Combining triazole ligation and enzymatic glycosylation on solid phase simplifies the synthesis of very long glycoprotein analogues

Solid phase chemical ligation followed by enzymatic glycosylation exploits the advantages of a solid support to minimize the purification steps, constituting a promising approach for the synthesis of complex glycoproteins.


ESI-HRMS
The coupling and Fmoc deprotection of the N-terminal Val residue were performed by automated SPPS on the peptide resin S7 (5 µmol). The activated carbonate N 3 -Esoc-ONp [7] (5 equiv., dissolved in DMF) and iPr 2 NEt (5 equiv.) were then added to the peptide resin (1 equiv., swelled in DMF prior to the reaction), and the resulting suspension was stirred overnight at room temperature. The resin was then thoroughly washed with DMF. The resin was further washed with CH 2 Cl 2 , and treated with TFA/iPr 3 SiH/1,3 dimethoxybenzene (90:5:5) for 2 h. Peptide 1 was precipitated by dilution with ice-cold diethyl ether, recovered by centrifugation, washed 3 times with diethyl ether then dried under reduced pressure. The crude mixture was purified by semi-preparative HPLC to yield peptide 1 as a white fluffy powder (7.4 mg, 1.6 µmol, 32%* isolated yield based on the original resin loading).
*: estimated considering a MW = 4595 g/mol taking into account 4 trifluoroacetate counter-anions, as predicted from the sequence: 2Arg, 2His = +4 overall charge. Water content is not taken into account. Supplementary scheme S6 -Solid-phase synthesis of peptide 2

ESI-HRMS
The coupling and Fmoc deprotection of the N-terminal Val residue were performed by automated SPPS on the peptide resin S7 (5 µmol). The activated carbonate S3 (5 equiv., dissolved in DMF) and iPr 2 NEt (5 equiv.) were then added to the peptide resin (1 equiv., swelled in DMF prior to the reaction), and the resulting suspension was stirred overnight at room temperature. The resin was then thoroughly washed with DMF. The resin was further washed with CH 2 Cl 2 , and treated with TFA/iPr 3 SiH/1,3 dimethoxybenzene (90:5:5) for 2 h. Peptide 2 was precipitated by dilution with ice-cold diethyl ether, recovered by centrifugation, washed 3 times with diethyl ether then dried under reduced pressure. The crude mixture was purified by semi-preparative HPLC to yield the peptide 2 as a white fluffy powder (3.1 mg, 0.7 µmol, 13%* isolated yield based on the original resin loading).
*: estimated considering a MW = 4642 g/mol taking into account 5 trifluoroacetate counter-anions, as predicted from the sequence: Linker, 2Arg, 2His = +5 overall charge. Water content is not taken into account. Supplementary scheme S7-Solid-phase synthesis of peptide 3

ESI-HRMS
The coupling and Fmoc deprotection of the N-terminal Val residue were performed by automated SPPS on the peptide resin S7 (5 µmol). The compound S4 (5 equiv., dissolved in DMF) was then added to the peptide resin (1 equiv., swelled in DMF prior to the reaction), and the resulting suspension was stirred overnight at room temperature. The resin was then thoroughly washed with DMF. The resin was further washed with CH 2 Cl 2 , and treated with TFA/iPr 3 SiH/1,3 dimethoxybenzene (90:5:5) for 2 h. Peptide 3 was precipitated by dilution with ice-cold diethyl ether, recovered by centrifugation, washed 3 times with diethyl ether then dried under reduced pressure. The crude mixture was purified by semi-preparative HPLC to yield peptide 3 as a white fluffy powder (6.3 mg, 1.4 µmol, 27%* isolated yield based on the original resin loading). *: estimated considering a MW = 4593 g/mol taking into account 4 trifluoroacetate counter-anions, as predicted from the sequence: 2Arg, 2His = +4 overall charge. Water content is not taken into account. Supplementary scheme S8 -Solid-phase synthesis of peptide 4

ESI-HRMS
Compound S6 (2 equiv., dissolved in DMF), HATU (2 equiv.) and iPr 2 NEt (3 equiv.) were added to the peptide resin S7 (5 µmol, 1 equiv., swelled in DMF prior to the reaction), and the resulting suspension was stirred overnight at room temperature. The resin was thoroughly washed with DMF. The resin was further washed with CH 2 Cl 2 , and treated with TFA/iPr 3 SiH/1,3 dimethoxybenzene (90:5:5) for 2 h. Peptide 4 was precipitated by dilution with ice-cold diethyl ether, recovered by centrifugation, washed 3 times with diethyl ether then dried under reduced pressure. The crude mixture was purified by semi-preparative HPLC and lyophilized to yield the peptide 4 as a white fluffy powder (7.1 mg, 1.5 µmol, 31%* isolated yield based on the original resin loading).
*: estimated considering a MW = 4611 g/mol taking into account 4 trifluoroacetate counter-anions, as predicted from the sequence: 2Arg, 2His = +4 overall charge. Water content is not taken into account.

