Convergent chemoenzymatic synthesis of a library of glycosylated analogues of pramlintide:

Transcription

1 Electronic Supplementary Material (ESI) for Organic & Biomolecular Chemistry. This journal is The Royal Society of Chemistry 2014 Convergent chemoenzymatic synthesis of a library of glycosylated analogues of pramlintide: structure-activity relationships of amylin receptor agonism. Renata Kowalczyk, a,b,d Margaret A. Brimble,* a,d Yusuke Tomabechi, c Madeleine Fletcher, b Debbie L. Hay* b,d Antony J. Fairbanks* c,d,e a The School of Chemical Sciences, University of Auckland, 23 Symonds St, Auckland 1010, New Zealand b The School of Biological Sciences, University of Auckland, 3 Symonds St, Auckland 1010, New Zealand c Department of Chemistry, University of Canterbury, Private Bag 4800, Christchurch 8140, New Zealand d Maurice Wilkins Centre for Molecular Biodiscovery, University of Auckland, Private Bag 92019, Auckland 1010, New Zealand e Biomolecular Interactions Centre, University of Canterbury, Private Bag 4800, Christchurch 8140, New Zealand m.brimble@auckland.ac.nz antony.fairbanks@canterbury.ac.nz dl.hay@auckland.ac.nz 1

2 Experimental section Abbreviations and nomenclature Penta refers to the core N-glycan pentasaccharide: [Man 3 (GlcNAc) 2 ]; SG (sialylglycopeptide) refers to the complex biantennary N-glycan: [(NeuAcGalGlcNAcMan) 2 Man(GlcNAc) 2 ]; Materials All reagents were purchased as reagent grade and used without further purification. O-(6- Chlorobenzotriazol-1-yl)-N,N,N,N -tetramethyluronium hexafluorophosphate (HCTU), N- [(dimethylamino)-1h-1,2,3-triazolo-[4,5-b]pyridin-1-ylmethylene]-n-methylmethanaminium hexafluorophosphate N-oxide (HATU), 4-[(R,S)-α-[1- (9H-floren-9-yl)]methoxycarbonylamino]- 2,4-dimethoxy]phenoxyacetic acid (Rink linker), and Fmoc-amino acids were purchased from GL Biochem (Shanghai, China). Fmoc-amino acids were supplied with the following side-chain protection: Fmoc Asn(Trt) OH, Fmoc Arg(Pbf) OH, Fmoc Cys(Trt) OH, Fmoc Gln(Trt) OH, Fmoc His(Trt) OH, Fmoc Lys(Boc) OH, Fmoc Ser(OtBu) OH, Fmoc Thr(OtBu) OH, Fmoc Tyr(OtBu) OH. N,N-Diisopropylethylamine (iprnet), 2,4,6-collidine, piperidine, hydrazine hydrate (NH 2 NH H 2 O), N,N -diisopropylcarbodiimide (DIC), 3,6-dioxa-1,8-octane-dithiol (DODT), formic acid, 1-methyl-2-pyrrolidinone (NMP) and triisopropylsilane (ipr 3 SiH) were purchased from Sigma-Aldrich (Sydney, Australia). N,N-Dimethylformamide (DMF) and acetonitrile (MeCN) were supplied from Scharlau (Barcelona, Spain). Dichloromethane (CH 2 Cl 2 ) was purchased from ECP Limited. Trifluoroacetic acid (TFA) was purchased from Halocarbon (River Edge, New Jersey), 2,2'-dipyridyl disulphide (DPDS) was sourced from Fluka (Switzerland), guanidinium hydrochloride (Gu HCl) was purchased from MP Biomedicals (Illkirch, France) and dimethyl sulphoxide (DMSO) was purchased from Romil Ltd (Cambridge, United Kingdom). Aminomethyl polystyrene resin, 1,2 N 4 -(2-acetamido-3,4,6-tri-O-acetyl-2-deoxy- -D-glucopyranosyl)-N 2 -(9- fluorenylmethoxycarbonyl)asparagine building block (27), 3 oxazoline 28 4 and 29 5,6 were synthesised following literature procedures. A commercial sample of glycopeptide 4 was sourced from Mimotopes. 7 The Endo-A enzyme was produced using the pet23d-endo-a plasmid, originally supplied by Professor Kaoru Takegawa (Kyushu University), as previously described. 8 The Endo-M N175Q mutant enzyme was purchased from Tokyo Chemical Industry Co., Ltd. Units of enzyme activity are defined as follows: one unit of N175Q Endo M converts 1.0 mol of pnp- GlcNAc to SG-GlcNAc-pNP per minute at 30 o C at ph

