cl2, 0 °C (88%); (c) TBDPSCl, imidazole, DMF, rt (91%); (d) LiOH, H2O2, THF, H2O, 0 °C → rt (95%); (e) BH3⋅Me2S, THF, 0 °C (93%); (f) Dess-Martin periodinane, CH2Cl2, rt (89%). (4R)-4-Benzyl-3-(3-phenyl-propionyl)-oxazolidin-2-one (IV). Triethylamine (1.0 mL, 7.20 mmol) was added to a solution of 3-phenylpropionic acid (0.98 g, 6.55 mmol) in THF (65 mL) cooled to -78 °C. The solution was stirred for 5 min, pivaloyl chloride (0.89 mL, 7.20 mmol) was then added followed by removal of the cooling bath and stirring for 40 min. The solution was again cooled to -78 °C and a mixture of (R)-4-benzyloxazolidin-2-one (1.16 g, 6.55 mmol) and n-BuLi (5.0 mL, 6.55 mmol, 1.3 M in hexanes) was transferred to this solution via a cannula. The mixture was stirred for 2 h at -78 °C, and NH4Cl (aq, satd) was then added followed by washing with brine. The combined aqueous layers were extracted with CH2Cl2, dried and concentrated under reduced pressure. Purification by flash chromatography (n-heptane/EtOAc 5:1→4:1) gave IV (1.40 g, 69%) as a white solid. 1H NMR (CDCl3): δ 7.37-7.16 (m, 10H, Ph), 4.71-4.63 (m, 1H, NCH), 4.18-4.14 (m, 2H, OCH2), 3.39-3.22 (m, 3H, CHCH2Ph and CH2CH2Ph), 3.12-2.99 (m, 2H, COCH2), 2.78 (dd, J = 13.5 and 9.5 Hz, 1H, CHCH2Ph); 13C NMR (CDCl3): δ 172.3 (COCH2), 153.3 (NCO), 140.4 (Ph), 135.1 (Ph), 129.3 (Ph), 128.8 (Ph), 128.5 (Ph), 128.4 (Ph), 127.2 (Ph), 126.2 (Ph), 66.1 (OCH2), 55.0 (NCH), 37.7 (CHCH2Ph), 37.0 (CH2CH2Ph), 30.2 (COCH2).

(4R)-4-Benzyl-3-((2S)-2-hydroxymethyl-3-phenyl-propionyl)-oxazolidin-2-one (V).10 Freshly distilled TiCl4 (0.74 mL, 6.71 mmol) was added dropwise to a stirred solution of IV (1.97 g, 6.37 mmol) in CH2Cl2 (30 mL) cooled to 0 °C. After stirring for 10 min, freshly distilled diisopropylamine (1.15 mL, 6.60 mmol) was added. Stirring was continued for 70 min at 0 °C, and then was s-trioxane (635 mg, 7.05 mmol) dissolved in CH2Cl2 (8 mL) added dropwise via a cannula followed. After 10 min, a second addition of TiCl4 (0.74 mL, 6.71 mmol) was made. After stirring for 3 h, NH4Cl (aq, satd) was added followed by extraction with CH2Cl2. The solution was dried and concentrated under reduced pressure. Purification by flash chromatography (n-heptane/EtOAc 4:1→2:1) gave V (1.90 g, 88%) as a slightly yellow

S15

oil. 1H NMR (CDCl3): δ 7.39-7.21 (m, 10H, Ph), 4.61-4.55 (m, 1H, NCH), 4.36-4.29 (m, 1H, COCH), 4.14 (dd, J = 9.1 and 2.3 Hz, 1H, OCH2), 4.05-3.99 (m, 1H, OCH2), 3.92 (dd, J = 11.3 and 3.9 Hz, 1H, CH2OH), 3.85 (dd, J = 11.3 and 6.8 Hz, 1H, CH2OH), 3.30 (dd, J = 13.5 and 3.4 Hz, 1H, NCHCH2Ph), 3.05 (dd, J = 13.3 and 7.5 Hz, 1H, COCHCH2Ph), 2.92 (dd, J = 13.3 and 7.9 Hz, 1H, COCHCH2Ph), 2.83 (dd, J = 13.5 and 9.5 Hz, 1H, NCHCH2Ph), 2.33 (br s, 1H, OH); 13C NMR (CDCl3): δ 175.3 (COCH2), 153.3 (NCO), 138.2 (Ph), 135.1 (Ph), 129.4 (Ph), 129.1 (Ph), 128.9 (Ph), 128.5 (Ph), 127.4 (Ph), 126.6 (Ph), 66.1 (OCH2), 63.0 (CH2OH), 55.5 (NCH), 47.0 (COCH2), 37.9 (NCHCH2Ph), 34.7 (COCH2CH2Ph).