Muc1
Supplementary scheme S9 -Solid-phase synthesis of peptide S8 Peptide 4 (5 µmol) and 4-pentynoic acid (5 mg, 50 µmol, 10 equiv.) were dissolved in 400 µL of a 2:8 mixture of MeOH and 100 mM HEPES buffer pH 7.5. The reaction mixture was introduced in a 2 mL microcentrifuged tube sealed with a rubber septum and was deoxygenated through several successive vacuum (15 mbar) / argon cycles. Then, a mixture containing copper(I) bromide dimethyl sulfide complex (10 equiv.) and THPTA (15 equiv.) dissolved under argon in 50 µL of deoxygenated NMP was added, the resulting mixture was further deoxygenated and was stirred for 1 h at room temperature. Reaction mixture was analyzed by HPLC to check the total consumption of the peptide 4. The crude mixture was purified by semi-preparative HPLC and lyophilized to yield the peptide 4' as a white fluffy powder (6.1 mg, 1.3 µmol, 26%* isolated yield based on the original resin loading).
*: estimated considering a MW = 4707 g/mol taking into account 4 trifluoroacetate counter-anions, as predicted from the sequence: 2Arg, 2His = +4 overall charge. Water content is not taken into account. Supplementary figure S11 -ESI-HRMS mass spectrum of peptide 4' Table S1 S9 Peptides 1, 2, 3 and 4 (100 µM final peptide concentration) were subjected to a set of varied conditions to evaluate the stability of the four linkers in solution. Stabilities of each linker were followed by analytical HPLC. The amount of remaining intact peptide linker was measured by integration of the HPLC peaks at l = 214 nm. Supplementary scheme S11-Solid-phase synthesis of peptide 3'

Proportion of intact linker
The coupling and Fmoc deprotection of the N-terminal Val residue were performed by automated SPPS on the peptide resin S7 (1 µmol). 2-Acetyl-5,5-dimethyl-1,3cyclohexanedione (5 equiv., dissolved in DMF) was then added to the peptide resin (1 equiv., swelled in DMF prior to the reaction), and the resulting suspension was stirred overnight at room temperature. The resin was then thoroughly washed with DMF. The resin was further washed with CH 2 Cl 2 , and treated with TFA/iPr 3 SiH/1,3 dimethoxybenzene (90:5:5) for 2 h. Peptide 3' was precipitated by dilution with ice-cold diethyl ether, recovered by centrifugation, washed 3 times with diethyl ether then dried under reduced pressure. The crude mixture was purified by semi-preparative HPLC to yield peptide 3' as a white fluffy powder (1.5 mg, 0.33 µmol, 33%* isolated yield based on the original resin loading). *: estimated considering a MW = 4512 g/mol taking into account 4 trifluoroacetate counter-anions, as predicted from the sequence: 2Arg, 2His = +4 overall charge. Water content is not taken into account.  Supplementary scheme S12 -Reduction of protected alkyne by aqueous hydrazine solution Peptide 4 was dissolved in a 1 M aqueous hydrazine solution (100 µM final peptide concentration, pH 9.5). Reaction was followed by analytical HPLC (column: Nucleosil ® , gradient: from 25% to 55% B over 30 min). The amount of resulting products was measured by integration of the HPLC peaks at l = 214 nm.

A B
Supplementary figure S17 -A) ESI-HRMS mass spectrum of peptide S9 B) ESI-HRMS mass spectrum of peptide S10

D.2 Case study of the reduction of a model TIPS-alkyne with aqueous hydrazine and hydroxylamine
Supplementary scheme S13 -Reduction of a model TIPS-alkyne with aqueous hydrazine and hydroxylamine 2-Phenyl-N- [3-(triisopropylsilyl)prop-2-ynyl] acetamide S11 (CAS N°: 1189342-28-6) [8] was subjected to a set of aqueous conditions to evaluate the kinetics of the reduction of the alkyne moiety by hydrazine and hydroxylamine. S11 was directly dissolved in the different aqueous mixtures (1 mM final concentration) and stirred under an open air atmosphere at room temperature. The reaction was followed by analytical HPLC (column: Chromolith ® ; gradient: from 25% to 55% B over 5 min). The amount of the products S12 and S13 resulting from the reduction of S11 was measured by integration of the HPLC peaks at l = 254 nm. Addition of sodium ascorbate prevented the reduction of TIPS-alkyne. We hypothesize that AscNa helps avoiding the air oxidation of hydrazine into diimide.  Supplementary figure S19 -Copy of the 1 H NMR spectrum of pure S11 and S13, and S12 in mixture