3 General procedure for peptide synthesis, purification and analysis Fmoc SPPS was performed on a 0.1 mmol scale using the Fmoc/tBu strategy and Liberty 12 Microwave Peptide Synthesiser (CEM Corporation, Mathews, NC). All amino acid couplings were performed as single coupling cycles, with the exception of Fmoc-Arg(Pbf) where a double coupling cycle was performed as part of a synthetic protocol recommended by CEM Microwave Technology. Protected amino acids were incorporated using Fmoc-AA (5.0 equiv, 0.2 M), HCTU (4.5 equiv, 0.45 M), and iprnet (10 equiv, 2 M) in DMF, for 5 min, at 25 W and maximum temperature of 75 ºC, except Fmoc-Arg(Pbf) that was initially coupled for 25 min at room temperature which was followed by the second coupling for 3 min, at 25 W and maximum temperature of 72 ºC. 2-Fold molar excess of Fmoc-Asn(GlcNAc(OAc) 3 ) (27) in the presence of HATU (1.9 equiv) and collidine (4 equiv) in DMF was used to incorporate N-glycan building block (15 min, 25 W, maximum temperature of 75 ºC). The Fmoc group was removed using 20% piperidine in DMF (30 s followed by a second deprotection for 3 min at 62 W and maximum temperature of 75 ºC). Resin cleavage and removal of the amino acid side-chain protecting groups was undertaken by incubating the resin in TFA/iPr 3 SiH/H 2 O/DODT (v/v/v/v; 94/1/2.5/2.5) cleavage cocktail for 2 h at room temperature. The crude peptides were precipitated and triturated with cold diethyl ether, isolated (centrifugation), dissolved in 20% MeCN (aq) containing 0.1% TFA and lyophilized. Analytical reverse phase high-performance liquid chromatography (RP-HPLC) was performed using either a Dionex P680 or a Dionex Ultimate U3000 system (flow rate of 1 ml/min), using analytical columns and gradient systems as indicated in synthesis section. The solvent system used was A (0.1% TFA in H 2 O) and B (0.1% TFA in MeCN) with detection at 210 nm, 254 nm, and 280 nm. A Hewlett Packard (HP) 1100MSD mass spectrometer, or an inline Thermo Finnegan MSQ mass spectrometer, or a Bruker maxis 3G UHR-TOF mass spectrometer were used for ESI-MS analysis in the positive mode. The ratio of products was determined by integration of spectra recorded at 210 nm or 214 nm. Peptide purification was performed using a Waters 600E system or a Dionex P680 system, using columns as indicated in synthesis section. Gradient systems were adjusted according to the elution profiles and peak profiles obtained from the analytical RP-HPLC chromatograms. Fractions were collected, analysed by either RP-HPLC or ESI-MS, pooled and lyophilised. General procedure for glycosylation reactions with Endo-A (glycopeptides 8-13) 3

