Peptidoglycan Modifications Tune the Stability and Function of the Innate Immune Receptor Nod2

Transcription

1 Peptidoglycan Modifications Tune the Stability and Function of the Innate Immune Receptor Nod2 James E. Melnyk, Vishnu Mohanan, Amy K. Schaefer, Ching-Wen Hou and Catherine Leimkuhler Grimes*,, Department of Chemistry and Biochemistry, University of Delaware, Newark, Delaware 19716, United States Department of Biological Sciences, University of Delaware, Newark, Delaware 19716, United States Supporting Information Table of Contents I. General Considerations S2 II. III. IV. Synthesis of Peptidoglycan Derivatives... S3 Biological Assays..S32 1 H and 13 C NMR Spectra S37 V. References.S77 S1

2 I. General Considerations All chemicals were purchased from Sigma Aldrich, Alfa Aesar, BA Chem, or Invivogen and used without further purification. Solvents were reagent grade and were further dried when necessary. Analytical thin-layer chromatography was performed on glass plates pre-coated with silica gel (250 µm, Sorbent Technologies). Flash chromatography was carried out on silica gel (60 Å, µm), purchased from Sorbent Technologies. Preparative HPLC was performed on an Agilent Series 1100 using a Phenomenex Luna 5 µm C18 column (250 x mm). NMR spectra were recorded on a Bruka AV 400 MHz and AV III 600 MHz spectrometers. IR spectra were obtained on an IR100 with an ATR Probe. Mass spectra (ESI) were obtained at the Mass Spectroscopy Facility at the Department of Chemistry, University of Delaware. S2

3 II. Synthesis of Peptidoglycan Derivatives Scheme S1. (2S,3R,4R,5S,6R)-6-(acetoxymethyl)-3-azidotetrahydro-2H-pyran-2,4,5-triyl triacetate (4): Synthesized from glucosamine hydrochloride according to literature precedent [S1],[S2]. (2R,3S,4R,5R,6R)-2-(acetoxymethyl)-5-azido-6-hydroxytetrahydro-2H-pyran-3,4-diyl diacetate (4.1): 4 (11.10 g, 29.7 mmol) and hydrazine acetate (3.29 g, 35.7 mmol) were dissolved in 30.5 ml of anhydrous dimethylformamide. The solution was stirred at 50 C for 20 mins under N 2. The reaction was cooled to room temperature and diluted with dichloromethane. The organic layer solution was washed with water, saturated sodium bicarbonate, brine, dried with sodium S3

4 sulfate and evaporated. The yellow oil was purified by column chromatography (2:3 ethyl acetate to hexanes) to yield a clear oil (7.85g, 80%). 1 H NMR (400 MHz, Chloroform-d) (Anomers 1.00α : 0.51β) δ 5.45 (dd, J = 10.5, 9.4 Hz, 1H, α 3-H), 5.30 (d, J = 3.4 Hz, 1H, α 1- H), (m, 3H, β 3-H, β 4-H, α 4-H), 4.72 (d, J = 8.0 Hz, 1H, β 1-H), (m, 3H, α 5-H, α 6-H, β 6-H), (m, 2H, α 6 -H, β 6 -H), 3.83 (ddd, J = 9.8, 4.7, 2.3 Hz, 1H, β 5-H), 3.45 (dd, J = 10.0, 8.1 Hz, 1H, β 2-H), 3.38 (dd, J = 10.6, 3.4 Hz, 1H, α 2-H), 2.06 (s, 3H, Ac), 2.05 (s, 3H, Ac), 2.04 (s, 3H, Ac), 2.03 (s, 3H, Ac), 2.02 (s, 3H, Ac), 1.99 (s, 3H, Ac). 13 C NMR (101 MHz, Chloroform-d) δ (carbonyl), (carbonyl), (carbonyl), (carbonyl), (carbonyl), (carbonyl), (β 1-C), (α 1- C), (β 3-C), (β 5-C), (α 3-C), (α 4-C), (β 4-C), (α 5-C), (β 2-C), (α 6-C), (β 6-C), (α 2-C), 20.67(methyl), (methyl), (methyl), (methyl x 2), (methyl). LRMS (ESI-Pos) for C 12 H 17 N 3 O 8 (331.10): [M+H] +. (2R,3S,4R,5R,6R)-2-(acetoxymethyl)-5-azido-6-(benzyloxy)tetrahydro-2H-pyran-3,4-diyl diacetate (4.2): 4.1 (2.10 g, 6.34 mmol) dissolved in 64 ml of anhydrous dichloromethane and a catalytic amount of anhydrous N,N-dimethylformamide (97.8 µl, 1.27 mmol) and stirred over 4 Å molecular sieves for 30 mins under N 2 gas. Oxalyl chloride (2 M, 3.81 ml) added and reaction stirred for 1 hr. The reaction was filtered and evaporated without heating. The oil was coevaporated 3x with toluene, subsequently dissolved in 73 ml of anhydrous dichloromethane, stirred over 4 Å molecular sieves for 15 mins under N 2 gas and subsequently added dropwise to a solution of benzyl alcohol (3.28 ml, 31.7 mmol), silver carbonate (17.5 g, 63.4 mmol) and silver triflate (375 mg, 1.46 mmol) that had been stirring in 171 ml of anhydrous dichloromethane S4

5 over 4 Å molecular sieves at 0 C for 15 mins. The reaction was allowed to slowly warm to room temperature and stirred for 15 hrs. The reaction was then filtered through celite and the organic layer was washed 3x with deionized water, dried with sodium sulfate and evaporated. The oily, white powder was purified by column chromatography (1:5 ethyl acetate to hexanes) to yield a white powder (2.27 g, 85%). 1 H NMR (600 MHz, DMSO-d6) (Anomers 1.00α : 0.61β) δ (m, 10H, aromatic), 5.28 (dd, J = 10.6, 9.5 Hz, 1H, α 3-H), 5.19 (d, J = 3.5 Hz, 1H, α 1-H), (m, 1H, β 3-H), 4.96 (t, J = 9.7 Hz, 1H, α 4-H), 4.87 (t, J = 9.7 Hz, 1H, β 4- H), (m, 2H, β 1-H, β benzyl methylene), 4.73 (d, J = 11.7 Hz, 1H, α benzyl methylene), 4.66 (d, J = 11.9 Hz, 1H, β benzyl methylene), 4.60 (d, J = 11.7 Hz, 1H, α benzyl methylene), 4.20 (dd, J = 12.3, 5.0 Hz, 1H, β 6 -H), 4.16 (dd, J = 12.3, 4.6 Hz, 1H, α 6 -H), (m, 3H, β 5-H, β 6-H, α 5-H, α 6-H), 3.80 (dd, J = 10.3, 8.1 Hz, 1H, β 2-H), 3.73 (dd, J = 10.7, 3.5 Hz, 1H, α 2-H) 2.03 (s, 1H, Ac), 2.03 (s, 3H, Ac), 2.02 (s, 3H, Ac), 2.02 (s, 3H, Ac), 1.99 (s, 3H, Ac), 1.97 (s, 3H, Ac). 13 C NMR (151 MHz, DMSO-d6) δ (carbonyl x 2), (carbonyl), (carbonyl), (carbonyl), (carbonyl), (aromatic), (aromatic), (aromatic), (aromatic), (aromatic), (aromatic), (aromatic), (aromatic), (β 1-C), (α 1-C), (β 3-C), (β 5-C), (β benzyl methylene), (α 3-C), (α benzyl methylene), (β 4-C), (α 4-C), (α 5-C), (β 2-C), (β 6-C), (α 6-C), (α 2-C), (methyl x 2), (methyl), (methyl), (methyl), (methyl). LRMS (ESI-Pos) for C 19 H 23 N 3 O 8 (421.15): [M+Na] +. (2R,3S,4R,5R,6R)-5-azido-6-(benzyloxy)-2-(hydroxymethyl)tetrahydro-2H-pyran-3,4-diol (4.3): 4.2 (1.80 g, 4.27 mmol) dissolved in 70 ml of anhydrous methanol under N 2 gas and a catalytic S5

6 amount of sodium methoxide (0.5 M, 5.98 ml) was added. The reaction was stirred for 4 hrs and quenched with Amberlite IR120, H form ion exchange resin. The reaction solution was filtered and evaporated. The residue was purified by column chromatography (10% methanol/dichloromethane) to yield a white powder (quantitative). 1 H NMR (600 MHz, Methanol-d4) (Anomers 1.00 α : 0.61 β) δ (m, 10H, aromatic), 4.97 (d, J = 3.5 Hz, 1H, α 1-H), 4.94 (d, J = 11.8 Hz, 1H, β benzyl methylene), 4.76 (d, J = 11.9 Hz, 1H, α benzyl methylene), 4.69 (d, J = 11.8 Hz, 1H, β benzyl methylene), 4.57 (d, J = 11.9 Hz, 1H, α benzyl methylene), 4.41 (d, J = 7.8 Hz, 1H, β 1-H), (m, 2H, α 3-H, β 6-H), 3.81 (dd, J = 11.8, 2.1 Hz, 1H, α 6-H), 3.70 (dt, J = 11.6, 5.4 Hz, 2H, α 6 -H, β 6 -H), 3.64 (ddd, J = 9.8, 5.5, 2.1 Hz, 1H, α 5-H), (m, 2H, α 4-H, β 4-H), (m, 3H, β 2-H, β 3-H, β 5-H), 3.12 (dd, J = 10.4, 3.5 Hz, 1H, α 2-H). 13 C NMR (151 MHz, Methanol-d4) δ (aromatic), (aromatic), (aromatic x 2), (aromatic), (aromatic), (aromatic), (aromatic), (β 1-C), (α 1-C), (β 5-C), (β 3-C), (α 5-C), (α 4-C), (α 3-C), (β benzyl methylene), (β 4-C), (α benzyl methylene), (β 2-C), (α 2-C), (β 6-C), (α 6-C). LRMS (ESI-Pos) for C 13 H 17 N 3 O 5 (295.12): [M+Na] +. (2S,4aR,6R,7R,8R,8aS)-7-azido-6-(benzyloxy)-2-phenylhexahydropyrano[3,2-d][1,3]dioxin-8-ol (5): 4.3 (1.65 g, 5.59 mmol), a catalytic amount of p-toluene sulfonic acid (0.213 g, 1.12 mmol) and benzaldehyde dimethyl acetal (2.51 ml, 16.8 mmol) were dissolved in 13.2 ml of anhydrous N,N-dimethylformamide and stirred at 60 C under a vacuum. After 1.5 hrs additional benzaldehyde dimethyl acetal (2.51 ml, 16.8 mmol) was added and the reaction was stirred for 1.5 hrs. The reaction was cooled to room temperature, quenched with 48 ml of S6