(4S)-4-Benzyl-3-[(2S)-2-(tert-butyl-dimethyl-silanyloxymethyl)-3-phenyl-propionyl]-oxazolidin-2-one (VI). Alcohol V (1.36 g, 4.00 mmol) and imidazole (363 mg, 5.33 mmol) were dissolved in DMF (10 mL) and cooled to 0 °C. tert-Butyldiphenylchlorosilane (1.1 mL, 4.23 mmol) was added followed by stirring for 2 h. H2O (8 mL) was added and the mixture was extracted with EtOAc. The combined organic layers were washed with brine, dried and concentrated under reduced pressure. Purification by flash chromatography (n-heptane/EtOAc 30:1→10:1) gave VI (2.10 g, 91%) as a white foam. 1H NMR (CDCl3): δ 7.70-7.66 (m, 3H, Ph), 7.46-7.36 (m, 4H, Ph), 7.32-7.14 (m, 8H, Ph), 4.63-4.54 (m, 1H, COCH), 4.53-4.46 (m, 1H, NCH), 4.09 (dd, J = 10.1 and 7.6 Hz, 1H, CH2OSi), 4.00 (dd, J = 8.9 and 2.6 Hz, 1H, COCH2), 3.89-3.80 (m, 2H, CH2OSi and COCH2), 3.31 (dd, J = 13.4 and 3.3 Hz, 1H, NCHCH2Ph), 2.96-2.83 (m, 2H, COCHCH2Ph), 2.55 (dd, J = 13.4 and 10.2 Hz, 1H, NCHCH2Ph), 1.06 (s, 9H, CH3); 13C NMR (CDCl3): δ 174.3 (COCH2), 152.9 (NCO), 138.5 (Ph), 135.6 (2C, Ph), 135.5 (Ph), 133.3 (Ph), 133.2 (Ph), 129.71 (Ph), 129.66 (Ph), 129.3 (Ph), 129.0 (Ph), 128.9 (Ph), 128.3 (Ph), 127.71 (Ph), 127.68 (Ph), 127.2 (Ph), 126.4 (Ph), 65.8 (COCH2), 64.6 (CH2OSi), 55.4 (NCH), 47.2 (COCH), 38.0 (NCHCH2Ph), 35.0 (COCH2CH2Ph), 26.8 (C(CH3)3), 19.2 (C(CH3)3). (2S)-2-(tert-Butyl-diphenyl-silanyloxymethyl)-3-phenyl-propionic acid (VII). H2O2 (1.1 mL, 30% (w/w) in H2O) was added to a solution of VI (614 mg, 1.06 mmol) in THF (6 mL) and H2O (1.1 mL) cooled to 0 °C. LiOH (102 mg, 4.25 mmol) dissolved in H2O (1.4 mL) was added and the resulting mixture was stirred at 0 °C for 45 min and then at rt for 4 h. After cooling to 0 °C, Na2SO3 (aq, satd) was added followed by stirring for 30 min. CH2Cl2 was added, the aqueous layer was acidified to pH 2 by addition of HCl (aq, 1M) followed by extraction with EtOAc. The combined organic layers were washed with brine, dried and concentrated under reduced pressure. Purification by flash chromatography (n-heptane/EtOAc/AcOH 6:1:0.1%) gave VII (425 mg, 95%) as a colorless oil. 1H NMR (CDCl3): δ 7.66-7.61 (m, 3H, Ph), 7.46-7.33 (m, 4H, Ph), 7.23-7.11 (m, 3H, Ph), 3.83 (d, J = 5.3 Hz, 2H, CH2O), 3.07-2.98 (m, 1H, CH2Ph), 2.95-2.86 (m, 2H, CH2Ph and COCH), 1.06 (s, 9H, CH3); 13C NMR (CDCl3): δ 179.1 (CO2H), 138.6 (Ph), 135.6 (Ph), 133.0 (splitted, Ph), 129.8 (Ph), 128.9 (Ph), 128.4 (Ph), 127.7 (Ph), 126.4 (Ph), 63.5 (CH2O), 49.6 (CH), 33.7 (CH2Ph), 26.7 (CH3), 19.2 (C(CH3)3). (2S)-2-(tert-Butyl-diphenyl-silanyloxymethyl)-3-phenyl-propan-1-ol (VIII). BH3⋅Me2S (2.0 mL, 4.0 mmol, 2 M in THF) was added to a stirred solution of VII (405 mg, 0.97 mmol)