4 A solution of the glycosyl donor (tetrasaccharide oxazoline 28, 15 mm) and the glycosyl acceptor {[Asn(GlcNAc) 3 ]pramlintide 2 (5 mm), [Asn(GlcNAc) 14 ]pramlintide 3 (5 mm), [Asn(GlcNAc) 21 ]pramlintide 4 (5 mm), [Asn(GlcNAc) 22 ]pramlintide 5 (5 mm), [Asn(GlcNAc) 31 ]pramlintide 6 (5 mm) or [Asn(GlcNAc) 35 ]pramlintide 7 (5 mm)} was incubated with Endo A (140 g/ ml) in sodium phosphate buffer (100 mm, ph 6.5) at 23 C. After incubation for 5 min, the mixture was added to 50 L of 0.2% aqueous TFA to quench the reaction. Purification of the crude reaction products was performed using RP-HPLC on a Dionex P680 HPLC instrument with a Phenomenex Jupiter 5 C18 300A column (5.0 μm, mm) at 40 C. The column was eluted with a linear gradient of MeCN at a flow rate of 1 ml/min using a gradient method (20-35% MeCN containing 0.05% TFA) for 16 min. Lyophilisation gave the product as a white powder. The yields were determined by integration of the product and acceptor peaks. General procedure for glycosylation reactions with N175Q Endo-M (glycopeptides 14-19) A solution of the glycosyl donor (decasaccharide oxazoline 29, 15 mm) and the glycosyl acceptor {[Asn(GlcNAc) 3 ]pramlintide 2 (5 mm), [Asn(GlcNAc) 14 ]pramlintide 3 (5 mm), [Asn(GlcNAc) 21 ]pramlintide 4 (5 mm), [Asn(GlcNAc) 22 ]pramlintide 5 (5 mm), [Asn(GlcNAc) 31 ]pramlintide 6 (5 mm) or [Asn(GlcNAc) 35 ]pramlintide 7 (5 mm)} was incubated with N175Q Endo M (160 U/ ml) in sodium phosphate buffer (100 mm, ph 6.5) at 23 C. After incubation for 3 h, the mixture was added to 50 L of a 0.2% aqueous TFA solution to quench the reaction. Purification of the crude products was performed using RP-HPLC on a Dionex P680 HPLC instrument with a Phenomenex Jupiter 5 C18 300A column (5.0 μm, mm) at 40 C. The column was eluted with a linear gradient of MeCN at a flow rate of 1 ml/min using a gradient method (20-35% MeCN containing 0.05% TFA) for 16 min. Lyophilisation gave the product as a white powder. The yields were determined by integration of the product and acceptor peaks. General procedure for measuring the agonist effect of pramlintide 1 and pramlintide analogues 2-19 at amylin receptor Pramlintide 1 and glycopeptides 2-19 were screened at amylin receptors. Cos 7 cells were transiently transfected with the necessary receptor components, and cyclic AMP production was 4

5 measured according to our published methods. 9,10 For this study we used untagged human RAMP1 and RAMP3, kindly provided by Dr S.M. Foord (GlaxoWellcome, Stevenage, UK) and the human CT (a) receptor with a single N-terminal HA tag (haemagglutinin, YPYDVPDYA) inserted into the pcdna 3.1 plasmid with leucine at the polymorphic amino acid position 447, as kindly provided by Professor Patrick Sexton (Monash University, Australia). For 1-7, peptides were weighed out and stock solutions made at 1 mm or 500 µm (diluted in sterile water), on the basis of peptide weight. Peptide purity was taken into account in these calculations. For analogues 8-19, a standard curve of 1 was prepared (1.0 mg/ml, 5.0 mg/ml and 15 mg/ml) and absorbance measured at 280 nm using a NanoDrop spectrophotometer. Samples of 8-19 were prepared in 100 µl of water and the absorbances measured; stock solutions of 8-19 were prepared using the standard curve. All peptide stock solutions were stored in siliconised or Lobind (Eppendorf, Hamburg, Germany) microcentrifuge tubes at -30 C in 2-6 µl aliquots to minimise freeze-thaw cycles. 5

6 Synthesis of pramlintide 1 Microwave enhanced Fmoc SPPS was used for the synthesis of linear pramlintide 20 which was followed by the resin cleavage using the conditions outlined in the general section to afford mg of crude 20; R t min; m/z (ESI-MS) ([M + 4H] 4+ requires 988.9), Figure S 1. Formation of disulphide bond of 20 was undertaken following conditions previously described. 11 Crude peptide 20 (61.2 mg, 15.5 x 10-3 mmol) was dissolved in 6.1 ml of DMSO, DPTS (10.2 mg, 46.5 x 10-3 mmol) was added and the mixture stirred at room temperature for 20 min during which time formation of oxidised product was complete as judged by analytical LCMS; R t min; m/z (ESI-MS) ([M + 4H] 4+ requires 988.4), (Figure S 2). Crude product 1 was purified by RP- HPLC on a preparative Grace Vydac219TP Diphenyl column (250 mm x 10 mm) at a flow rate of 5 ml/min, using a linear gradient of 5%B to 15%B over 10 min (ca. 1%B per minute), followed by 15%B to 65%B over 500 min (ca. 0.1%B per minute). Fractions were collected at 0.5 min intervals, analysed (MS and HPLC), pooled and lyophilised to give the title compound 1 as a white amorphous solid (6.84 mg); R t min; m/z (ESI-MS) ([M + 3H] 3+ requires ), Figure S 3. Figure S 1 LCMS traces of crude pramlintide 20 (ca 49% as judged by peak area of RP-HPLC at 214 nm); Hewlett Packard (HP) 1100MSD, Zorbax 300SB C3 3.5μ; 3.0 x 150 mm) column (Agilent), column, linear gradient of 5%B to 65%B over 20 min, ca. 3%B per minute at 40 ºC. 6