7 aqueous saturated sodium bicarbonate solution and stirred for 15 mins. The solution was extracted 3x with dichloromethane and the organic phase was then washed with aqueous 1 N hydrochloride. The organic layer was dried with sodium sulfate and evaporated. The residue was purified by column chromatography (1% methanol/dichloromethane) to yield a white powder (2.02 g, 95%). (Anomers 1.00α : 0.54β) 1 H NMR (600 MHz, Chloroform-d) δ (m, 4H, aromatic), (m, 13H, aromatic), (m, 3H, aromatic), 5.55 (s, 1H, β 4,6-benzylidene C-H), 5.54 (s, 1H, α 4,6-benzylidene C-H), 5.01 (d, J = 3.7 Hz, 1H, α 1- H), 4.95 (d, J = 11.7 Hz, 1H, β benzyl methylene), 4.78 (d, J = 12.0 Hz, 1H, α benzyl methylene), 4.71 (d, J = 11.7 Hz, 1H, β benzyl methylene), 4.63 (d, J = 12.0 Hz, 1H, α benzyl methylene), 4.52 (d, J = 8.0 Hz, 1H, β 1-H), 4.38 (dd, J = 10.5, 5.0 Hz, 1H, β 6-H), 4.28 (td, J = 10.8, 10.0, 1.6 Hz, 1H, α 3-H), 4.24 (dd, J = 10.3, 4.9 Hz, 1H, α 6-H), 3.91 (td, J = 10.0, 4.9 Hz, 1H, α 5-H), 3.82 (t, J = 10.3 Hz, 1H, β 6 -H), 3.74 (t, J = 10.3 Hz, 1H, α 6 -H), 3.66 (td, J = 9.4, 1.9 Hz, 1H, β 3-H), 3.58 (t, J = 9.3 Hz, 1H, β 4-H), 3.54 (t, J = 9.4 Hz, 1H, α 4-H), 3.48 (dd, J = 9.5, 8.1 Hz, 1H, β 2-H), 3.42 (td, J = 9.7, 5.0 Hz, 1H, β 5-H), 3.31 (dd, J = 10.0, 3.7 Hz, 1H, α 2- H), 2.66 (d, J = 2.1 Hz, 1H, α 3-OH), 2.64 (d, J = 2.2 Hz, 1H, β 3-0H). 13 C NMR (151 MHz, Chloroform-d) δ (aromatic), (aromatic), (aromatic), (aromatic), (aromatic), (aromatic), (aromatic), (aromatic), (aromatic), (aromatic), (aromatic), (aromatic), (aromatic), (aromatic), (α 4,6-benzylidene C-H), (β 4,6-benzylidene C-H), (β 1-C), (α 1-C), (α 4-C), (β 4-C), (β 3-C), (β benzyl methylene), (α benzyl methylene), (α 3-C), (α 3-C), (β 3-C), (β 2-C), (β 2-C), (α 2-C), (α 5-C). LRMS (ESI-Pos) for C 20 H 21 N 3 O 5 (383.15): [M+H] +. S7

8 (R)-2-(((2S,4aR,6R,7R,8R,8aS)-7-azido-6-(benzyloxy)-2-phenylhexahydropyrano[3,2- d][1,3]dioxin-8-yl)oxy)propanoic acid (6): 5 (1.87 g, 4.88 mmol) dissolved in 25 ml anhydrous N,N-dimethylformamide under N 2 gas. Sodium hydride (60% in oil, 1.22 g, 30.5 mmol) added, H 2 gas evolved and the reaction was stirred for 30 mins. (S)-(-)-2-chloropropanoic acid was added dropwise to the reaction as H 2 gas evolved and the mixture was allowed to stir for 30 mins. A second addition of sodium hydride (60% in oil, 1.22 g, 30.5 mmol) was then added and H 2 gas evolved, after which the reaction was allowed to stir for 20hrs. The reaction was quenched with deionized water and acidified to a ph of 4-5 with 1 N hydrochloride and a precipitate formed. The precipitate was filtered and dried to a tan, oily powder which was purified by column chromatography (2.5% methanol/ dichloromethane with 0.01% acetic acid then 5% methanol/dichloromethane with 0.01% acetic acid) to yield a white powder (1.68 g, 76% yield). 1 H NMR (600 MHz, Methanol-d4) (Anomers 1.00α : 0.28β) δ (m, 5H, aromatic), (m, 3H, aromatic), (m, 10H, aromatic), (m, 2H, aromatic), 5.61 (s, 1H, β 4,6-benzylidene C-H), 5.61 (s, 1H, α 4,6-benzylidene), 5.02 (d, J = 3.7 Hz, 1H, α 1-H), 4.88 (d, J = 12.0 Hz, 1H, β benzyl methylene), 4.74 (d, J = 12.0 Hz, 1H, α benzyl methylene), 4.68 (d, J = 11.8 Hz, 1H, β benzyl methylene), 4.61 (d, J = 12.0 Hz, 1H, α benzyl methylene), 4.55 (d, J = 8.1 Hz, 1H, β 1-H), 4.49 (q, J = 6.9 Hz, 1H, α C-H), 4.39 (q, J = 6.9 Hz, 1H, β C-H), 4.31 (dd, J = 10.3, 5.0 Hz, 1H, β 6-H), 4.15 (dd, J = 9.7, 4.4 Hz, 1H, α 6-H), 4.07 (t, J = 9.4 Hz, 1H, α 3-H), (m, 2H, β 6 -H, α 5-H), (m, 3H, α 6 -H, α 4-H, β 4-H), 3.61 (t, J = 9.2 Hz, 1H, β 3-H), (m, 1H, β 2-H, β 5-H, α 2-H), 1.37 (d, J = 6.9 Hz, 3H, β methyl), 1.36 (d, J = 6.9 Hz, 3H, α methyl). 13 C NMR (151 MHz, Methanol-d4) δ (carbonyl), (carbonyl), (aromatic), (aromatic), (aromatic), (aromatic), (aromatic), (aromatic), (aromatic), (aromatic), S8

9 (S)-methyl (aromatic), (aromatic), (aromatic), (aromatic), (aromatic), (aromatic), (aromatic), (α 4,6-benzylidene C-H), (β 4,6- benzylidene C-H), (β 1-C), (α 1-C), (α 4-C), (β 4-C), (β 3-C), (β C-H), (α 3-C), (α C-H), (β benzyl methylene), (α benzyl methylene), (α 6-C), (β 6-C), (β 2-C), (β 5-C), (α 5-C), (α 2-C), (β methyl), (α methyl). LRMS (ESI-Neg) for C 23 H 25 N 3 O 7 (455.17): [M-H] -. IR (ATR probe): Azide cm -1 (medium). 2-((R)-2-(((2S,4aR,6R,7R,8R,8aS)-7-azido-6-(benzyloxy)-2- phenylhexahydropyrano[3,2-d][1,3]dioxin-8-yl)oxy)propanamido)propanoate (6.1): 6 (300.0 mg, mmol) dissolved in 6 ml anhydrous N,N-dimethylformamide under N 2 gas. N- methylmorpholine (218.0 µl, 1.98 mmol) and N,N,N,N -Tetramethyl-O-(1H-benzotriazol-1- yl)uronium hexafluorophosphate (HBTU) (500.6 mg, 1.32 mmol) were subsequently added and the reaction was allowed to stir for 10 mins before L-alanine methyl ester hydrochloride (184.0 mg, 1.32 mmol) was added. The reaction stirred for 20 hours and was quenched with 6 ml water and then diluted with ethyl acetate. The phases were separated and the organic phase was washed with 1 N hydrochloride, saturated sodium bicarbonate, brine and was dried with sodium sulfate. The organic phase was evaporated and purified by column chromatography (2% methanol/dichloromethane) to yield a white powder (286.3 mg, 81%). 1 H NMR (600 MHz, Chloroform-d) (Anomers 1.00 α : 0.48 β) δ 7.92 (d, J = 7.7 Hz, 1H, β N-H), 7.69 (d, J = 7.6 Hz, 1H, α N-H), (m, 5H, aromatic), (m, 15H, aromatic), 5.57 (s, 1H, β 4,6-benzylidene C-H), 5.56 (s, 1H, α 4,6-benzylidene C-H), 5.11 (d, J = 3.6 Hz, 1H, α 1-H), 4.96 (d, J = 11.5 Hz, 1H, β benzyl methylene), 4.77 (d, J = 11.8 Hz, 1H, α benzyl methylene), 4.72 (d, S9