D.3 Case study of the reduction of a model terminal alkyne with aqueous hydrazine and hydroxylamine
Supplementary scheme S14 -Reduction of a model terminal alkyne with aqueous hydrazine and hydroxylamine N-Propargyl-2-phenylacetamide S14 (CAS N°: 174271-37-5) was subjected to a set of aqueous conditions to evaluate the kinetics of the reduction of the alkyne moiety by hydrazine and hydroxylamine. S14 was directly dissolved in the different aqueous mixtures (1 mM final concentration) and stirred under an open air atmosphere at room temperature. The reaction was followed by analytical HPLC (column: Chromolith ® ; gradient: from 20% to 50% B over 5 min). The amount of the products S15 and S16 resulting from the reduction of S14 was measured by integration of the HPLC peaks at l = 254 nm.  a e S14 S15 S16 Supplementary figure S21 -Copy of the 1 H NMR spectrum (600 MHz, CDCl 3 ) of S14 and S15 and S16 in mixture Selected data for S14 (not isolated from the mixture): 1 H NMR (600 MHz, CDCl 3 ) δ = 3.93 (dd, J = 5.3, 2.6 Hz, 2H), 3.51 (s, 2H), 2.11 (t, J = 2.6
Selected data for S15 (not isolated from the mixture):
[ 9 ] A 65% elongation yield was obtained, determined by the titration of the first and last ( 2 Thr) Fmoc group deprotection (UV, 301 nm). Peptide resin (25 µmol, 1 equiv., swelled in DMF prior to the reaction) was further washed with CH 2 Cl 2 , and treated with TFA/iPr 3 SiH/1,3 dimethoxybenzene (90:5:5) for 2 h. Peptide 5 was precipitated by dilution with ice-cold diethyl ether, recovered by centrifugation, washed 3 times with diethyl ether then dried under reduced pressure. The crude mixture was purified by semi-preparative HPLC and lyophilized to yield 5 as a white fluffy powder (30 mg, 5.7 µmol, 23%* isolated yield based on the original resin loading).
*: estimated considering a MW = 5267 g/mol taking into account 4 trifluoroacetate counter-anions, as predicted from the sequence: 2Arg, 2His = +4 overall charge. Water content is not taken into account.  Purified glycopeptide 5 (2 mmol) was dissolved in anhydrous methanol (2 mL) under an argon atmosphere. Freshly prepared 1% MeONa/MeOH solution was added dropwise at 0 °C until pH 10 was reached when spotting an aliquot of the solution on a wet pH indicator paper. After 1 h stirring at room temperature and confirmation of the completion of the reaction by RP-HPLC analysis, the reaction mixture was neutralized with acetic acid (10 µL). Volatiles were removed under reduced pressure and the residue was dissolved in mQ water (1 mL). Lyophilization yielded glycopeptide 6 as a white fluffy powder. Crude lyophilized glycopeptide 6 (100 nmol) was dissolved in anhydrous DMF (50 µL) under an argon atmosphere. 50 µL of a 200 mM tetrabutylammonium fluoride trihydrate in anhydrous DMF was added to the mixture. After 1 h stirring at room temperature and confirmation of the completion of the reaction by RP-HPLC, the reaction mixture was neutralized with acetic acid (10 µL) and diluted with water (2 mL). DMF and salts were removed using standard hydrophobic SPE cartridge. Lyophilization yielded glycopeptide 7 as a white fluffy powder. Crude lyophilized glycopeptide 7 (100 nmol) was dissolved in water (50 µL) and a 2M hydroxylamine solution (50 µL) was added to the mixture. After 2 h stirring at room temperature and confirmation of the completion of the reaction by RP-HPLC, the reaction mixture was neutralized with acetic acid (20 µL) and diluted with water (2 mL). Salts and hydroxylamine were removed using standard hydrophobic SPE cartridge. Lyophilization yielded glycopeptide 8 as a white fluffy powder.    Controlled Pore Glass (aminopropyl CPG beads TRISOPERL ® ) or polymethacrylate resin (SPRINBeads AH130) [10] was introduced in a syringe equipped with a polypropylene frit and a teflon stopcock and washed successively with DMF (3x), 10% iPr 2 NEt in CH 2 Cl 2 (3x), DMF (3x) and peptide synthesis-grade DMF (3x). Then, Fmoc-spacer-OH (2 equiv., see Table S3 Fmoc-NH-X-COOH,) and HATU (2 equiv.) dissolved in a minimum amount of DMF, were transferred by suction followed by iPr 2 NEt (4 equiv.). The resin suspension was mixed by rotation for 16 h. In case of incomplete reaction (positive Kaiser's test), this protocol was repeated once followed by capping with acetic anhydride. The Fmoc group was removed by a treatment by 20% piperidine in DMF (3 x 3min), followed by thorough wash with DMF. Finally, pentynoic acid was coupled using HCTU (10 equiv.) and iPr 2 NEt (20 equiv.) in DMF to give the respective alkyne-containing solid support.