7 Figure S 2 LCMS traces of crude pramlintide 1; Hewlett Packard (HP) 1100MSD, Zorbax 300SB C3 3.5μ; 3.0 x 150 mm) column (Agilent), linear gradient of 5%B to 65%B over 20 min, ca. 3%B per minute at 40 ºC, 214 nm. Figure S 3 RP-HPLC and ESI-MS traces of pure pramlintide 1; Dionex Ultimate U3000, VydacTP Diphenyl (5μ; mm) column (Grace), linear gradient of 5%B to 65%B over 60 min, ca. 1%B per minute at 40 ºC; Thermo Finnegan MSQ mass spectrometer. Synthesis of [Asn(GlcNAc) 3 ]pramlintide 2 Microwave enhanced Fmoc SPPS was used for the synthesis of linear pramlintide 21 which was followed by the resin cleavage using the conditions outlined in the general section to afford mg of crude 21; R t min; m/z (ESI-MS) ([M + 4H] 4+ requires ), Figure S 4. Crude peptide 21 (62.5 mg, 14.6 x 10-3 mmol) was dissolved in 20 ml of DMSO, DPTS (9.6 mg, 43.7 x 10-3 mmol) was added and the mixture stirred at room temperature for 1 h during which time formation of oxidised product was complete as judged by analytical LCMS; R t min; m/z (ESI- MS) ([M + 4H] 4+ requires ), (Figure S 5). NH 2 NH H 2 O (3.1 ml) was then added and reaction mixture was further stirred for 2 h during which time formation of the acetate deprotected product 2 was finished as indicated by LCMS analysis; R t min; m/z (ESI-MS) ([M + 4H] 4+ requires ), (Figure S 6). Crude product 2 was purified by RP-HPLC on a preparative Phenomenex Gemini C18 column (110 Å, 250 mm x 10 mm x 5 µ) at a flow rate of 5 ml/min, using a linear gradient of 1%B to 18%B over 17 min (ca. 1%B per minute), followed by 7

8 18%B to 61%B over 430 min (ca. 0.1%B per minute). Fractions were collected at 0.5 min intervals, analysed (MS and HPLC), pooled and lyophilised to give the title compound 2 as a white amorphous solid (3.9 mg); R t min; m/z (ESI-MS) ([M + 4H] 4+ requires ), Figure S 7. Figure S 4 LCMS traces of crude pramlintide analogue 21; Hewlett Packard (HP) 1100MSD, Zorbax 300SB C3 3.5μ; Figure S 5 LCMS traces of crude and Cys2/Cys7 oxidized pramlintide analogue 21; Hewlett Packard (HP) 1100MSD, Zorbax 300SB C3 3.5μ; 3.0 x 150 mm) column (Agilent), linear gradient of 5%B to 65%B over 60 min, ca. 1%B per minute at 40 ºC, 214 nm. Figure S 6 LCMS traces of crude pramlintide analogue 2; Hewlett Packard (HP) 1100MSD, Zorbax 300SB C3 3.5μ; 8