10 J = 11.5 Hz, 1H, β 4,6 benzylidene), (m, 4H, α 4,6-benzylidene, α and β C-H alanine, β 1-H), (m, 3H, β 6-H, α and β C-H), 4.24 (dd, J = 10.3, 4.9 Hz, 1H, α 6-H) (m, 2H, α 3-H, α 5-H), 3.82 (t, J = 10.3 Hz, 1H, β 6 -H), 3.75 (m, 7H, α 6 -H, α and β methyl ester), 3.67 (t, J = 9.2 Hz, 1H, β 4-H), 3.62 (t, J = 9.3 Hz, 1H, α 4-H), 3.55 (dd, J = 9.5, 7.8 Hz, 1H, β 2-H), (m, 3H, α 2-H, β 5-H, β 5-H), 1.45 (d, J = 7.2 Hz, 3H, α alanine methyl), 1.44 (d, J = 7.1 Hz, 3H, β alanine methyl), 1.42 (d, J = 6.8 Hz, 3H, α methyl), 1.41 (d, J = 6.9 Hz, 3H, β methyl). 13 C NMR (151 MHz, Chloroform-d) δ (carbonyl), (carbonyl), (carbonyl), (carbonyl), (aromatic), (aromatic), (aromatic), (aromatic), (aromatic), (aromatic), (aromatic), (aromatic), (aromatic), (aromatic), (aromatic), (aromatic), (aromatic), (aromatic), (aromatic), (aromatic), (α 4,6-benzylidene C-H), (β 4,6-benzylidene C-H), (β 1-C), (α 1-C), (α 4-C), (β 4- C), (α C-H), (β 3-C), (β C-H), (α 3-C), (β benzyl methylene), (α benzyl methylene), (α 6-C), (β 6-C), (β 5-C), (β 2-C), (α 5-C), (α 2-C), (β methyl ester), (α methyl ester), (β alanine C-H), (α alanine C-H), (α methyl), (β methyl), (β alanine methyl), (α alanine methyl). LRMS (ESI-Pos) for C 27 H 32 N 4 O 7 (540.22): [M+H] +. (R)-benzyl 5-amino-4-((S)-2-((R)-2-(((2S,4aR,6R,7R,8R,8aS)-7-azido-6-(benzyloxy)-2- phenylhexahydropyrano[3,2-d][1,3]dioxin-8-yl)oxy)propanamido)propanamido)-5- oxopentanoate (7): 6.1 (119.0 mg, mmol) dissolved in 7 ml methanol. Subsequently 2 ml of 0.5M of potassium hydroxide was added and the reaction was monitored by TLC (7% methanol/dichloromethane) until complete. The solvent was then evaporated and the white solid S10

11 was co-evaporated with toluene (3x) and subsequently dissolved in 4 ml of anhydrous N,Ndimethylformamide under N 2 gas. 1-Hydroxybenzotriazole hydrate (wetted with not less than 20 wt % water) (44.6 mg, mmol), 2,4,6-trimethylpyridine (87.2 µl, mmol) and then 1- ethyl-3-(3-dimethylaminopropyl)carbodiimide (63.3 mg, mmol) were subsequently added and the reaction was allowed to stir for 2 mins before D-γ-benzyl Isoglutamine perchlorate [S3] (104.0 mg, mmol) was added. The reaction stirred 16 hrs and was quenched with 4 ml water and then diluted with ethyl acetate. The phases were separated and the organic phase was washed with 1 N hydrochloride, saturated sodium bicarbonate, brine and was dried with sodium sulfate. The organic phase was evaporated and purified by column chromatography (5% methanol/dichloromethane) to yield a white powder (146.1 mg, 89% yield). 1 H NMR (600 MHz, Chloroform-d) (Anomers 1.00 α : 0.61 β) δ 7.85 (d, J = 6.0 Hz, 1H, β alanine N-H), 7.65 (d, J = 6.1 Hz, 1H, α alanine N-H), (m, 30H, aromatic), 7.14 (d, J = 7.5 Hz, 2H, α and β isoglutamine N-H), 5.56 (s, 1H, β 4,6-benzylidene C-H), 5.55 (s, 1H, α 4,6-benzylidene C-H), (m, 3H, α 1-H, α and β isoglutamine benzyl methylene), 4.95 (d, J = 11.5 Hz, 1H, β benzyl methylene), 4.77 (d, J = 11.8 Hz, 1H, α benzyl methylene), 4.71 (d, J = 11.5 Hz, 1H, β benzyl methylene), 4.62 (d, J = 11.8 Hz, 1H, α benzyl methylene), 4.56 (d, J = 8.0 Hz, 1H, β 1- H), 4.46 (td, J = 8.2, 4.8 Hz, 2H, α and β isoglutamine C-H), 4.38 (dd, J = 10.6, 5.0 Hz, 1H, β 6- H), 4.33 (q, J = 6.8 Hz, 1H, β C-H), (m, 4H, α C-H, α and β alanine C-H, α 6-H), (m, 2H, α 3-H, α 5-H), 3.82 (t, J = 10.3 Hz, 1H, β 6 -H), 3.75 (t, J = 10.3 Hz, 1H, α 6 -H), 3.66 (t, J = 9.2 Hz, 1H, β 4-H), 3.61 (t, J = 9.3 Hz, 1H, α 4-H), 3.54 (dd, J = 9.8, 8.1 Hz, 1H, β 2-H), (m, 3H, α 2-H, β 5-H, β 3-H), 2.58 (ddd, J = 17.3, 9.1, 5.0 Hz, 2H, α and β isoglutamine methylene), (m, 2H, α and β isoglutamine methylene), 2.22 (dddd, J = 14.4, 12.2, 7.8, 3.7 Hz, 2H, α and β isoglutamine methylene), (m, 2H, α and β S11

12 isoglutamine methylene), 1.40 (d, J = 7.0 Hz, 6H, α and β alanine methyl), 1.38 (d, J = 6.9 Hz, 6H, α and β methyl). 13 C NMR (151 MHz, Chloroform-d) δ (carbonyl), (carbonyl), (carbonyl), (carbonyl), (carbonyl), (carbonyl), (carbonyl), (carbonyl), (aromatic), (aromatic), (aromatic), (aromatic), (aromatic), (aromatic), (aromatic), (aromatic), (aromatic), (aromatic), (aromatic), (aromatic), (aromatic), (aromatic), (aromatic), (aromatic), (aromatic), (aromatic), (aromatic), (α 4,6-benzylidene C-H), (β 4,6-benzylidene C-H), (β 1-C), (α 1-C), (α 4-C), (β 4-C), (β 3-C), (α C-H), (β C-H), (α 3-C), (β benzyl methylene), (α benzyl methylene), (α 6-C), (β 6-C), (α isoglutamine benzyl methylene), (β isoglutamine benzyl methylene), (β 5- C), (β 2-C), (α 5-C), (α 2-C), (α isoglutamine C-H), (β isoglutamine C-H), (α and β alanine C-H), (α and β isoglutamine methylene), (β isoglutamine methylene), (α isoglutamine methylene), (α methyl), (β methyl), (β alanine methyl), (α alanine methyl). LRMS (ESI-Pos) for C 38 H 44 N 6 O 10 (744.31): [M+H] +. IR (ATR probe): Azide cm -1 (medium). (R)-5-amino-4-((S)-2-((R)-2-(((2R,3R,4R,5S,6R)-3-amino-2,5-dihydroxy-6- (hydroxymethyl)tetrahydro-2h-pyran-4-yl)oxy)propanamido)propanamido)-5-oxopentanoic acid (3): 7 (108.0 mg, mmol) dissolved in 6.4 ml water, 4.8 ml methanol and 0.80 ml acetic acid with stirring at room temperature. 10% palladium on carbon (60.0 mg, mmol) added and the reaction was degassed and stirred under H 2 gas for 17 hrs. The reaction was monitored by mass-spectrometry and filtered through celite upon completion. Purified by HPLC (5% S12

