Supporting Information

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1 Supporting Information Wiley-VCH Weinheim, Germany

2 A Convergent Synthesis of N-Linked Glycopeptides Clyde M. Kaneshiro, Katja Michael* 1. LC-MS analysis of crude glycopeptide ESI-TOF of glycopeptide 16 (zoomed in). 3. 1D- 1 H-NMR spectrum of purified glycopeptide 16 in [D 6 ]DMSO. 4. DQF-COSY spectrum of 16 in [D 6 ]DMSO. 5. TOCSY spectrum of the GlcNAc moiety in glycopeptide 16 in [D 6 ]DMSO/D 2 O. 6. NMR signal assignment for glycopeptide ESI-TOF spectrum of fully deprotected glycopeptide Experimental Section (Syntheses and characterization of 11, 17, and 18).

3 LC-MS analysis of crude glycopeptide 16: top) total ion current diagram; middle) ion extraction diagram of the mass range ; bottom) masses of the signal eluting at min. 2

4 ESI-TOF mass spectrum of glycopeptide 16, zoomed into the mass region It shows the mass signals for [M+H] + (left); [M+Na] + (middle), and a weak signal for [M+K] + (right). 3

5 1D- 1 H NMR spectrum of glycopeptide 16 in [D 6 ]DMSO. 4

6 DQF-COSY spectrum of glycopeptide 16 in [D 6 ]DMSO. 5

7 TOCSY spectrum of the GlcNAc moiety of glycopeptide 16 in [D 6 ]DMSO/D 2 O with presaturation of the water signal at ~ 3.7 ppm. Irradiation took place at 3.07 ppm (H-4, H-5). The signal overlap of H-4 and H-5 in unprotected GlcNAc residues is well documented. [21b] 6

8 NMR signal assignment for glycopeptide 16: 1 H-NMR (500 MHz, [D 6 ]DMSO, 25 C) δ=8.28 [d, 3 J (NH,α)=7.7 Hz,1H; NH (Asn)]; 8.10 [d, 3 J (NH,1)=9.3 Hz, 1H; NH-1]; 7.87 [d, 3 J=7.4 Hz, 2H; Fmoc,]; [m, 5H; NH (Val), NH-2, Fmoc, NH (Thr)]; 7.58 [m, 2H; NH (Ile), NH (Glu)]; 7.39 [t, 2H; Fmoc]; [m, 7H; Fmoc (2), Bn (5)]; 7.24 [s, 1H; NH (C-term. NH 2 )]; 6.82 [s, 1H; NH' (C-term. NH 2 )]; 5.07 [s, 2H; CH 2 (Bn)]; 4.78 [dd, 3 J (1,2)=9.3 Hz, 1H; H-1]; 4.56 [m, 1H; H-α (Asn)]; [m, 7H; H-α (Val), H-α (Thr), H-α (Ile), H-α (Pro), CH, CH 2 (Fmoc)]; 4.06 [m, 1H; H-α (Glu)]; 3.89 [m,1h; H-β (Thr)]; 3.67 [m, 1Hp; H-δ (Pro)]; 3.54 [m, 1H; H-δ' (Pro)]; 2.63 [dd, 2 J (β,β')=16.2 Hz, 3 J (α,β)=6.1 Hz, 1H; H-β (Asn)]; 2.39 [m, 2H; H-γ, H-γ' (Glu)]; 2.32 [dd, 3 J (α,β')=6.9 Hz, 1H; H-β' (Asn)]; [m, 4H; H-β (Val), H-β (Glu), H-β (Pro), H-γ (Pro)]; [m, 6H; Ac, H-β' (Glu), H-β' (Pro), H-γ' (Pro)]; 1.69 [m, 1H; H-β (Ile)]; 1.35 [m, 1H; H-γ (Ile)]; 1.11 [s, 9H; tbu]; 0.97 [ m, 4H; H-γ' (Ile), 3 H-γ (Thr)]; 0.92 [d, 3 J (β,γ)=6.7 Hz, 3H; 3 H-γ (Val)]; 0.86 [d, 3 J (β,γ')=8.2 Hz, 3H; 3 H-γ' (Val)] [m, 6H; 3 H-γ'' (Ile), 3 H-δ (Ile)] ppm. GlcNAc moiety of 16: 1 H-NMR (500 MHz, [D 6 ]DMSO/D 2 O, 25 C) δ=3.59 [d, 2 J (6,6 )=12.2 Hz, 1H; H-6]; 3.48 [dd, 3 J (2,3)= 9.3 Hz, 1H; H-2]; 3.40 [m, 1H, H-6 ]; 3.29 [m, 1H, H-3]; 3.07 [m, 2H, H-4, H-5] ppm. 7