9 Figure S 7 RP-HPLC and ESI-MS traces of pure pramlintide analogue 2; Dionex P680, VydacTP Diphenyl (5μ; mm) column (Grace), linear gradient of 5%B to 65%B over 60 min, ca. 1%B per minute at 40 ºC; Hewlett Packard (HP) 1100MSD. Synthesis of [Asn(GlcNAc) 14 ]pramlintide 3 Microwave enhanced Fmoc SPPS was used for the synthesis of linear pramlintide 22 which was followed by the resin cleavage using the conditions outlined in the general section to afford mg of crude 22; R t min; m/z (ESI-MS) ([M + 4H] 4+ requires ), Figure S 8. Crude peptide 22 (64.0 mg, 14.9 x 10-3 mmol) was dissolved in 21 ml of DMSO, DPTS (9.9 mg, 44.8 x 10-3 mmol) was added and the mixture stirred at room temperature for 1 h during which time formation of oxidised product was complete as judged by analytical LCMS; R t min; m/z (ESI- MS) ([M + 4H] 4+ requires ), (Figure S 9). NH 2 NH H 2 O (3.2 ml) was then added and reaction mixture was further stirred for 2 h during which time formation of the acetate deprotected product 3 was finished as indicated by LCMS analysis; R t min; m/z (ESI-MS) ([M + 4H] 4+ requires ), (Figure S 10). Crude product 3 was purified by RP-HPLC on a preparative Phenomenex Gemini C18 column (110 Å, 250 mm x 10 mm x 5 µ) at a flow rate of 5 ml/min, using a linear gradient of 1%B to 18%B over 17 min (ca. 1%B per minute), followed by 18%B to 61%B over 430 min (ca. 0.1%B per minute). Fractions were collected at 0.5 min intervals, analysed (MS and HPLC), pooled and lyophilised to give the title compound 3 as a white amorphous solid (2.8 mg); R t min; m/z (ESI-MS) ([M + 4H] 4+ requires ), Figure S 11. 9

10 Figure S 8 LCMS traces of crude pramlintide analogue 22; Hewlett Packard (HP) 1100MSD, Zorbax 300SB C3 3.5μ; Figure S 9 LCMS traces of crude and Cys2/Cys7 oxidized pramlintide analogue 22; Hewlett Packard (HP) 1100MSD, Zorbax 300SB C3 3.5μ; 3.0 x 150 mm) column (Agilent), linear gradient of 5%B to 65%B over 60 min, ca. 1%B per minute at 40 ºC, 214 nm. Figure S 10 LCMS traces of crude pramlintide analogue 3; Hewlett Packard (HP) 1100MSD, Zorbax 300SB C3 3.5μ; 10

11 Figure S 11 LCMS traces of pure pramlintide analogue 3; Hewlett Packard (HP) 1100MSD, Zorbax 300SB C3 3.5μ; Synthesis of [Asn(GlcNAc) 21 ]pramlintide 4 Microwave enhanced Fmoc SPPS was used for the synthesis of linear pramlintide 23 which was followed by the resin cleavage using the conditions outlined in the general section to afford mg of crude 23; R t min; m/z (ESI-MS) ([M + 4H] 4+ requires ), Figure S 12. Crude peptide 23 (61.6 mg, 14.4 x 10-3 mmol) was dissolved in 21 ml of DMSO, DPTS (9.5 mg, 43.2 x 10-3 mmol) was added and the mixture stirred at room temperature for 1 h during which time formation of oxidised product was complete as judged by analytical LCMS; R t min; m/z (ESI- MS) ([M + 4H] 4+ requires ), (Figure S 13). NH 2 NH H 2 O (3.2 ml) was then added and reaction mixture was further stirred for 2 h during which time formation of the acetate deprotected product 4 was finished as indicated by LCMS analysis; R t min; m/z (ESI-MS) ([M + 4H] 4+ requires ), (Figure S 14). Crude product 4 was purified by RP-HPLC on a preparative Phenomenex Gemini C18 column (110 Å, 250 mm x 10 mm x 5 µ) at a flow rate of 5 ml/min, using a linear gradient of 1%B to 61%B over 60 min (ca. 1%B per minute). Fractions were collected at 0.5 min intervals, analysed (MS and HPLC), pooled and lyophilised to give 3 (10.6 mg, 47% purity). Compound 3 was further purified by RP-HPLC on an analytical Grace VydacTP Diphenyl column (250 mm x 4.6 mm x 5 μ) at a flow rate of 1 ml/min, using a linear gradient of 1%B to 11%B over 10 min (ca. 1%B per minute), followed by 11%B to 61%B over 500 min (ca. 0.1%B per minute). Fractions were collected at 0.5 min intervals, analysed (MS and HPLC), pooled and lyophilised to give the title compound 4 as a white amorphous solid (0.2 mg); R t min; m/z (ESI-MS) ([M + 4H] 4+ requires ), Figure S