F.2 Typical procedure for the release of the peptide
The peptide was released by cleaving the Dtpp linker through treatment with 1 M NH 2 -OH aqueous solution (1:10 v/v swollen resin/total volume), for 60 min at room temperature (addition of 100 mM AscNa is required for TIPS-protected alkynes). [11] This protocol was repeated once and the solid support was finally washed with de-ionized water. Released peptide was isolated from the mixture using standard procedures such as hydrophobic SPE cartridge.
[11] Note that contrary to TIPS-protected alkynes, terminal alkynes were much less sensitive to reduction during Dtpp cleavage, and did not require addition of ascorbate S54

S8
release of an analytical sample for HPLC /MS control 9a-c 17a-b X Support Muc1 X Support Muc1 S18a: Support = CPG; X = PAL S18b: Support = CPG; X = PEG300 S18c: Support = CPG; X = PEG3000 S18d: Support = SPRINbeads S18e: Support = SPRINbeads; X = PEG300 4, CuBr.Me 2 S, THPTA, HEPES (pH 7.5) / NMP Supplementary scheme S22 -Grafting of peptide 4 on alkyne functionalized resin A solution of the azide containing peptide (0.5 µmol) dissolved in 400 µL of a 1:1 mixture of NMP and 200 mM HEPES buffer pH 7.5 was added to a 2 mL microcentrifuge tube containing the alkyne functionalized solid support. To the suspension were subsequently added a 1 M aqueous aminoguanidine solution (15 µL, 30 equiv.), a 1 M aqueous tert-butanol solution (15 µL, 30 equiv.). The tube was sealed with a rubber septum and the resulting mixture was further deoxygenated through several successive vacuum (15 mbar) / argon cycles. Then, a mixture containing copper(I) bromide-dimethyl sulfide complex (30 equiv.) and THPTA (40 equiv.) dissolved under argon in 50 µL of deoxygenated NMP was added, the resulting suspension was further deoxygenated and was stirred for 5 h at 37 °C. The supernatant was analyzed by HPLC to check the total consumption of the azidopeptide. Capping of remaining solid-supported terminal alkyne was performed by adding 2-(2-azidoethoxy)-ethanol (100 equiv.). The mixture was stirred an additional hour at 37 °C. The resin was then extensively washed with NMP, de-ionized water, then repeatedly treated (3x) with a pH 7 aqueous buffer containing 6 M guanidinium chloride, 0.1 M EDTA and 0.1 M sodium dihydrogenophosphate for a few minutes then drained and finally extensively washed with deionized water.
A few beads of the resulting solid supported peptide were cleaved using the general procedure (p S53) to give crude peptide S8 which was analyzed by RP-HPLC and HRMS.  10a: Support = CPG; X = PAL 10b: Support = CPG; X = PEG300 10c: Support = CPG; X = PEG3000 S19a: Support = SPRINbeads S19b: Support = SPRINbeads; X = PEG300 TBAF/DMF S18a-e Supplementary scheme S23 -Solid supported deprotection of the TIPS group The solid supported peptide was introduced in a syringe fitted with a polypropylene frit and a teflon stopcock and was washed with anhydrous DMF (4x). A mixture of TBAF (100 equiv.) dissolved in anhydrous DMF (final concentration 100 mM) was added and the reaction mixture was stirred by syringe rotation for 15 min at room temperature. This protocol was repeated once and the resin was then extensively washed with DMF, a 0.1% aqueous TFA solution and finally de-ionized water. A few beads of the resulting solid supported peptide were cleaved to give crude peptide 11a which was analyzed by RP-HPLC and HRMS.  Muc1 Support Muc1 S18a: Support = CPG; X = PAL S18b: Support = CPG; X = PEG300 S18c: Support = CPG; X = PEG3000 S18d: Support = SPRINbeads S18e: Support = SPRINbeads; X = PEG300 Purified azido peptide 4 (5.01 mg, 1.09 µmol [a] ) was grafted on alkyne support 9a (100 mg) following the typical procedure (p S54). Then, solid-supported deprotection of the TIPS group was performed following the typical procedure (p S57). The whole resulting solid supported peptide were cleaved using the general procedure (p S53). A third cleavage with 1M aqueous NH 2 OH followed by a TFA cleavage was performed but no trace of peptide was observed by HPLC analysis. Released peptide was isolated from the mixture using a hydrophobic SPE cartridge to give pure peptide 11a as a white fluffy powder (4.14 mg, 0.99 µmol, 91% [b] ).