S16

in THF (15 mL) cooled to 0 °C. After 3 h 45 min at 0 °C, MeOH (10 mL) was carefully added followed by stirring for 40 min. The solution was concentrated under reduced pressure followed by purification by flash chromatography (n-heptane/EtOAc 6:1), which afforded VIII (366 mg, 93%) as a colorless oil. 1H NMR (CDCl3): δ 7.70-7.65 (m, 3H, Ph), 7.48-7.37 (m, 4H, Ph), 7.28-7.12 (m, 3H, Ph), 3.83-3.75 (m, 2H, CH2OH and CH2OSi), 3.73-3.66 (m, 2H, CH2OH and CH2OSi), 2.64 (d, J = 7.5 Hz, 2H, CH2Ph), 2.23 (br s, 1H OH), 2.11-2.01 (m, 1H, CH), 1.10 (s, 9H, CH3); 13C NMR (CDCl3): δ 140.0 (Ph), 135.6 (splitted, Ph), 133.1 (splitted, Ph), 129.8 (Ph), 129.0 (Ph), 128.3 (Ph), 127.8 (splitted, Ph), 125.9 (Ph), 65.9 and 64.8 (CH2OH and CH2OSi), 44.3 (CH), 34.0 (CH2Ph), 26.9 (CH3), 19.2 (C(CH3)3).

(2S)-2-(tert-Butyl-diphenyl-silanyloxymethyl)-3-phenyl-propionaldehyde (22). Dess-Martin periodinane (2.5 mL, 1.18 mmol, 15 wt.% solution in CH2Cl2) was added to a stirred solution of VIII (318 mg, 0.79 mmol) in CH2Cl2 (5 mL) at rt. After 45 min, Na2S2O5 (1.55 g, 8.15 mmol) dissolved in NaHCO3 (aq, satd) was added followed by stirring for 5 min. The phases were separated and the aqueous phase was extracted with CH2Cl2. The combined organic layers were washed with NaHCO3 (aq, satd) and brine, dried and concentrated under reduced pressure. Purification by flash chromatography (n-heptane/EtOAc 15:1→10:1) gave 22 (283 mg, 89%) as a colorless oil. 1H NMR (CDCl3): δ 9.81 (d, J = 1.3 Hz, 1H, CHO), 7.63-7.59 (m, 4H, Ph), 7.46-7.35 (m, 6H, Ph), 7.27-7.16 (m, 3H, Ph), 7.14-7.09 (m, 2H, Ph), 3.94 (dd, J = 10.6 and 4.2 Hz, 1H, CH2O), 3.82 (dd, J = 10.6 and 5.5 Hz, 1H, CH2O), 3.10 (dd, J = 14.0 and 6.4 Hz, 1H, CH2Ph), 2.86 (dd, J = 14.0 and 8.2 Hz, 1H, CH2Ph), 2.76-2.69 (m, 1H, CH), 1.06 (s, 9H, CH3); 13C NMR (CDCl3): δ 203.5 (CO), 138.7 (Ph), 135.59 (Ph), 135.57 (Ph), 133.0 (Ph), 132.9 (Ph), 129.84 (Ph), 129.80 (Ph), 129.0 (Ph), 128.5 (Ph), 127.8 (Ph), 127.7 (Ph), 126.3 (Ph), 61.5 (CH2O), 55.7 (CH), 31.2 (CH2Ph), 26.8 (CH3), 19.2 (C(CH3)3).