12 Figure S 12 LCMS traces of crude pramlintide analogue 23; Hewlett Packard (HP) 1100MSD, Zorbax 300SB C3 3.5μ; Figure S 13 LCMS traces of crude and Cys2/Cys7 oxidized pramlintide analogue 23; Hewlett Packard (HP) 1100MSD, Zorbax 300SB C3 3.5μ; 3.0 x 150 mm) column (Agilent), linear gradient of 5%B to 65%B over 60 min, ca. 1%B per minute at 40 ºC, 214 nm. Figure S 14 LCMS traces of crude pramlintide analogue 4; Hewlett Packard (HP) 1100MSD, Zorbax 300SB C3 3.5μ; 12

13 Figure S 15 RP-HPLC and ESI-MS traces of pure pramlintide analogue 4; Dionex P680, VydacTP Diphenyl (5μ; mm) column (Grace), linear gradient of 5%B to 65%B over 60 min, ca. 1%B per minute at 40 ºC; Hewlett Packard (HP) 1100MSD. Synthesis of [Asn(GlcNAc) 22 ]pramlintide 5 Microwave enhanced Fmoc SPPS was used for the synthesis of linear pramlintide 24 which was followed by the resin cleavage using the conditions outlined in the general section to afford mg of crude 24; R t min; m/z (ESI-MS) ([M + 4H] 4+ requires ), Figure S 16. Crude peptide 24 (60.0 mg, 14.4 x 10-3 mmol) was suspended in 20 ml mixture of 5% NH 2 NH H 2 O, 10 % DMSO and 85% 6 M Gu HCl and shaken for 17 h during which time formation of oxidised and the acetate deprotected product 5 was complete as judged by analytical LCMS; R t min; m/z (ESI-MS) ([M + 4H] 4+ requires ), (Figure S 17). Crude product 5 was purified by RP-HPLC on a preparative Waters XTerra Prep. C 18 column (300 mm x 19 mm x 10 µ) at a flow rate of 10 ml/min, using a linear gradient of 1%B to 15%B over 14 min (ca. 1%B per minute), followed by 15%B to 61%B over 460 min (ca. 0.1%B per minute). Fractions were collected at 0.5 min intervals, analysed (MS and HPLC), pooled and lyophilised to give the title compound 5 as a white amorphous solid (4.8 mg); R t min; m/z (ESI-MS) ([M + 4H] 4+ requires ), Figure S

14 Figure S 16 LCMS traces of crude pramlintide analogue 24; Hewlett Packard (HP) 1100MSD, Zorbax 300SB C3 3.5μ; Figure S 17 LCMS traces of crude pramlintide analogue 5; Hewlett Packard (HP) 1100MSD, Zorbax 300SB C3 3.5μ; Figure S 18 LCMS traces of pure pramlintide analogue 5; Hewlett Packard (HP) 1100MSD, Zorbax 300SB C3 3.5μ; Synthesis of [Asn(GlcNAc) 31 ]pramlintide 6 Microwave enhanced Fmoc SPPS was used for the synthesis of linear pramlintide 25 which was followed by the resin cleavage using the conditions outlined in the general section to afford mg of crude 25; R t min; m/z (ESI-MS) ([M + 4H] 4+ requires ), Figure S 19. Crude peptide 25 (66.0 mg, 15.4 x 10-3 mmol) was dissolved in 22 ml of DMSO, DPTS (10.2 mg, 46.2 x 10-3 mmol) was added and the mixture stirred at room temperature for 1 h during which time formation of oxidised product was complete as judged by analytical LCMS; R t min; m/z (ESI- MS) ([M + 4H] 4+ requires ), (Figure S 20). NH 2 NH H 2 O (1.2 ml) was then added and reaction mixture was further stirred for 4 h during which time formation of the acetate deprotected product 6 was finished as indicated by LCMS analysis; R t min; m/z (ESI-MS) 14