ESI-HRMS
[a]: estimated considering a MW = 4611 g/mol taking into account 4 trifluoroacetate counter-anions, as predicted from the sequence: 2Arg, 2His = +4 overall charge. Water content is not taken into account.
[b]: estimated considering a MW = 4192 g/mol taking into account 4 trifluoroacetate counter-anions, as predicted from the sequence: 2Arg, 2His = +4 overall charge. Water content is not taken into account. Solid supported peptide (20 nmol) was introduced in a 2 mL microcentrifuge tube and washed with de-ionized water (2 x 500 µL). To the solid was added 100 µL of an aqueous solution containing recombinant α-N-acetylgalactosaminyl transferase T1 [ 12 ] (GalNAc-T1, 6 mU), MES (50 mM, pH 6.5), MnCl 2 (15 mM), Bovine Serum albumin (BSA, 100 ng), DTT (1 mM) and UDP-GalNAc (2mM). The resulting suspension was mixed at 37 °C for 18 h under moderate agitation. Beads were then washed with de-ionized water (2 x 500 µL), in order to remove the UDP formed during the transfer reaction since it inhibits GalNAc-T1 activity. Solid supported peptide was then incubated under the same conditions as above for another 18 h and the resin was washed with de-ionized water. The resulting solid supported peptide was released under typical procedure (p S53) to give crude peptide which was analyzed by RP-HPLC and HRMS.

F.8 Kinetics of enzymatic glycosylation
Kinetics experiments were performed with solid supported peptide 10c. Conditions used for kinetics experiments on beads were identical to those described above, and reactions were stopped at different time points by washing the beads with water. Addition of freshly prepared enzyme and substrate solution was performed after 1 h, 3

F.9 Optimization of reaction conditions for enzymatic glycosylation
Attempts realized in order to improve the glycosylation score by addition of 0.1% (v/v) of Triton TM -X100 (Sigma-Aldrich) to the enzymatic mixture, showed no significative change in GalNAc incorporation.

F.10 Glycosylation site identification
After release from solid-support, glycopeptide 11g was purified by semi-preparative HPLC and lyophilized. O-glycosylation sites of the glycopeptides were identified by electron transfer dissociation (ETD) ESI-MS/MS analysis by a mass spectrometry as described above.  A solution of the azide-containing glycopeptide 6 (1 µmol) dissolved in 800 µL of a 1:1 mixture of NMP and 200 mM HEPES buffer pH 7.5 was added to a 2 mL microcentrifuge tube containing the alkyne functionalized solid support 9c (100 mg). To the suspension were subsequently added a 1 M aqueous aminoguanidine solution (30 µL, 30 equiv.), a 1 M aqueous tert-butanol solution (30 µL, 30 equiv.). The tube was sealed with a rubber septum and the resulting mixture was further deoxygenated through several successive vacuum (15 mbar) / argon cycles. Then, a mixture containing copper(I) bromide-dimethyl sulfide complex (30 equiv.) and THPTA (40 equiv.) dissolved under argon in 50 µL of deoxygenated NMP was added, the resulting suspension was further deoxygenated and was stirred for 5 h at 37 °C. The supernatant was analyzed by HPLC to check the total consumption of the azidopeptide. Capping of remaining solid-supported terminal alkyne was performed by adding 2-(2-azido-ethoxy)-ethanol (100 equiv.). The mixture was stirred an additional hour at 37 °C. The resin was then extensively washed with NMP, de-ionized water, then repeatedly treated (3x) with a pH 7 aqueous buffer containing 6 M guanidinium chloride, 0.1 M EDTA and 0.1 M sodium dihydrogenophosphate for a few minutes then drained and finally extensively washed with de-ionized water.
A few beads of the resulting solid supported peptide were cleaved to give crude glycopeptide S20' which was analyzed by RP-HPLC and HRMS. The solid supported glycopeptide S20 was introduced in a syringe fitted with a polypropylene frit and a teflon stopcock and was washed with anhydrous DMF (4x). A mixture of TBAF (100 equiv.) dissolved in anhydrous DMF (final concentration 100 mM) was added and the reaction mixture was stirred by syringe rotation for 15 min at room temperature. This protocol was repeated once and the resin was then extensively washed with DMF, a 0.1% aqueous TFA solution and finally de-ionized water to give the solid supported peptide 15.