S17

NH

PPh3

21

F3C

O

NH

F3C

O

H

O

Br

a

IXX

Scheme S3. Phosphonium salt 21 and isobutyraldehyde (IX) were used as a test system in attempts to improve the conditions for the reaction between phosphonium salt 21 with chiral aldehyde 22 (Scheme 3 in the Article) in the synthesis towards the Gly-Phe isostere derivatives. Different co-solvents and additives, including DME, DMPU, DMSO and TMEDA, were explored. Compound 21 was completely dissolved in THF/DMPU 1:1, but the Wittig reaction with IX then gave poor (E)-selectivity and a complex product mixture. The other co-solvents and additives did not significantly improve the solubility of 21, but because THF/DME 95:5 gave the highest (E)-selectivity for the test system, this solvent mixture was employed with chiral aldehyde 22. However, the selectivity was unchanged (E/Z 14:10) and the conversion was only slightly improved, giving approximately 20% of (E)-23.

S18

MHC-binding assay The binding of the glycopeptides to Aq was determined relative to a biotinylated marker

peptide in a competitive inhibition assay performed as described elsewhere with minor modifications.11,12 Briefly, a mixture of a fixed concentration of purified soluble recombinant Aq (0.5 µM), non-glycosylated and biotinylated CII259-273 (3 µM), and various concentrations of competitor glycopeptides 1 or 31-36 (0, 4, 20, 100, 500 and 2500 µM) was incubated in PBS containing a protease inhibitor cocktail (Complete, Boehringer, Mannheim, Germany) at room temperature for 48 h. This mixture (100 µL) was transferred to mAb precoated microtiter assay plates prepared as described below, and the plates were incubated at room temperature for 2 h or at 4 °C overnight to capture the class II MHC molecules. The plates were washed with PBS containing 0.1% Tween 20 to remove excess peptides and the amount of bound biotinylated marker peptide was detected and quantified by the dissociation-enhanced lanthanide fluoroimmunoassay (DELFIA®) system based on the time-resolved fluoroimmunoassay technique with europium-labeled streptavidin (Wallac, Turku, Finland), according to the manufacturer’s instructions. The Aq experiments were performed in triplicate. Microtiter assay plates precoated with mAb were prepared by incubation with 10 µg/mL Y3P mAb at room temperature for 2 h or at 4 °C overnight and blocked with PBS containing 2% low fat milk.

T-cell activation assay T-cell recognition by the peptide-MHC complexes was investigated using the Aq-restricted

T-cell hybridomas HCQ.3, HCQ.10, HM1R.2, and 22a1-7E, whose generation has been reported elsewhere.13,14 IL-2 production by T-cell hybridomas, following incubation with antigen and Aq-expressing antigen-presenting spleen cells, was measured in 96-well flat-bottom microtiter plates essentially as described elsewhere15 but with slight modifications. Briefly, T-cell hybridoma cells (5 × 104) and Aq-expressing syngeneic spleen cells (5 × 105) were co-cultured with various concentrations of glycopeptides 1 or 31-36 (0, 2.34, 4.69, 9.38, 18.75, 37.5, 75, and 150 µM) in a total volume of 200 µL. After 24 h, 140 µL portions of the supernatants were removed, spun down to avoid transfer of T cell hybridoma cells, and 100 µL supernatant was assayed for IL-2 production by a sandwich ELISA (capturing mAb, purified rat anti-mouse IL-2, JES6-IA 12; detecting mAb, biotinylated anti-Mouse Interleukin-2 mAb 5H4, Mabtech AB) using the DELFIA® system (Wallac, Turku, Finland) according to the manufacturer’s instructions. Recombinant mouse IL-2 was used as a positive control. The fluorescence value for 36 at 9.38 µM for the HCQ.3 hybridoma was not reported due to a technical error when performing the assay.