15 ([M + 4H] 4+ requires ), (Figure S 21). Crude product 6 was purified by RP-HPLC on a preparative Grace Vydac219TP Diphenyl column (250 mm x 10 mm) at a flow rate of 5 ml/min, using a linear gradient of 1%B to 61%B over 60 min (ca. 1%B per minute). Fractions were collected at 0.5 min intervals, analysed (MS and HPLC), pooled and lyophilised to give the title compound 6 as a white amorphous solid (1.4 mg); R t min; m/z (ESI-MS) ([M + 4H] 4+ requires ), Figure S 22. Figure S 19 LCMS traces of crude pramlintide analogue 25; Hewlett Packard (HP) 1100MSD, Zorbax 300SB C3 3.5μ; Figure S 20 LCMS traces of crude and Cys2/Cys7 oxidized pramlintide analogue 25; Hewlett Packard (HP) 1100MSD, Zorbax 300SB C3 3.5μ; 3.0 x 150 mm) column (Agilent), linear gradient of 5%B to 65%B over 20 min, ca. 3%B per minute at 40 ºC, 214 nm. Figure S 21 LCMS traces of crude pramlintide analogue 6; Hewlett Packard (HP) 1100MSD, Zorbax 300SB C3 3.5μ; 15

16 Figure S 22 RP-HPLC and ESI-MS traces of pure pramlintide analogue 6; Dionex P680, VydacTP Diphenyl (5μ; mm) column (Grace), linear gradient of 5%B to 65%B over 60 min, ca. 1%B per minute at 40 ºC; Hewlett Packard (HP) 1100MSD. Synthesis of [Asn(GlcNAc) 35 ]pramlintide 7 Microwave enhanced Fmoc SPPS was used for the synthesis of linear pramlintide 26 which was followed by the resin cleavage using the conditions outlined in the general section to afford mg of crude 26; R t min; m/z (ESI-MS) ([M + 4H] 4+ requires ), Figure S 23. Crude peptide 26 (53.1 mg, 12.4 x 10-3 mmol) was dissolved in 17.6 ml of DMSO, DPTS (8.2 mg, 37.2 x 10-3 mmol) was added and the mixture stirred at room temperature for 1.5 h during which time formation of oxidised product was complete as judged by analytical LCMS; R t min; m/z (ESI-MS) ([M + 4H] 4+ requires ), (Figure S 24). NH 2 NH H 2 O (0.9 ml) was then added and reaction mixture was further stirred for 4 h during which time formation of the acetate deprotected product 7 was finished as indicated by LCMS analysis; R t min; m/z (ESI-MS) ([M + 4H] 4+ requires ), (Figure S 25). Crude product 7 was purified by RP-HPLC on a preparative Grace Vydac219TP Diphenyl column (250 mm x 10 mm) at a flow rate of 5 ml/min, using a linear gradient of 1%B to 61%B over 240 min (ca. 0.25%B per minute). Fractions were collected at 0.5 min intervals, analysed (MS and HPLC), pooled and lyophilised to give the title compound 7 as a white amorphous solid (2.6 mg); R t min; m/z (ESI-MS) ([M + 4H] 4+ requires ), Figure S

17 Figure S 23 LCMS traces of crude pramlintide analogue 26; Hewlett Packard (HP) 1100MSD, Zorbax 300SB C3 3.5μ; Figure S 24 LCMS traces of crude and Cys2/Cys7 oxidized pramlintide analogue 26; Hewlett Packard (HP) 1100MSD, Zorbax 300SB C3 3.5μ; 3.0 x 150 mm) column (Agilent), linear gradient of 5%B to 65%B over 20 min, ca. 3%B per minute at 40 ºC, 214 nm. Figure S 25 LCMS traces of crude pramlintide analogue 7; Hewlett Packard (HP) 1100MSD, Zorbax 300SB C3 3.5μ; 17