ESI-HRMS
A few beads of the resulting solid supported peptide were cleaved to give crude glycopeptide 8 which was analyzed by RP-HPLC and HRMS.  Solid supported peptide 10c (100 nmol) was introduced in a 2 mL microcentrifuge tube and washed with de-ionized water (2 x 500 µL). A solution of N 3 -PEG 3 -biotin (450 µg, 1 µmol, 10 equiv) dissolved in 100 µL of a 1:1 mixture of NMP and 200 mM HEPES buffer pH 7.5 was then added. To the suspension were subsequently added a 1 M aqueous aminoguanidine solution (3 µL, 30 equiv.), a 1 M aqueous tert-butanol solution (3 µL, 30 equiv.). The tube was sealed with a rubber septum and the resulting mixture was further deoxygenated through several successive vacuum (15 mbar) / argon cycles. Then, a mixture containing copper(I) bromide-dimethyl sulfide complex (30 equiv.) and THPTA (40 equiv.) dissolved under argon in 10 µL of deoxygenated NMP was added, the resulting suspension was further deoxygenated and was stirred for 5 h at 37 °C. The resin was then extensively washed with NMP, de-ionized water, then repeatedly treated (3x) with a pH 7 aqueous buffer containing 6 M guanidinium chloride, 0.1 M EDTA and 0.1 M sodium dihydrogenophosphate for a few minutes then drained and finally extensively washed with de-ionized water.

ESI-HRMS
A few beads of the resulting solid supported peptide were cleaved to give crude peptide S23' which was analyzed by RP-HPLC and HRMS. Solid supported glycopeptide S21 (20 nmol) was introduced in a 2 mL microcentrifuge tube and washed with de-ionized water (2 x 500 µL). A solution of N 3 -PEG 3 -Biotin (450 µg, 1 µmol, 50 equiv) dissolved in 100 µL of a 1:1 mixture of NMP and 200 mM HEPES buffer pH 7.5 was then added. To the suspension were subsequently added a 1 M aqueous aminoguanidine solution (3 µL, 150 equiv.), a 1 M aqueous tert-butanol solution (3 µL, 150 equiv.). The tube was sealed with a rubber septum and the resulting mixture was further deoxygenated through several successive vacuum (15 mbar) / argon cycles. Then, a mixture containing copper(I) bromide-dimethyl sulfide complex (30 equiv.) and THPTA (40 equiv.) dissolved under argon in 10 µL of deoxygenated NMP was added, the resulting suspension was further deoxygenated and was stirred for 2 h at 37 °C. The resin was then extensively washed with NMP, de-ionized water, then repeatedly treated (3x) with a pH 7 aqueous buffer containing 6 M guanidinium chloride, 0.1 M EDTA and 0.1 M sodium dihydrogenophosphate for a few minutes then drained and finally extensively washed with de-ionized water.

ESI-HRMS
A few beads of the resulting solid supported peptide were cleaved to give crude glycopeptide 18 which was analyzed by RP-HPLC and HRMS.