S19

Molecular dynamics simulations All MD simulations were performed using the Desmond software package.16 OPLS-AA

2005 force field parameters assigned by Maestro17 were used in modeling the protein and ligand interactions and the TIP3P model was used for water. The initial atomic coordinates were taken from a comparative model3 of the Aq protein in complex with the CII259-270 glycopeptide.18 Only the α1 and β1 domains of Aq (i.e., residues α4-α84 and β3-β94) were taken into account in the MD simulations.19 The CII259-270 glycopeptide was manually mutated into the isostere-modified glycopeptides 31-36 and the complexes were energy minimized in two steps using MacroModel within Maestro.17 First, the complexes were minimized with the protein backbone atoms constrained with a force constant of 100 kJ*mol-

1*Å2 and the maximum number of iterations set to 1000. Second, minimization was performed without any constraints and the maximum number of iterations set to 5000. All other parameters were at their default settings. The Desmond16 software integrated in the Maestro17 environment was used for preparing the input structure files for the systems, whereas the MD simulations were run using the Desmond MD code implemented on the High Performance Computing Center North20 (HPC2N). The minimized complexes were neutralized by adding counter ions (Na+) and solvated using a cubic box shape with a layer of explicit TIP3P water molecules and a salt concentration of 0.15 M NaCl. The distance between the edges of the box and the closest atom in the complex was 15 Å. The model systems were relaxed prior to the simulations using a default relaxation protocol in Maestro17 that includes both restrained and unrestrained minimizations followed by four short MD simulations where restraints were gradually removed. A NVT simulation of 18 ns was then performed at 300 K and a recording interval of 2 ps for both the trajectory and the energy. All simulations were carried out under periodic boundary conditions. The Nose-Hoover thermostat method was used to maintain a constant temperature of 300 K with a relaxation time of 1.0 ps. Non-bonded interactions were modeled using a short range cutoff method, with a cutoff radius of 9.0 Å, and a long range Smooth Particle Mesh Ewald method, with Ewald tolerance of 1e-9. All other parameters were at their default settings. The hydrogen bond occupancies were calculated with the Hydrogen Bond extension in VMD21. A hydrogen bond was defined by a donor-acceptor distance of less than 3.3 Å and a donor-H…acceptor angle of less than 20 degrees. A strong hydrogen bond was defined as having an occupancy > 40%. Only occupancies of > 5% are reported.

S20

NH

OH

9

F3C

O

NH

OH

9

F3C

O

S21

10

NH

BrF3C

O

10

NH

BrF3C

O

S22

11

NH

PPh3 BrF3C

O

11

NH

PPh3 BrF3C

O

S23

NH

F3C

O

OTBDPS

13Me

NH

F3C

O

OTBDPS

13Me

S24

NH

F3C

O

OH

14Me

NH

F3C

O

OH

14Me

S25

NH

F3C

O

OH

15

O

Me

NH

F3C

O

OH

15

O

Me

S26

NH

F3C

O

16O

NH

F3C

O

16O

S27

FmocHN OH

2

O

Me

FmocHN OH

2

O

Me

S28

FmocHN OH

3

O

Me

FmocHN OH

3

O

Me

S29

NH

Me

F3C

O

OH

19

NH

Me

F3C

O

OH

19

S30

NH

Me

OH

20

O

F3C

O

NH

Me

OH

20

O

F3C

O

S31

FmocHN

Me

OH

5

O

FmocHN

Me

OH

5

O

S32

27

BocHN N

O

O

O

Me Ph

27

BocHN N

O

O

O

Me Ph

S33

N N

O

O

O

Me Ph28

Ph

Ph

N N

O

O

O

Me Ph28

Ph

Ph

S34

N N

O

O

O

Me Ph29

Ph

Ph

Ph

N N

O

O

O

Me Ph29

Ph

Ph

Ph

S35

FmocHN OH

O

6

Ph

FmocHN OH

O

6

Ph

S36

FmocHN OH

O

7

Ph

FmocHN OH

O

7

Ph

S37

NH

BrF3C

O

I

NH

BrF3C

O

I

S38

NH

PPh3

21

F3C

OBr

NH

PPh3

21

F3C

OBr

S39

N

O

O

O

Ph IV

N

O

O

O

Ph IV

S40

N

O

O

O

PhOH

V

N

O

O

O

PhOH

V

S41

N

O

O

O

PhOTBDPS

VI

N

O

O

O

PhOTBDPS

VI

S42

HO

OTBDPS

O

VII

HO

OTBDPS

O

VII

S43

HO

OTBDPS

VIII

HO

OTBDPS

VIII

S44

OTBDPSH

O

22 Ph

OTBDPSH

O

22 Ph

S45

Figure S1. Aq binding graphs showing the triplicate curves for each glycopeptide.