13 acetonitrile in water with 0.1% trifluoroacetic acid isocratic flow for 30 mins). White solid (quantitative yield). 1 H NMR (600 MHz, Deuterium Oxide) (Anomers 1.00α : 0.61β) δ 5.41 (d, J = 3.5 Hz, 1H, α 1-H), 4.91 (d, J = 8.4 Hz, 1H, β 1-H), 4.72 (q, J = 6.9 Hz, 1H, β C-H), 4.67 (q, J = 6.7 Hz, 1H, α C-H), (m, 4H, α and β alanine C-H, α and β isoglutamine C-H), (m, 2H, α 5-H, β 6-H), 3.82 (dd, J = 12.3, 2.0 Hz, 1H, α 6-H), 3.76 (dd, J = 12.3, 5.0 Hz, 1H, α 6 -H), (m, 2H, α 3-H, β 6 -H), (m, 2H, α 4-H, β 6-H), (m, 1H, β 3-H), 3.48 (ddd, J = 9.8, 5.6, 1.9 Hz, 1H, β 5-H), 3.30 (dd, J = 10.4, 3.5 Hz, 1H, α 2-H), 3.01 (dd, J = 10.4, 8.6 Hz, 1H, β 2-H), (m, 4H, α and β isoglutamine methylene), (m, 2H, α and β isoglutamine methylene), (m, 2H, α and β isoglutamine methylene), (m, 12H, α and β methyl, α and β alanine methyl). 13 C NMR (151 MHz, Deuterium Oxide) δ (carbonyl), (carbonyl), (carbonyl), (carbonyl), (carbonyl), (carbonyl), (carbonyl), (carbonyl), (β 1-C), (α 1-C), (β 3-C), (α 3-C), (α C-H), (β C-H), (β 5-C), (α 5-C), (β 4-C), (α 4-C), (β 6-C), (α 6-C), (β 2-C), (α 2-C), (α isoglutamine C-H), (β isoglutamine C-H), (α alanine C-H), (β alanine C-H), ( α and β isoglutamine methylene), (β isoglutamine methylene)26.00 (α isoglutamine methylene)18.99 (β methyl), (α methyl), (β alanine methyl), (α alanine methyl). HRMS (ESI-Pos) for C 17 H 30 N 4 O 10 ( ): [M+H] +. (R)-4-((S)-2-((R)-2-(((2R,3R,4R,5S,6R)-3-acetamido-2,5-dihydroxy-6- (hydroxymethyl)tetrahydro-2h-pyran-4-yl)oxy)propanamido)propanamido)-5-amino-5- oxopentanoic acid (1): 3 (24.0 mg, mmol) and sodium bicarbonate (10.7 mg, mmol) S13

14 dissolved in 1.0 ml water with stirring at room temperature. Acetic anhydride (5.5 µl, mmol) added dropwise and reaction stirred for 1.5 hrs. The reaction was subsequently quenched with Amberlite IR120, H form ion exchange resin. The mixture was filtered and evaporated. White solid (quantitative yield). NMR data matches previously published spectra [S4]. 1 H NMR (600 MHz, Methanol-d4) (Anomers 1.00α : 0.45β) δ 8.37 (d, J = 7.8 Hz, 1H, α N-H), 8.28 (d, J = 8.0 Hz, 1H, β N-H), 7.95 (d, J = 5.9 Hz, 1H, α N-H), 7.87 (d, J = 5.9 Hz, 1H, β N-H), 5.17 (d, J = 3.3 Hz, 1H, α 1-H), 4.56 (d, J = 8.3 Hz, 1H, β 1-H), (m, 4H, α and β C-H, α and β isoglutamine methyne), (m, 2H, α and β C-H alanine), (m, 2H, α 2-H, β 6-H), (m, 2H, α 5-H, α 6-H), (m, 4H, α 6 -H, β 6 -H, β 2-H, β 4-H), (dd, J = 10.5 Hz, 8.9 Hz, 1H, α 3-H), 3.49 (t, J = 9.4 Hz, 1H, α 4-H), 3.43 (t, J = 9.2 Hz, 1H, β 3-H), (m, 1H, β H-5), 2.40 (q, J = 7.6 Hz, 4H, α and β isoglutamine methylene), 2.21 (ddd, J = 21.0, 7.8, 4.8 Hz, 2H, α and β isoglutamine methylene), 1.96 (s, 3H β acetyl), 1.96 (s, 3H, α acetyl), (m, 2H, α and β isoglutamine methylene), (m, 16H, α and β methyl, α and β methyl alanine). 13 C NMR (151 MHz, Methanol-d4) δ (carbonyl), (carbonyl), (carbonyl), (carbonyl), (carbonyl), (carbonyl), (carbonyl), (carbonyl), (carbonyl), (carbonyl), (β 1-C), (α 1-C), (β 3-C), (α 3-C), (β methyl), (α methyl), (β 5-C), (α 5-C), (α 4-C), (β 4-C), (β 6-C), (α 6-C), (β 2-C), (α 2-C), (α isoglutamine methyne), (β isoglutamine methyne), (α C-H alanine), (β C-H alanine), (α isoglutamine methylene), (β isoglutamine methylene), (α isoglutamine methylene), (β isoglutamine methylene), (β acetyl), (α acetyl), (α methyl), (β methyl), (β methyl alanine), (α methyl alanine). LRMS (ESI-Pos) for C 19 H 32 N 4 O 11 (492.21): [M+H] +. S14

15 Scheme S2. (R)-5-amino-4-((S)-2-((R)-2-(((2R,3R,4R,5S,6R)-2,5-dihydroxy-3-(2-hydroxyacetamido)-6- (hydroxymethyl)tetrahydro-2h-pyran-4-yl)oxy)propanamido)propanamido)-5-oxopentanoic acid (2): 3 (4.2 mg, mmol) and sodium carbonate (4. 9 mg, mmol) suspended in 150 μl wet methanol with stirring under nitrogen. Acetoxyacetic acid 2,5-Dioxopyrrolidin-1-yl ester [S5] (2.0 mg, mmol) added 5 times with 15 min intervals between additions and the reaction was subsequently allowed to stir for a total of 16 hrs. The reaction was quenched with Amberlite IR120, H form ion exchange resin. The mixture was filtered, evaporated and the residue was suspended in 5% methanol/dichloromethane. The suspension was filtered and the white precipitate was collected. White solid (4.0 mg, 85%). NMR data matches previously published spectra [S6]. 1 H NMR (600 MHz, Methanol-d4) (Anomers 1.00α : 0.19β) δ 5.20 (d, J = 3.4 Hz, 1H, α 1-H), 4.50 (q, J = 6.7 Hz, 1H, α C-H), 4.29 (q, J = 7.1 Hz, 1H, α alanine C-H), 4.22 (dd, J = 9.0, 4.1 Hz, 1H, α isoglutamine methyne), 4.00 (s, 2H, α glycolyl methylene), 3.90 (dd, J = 10.6, 3.4 Hz, 1H, α 2-H), 3.82 (dq, J = 6.9, 2.1 Hz, 1H, α 5-H), (m, 3H, α 3-H, 6-H, 6 -H), (m, 1H, 4-H), (m, 2H, α isoglutamine methylene), 2.15 (dtq, J = 15.3, 7.6, 3.9 Hz, 1H, α isoglutamine methylene), (m, 1H, α isoglutamine methylene), 1.39 (d, J = 7.1 Hz, 3H, α alanine methyl), 1.38 (d, J = 6.8 Hz, 3H, α methyl). 13 C NMR (151 MHz, Methanol-d4) δ (carbonyl), (carbonyl), (carbonyl), S15

16 (carbonyl), (carbonyl), (α 1-C), (α 3-C), (α C-H), (α 5- C), (α 4-C), (α glycolyl methylene), (α 6-C), (α isoglutamine methyne), (α 2-C), (α alanine C-H), (α isoglutamine methylene), (α isoglutamine methylene), (α methyl), (α alanine methyl). LRMS (ESI-Neg) for C 19 H 32 N 4 O 12 (508.20): [M-H] -. Scheme S3. (R)-4-((S)-2-((R)-2-(((2R,3R,4R,5S,6R)-3-(2-acetoxyacetamido)-2,5-dihydroxy-6- (hydroxymethyl)tetrahydro-2h-pyran-4-yl)oxy)propanamido)propanamido)-5-amino-5- oxopentanoic acid (8): 3 (4.7 mg, mmol) suspended in 200 μl water and ph raised to 9 with aqueous saturated sodium bicarbonate. Acetoxyacetic acid 2,5-Dioxopyrrolidin-1-yl ester [S5] (2.3 mg, mmol) added 4 times with 15 min intervals between additions (1 hr total). The reaction was subsequently quenched with Amberlite IR120, H form ion exchange resin. The mixture was filtered, evaporated and the residue was suspended in 5% methanol/dichloromethane. The suspension was filtered and the white precipitate was collected. White solid (2.7 mg, 48%). 1 H NMR (600 MHz, Methanol-d4) (Anomers 1.00α : 0.15β) δ 5.26 (d, J = 3.3 Hz, 1H, α 1-H), (m, 2H, α acetoxycetic methylene, α C-H), 4.34 (dd, J = 9.4, 4.7 Hz, 1H, α isoglutamine methyne), (m, 1H, α alanine C-H), (m, 3H, α 2-H, α 5-H, α 6-H), 3.72 (dd, J = 11.8, 5.1 Hz, 1H, α 6 -H), (m, 1H, α 3-H), S16

17 3.50 (t, J = 9.4 Hz, 1H, α 4-H), 2.42 (t, J = 7.5 Hz, 2H. α isoglutamine methylene), 2.21 (ddt, J = 14.4, 7.7, 3.7 Hz, 1H, α isoglutamine methylene), 2.15 (s, 3H, α acetyl), 1.92 (ddt, J = 12.5, 9.6, 6.3 Hz, 1H, α isoglutamine methylene), 1.39 (d, J = 7.3 Hz, 3H, α alanine methyl), 1.37 (d, J = 6.8 Hz, 3H, α methyl). 13 C NMR (151 MHz, Methanol-d4) δ (carbonyl), (carbonyl), (carbonyl), (carbonyl), (carbonyl), (carbonyl), (α 1-C), (α 3-C), (α C-H), (α 5-C), (α 4-C), (α acetoxyacetic methylene), (α 6-C), (α 2-C), (α isoglutamine methyne), (α alanine C- H), (α isoglutamine methylene), (α isoglutamine methylene), (α acetyl), (α methyl), (α alanine methyl). LRMS (ESI-Neg) for C 21 H 34 N 4 O 13 (550.21): [M- H] -. Scheme S4. (R)-5-amino-4-((S)-2-((R)-2-(((2R,3R,4R,5S,6R)-2,5-dihydroxy-6-(hydroxymethyl)-3-(2- methoxyacetamido)tetrahydro-2h-pyran-4-yl)oxy)propanamido)propanamido)-5-oxopentanoic acid (9): 3 (7.0 mg, mmol) and sodium carbonate (6.8 mg, mmol) suspended in 200 μl anhydrous methanol with stirring and under nitrogen. The reaction was stirred for a total of 2.5 hours and subsequently quenched with Amberlite IR120, H form ion exchange resin. The mixture was filtered, evaporated and the residue was suspended in 5% methanol/dichloromethane. The suspension was filtered and the white precipitate was collected. S17