I Fluorescence labeling of CPG supported peptides
All the differently substituted beads (9c, 10c, S23, S21 and 17) were washed with water (2x), PBS containing BSA (2 mg/mL). Substituted beads were subsequently incubated with labelling agent at room temperature, with moderate agitation. At the end of the incubation periods, all beads were washed in PBS, mounted on glass slides in a 1:1 mixture of PBS/mounting medium (Vectashield, Vector Laboratories, Burlingame, CA, USA) and observed with a Zeiss Axiovert 200 M microscope coupled with a Zeiss LSM 510 scanning device (Carl Zeiss Co. Ltd., Jena, Germany). Mouse anti human MUC1 antibodies (clones C595 and CB2, Serotec, Cergy, France) were used together at 0.5 µg/mL in PBS-BSA. After 45 min incubation followed by 3 washes in PBS, a secondary antibody (FITC-conjugated goat anti mouse IgG, Jackson ImmunoResearch, West Grove, PA, USA) diluted 7 µg/mL in PBS-BSA was added to the beads for 45 min. Incubations with the GalNAc-specific Vica villosa lectin (VVL) were routinely carried out for 45 min in PBS-BSA with 20 µg/mL biotin-labeled lectin or FITC-conjugated lectin (Vector Laboratories). The beads were washed in PBS and for the biotin-VVL treatment, further incubated with streptavidin Alexa-fluor 568 (Molecular Probes, Eugene, OR, USA) at 4 µg/mL in PBS-BSA for 45 min. Biotin-substituted beads were incubated with streptavidin Alexa-fluor 568 (Molecular Probes) at 4 µg/mL in PBS-BSA for 45 min. Whenever they were doubly labeled, the beads were incubated in the same conditions as above first with the antibodies and then with the couple biotin-VVL / streptavidin-Alexa-fluor 568, or first with FITC-VVL and then streptavidin-Alexa-fluor 568, or with the antibodies and then streptavidin Alexa-fluor 568. For an extensive labeling of the beads, FITC-VVL was used at 2 mg/mL for 18 h, followed by washes in PBS (6x). [1] no fluorescent labeling, [2] not determined, [3] positive FITC labeling, The backbone amide linker 4-(4-formyl-3,5-dimethoxyphenoxy)butyric acid, (34 mg, 125 µmol, 2.5 equiv.) was loaded on Rink amide ChemMatrix ® resin (96 mg, 0.52 mmol/g, 50 µmol) using HATU (47 mg, 125 µmol, 2.5 equiv.) and iPr 2 NEt (43 µL, 250 µmol, 5 equiv.) in DMF for 2 h at room temperature. The resin was then thoroughly washed with DMF. In case of incomplete reaction (positive Kaiser's test), was repeated once. 3-(Triisopropylsilyl)prop-2-yn-1-amine (53 mg, 250 µmol, 5 equiv.) and NaBH 3 CN (16 mg, 250 µmol, 5 equiv.) dissolved in 5 mL of DMF/MeOH/AcOH (7:2:1) were added to the resin (1 equiv., swelled in DMF prior to the reaction), and the resulting suspension was mixed overnight at 50 °C with N 2 bubbling. The resin was then thoroughly washed with DMF and CH 2 Cl 2 . Fmoc-His(Trt)-OH (310 mg, 0.5 mmol, 10 equiv.) was coupled onto the resulting resin with HATU (190 mg, 0.5 mmol, 10 equiv.) and iPr 2 NEt (175 µL, 1 mmol, 20 equiv.) in DMF overnight at room temperature. The elongation of the peptide was performed by standard automated solid phase synthesis (p S4) up to 2 Thr. Prolines 4 and 5 were coupled twice following the automated program for double couplings. A 85% elongation yield was obtained, determined by the titration of the first (BAL) and last ( 2 Thr) Fmoc group deprotection (UV, 301 nm). At this point, the resin was divided in two equal portions, for the production of both S24 and S25.
Compound S6 (2 equiv., dissolved in DMF), HATU (2 equiv.) and iPr 2 NEt (3 equiv.) were added to the peptide resin (25 µmol, 1 equiv., swelled in DMF prior to the reaction), and the resulting suspension was stirred overnight at room temperature. The resin was thoroughly washed with DMF. The resin was further washed with CH 2 Cl 2 , and treated with TFA/iPr 3 SiH/1,3 dimethoxybenzene (90:5:5) for 2 h. Peptide S24 was precipitated by dilution with ice-cold diethyl ether, recovered by centrifugation, washed 3 times with diethyl ether, dried under reduced pressure and lyophilized to yield crude peptide S24 as a white fluffy powder (42.7 mg, 17 µmol, 68%* isolated yield based on the original resin loading). This peptide was used without any further purification.
*: estimated considering a MW = 2514 g/mol taking into account 4 trifluoroacetate counter-anions, as predicted from the sequence: 1Arg, 1His = +2 overall charge. Water content is not taken into account. The solid phase elongation of S25 up to 2 Thr is described in the synthesis of S24 (pS82). N 3 Val-OH (7 mg, 50 µmol, 2 equiv., dissolved in DMF), HATU (19 mg, 50 µmol, 2 equiv.) and iPr 2 NEt (13 µL, 75 µmol, 3 equiv.,) were dissolved in DMF. The mixture was then added to the peptide resin (25 µmol, 1 equiv., swelled in DMF prior to the reaction), and the resulting suspension was stirred by syringe rotation for 2 h at room temperature. The resin was then thoroughly washed with DMF. The resin was further washed with CH 2 Cl 2 , and treated with TFA/iPr 3 SiH/1,3 dimethoxybenzene (90:5:5) for 2 h. Peptide S25 was precipitated by dilution with ice-cold diethyl ether, recovered by centrifugation, washed 3 times with diethyl ether, dried under reduced pressure and lyophilized to yield crude peptide S25 as a white fluffy powder (39 mg, 17 µmol, 69%* isolated yield based on the original resin loading). This peptide was used without any further purification.

ESI-HRMS
A few beads of the resulting solid supported peptide were cleaved using the general procedure (p S53) to give crude peptide S26' which was analyzed by RP-HPLC and HRMS. The solid supported peptide S26 was introduced in a syringe fitted with a polypropylene frit and a teflon stopcock and was washed with anhydrous DMF (4x). A mixture of TBAF (100 equiv.) dissolved in anhydrous DMF (final concentration 100 mM) was added and the reaction mixture was stirred by syringe rotation for 15 min at room temperature. This protocol was repeated once and the resin was then extensively washed with DMF, a 0.1% aqueous TFA solution and finally de-ionized water to give the solid supported peptide S27.