S46

References 1. Ley, S. V.; Anthony, N. J.; Armstrong, A.; Brasca, M. G.; Clarke, T.; Culshaw, D.;

Greck, C.; Grice, P.; Jones, A. B.; Lygo, B.; Madin, A.; Sheppard, R. N.; Slawin, A. M. Z.; Williams, D. J. Tetrahedron 1989, 45, 7161-7194.

2. Guan, Y. C.; Wu, J. L.; Sun, L.; Dai, W. M. J. Org. Chem. 2007, 72, 4953-4960. 3. Andersson, I. E.; Dzhambazov, B.; Holmdahl, R.; Linusson, A.; Kihlberg, J. J. Med.

Chem. 2007, 50, 5627-5643. 4. Allan, R. D.; Dickenson, H. W.; Johnston, G. A. R.; Kazlauskas, R.; Tran, H. W. Aust. J.

Chem. 1985, 38, 1651-1656. 5. Syed, B. M.; Gustafsson, T.; Kihlberg, J. Tetrahedron 2004, 60, 5571-5575. 6. Kofron, W. G.; Baclawski, L. M. J. Org. Chem. 1976, 41, 1879-1880. 7. Holm, B.; Bäcklund, J.; Recio, M. A. F.; Holmdahl, R.; Kihlberg, J. ChemBioChem 2002,

3, 1209-1222. 8. Broddefalk, J.; Forsgren, M.; Sethson, I.; Kihlberg, J. J. Org. Chem. 1999, 64, 8948-

8953. 9. Krchnak, V.; Vagner, J.; Lebl, M. Int. J. Pept. Prot. Res. 1988, 32, 415-416. 10. Evans, D. A.; Urpi, F.; Somers, T. C.; Clark, J. S.; Bilodeau, M. T. J. Am. Chem. Soc.

1990, 112, 8215-8216. 11. Hill, C. M.; Liu, A.; Marshall, K. W.; Mayer, J.; Jorgensen, B.; Yuan, B.; Cubbon, R. M.;

Nichols, E. A.; Wicker, L. S.; Rothbard, J. B. J. Immunol. 1994, 152, 2890-2898. 12. Kjellén, P.; Brunsberg, U.; Broddefalk, J.; Hansen, B.; Vestberg, M.; Ivarsson, I.;

Engström, Å.; Svejgaard, A.; Kihlberg, J.; Fugger, L.; Holmdahl, R. Eur. J. Immunol. 1998, 28, 755-767.

13. Corthay, A.; Bäcklund, J.; Broddefalk, J.; Michaëlsson, E.; Goldschmidt, T. J.; Kihlberg, J.; Holmdahl, R. Eur. J. Immunol. 1998, 28, 2580-2590.

14. Holm, L.; Bockermann, R.; Wellner, E.; Bäcklund, J.; Holmdahl, R.; Kihlberg, J. Bioorg. Med. Chem. 2006, 14, 5921-5932.

15. Michaëlsson, E.; Andersson, M.; Holmdahl, R.; Engström, Å. Eur. J. Immunol. 1992, 22, 1819-1825.

16. Bowers, K. J.; Chow, E.; Xu, H.; Dror, R. O.; Eastwood, M. P.; Gregersen, B. A.; Klepeis, J. L.; Kolossváry, I.; Moraes, M. A.; Sacerdoti, F. D.; Salmon, J. K.; Shan, Y.; Shaw, D. E. In Scalable Algorithms for Molecular Dynamics Simulations on Commodity Clusters, Proceedings of the ACM/IEEE Conference on Supercomputing (SC06), Tampa, Florida, November 11–17, 2006; Tampa, Florida, 2006.