18 Figure S 26 LCMS traces of pure pramlintide analogue 7; Hewlett Packard (HP) 1100MSD, Zorbax 300SB C3 3.5μ; Synthesis of [Asn(penta) 3 ]pramlintide 8 using Endo-A Buffer 53.4 L; Title compound 8 (0.390 mg, 49%); analytical HPLC: R t = min; ESI-MS: calculated for C 205 H 324 N 53 O 78 S 2 [M+H] + : Found: (Figure S 27). Figure S 27 Synthesis of [Asn(penta) 14 ]pramlintide 9 using Endo-A Buffer 46.1 L; Title compound 9 (0.300 mg, 87%); analytical HPLC: R t = min; ESI-MS: calculated for C 205 H 324 N 53 O 78 S 2 [M+H] + : Found: (Figure S 28). 18

19 Figure S 28 Synthesis of [Asn(penta) 21 ]pramlintide 10 using Endo-A Buffer 53.4 L; Title compound 10 (0.390 mg, 61%); analytical HPLC: R t = min; ESI-MS: calculated for C 205 H 324 N 53 O 78 S 2 [M+H] + : Found: (Figure S 29). Figure S 29 Synthesis of [Asn(penta) 22 ]pramlintide 11 using Endo-A 19

20 Buffer 54.1 L; Title compound 11 (0.260 mg, 82%); analytical HPLC: R t = min; ESI-MS: calculated for C 205 H 324 N 53 O 78 S 2 [M+H] + : Found: (Figure S 30). Figure S 30 Synthesis of [Asn(penta) 31 ]pramlintide 12 using Endo-A Buffer 31.0 L; Title compound 12 (0.240 mg, 93%); analytical HPLC: R t = min; ESI-MS: calculated for C 205 H 324 N 53 O 78 S 2 [M+H] + : Found: (Figure S 31). Figure S 31 Synthesis of [Asn(penta) 35 ]pramlintide 13 using Endo-A 20

21 Buffer 32.4 L; Title compound 13 (0.250 mg, 90%); analytical HPLC: R t = min; ESI-MS: calculated for C 205 H 324 N 53 O 78 S 2 [M+H] + : Found: (Figure S 32). Figure S 32 Synthesis of [Asn(SG) 3 ]pramlintide 14 using N175Q Endo-M Buffer 54.9 L; Title compound 14 (0.225 mg, 35%); analytical HPLC: R t = min; ESI-MS: calculated for C 255 H 404 N 57 O 114 S 2 [M+H] + : Found: (Figure S 33). 21

22 Figure S 33 Synthesis of [Asn(SG) 14 ]pramlintide 15 using N175Q Endo-M Buffer 47.5 L; Title compound 15 (0.190 mg, 46%); analytical HPLC: R t = min; ESI-MS: calculated for C 255 H 404 N 57 O 114 S 2 [M+H] + : Found: (Figure S 34). Figure S 34 Synthesis of [Asn(SG) 21 ]pramlintide 16 using N175Q Endo-M 22

23 Buffer 55.2 L; Title compound 16 (0.195 mg, 34%); analytical HPLC: R t = min; ESI-MS: calculated for C 255 H 404 N 57 O 114 S 2 [M+H] + : Found: (Figure S 35). Figure S 35 Synthesis of [Asn(SG) 22 ]pramlintide 17 using N175Q Endo-M Buffer 55.6 L; Title compound 17 (0.080 mg, 28%); analytical HPLC: R t = min; ESI-MS: calculated for C 255 H 404 N 57 O 114 S 2 [M+H] + : Found: (Figure S 36). 23

24 Figure S 36 Synthesis of [Asn(SG) 31 ]pramlintide 18 using N175Q Endo-M Buffer 31.9 L; Title compound 18 (0.085 mg, 41%); analytical HPLC: R t = min; ESI-MS: calculated for C 255 H 404 N 57 O 114 S 2 [M+H] + : Found: (Figure S 37). Figure S 37 Synthesis of [Asn(SG) 35 ]pramlintide 19 using N175Q Endo-M 24

25 Buffer 33.4 L; Title compound 19 (0.030 mg, 34%); analytical HPLC: R t = min; ESI-MS: calculated for for C 255 H 404 N 57 O 114 S 2 [M+H] + : Found: (Figure S 38). Figure S 38 25

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