18 White solid (6.3 mg, 78% yield). 1 H NMR (600 MHz, Methanol-d4) (Anomers 1.00α : 0.31β) δ 5.20 (d, J = 3.1 Hz, 1H, α 1-H), 4.66 (d, J = 8.3 Hz, 1H, β 1-H), (m, 2H, α and β C- H), 4.33 (dd, J = 9.6, 4.6 Hz, 2H, α and β isoglutamine methyne), (m, 2H, α and β alanine C-H), (m, 5H, α 2-H, α 5-H, β 6-H, α and β methoxyacetic methylene), (m, 2H, α 6-H, β 2-H), 3.73 (dd, J = 11.9, 5.2 Hz, 1H, α 6 -H), (m, 2H, α 3-H, β 6 -H), (m, 1H, β 3-H), 3.50 (t, J = 9.4 Hz, 1H, α 3-H), 3.45 (t, J = 9.2 Hz, 1H, β 4- H), 3.41 (s, 6H, α and β O-methyl), (m, 1H, β 5-H), 2.41 (td, J = 14.2, 12.3, 7.9 Hz, 4H, α and β isoglutamine methylene), 2.21 (ddt, J = 15.6, 7.9, 4.8 Hz, 2H, α and β isoglutamine methylene), (m, 2H, α and β isoglutamine methylene), 1.39 (d, J = 7.2 Hz, 6H α and β alanine methyl), 1.38 (d, J = 6.9 Hz, 6H, α and β methyl). 13 C NMR (151 MHz, Methanol-d4) δ (carbonyl), (carbonyl), (carbonyl), (carbonyl), (carbonyl), (carbonyl), (carbonyl), (carbonyl), (carbonyl), (carbonyl), (β 1-C), (α 1-C), (β 3-C), (α 3-C), (β 5-C), (β C-H), (α C-H), (α 5-C), (β methoxyacetic methylene), (α methoxyacetic methylene), (α 4-C), (β 4-C), (β 6-C), (α 6-C), (α O-methyl), (β O- methyl), (β 2-C), (α 2-C), (α isoglutamine methyne), (β isoglutamine methyne), (α alanine C-H), (β alanine C-H), (α isoglutamine methylene), (β isoglutamine methyne), (α isoglutamine methyne), (β isoglutamine methyne), (α methyl), (β methyl), (α alanine methyl), (β alanine methyl). LRMS (ESI-Pos) for C 20 H 34 N 4 O 12 (522.22): [M+H] +. S18

19 Scheme S5. (R)-5-amino-4-((S)-2-((R)-2-(((2R,3R,4R,5S,6R)-2,5-dihydroxy-6-(hydroxymethyl)-3-(2,2,2- trifluoroacetamido)tetrahydro-2h-pyran-4-yl)oxy)propanamido)propanamido)-5-oxopentanoic acid (10): 3 (9.1 mg, mmol) and sodium carbonate (12.9 mg, mmol) suspended in 200 μl anhydrous methanol and stirred for 10 mins under nitrogen. Ethyl trifluoroacetate (14.4 µl, mmol) added dropwise and reaction stirred for 17 hrs. The reaction was subsequently quenched with Amberlite IR120, H form ion exchange resin. The mixture was filtered, evaporated and then co-evaporated with toluene three times. Clear, flakey solid (10.1 mg, 92% yield). 1 H NMR (600 MHz, Methanol-d4) (Anomers 1.00α : 0.33β) δ 5.35 (d, J = 3.2 Hz, 1H, α 1-H), 4.66 (d, J = 8.3 Hz, 1H, β 1-H), 4.55 (q, J = 6.8 Hz, 1H, α C-H), 4.36 (dt, J = 9.4, 4.2 Hz, 1H, β isoglutamine methyne), (m, 2H, α alanine C-H, α isoglutamine methyne), (m, 1H, β alanine C-H), 4.17 (q, J = 6.9 Hz, 1H, β C-H), 3.88 (dd, J = 11.9, 2.3 Hz, 1H, β 6-H), (m, 1H, β 2-H), (m, 3H, α 2-H, α 5-H, α 6-H), (m, 3H, α 3-H, α 6 -H, β 6 -H), 3.52 (t, J = 9.3 Hz, 1H, α 4-H), (m, 2H, β 3-H, β 4-H), (m, 1H, β 5-H), (m, 4H, α and β isoglutamine methylene), 2.21 (ddt, J = 15.9, 7.9, 4.8 Hz, 2H, α and β isoglutamine methylene), (m, 2H, α and β isoglutamine methylene), (m, 12H, α and β methyl, α and β methyl alanine). 13 C NMR (151 MHz, Methanol-d4) δ (carbonyl), (carbonyl), (carbonyl), (carbonyl), (carbonyl) (carbonyl), (carbonyl), (carbonyl), (carbonyl), S19

20 (carbonyl), (q, J = H, α and β CF 3 ) (β 1-C), (α 1-C), (β 3- C), (β C-H), (α 3-C), (β 5-C), (α C-H), (α 5-C), (α 4-C), (β 4-C), (β 6-C), (α 6-C), (β 2-C), (α 2-C), (α isoglutamine methyne), (β isoglutamine methyne), (α alanine C-H), (β alanine C-H), (α isoglutamine methylene), (β isoglutamine methylene), (β isoglutamine methyne), (α isoglutamine methylene), (α methyl), (β methyl), (β alanine methyl), (α alanine methyl). LRMS (ESI-Pos) for C 19 H 29 F 3 N 4 O 11 (546.18): [M+H] +. Scheme S6. (R)-5-amino-4-((S)-2-((R)-2-(((2R,3R,4R,5S,6R)-3-(3-carboxypropanamido)-2,5-dihydroxy-6- (hydroxymethyl)tetrahydro-2h-pyran-4-yl)oxy)propanamido)propanamido)-5-oxopentanoic acid (11): 3 (8.5 mg, mmol) and sodium carbonate (8.0 mg, mmol) suspended in 100 μl anhydrous methanol and 20 μl acetone with stirring and under nitrogen. Succinic anhydride (2.2 mg, mmol) added and reaction stirred for 24 hrs. The reaction was subsequently quenched with Amberlite IR120, H form ion exchange resin. The mixture was filtered, evaporated and the residue was suspended in 5% methanol/dichloromethane. The suspension was filtered and the white precipitate was collected. White solid (6.9 mg, 66% yield). 1 H NMR (600 MHz, Methanol-d4) (Anomers 1.00α : 0.21β) δ 5.17 (d, J = 3.3 Hz, 1H, α 1-H), 4.59 (d, J = 8.3 Hz, 1H, β 1-H), 4.42 (q, J = 6.6 Hz, 2H, α and β C-H), (m, 2H, α and β S20

21 isoglutamine methyne), 4.28 (q, J = 7.0 Hz, 2H, α and β alanine C-H), 3.86 (dd, J = 10.7, 3.3 Hz, 2H, α 2-H, β 6-H), (m, 2H, α 5-H, α 6-H), 3.72 (dd, J = 11.9, 5.2 Hz, 1H, α 6 -H), (m, 2H, β 2-H, β 6 -H), 3.65 (dd, J = 10.5, 8.9 Hz, 1H, α 3-H), (m, 1H, α 4-H), (m, 2H, β 3-H, β 4-H), 3.31 (m, 1H, β 5-H), (m, 4H, α and β N- succinyl methylene), (m, 4H, α and β N-succinyl methylene), (m, 4H, α and β isoglutamine methylene), (m 2H, α and β isoglutamine methylene), (m, 2H, α and β isoglutamine methylene), 1.40 (d, J = 7.2 Hz, 6H, α and β alanine methyl), 1.38 (d, J = 6.8 Hz, 6H, α and β methyl). 13 C NMR (151 MHz, Methanol-d4) δ (carbonyl), (carbonyl), (carbonyl x2), (carbonyl), (carbonyl), (carbonyl), (carbonyl), (carbonyl x2), (carbonyl), (carbonyl), (β 1-C), (α 1-C), (β 3-C), (α 3-C), (β C-H), (β 5-C), (α C- H), (α 5-C), (α 4-C), (β 4-C), (β 6-C), (α 6-C), (β 2-C), (α 2-C), (α isoglutamine methyne), (β isoglutamine methyne), (α alanine C-H), (β alanine C-H), (β succinyl methylene), (α succinyl methylene), (α isoglutamine methylene), (β isoglutamine methylene), (α succinyl methylene), (β succinyl methylene), (α isoglutamine methylene), (β isoglutamine methylene), (α methyl), (β methyl), (β alanine methyl), (α alanine methyl). LRMS (ESI-Pos) for C 21 H 34 N 4 O 13 (550.21): [M+H] +. S21