17. Maestro, version 9.0; Schrödinger, LLC, New York, NY. 18. Andersson, I. E.; Batsalova, T.; Dzhambazov, B.; Edvinsson, L.; Holmdahl, R.; Kihlberg,

J.; Linusson, A. Org. Biomol. Chem. 2010, 8, 2931-2940. 19. Omasits, U.; Knapp, B.; Neumann, M.; Steinhauser, O.; Stockinger, H.; Kobler, R.;

Schreiner, W. Mol. Simulat. 2008, 34, 781-793. 20. High Performance Computing Center North (HPC2N). www.hpc2n.umu.se/ 21. Humphrey, W.; Dalke, A.; Schulten, K. J. Molec. Graphics 1996, 14, 33-38. Complete ref 20 in article: Bolin, D. R.; Swain, A. L.; Sarabu, R.; Berthel, S. J.; Gillespie, P.; Huby, N. J. S.; Makofske, R.; Orzechowski, L.; Perrotta, A.; Toth, K.; Cooper, J. P.; Jiang, N.; Falcioni, F.; Campbell, R.; Cox, D.; Gaizband, D.; Belunis, C. J.; Vidovic, D.; Ito, K.; Crowther, R.; Kammlott, U.; Zhang, X.; Palermo, R.; Weber, D.; Guenot, J.; Nagy, Z.; Olson, G. L. J. Med. Chem. 2000, 43, 2135-2148.

S47

Table S7. Average RMSD for the Aq/glycopeptide complexes for the MD trajectory between

9 to 18 ns.

RMSD Aq/glycopeptide complexes (Å)a

Glycopeptide

Average ±sd 1 (native) 2.91 0.31 31 (Ile-Ala (E)-alkene) 2.85 0.21 32 (Ile-Ala ethylene) 3.55 0.29 33 (Ala-Gly amide) 2.91 0.20 34 (Ala-Gly (E)-alkene) 3.18 0.25 35 (Ala-Gly ethylene) 2.26 0.28 36 (Gly-Phe amide) 2.61 0.21

a RMSD calculated with frame 1 as reference in the respective simulation.

S48

A)

B)

C)

Figure S2. C and N backbone RMSD versus simulation time for the glycopeptide/Aq complexes of 1 and:A) Ile260-Ala261 (E)-alkene 31 and Ile260-Ala261 ethylene 32, B) Ala261-Gly262 (E)-alkene 33 and Ala261-Gly262

ethylene 34, and C) Gly262-Phe263 (E)-alkene 35 and Gly262-Phe263 ethylene 36.

0 2000 4000 6000 8000 10000 12000 14000 16000 180000

0,51

1,52

2,53

3,54

4,55

13334

Simulation time /ps

rmsd

/Å

0 2000 4000 6000 8000 10000 12000 14000 16000 180000

0,51

1,52

2,53

3,54

4,55

13132

Simulation time /ps

rmsd

/Å

0 2000 4000 6000 8000 10000 12000 14000 16000 180000

0,5

1

1,5

2

2,5

3

3,5

4

4,5

5

13536

Simulation time /ps

rmsd

/Å

S49

A)

B)

C)

Figure S3. Backbone dihedral angle (defined by the atoms Cα-C´-N-Cα) for the glycopeptide 1 and: A)Ile260-Ala261 (E)-alkene 31, B) Ala261-Gly262 (E)-alkene 33, and C) Gly262-Phe263 (E)-alkene 35.

0 2 4 6 8 10 12 14 16 1860

100

140

180

220

260

300

31 (Ile-Ala)1 (Ile-Ala)

Simulation time /ns

Dih

edra

l ang

le /

º

0 2 4 6 8 10 12 14 16 1860

100

140

180

220

260

300

33 (Ala-Gly)1 (Ala-Gly)

Simulation time /ns

Dih

edra

l ang

le /

º

0 2 4 6 8 10 12 14 16 1860

100

140

180

220

260

300

35 (Gly-Phe)1 (Gly-Phe)

Simulation time /ns

Dih

edra

l ang

le /

º

S50

A)

B)

C)

Figure S4. Backbone dihedral angle (defined by the atoms Cα-C´-N-Cα) for the glycopeptide 1 and: A)Ile260-Ala261 ethylene 32, B) Ala261-Gly262 ethylene 34, and C) Gly262-Phe263 ethylene 36.