9 LC-MS analysis of glycopeptide 19: top) ion extraction diagram for the mass range ; bottom) region of the doubly charged molecular ions, left [M+2H] 2+, middle [M+H+Na] 2+, right [M+2Na] 2+. 8

10 Experimental Section Synthesis of glycopeptide 11: peptide 2 (50.1 mg, mmol), and glycosylamine 5 (28.0 mg, mmol) were dissolved in 4 ml anhydrous CH 2 Cl 2, and passed through a syringe with powdered molecular sieves (4Å) into a Pyrex test tube with screw cap under argon. The mixture was irradiated in a Rayonet photoreactor at 26 C under magnetic stirring. After 54 h, HPLCanalysis indicated 95% conversion. The solvent was evaporated and the crude was chromatographed on silica with CHCl 3 /MeOH 15:1, which gave 36 mg of 11 (70% yield). R f =0.31 in CHCl 3 /MeOH 9:1. 1 H NMR: (500 MHz, [D 6 ]DMSO, 25 C): δ=8.52 (d, 3 J(NH,1)=9.4 Hz, 1H; NH-1), 8.30 [d, 3 J(NH,α)=7.8 Hz, 1H; NH (Asn)], 7.88 (d, 2H; Fmoc), 7.84 (d, 3 J(NH,2)=9.2 Hz, 1H; NH-2), 7.76 [d, 3 J(NH,α)=7.8 Hz, 1H; NH (Val)], [m, 3H; Fmoc (2); NH (Thr)], 7.57 [d, 3 J(NH,α=8.8 Hz, 1H, NH (Ile)], 7.52 [d, 3 J(NH,α=8.1 Hz, 1H; NH (Glu)], 7.41 (t, 2H; Fmoc), 7.31 (m, 2H; Fmoc), 7.24 [s, 1H, NH (C-term. NH 2 )], 6.83 [s, 1H, NH' (C-term. NH 2 )], 5.14 (dd, 3 J(1,2)=9.4 Hz, 1H; H-1), 5.08 (dd, 3 J(2,3)= 3 J(3,4)=9.8 Hz, 1H; H-3), 4.81 (dd, 3 J(4,5)=9.8 Hz, 1H; H-4), 4.60 [m, 1H; H-α (Asn)], [m, 4H; H- α(val); H-α(Ile); H-α(Thr); Fmoc (1)], [m, 4H; Fmoc (2); H-α(Pro); H-6], 4.02 [m, 1H; H-α(Glu)], [m, 3H; H-6'; H-β (Thr); H-2], 3.76 (1H, m; H-5), 3.69 [m, 1H; H-δ (Pro)], 3.57 [m, 1H; H-δ' (Pro)], 2.67 [m, 1H; H-β (Asn)], 2.35 [dd, 2 J(β,β')=16.3 Hz, 3 J(α,β')=7.3 Hz, 1H; H-β' (Asn)], 2.21 [m, 2H; H-γ, H-γ' (Glu)], [m, 9H; H-β (Pro), H- β (Val), H-β (Glu), 2 Ac], [m, 4H; H-γ (Pro), Ac], [m, 7H; H-β' (Pro), H-γ' (Pro), H-β' (Glu), H-β (Ile), Ac], 1.37 [m, 10H; H-γ (Ile), tbu (Glu)], 1.11 [s, 9H; tbu (Thr)], 1.03 [m, 1H; H-γ' (Ile)], 0.98 [d, 3H; 3 H-γ (Thr), 3 J(β,γ)=6.2 Hz]; 0.92 [d, 3 J(β,γ)=6.6 Hz, 3H; 3 H-γ (Val)], 0.86 [d, 3 J (β,γ')=6.7 Hz, 3H; 3 H-γ' (Val)], ppm [m, 6H, 3 H-γ'' (Ile), 3 H-δ (Ile)]. Synthesis of glycopeptide 17: peptide 2 (76 mg, mmol), and glycosylamine 8 (70% pure, 29 mg, mmol) were dissolved in 5 ml anhydrous DMSO, and passed through a syringe with powdered molecular sieves (4Å) into a Pyrex test tube with screw cap under argon. The