22 Scheme S7. (R)-5-amino-4-((S)-2-((R)-2-(((2R,3R,4R,5S,6R)-2,5-dihydroxy-6-(hydroxymethyl)-3-(4- oxopentanamido)tetrahydro-2h-pyran-4-yl)oxy)propanamido)propanamido)-5-oxopentanoic acid (12): Levulinic acid 2,5-Dioxopyrrolidin-1-yl ester prepared as follows: Levulinic acid (100 mg, mmol) and N-hydroxysuccinimide (104 mg, mmol) dissolved in 2 ml anhydrous dimethylformamide with stirring under nitrogen. 1-ethyl-3-(3- dimethylaminopropyl)carbodiimide (198 mg, 1.03 mmol) was subsequently added and the reaction stirred 16 hrs and then was diluted with dichloromethane. The reaction was washed with water, aqueous 1N HCl (3x) and aqueous saturated sodium chloride. The organic layer was dried with sodium sulfate and evaporated to yield a clear, semi-viscous oil. Anhydrous diethyl ether was added to the oil to yield a white precipitate. The ether layer was decanted and the precipitate was dried. White powder (164 mg, 89%). 1 H NMR (600 MHz, Chloroform-d) δ (m, 4H, levulinyl methylene (x2)), 2.82 (s, 4H, NHS methylene (x2)), 2.20 (s, 3H, methyl). 13 C NMR (151 MHz, Chloroform-d) δ (carbonyl), (carbonyl), (carbonyl), (levulinyl methylene), (levulinyl methylene), (NHS methylene), (methyl). 3 (3.5 mg, mmol) and sodium carbonate (4.1 mg, mmol) suspended in 150 μl anhydrous methanol with stirring under nitrogen. Levulinic acid 2,5-Dioxopyrrolidin-1-yl ester (1.7 mg, mmol) added 5 times with 15 min intervals between additions and the reaction was subsequently allowed to stir for a total of 2.5 hrs. The reaction was quenched with Amberlite IR120, H form ion exchange resin. The mixture was filtered, evaporated and the residue was suspended in 5% methanol/dichloromethane. The suspension was filtered and the white precipitate was collected. White solid (3.3 mg, 77%). 1 H NMR (600 MHz, Methanol-d4) S22

23 (Anomers 1.00α : 0.35β) δ 5.17 (d, J = 3.3 Hz, 1H, α 1-H), 4.46 (q, J = 6.7 Hz, 1H, α C-H), 4.31 (dd, J = 9.3, 4.3 Hz, 1H, α isoglutamine methyne), 4.28 (q, J = 7.0 Hz, 1H, α alanine C-H) 3.82 (dd, J = 10.7, 3.4 Hz, 1H, α 2-H), (m, 1H, α 5-H, α 6-H), 3.72 (dd, J = 12.0, 5.2 Hz, 1H, α 6 -H), (m, 1H, α 3-H), (m, 1H, α 4-H), 2.76 (qq, J = 13.2, 6.8 Hz, 2H, α levulinyl methylene), 2.47 (qq, J = 13.6, 6.7 Hz, 2H, α levulinyl methylene), (m, 2H, α isoglutamine methylene), (m, 1H, α isoglutamine methylene), 2.16 (s, 3H, α levulinyl methyl), (m, 1H, α isoglutamine methylene), 1.40 (d, J = 7.1 Hz, 3H, α alanine methyl), 1.38 (d, J = 6.7 Hz, 3H, α methyl). 13 C NMR (151 MHz, Methanol-d4) δ (carbonyl), (carbonyl), (carbonyl), (carbonyl), (carbonyl), (carbonyl), (α 1-C), (α 3-C), (α C-H), (α 5-C), (α 4-C), (α 6-C), (α 2-C), (α isoglutamine methyne), (α alanine C-H), (α levulinyl methylene), (α levulinyl methylene), (α levulinyl methyl), (α isoglutamine methylene), (α isoglutamine methylene), (α methyl), (α alanine methyl). LRMS (ESI-Pos) for C 22 H 36 N 4 O 12 (548.23): [M+H] +. Scheme S8. (R)-5-amino-4-((S)-2-((R)-2-(((2R,3R,4R,5S,6R)-3-(2-azidoacetamido)-2,5-dihydroxy-6- (hydroxymethyl)tetrahydro-2h-pyran-4-yl)oxy)propanamido)propanamido)-5-oxopentanoic acid (13): 2-azidoacetic acid 2,5-Dioxopyrrolidin-1-yl ester prepared as follows: 2-azidoacetic acid S23

24 (135 mg, mmol) and N-hydroxysuccinimide (119.6 mg, 1.04 mmol) dissolved in 2 ml anhydrous dimethylformamide with stirring under nitrogen. 1-ethyl-3-(3- dimethylaminopropyl)carbodiimide (227.6 mg, 1.18 mmol) was subsequently added and the reaction stirred 16 hrs and then was diluted with dichloromethane. The reaction was washed with water, aqueous 1N HCl (3x) and aqueous saturated sodium chloride. The organic layer was dried with sodium sulfate and evaporated to yield a clear, semi-viscous oil. Anhydrous diethyl ether was added to the oil to yield a white precipitate. The ether layer was decanted and the precipitate was dried. White powder (106.4 mg, 54%). 1 H NMR (600 MHz, Chloroform-d) δ 4.24 (s, 2H, 2-azidoacetic acid methylene), 2.88 (s, 4H, NHS methylene). 13 C NMR (151 MHz, Chloroform-d) δ (carbonyl), (carbonyl), (2-azidoacetic acid methylene), (NHS methylene). 3 (5.0 mg, mmol) and sodium carbonate (4.7 mg, mmol) suspended in 150 μl anhydrous methanol with stirring under nitrogen. 2-azidoacetic acid 2,5-Dioxopyrrolidin-1-yl ester (2.2 mg, mmol) was added in 5 aliquots with 15 min intervals between additions and the reaction was allowed to stir for a total of 2 hrs. The reaction was quenched with Amberlite IR120, H form ion exchange resin. The mixture was filtered, evaporated and the residue was suspended in 5% methanol/dichloromethane. The suspension was filtered and the white precipitate was collected. White solid (5.2 mg, 88%). 1 H NMR (600 MHz, Methanol-d4) (Anomers 1.00α : 0.16β) δ 5.22 (d, J = 3.4 Hz, 1H, α 1-H), 4.45 (q, J = 6.8 Hz, 1H, α C-H), 4.32 (dd, J = 9.3, 4.4 Hz, 1H, α isoglutamine), 4.30 (q, J = 7.1, 6.5 Hz, 1H, α alanine C-H), 3.91 (d, J = 15.9 Hz, 1H, α 2-azidoacetic acid methylene), 3.86 (d, J = 16.0 Hz, 1H, α 2-azidoacetic acid methylene), 3.86 (dd, J = 10.6, 3.3 Hz, 1H, α 2-H), (m, 2H, α 5-H, α 6-H), 3.72 (dd, J = 11.9, 5.2 Hz, 1H, α 6 -H), (m, 1H, α 3-H), (m, 1H, α 4-H), 2.46 S24

25 2.32 (m, 2H, α isoglutamine methylene), (m, 1H, α isoglutamine methylene), (m, 1H, α isoglutamine methylene), 1.41 (d, J = 7.1 Hz, 1H, α alanine methyl), 1.38 (d, J = 6.7 Hz, 1H, α methyl). 13 C NMR (151 MHz, Methanol-d4) δ (carbonyl), (carbonyl), (carbonyl), (carbonyl), (carbonyl), (α 1-C), (α 3- C), (α C-H), (α 5-C), (α 4-C), (α 6-C), (α 2-C), (α isoglutamine methyne), (α 2-azidoacetic acid methylene), (α alanine C-H), (α isoglutamine methylene), (α isoglutamine methylene), (α methyl), (α alanine methyl). LRMS (ESI-Pos) for C 19 H 31 N 7 O 11 (533.21): [M+H] +. IR (ATR probe): Azide cm -1 (medium). Scheme S9. (R)-5-amino-4-((S)-2-((R)-2-(((2R,3R,4R,5S,6R)-3-(5-(dimethylamino)naphthalene-1- sulfonamido)-2,5-dihydroxy-6-(hydroxymethyl)tetrahydro-2h-pyran-4- yl)oxy)propanamido)propanamido)-5-oxopentanoic acid (14): 3 (4.0 mg, mmol) dissolved in 200 μl water with stirring and the ph was raised to 9 with an aqueous saturated sodium bicarbonate solution. The reaction was cooled to 4 C and a solution of 12 mg of dansyl chloride in 150 μl dimethyl formamide was prepared and was added to the reaction in 30 μl aliquots once every hour over a 5 hour period after which the reaction was allowed to continue stirring at 4 C for a total of 24 hours. Purified by HPLC (5% acetonitrile in water with 0.1% S25