0 2 4 6 8 10 12 14 16 1860

100

140

180

220

260

300

1 (Ile-Ala)32 (Ile-Ala)

Simulation time /ns

Dih

edra

l ang

le /

º

0 2 4 6 8 10 12 14 16 1860

100

140

180

220

260

300

1 (Ala-Gly)34 (Ala-Gly)

Simulation time /ns

Dih

edra

l ang

le /

º

0 2 4 6 8 10 12 14 16 1860

100

140

180

220

260

300

1 (Gly-Phe)36 (Gly-Phe)

Simulation time /ns

Dih

edra

l ang

le /

º

S51

A)

B)

C)

Figure S5. Heavy atom RMSD of the glycopeptide Ile260-Phe263 sequence versus simulation time forglycopeptide 1 and: A) Ile260-Ala261 (E)-alkene 31 and Ile260-Ala261 ethylene 32, B) Ala261-Gly262 (E)-alkene 33and Ala261-Gly262 ethylene 34, and C) Gly262-Phe263 (E)-alkene 35 and Gly262-Phe263 ethylene 36.

0 2000 4000 6000 8000 10000 12000 14000 16000 180000

0,5

1

1,5

2

2,5

3

3,5

13132

Simulation time /ps

rmsd

/Å

0 2000 4000 6000 8000 10000 12000 14000 16000 180000

0,5

1

1,5

2

2,5

3

3,5

13334

Simulation time /ps

rmsd

/Å

0 2000 4000 6000 8000 10000 12000 14000 16000 180000

0,5

1

1,5

2

2,5

3

3,5

13536

Simulation time /ps

rmsd

/Å

S52

A)

B)

C)

Figure S6. The dihedral angle χ1 in the Phe263 residue versus simulation time (from 6 to 18 ns) forglycopeptide 1 and: A) Ile260-Ala261 (E)-alkene 31 and Ile260-Ala261 ethylene 32, B) Ala261-Gly262 (E)-alkene 33and Ala261-Gly262 ethylene 34, and C) Gly262-Phe263 (E)-alkene 35 and Gly262-Phe263 ethylene 36.

6 8 10 12 14 16 18-180

-160

-140

-120

-100

-80

-60

-40

-20

13536

Simulation time /ns

Dih

edra

l ang

le /

º

6 8 10 12 14 16 18-180

-160

-140

-120

-100

-80

-60

-40

-20

13334

Simulation time /ns

rmsd

/Å

6 8 10 12 14 16 18-180

-160

-140

-120

-100

-80

-60

-40

-20

13132

Simulation time /ns

rmsd

/Å

S53

A)

B)

C)

Figure S7 RMSD of the α1 helix of Aq versus simulation time for the glycopeptide/Aq complexes of 1 and:A) Ile260-Ala261 (E)-alkene 31 and Ile260-Ala261 ethylene 32, B) Ala261-Gly262 (E)-alkene 33 and Ala261-Gly262


9000 10000 11000 12000 13000 14000 15000 16000 17000 180000

0,51

1,52

2,53

3,54

4,55

13132

Simulation time /ps

rmsd

/Å

9000 10000 11000 12000 13000 14000 15000 16000 17000 180000

0,51

1,52

2,53

3,54

4,55

13334

Simulation time /ps

rmsd

/Å

9000 10000 11000 12000 13000 14000 15000 16000 17000 180000

0,51

1,52

2,53

3,54

4,55

13536

Simulation time /ps

rmsd

/Å

S54

A)

B)

C)

Figure S8. RMSD of the β1 helix of Aq versus simulation time for the glycopeptide/Aq complexes of 1 and:A) Ile260-Ala261 (E)-alkene 31 and Ile260-Ala261 ethylene 32, B) Ala261-Gly262 (E)-alkene 33 and Ala261-Gly262


9000 10000 11000 12000 13000 14000 15000 16000 17000 180000

0,5

1

1,5

2

2,5

3

13132

Simulation time /ps

rmsd

/Å

9000 10000 11000 12000 13000 14000 15000 16000 17000 180000

0,5

1

1,5

2

2,5

3

13334

Simulation time /ps

rmsd

/Å

9000 10000 11000 12000 13000 14000 15000 16000 17000 180000

0,5

1

1,5

2

2,5

3

13536

Simulation time /ps

rmsd

/Å

S55

e)-alkene and ethylene isosteres substantially alter the ... · 2 (84 ml), and triethylamine (8.4...

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