ESI-HRMS
A few beads of the resulting solid supported peptide were cleaved using the general procedure (p S53) to give crude peptide S27' which was analyzed by RP-HPLC and HRMS. Azido peptide S25 (3.4 mg, 1.5 µmol, 1.5 equiv.) was grafted on peptide resin S27 (1 µmol) following the typical procedure (p S50)

ESI-HRMS
A few beads of the resulting solid supported peptide were cleaved using the general procedure (p S53) to give crude peptide S28' which was analyzed by RP-HPLC and HRMS. The solid supported peptide S28 was introduced in a syringe fitted with a polypropylene frit and a teflon stopcock and was washed with anhydrous DMF (4x). A mixture of TBAF (100 equiv.) dissolved in anhydrous DMF (final concentration 100 mM) was added and the reaction mixture was stirred by syringe rotation for 15 min at room temperature. This protocol was repeated once and the resin was then extensively washed with DMF, a 0.1% aqueous TFA solution and finally de-ionized water to give the solid supported peptide S29.

ESI-HRMS
A few beads of the resulting solid supported peptide were cleaved using the general procedure (p S53) to give crude peptide S30a which was analyzed by RP-HPLC and HRMS. Solid supported alkyne peptide (1 µmol) was introduced in a 2 mL microcentrifuge tube and washed with de-ionized water (2 x 500 µL). A solution of crude 19 (6.6 mg, 1.5 µmol, 1.5 equiv) dissolved in 100 µL of a 1:1 mixture of NMP and 200 mM HEPES buffer pH 7.5 was then added. To the suspension were subsequently added a 1 M aqueous aminoguanidine solution (3 µL, 30 equiv.) and a 1 M aqueous tert-butanol solution (3 µL, 30 equiv.). The tube was sealed with a rubber septum and the resulting mixture was further deoxygenated through several successive vacuum (15 mbar) / argon cycles. Then, a mixture containing copper(I) bromide-dimethyl sulfide complex (30 equiv.) and THPTA (40 equiv.) dissolved under argon in 10 µL of deoxygenated NMP was added, the resulting suspension was further deoxygenated and was stirred for 5 h at 37 °C. The resin was then extensively washed with NMP, de-ionized water, then repeatedly treated (3x) with a pH 7 aqueous buffer containing 6 M guanidinium chloride, 0.1 M EDTA and 0.1 M sodium dihydrogenophosphate for a few minutes then drained and finally extensively washed with de-ionized water.

ESI-HRMS
ii) Typical procedure for solid-supported deprotection of the TIPS group: The solid supported TIPS-alkyne peptide was introduced in a syringe fitted with a polypropylene frit and a teflon stopcock and was washed with anhydrous DMF (4x). A mixture of TBAF (100 equiv.) and phenol [15] (110 equiv.) dissolved in anhydrous DMF (final TBAF concentration: 100 mM) was added and the reaction mixture was stirred by syringe rotation for 2 h at room temperature. This protocol was repeated once and the resin was then extensively washed with DMF, a 0.1% aqueous TFA solution and finally de-ionized water to give the solid supported alkyne peptide.
[15] Phenol was introduced to reduce the basic condition of the reaction. Better results in term of purity were observed with an equimolar addition of phenol during TIPS deprotection S108

Supplementary scheme S39 -Introduction of a biotin probe on solid supported peptide 10c
The N-to-C elongation of 10c (1 µmol) through multiple solid phase PTLs was performed by repeating three times a succession of i) solid-supported peptidomimetic triazole ligations with azido alkyne 19 and ii) solid-supported deprotection of the TIPS group.
After each step, a few beads of the resulting solid supported peptide were cleaved using the general procedure (p S53) and the released peptide was analyzed by RP-HPLC and HRMS. S109  Analysis of S31' Supplementary scheme S40 -Enzymatic glycosylation and release of solid supported peptide Solid supported glycopeptides S32, S34 and 20 (20 nmol) were introduced in a 2 mL microcentrifuge tube and washed with de-ionized water (2 x 500 µL). To the solid was added 100 µL of an aqueous solution containing GalNAc-T1 (6 mU), MES (50 mM, pH 6.5), MnCl 2 (15 mM), BSA (100 ng), DTT (1 mM) and UDP-GalNAc (2 equiv. per glycosylation sites). The resulting suspension was mixed at 37 °C for 48 h under moderate agitation. Beads were then washed with de-ionized water (2 x 500 µL), in order to remove the UDP formed during the transfer reaction since it inhibits GalNAc-T1 activity. Solid supported glycopeptide was then incubated under the same conditions as above for another 48 h and the resin was washed with de-ionized water. The resulting solid supported glycopeptides were released under typical procedure (p S53) to give glycopeptide mixture which was purified by semi-preparative HPLC and lyophilized to yield pure homogeneous glycopeptide containing 3 O-GalNAc per Muc1 tandem repeat. S116  Analysis of S36 VTSAPDTRPAPGSTAPPAHGVTSAPDTRPAPGSTAPPAH VTSAPDTRPAPGSTAPPAHGVTSAPDTRPAPGSTAPPAH t/min 