26 trifluoroacetic acid isocratic flow for 5 mins then 30 min gradient to 100% acetonitrile with 0.1% trifluoroacetic acid). Light yellow solid (2.9 mg, 48%). 1 H NMR (600 MHz, Methanold4) δ 8.54 (d, J = 8.6 Hz, 1H, aromatic), 8.50 (d, J = 8.7 Hz, 1H, aromatic), 8.29 (d, J = 6.3 Hz, 1H, aromatic), (m, 1H, aromatic), (m, 1H, aromatic), 7.41 (d, J = 7.6 Hz, 1H, aromatic), 4.89 (d, J = 3.2 Hz, 1H, α 1-H), 4.47 (q, J = 7.0 Hz, 1H, α C-H), (m, 2H, α alanine methyl, α isoglutamine methyne), (m, 2H, α 5-H, α 6-H), (m, 1H, α 6 -H), 3.47 (dd, J = 10.3, 8.8 Hz, 1H, α 3-H), 3.25 (t, J = 9.2 Hz, 1H, α 4-H), 2.98 (s, 6H, N-methyl), 2.95 (dd, J = 10.4, 3.2 Hz, 1H, α 2-H), (m, 2H, α isoglutamine methylene), (m, 1H, α isoglutamine methylene), (m, 1H, α isoglutamine methylene), 1.43 (d, J = 7.1 Hz, 3H, α alanine methyl), 0.93 (d, J = 6.8 Hz, 3H, methyl). 13 C NMR (151 MHz, Methanol-d4) δ (carbonyl), (carbonyl), (carbonyl), (carbonyl), (aromatic), (aromatic), (aromatic), (aromatic), (aromatic), (aromatic), (aromatic), (aromatic), (aromatic), (aromatic), (α 1-C), (α 3-C), (α C-H), (α 5-C), (α 4-C), (α 6-C), (α 2-C), (α isoglutamine methylene), (α alanine C-H), (α N-methyl), (α isoglutamine methylene), (α isoglutamine methylene), (α methyl), (α alanine methyl). LRMS (ESI-Pos) for C 29 H 41 N 5 O 12 S (683.25): [M+H] +. S26

27 Scheme S10. (R)-5-amino-4-((S)-2-((R)-2-(((2R,3R,4R,5S,6R)-2,5-dihydroxy-6-(hydroxymethyl)-3-(5- ((3aS,4S,6aR)-2-oxohexahydro-1H-thieno[3,4-d]imidazol-4-yl)pentanamido)tetrahydro-2Hpyran-4-yl)oxy)propanamido)propanamido)-5-oxopentanoic acid (15): 3 (5.1 mg, mmol) and sodium carbonate (3.5 mg, mmol) suspended in 200 μl anhydrous methanol with stirring under nitrogen. Succinimidyl D-Biotin 3.8 mg, mmol) added 3 times with 20 min intervals between additions and the reaction was allowed to stir for a total of 3 hrs. The reaction was quenched with Amberlite IR120, H form ion exchange resin. The mixture was filtered, evaporated and the residue was suspended in 5% methanol/dichloromethane. The suspension was filtered and the white precipitate was collected. White solid (6.1 mg, 79%). 1 H NMR (600 MHz, Methanol-d4) (Anomers 1.00α : 0.21β) δ 5.21 (d, J = 3.4 Hz, 1H, α 1-H), 4.50 (dd, J = 7.7, 5.0 Hz, 1H,α 9-position), 4.45 (q, J = 6.7 Hz, 1H, α C-H), (m, 3H, α 10-position biotin, α isoglutamine methyne, α alanine C-H), 3.84 (dd, J = 10.7, 3.4 Hz, 1H, α 2-H), 3.79 (m, 3H, α 5-H, α 6-H), 3.72 (dd, J = 11.6, 5.0 Hz, 1H, α 6 -H), 3.65 (dd, J = 10.5, 8.9 Hz, 1H, α 3- H), (m, 1H, α 4-H), (m, 1H, α 6-position biotin), 2.93 (dd, J = 12.7, 5.0 Hz, 1H, α 8-position biotin), (m, 1H, α 8-position biotin), (m, 2H, α isoglutamine methlene), 2.24 (t, J = 7.5 Hz, 2H, α 2-position biotin), (m, 1H, α isoglutamine methylene), 1.92 (dtd, J = 14.3, 8.8, 8.1, 4.6 Hz, 1H, isoglutamine methylene), (m, 4H, α 3-position biotin, α 5-position biotin), (m, 2H, α 4-position biotin), 1.40 (d, J = 7.1 Hz, 3H, α alanine methyl), 1.38 (d, J = 6.8 Hz, 3H, α methyl). 13 C NMR (151 MHz, Methanol-d4) δ (carbonyl), (carbonyl), (carbonyl), (carbonyl), (carbonyl), (carbonyl), (α 1-C), (α 3-C), (α C-H), (α 5-C), (α 4-C), (α 10-position biotin), (α 6-C), (α 9-position S27

28 biotin), (α 6-position biotin), (α 2-C), (α isoglutamine methyne), (α alanine C-H), (α 8-position biotin), (α 2-position biotin), (α isoglutamine methylene), (α 4-position biotin), (α 3-position biotin), (α isoglutamine methylene), (α 5-position methylene), (α methyl), (α alanine methyl). LRMS (ESI-Pos) for C 27 H 44 N 6 O 12 S (676.27): [M+H] +. Scheme S11. (R)-4-((S)-2-((R)-2-(((2R,3R,4R,5S,6R)-3-acetamido-2,5-dihydroxy-6- (hydroxymethyl)tetrahydro-2h-pyran-4-yl)oxy)propanamido)propanamido)-n 1 -(2-(5- (dimethylamino)naphthalene-1-sulfonamido)ethyl)pentanediamide (16): 1 (5.0 mg, mmol) and 5-Dimethylaminonaphthalene-1-(N-(2-Aminoethyl))sulfonamide (5.9 mg, mmol) dissolved in 250 μl of anhydrous methanol under N 2 gas. 1-Hydroxybenzotriazole hydrate (wetted with not less than 20 wt % water) (2.0 mg, mmol), 2,4,6-trimethylpyridine (4.0 µl, mmol) and then 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide (2.9 mg, mmol) were subsequently. The reaction stirred 18 hrs and was evaporated. Purified by HPLC (5% acetonitrile in water with 0.1% trifluoroacetic acid isocratic flow for 5 mins then 30 min gradient to 100% acetonitrile with 0.1% trifluoroacetic acid). Product can also be purified by S28

29 column chromatography with C2 silica (10% methanol/dichloromethane then 20% methanol/dichloromethane once produce begins to elute) Light yellow powder (quantitative). 1 H NMR (600 MHz, Methanol-d4) (Anomers 1.00α : 0.15β) δ 8.46 (d, J = 8.5 Hz, 1H, aromatic), 8.26 (d, J = 8.6 Hz, 1H, aromatic), 8.10 (d, J = 8.1 Hz, 1H, aromatic), (m, 2H, aromatic), 7.22 (d, J = 7.5 Hz, 1H, aromatic), 5.06 (d, J = 3.4 Hz, 1H, α 1-H), 4.29 (q, J = 6.7 Hz, 1H, α C-H), (m, 2H, α alanine C-H, α isoglutamine methyne), 3.77 (dd, J = 10.6, 3.4 Hz, 1H, α 2-H), (m, 2H, α 5-H, α 6-H), 3.61 (dd, J = 11.9, 5.3 Hz, 1H, α 6 -H), 3.54 (dd, J = 10.5, 8.9 Hz, 1H, α 3-H), (m, 1H, α 4-H), 3.12 (dt, J = 12.3, 6.1 Hz, 1H, α ethylene linker methylene), 3.06 (dt, J = 13.5, 6.2 Hz, 1H, α ethylene linker methylene), 2.84 (t, J = 6.2 Hz, 2H, α ethylene linker methylene), 2.82 (s, 6H, α N-methyl), (m, 3H, α isoglutamine methylene, α isoglutamine methylene), 1.85 (s, 2H, acetyl), (m, 1H, α isoglutamine methylene), 1.30 (d, J = 7.2 Hz, 3H, α Alanine methyl), 1.29 (d, J = 6.8 Hz, 3H, α methyl). 13 C NMR (151 MHz, Methanol-d4) δ (carbonyl), (carbonyl), (carbonyl), (carbonyl), (carbonyl), (aromatic), (aromatic), (aromatic), (aromatic), (aromatic), (aromatic), (aromatic), (aromatic), (aromatic), (aromatic), (α 1-C), (α 3-C), (α C-H), (α 5-C), (α 4-C), (α 6-C), (α 2-C), (α isoglutamine methyne), (α alanine C-H), (N-methyl), (α ethylene linker methylene), (α ethylene linker methylene), (α isoglutamine methylene), (α isoglutamine methylene), (α acetyl), (α methyl), (α alanine methyl). LRMS (ESI-Pos) for C 33 H 49 N 7 O 12 S (767.32): [M+H] +. S29

30 Scheme S12. (R)-5-amino-4-((S)-2-((R)-2-(((2R,3R,4R,5S,6R)-2,5-dihydroxy-3-(2-hydroxyacetamido)-6- (hydroxymethyl)tetrahydro-2h-pyran-4-yl)oxy)propanamido)propanamido)-n 1 -(2-(5- (dimethylamino)naphthalene-1-sulfonamido)ethyl)pentanediamide (17): 2 (5.0 mg, mmol) and 5-Dimethylaminonaphthalene-1-(N-(2-Aminoethyl))sulfonamide (5.9 mg, mmol) dissolved in 250 μl of anhydrous methanol under N 2 gas. 1-Hydroxybenzotriazole hydrate (wetted with not less than 20 wt % water) (2.0 mg, mmol), 2,4,6-trimethylpyridine (4.0 µl, mmol) and then 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide (2.9 mg, mmol) were subsequently. The reaction stirred 16 hrs and was evaporated. Product can also be purified by column chromatography with C2 silica (10% methanol/dichloromethane then 20% methanol/dichloromethane once produce begins to elute) Light yellow powder (quantitative). 1 H NMR (600 MHz, Methanol-d4) (Anomers 1.00α : 0.27β) δ 8.57 (d, J = 8.5 Hz, 1H, aromatic), 8.33 (d, J = 8.6 Hz, 1H, aromatic), 8.19 (d, J = 7.2 Hz, 1H, aromatic), 7.59(q, J = 7.9 Hz, 2H, aromatic), 7.28 (d, J = 7.5 Hz, 1H, aromatic), 5.16 (d, J = 3.2 Hz, 1H, α 1-H), 4.40 (q, J = 6.7 Hz, 1H, α C-H), (m, 2H, α alanine C-H, α isoglutamine methyne), 3.99 (s, 1H, α glycolyl methylene), 3.96 (dd, J = 10.5, 3.2 Hz, 1H, α 2-H), (m, 2H, α 5-H, α 6-H), S30