11 mixture was irradiated in a Rayonet photoreactor at 26 C under magnetic stirring. After 72 h, HPLC-analysis indicated 84% conversion. The reaction mixture was concentrated in vacuum and purified by RP-HPLC, which gave 21 mg of 17 (45% yield). 1 H NMR: (500 MHz, [D 6 ]DMSO, 25 C): δ=8.28 [d, 3 J(NH,α)=7.5 Hz, 1H; NH (Asn)], 8.16 (d, 1H; NH-1), 7.88 (d, 2H; Fmoc), 7.84 (d, 3 J(NH,2)=7.8 Hz, 1H; NH-2), [m, 5H; NH (Val), Fmoc (2), NH (Thr), (NH- 2)], 7.53 [m, 2H; NH (Glu), NH (Ile)], 7.40 (t, 2H; Fmoc), 7.32 (m, 2H; Fmoc), 7.24 [s, 1H, NH (C-term. NH 2 )], 6.83 [s, 1H, NH' (C-term. NH 2 )], 4.81 (dd, 3 J(1,2)= 3 J(1,NH)=9.5 Hz, 1H; H-1), 4.56 [m, 1H; H-α (Asn)], [m, 5H; H-α (Val), H-α(Ile), H-α (Thr), Fmoc (1), (H-1)], [m, 3H; Fmoc (2), H-α (Pro)], 4.02 [m, 1H; H-α (Glu)], 3.88 [m, 1H; H-β (Thr)], [m, 2H; (H-6), H-δ (Pro)], [m, 3H; H-2, H-6, H-δ' (Pro)], [m, 3H; H-3, H-6', (H-2)], 3.36 [dd, 2 J(6,6')=11.5 Hz, 1H; (H-6')], [m, 2H; (H-3), H-4], 3.16 [m, 1H; (H-5)], 3.11 (m, 1H; H-5), 3.02 [dd, 1H; (H-4), 3 J(3,4)= 3 J(4,5)=9.2 Hz], 2.64 [dd, 2 J(β,β')=16.3 Hz, 3 J(α,β)=5.8 Hz, 1H; H-β (Asn)], 2.31 [dd, 3 J(α,β')=6.3 Hz, 1H; H-β' (Asn)], 2.21 [m, 2H; H-γ, H-γ' (Glu)], [m, 14H; H-β (Pro), H-β' (Pro), H-β (Val), H-β (Glu), H-β' (Glu), H-γ (Pro), H-γ' (Pro), H-β (Ile), 2 Ac], 1.37 [m, 10H; H-γ (Ile), tbu (Glu)], 1.11 [s, 9H; tbu (Thr)], 1.02 [m, 1H; H-γ' (Ile)], 0.98 [d, 3 J(β,γ)=6.2 Hz, 3H; 3 H-γ (Thr)], 0.92 [d, 3 J(β,γ)=6.7 Hz, 3H; 3 H-γ (Val)], 0.86 [d, 3 J(β,γ')=6.8 Hz, 3H; 3 H-γ' (Val)], ppm [m, 6H; 3 H-γ'' (Ile), 3 H-δ (Ile)]. All signals of the terminal GlcNAc moiety are listed in parenthesis. FAB-MS: calcd. for C 68 H 102 N 10 O 22, ; found, m/z [M+H] +, [M+Li] +, [M+Na] +. Synthesis of glycopeptide 18: peptide 2 (7.5 mg, 6 µmol), glycosylamine 9 (6.2 mg, 3 µmol), which contains ca. 20% hemiacetal after multiple lyophilizations, and HOBt (0.8 mg, 6 µmol), powdered molecular sieves, (4Å) (6 mg) and anhydrous DMSO (650 µl) were placed into a 5 mm NMR tube under argon. The tube was tightly capped, and irradiated with a 200 W mercury lamp under water cooling. RP HPLC indicated the consumption of peptide 2 after 12 h. The crude mixture was analyzed by RP HPLC, and the isolated glycopeptide 18 was characterized by FAB-MS: calcd. for C 114 H 178 N 12 O 57, ; found, m/z [M+Na] +.

12 MS Characterization of other key compounds: 2: calcd. for C 60 H 80 BrN 9 O 14, ; FAB-MS: m/z [M+H] + ; [M+Na] + ; 4: calcd. for C 61 H 85 BrN 10 O 14, ; FAB-MS: m/z [M+H] + ; 13: calcd. for C 67 H 100 N 10 O 20, ; FAB-MS: m/z [M+H] + ; [M+Li] + ; [M+Na] + ; 16: calcd. for C 63 H 87 N 9 O 17, ; FAB-MS: m/z [M+H] + ; [M+Li] + ; [M+Na] + ; 19: calcd. for C 91 H 152 N 12 O 55, ; ESI-MS, m/z [M+2H] 2+ ; [M+H+Na] 2+ ; [M+2Na] 2+ ; [M+2H+Na] 3+ ; [M+3Na] 3+.