31 3.73 (dd, J = 11.9, 5.1 Hz, 1H, α 6 -H), (m, 1H, α 3-H), 3.51 (t, J = 9.4 Hz, 1H, α 4- H), (m, 2H, α ethylene linker methylene), 2.94 (t, J = 6.1 Hz, 2H, α ethylene linker methylene), 2.88 (s, 6H, α N-methyl), (m, 3H, α isoglutamine methylene, α isoglutamine methylene), (m, 1H, α isoglutamine methylene), 1.40 (d, J = 7.1 Hz, 3H, α Alanine methyl), 1.38 (d, J = 6.8 Hz, 3H, α methyl). 13 C NMR (151 MHz, Methanol-d4) δ (carbonyl), (carbonyl), (carbonyl), (carbonyl), (carbonyl), (aromatic), (aromatic), (aromatic), (aromatic), (aromatic), (aromatic), (aromatic), (aromatic), (aromatic), (aromatic), (α 1-C), (α 3-C), (α C-H), (α 5-C), (α 4-C), (α glycolyl methylene), (α 6-C), (α 2-C), (α isoglutamine methyne), (α alanine C- H), (N-methyl), (α ethylene linker methylene), (α ethylene linker methylene), (α isoglutamine methylene), (α isoglutamine methylene), (α methyl), (α alanine methyl). LRMS (ESI-Pos) for C 33 H 49 N 7 O 13 S (783.31): [M+H] +. S31

32 III. Biological Assays Cell Culture All cell lines used were obtained from American Type Culture Collection (ATCC, Manassas, VA). Phoenix-E cells were used for the generation of retroviruses. HEK293T or HEK293T- Nod2-myc/Tet-op cells [S7] were cultured in DMEM, 10% FBS (Atlantic Biologicals), 2 mmglutamine, penicillin/streptomycin (1%) and grown in a humidified incubator at 37 C and 5% CO 2. NF-κB Activation Assay HEK293T cells were transfected for 16 hours with Lipofectamine LTX reagent (Invitrogen), 1.0 ng of WT Nod2 or empty plasmid (CMV empty vector), 10.0 ng of pgl4.10 NF-κB luciferase reporter plasmid and 1.0 ng of Renilla luciferase plasmid. Activity was induced by incubating 20 µm of compounds 1-3, 9 17 with the transfected cells. After 12 hours of treatment, NF-κB luciferase activity was measured using the Dual-Luciferase Reporter Assay (Promega) according to the manufacturer s instructions and normalized to Renilla luciferase activity. Relative NF-κB activation (RLU) (-) Nod2 (+) Nod2 Ctrl 1.0 ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± 0.4 Table S1. Tabulated relative NF-κB activation (RLU) from Figure 2. S32

33 Figure S1. Select compounds were tested at a range of concentrations in the NF-κB luciferase reporter assay overexpressing Nod2. The graph shows the relative responses normalized to the response from 20 µm MDP. All compounds were tested well above the apparent K D, and compounds 1, 2, 12, and 13 were tested above the apparent EC 50. Compound 3 only demonstrated activity at 200 µm, and the detected activity at this concentration is significantly lower than activity of 20 µm natural ligand. Construction of Tetracycline-regulated NOD2-expressing Stable Cell Lines Stable cell lines of HEK293T cells that express tetracycline-inducible NOD2Myc were established as previously reported [S7]. Antibodies Mouse monoclonal anti-myc, mouse monoclonal anti-β-actin antibody, and anti-mouse IgG HRP linked secondary antibody were purchased from Cell Signaling Technology. Immunoblotting Cells were rinsed with 1X PBS and appropriate amount of lysis buffer (1% triton-x, 2 mm EDTA, 4 mm Na 3 PO 4, 100 mm NaCl, 10 mm MES ph 5.8, 10 mm NaF, 1 protease inhibitor tablet) was added and lysed using 21 gauge needle. Protein quantification was performed using Bradford assay using BioRad Protein Assay Dye Reagent Concentrate according to the manufacturer s instruction. Samples were prepared using 5X loading buffer (250 mm TrisHCl ph 6.8, 10% SDS, 30% Glycerol 0.02% bromophenol blue, 5% BME) and boiled for 5 min. The S33

34 samples were electrophoresed in 7.5% polyacrylamide gels containing 0.1% sodium dodecyl sulfate. Western transfer onto PVDF membrane was carried out using semi-dry transfer at 25 V for 1 h. 10% nonfat dry milk in TBS-Tween (T) was used to block the membrane for 1 h and washed in TBS-T three times for 5 min each. The blots were incubated overnight at 4 C with the appropriate amount of antibodies prepared in 1% milk. After three washes, the membrane was incubated with HRP-conjugated secondary antibody for 60 min at room temperature. Following secondary incubation, the blot was washed three times in TBS-T and incubated with the substrate (GE) according to the manufacturer s instruction. The blots were exposed to Fuji Super RX-U Half Speed Blue films (Brandywine Imaging Inc) in the dark room. All western blots were performed at least three times independently. Using the replicates, the data were analyzed using Image Lab TM. Half-life Determination Cycloheximide was used at a concentration of 100 μg/ml, and the lysates were collected every 4 hours. Compounds of interest were incubated with cycloheximide starting at 0 hours. Cells were rinsed with 1X PBS and 140 L of lysis buffer (1% triton-x, 2 mm EDTA, 4 mm Na 3 PO 4, 100 mm NaCl, 10 mm MES ph 5.8, 10 mm NaF, 1 protease inhibitor tablet) was added on to the 12 well dishes and lysed using 21 gauge needle. Protein quantification was performed using Bradford assay using BioRad Protein Assay Dye Reagent Concentrate according to the manufacturer s instruction. Briefly, 1mL of 1X dye was mixed with 2 L of protein lysates and the absorbance was determined at 595nm. Absorbance values were converted to concentration using the standard curve obtained using the known concentrations of bovine serum albumin. 30 Lof protein samples were prepared using 5X loading buffer (250 mm TrisHCl ph 6.8, 10% S34

35 SDS, 30% Glycerol 0.02% bromophenol blue, 5% BME) and boiled for 5 min. Equal amount of protein lysates were subjected to western blot. The protein bands for Nod2 and Actin were quantified using Image Lab 5.0. Actin is used as the loading control and the ratio of the intensity of Nod2 to actin bands (I r ) were used to analyze the half-life values as previously described [S8]. Briefly, relative Nod2 band intensities were plotted against time assuming first-order decay (ln(i r ) vs. time). The rate constant was calculated using the negative slope of the line (k = - slope), and the corresponding half-life was calculated (T 1/2 = ln(2)/k). This was done for each replicate of each compound, the replicates were averaged and subjected to appropriate Student s t-tests against the control values. Compounds with a p-value < 0.05 compared to the control were considered to significantly improve Nod2 half-life. Half-life Gels Figure S2 Figure S3 S35

36 Figure S4 Figure S5 Figure S6 Figure S7 Figure S8 S36

37 IV. 1 H and 13 C NMR Spectra 1 H and 13 C NMR Spectra for 4.1 (Methanol d4) S37

38 S38

39 1 H and 13 C NMR Spectra for 4.2 (DMSO d6) S39

40 S40

41 1 H and 13 C NMR Spectra for 4.3 (Methanol d4) S41

42 S42

43 1 H and 13 C NMR Spectra for 5 (Methanol d4) S43

44 S44

45 1 H and 13 C NMR Spectra for 6 (Chloroform d) S45

46 S46

47 1 H and 13 C NMR Spectra for 6.1 (Chloroform d) S47

48 S48

49 1 H and 13 C NMR Spectra for 7 (Chloroform d) S49

50 S50

51 1 H and 13 C NMR Spectra for 3 (Deuterium Oxide) S51

52 S52

53 1 H and 13 C NMR Spectra for 1 (Methanol d4) S53

54 S54

55 1 H and 13 C NMR Spectra for 2 (Methanol d4) S55

56 S56

57 1 H and 13 C NMR Spectra for 8 (Methanol d4) S57

58 S58

59 1 H and 13 C NMR Spectra for 9 (Methanol d4) S59

60 S60

61 1 H and 13 C NMR Spectra for 10 (Methanol d4) S61

62 S62

63 1 H and 13 C NMR Spectra for 11 (Methanol d4) S63

64 S64

65 1 H and 13 C NMR Spectra for 12 (Methanol d4) S65

66 S66

67 1 H and 13 C NMR Spectra for 13 (Methanol d4) S67

68 S68

69 1 H and 13 C NMR Spectra for 14 (Methanol d4) S69

70 S70

71 1 H and 13 C NMR Spectra for 15 (Methanol d4) S71

72 S72

73 1 H and 13 C NMR Spectra for 16 (Methanol d4) S73

74 S74

75 1 H and 13 C NMR Spectra for 17 (Methanol d4) S75