Convergent chemoenzymatic synthesis of a library of ...

1

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 Antony J. Fairbanks*

c,d,e

Madeleine Fletcher,b Debbie L. Hay*

b,d

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

e-mail: [email protected]



Electronic Supplementary Material (ESI) for Organic & Biomolecular Chemistry.This journal is © The Royal Society of Chemistry 2014

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Experimental section

Abbreviations and nomenclature

‘Penta’ refers to the core N-glycan pentasaccharide: [Man3(GlcNAc)2];

‘SG’ (sialylglycopeptide) refers to the complex biantennary N-glycan:

[(NeuAcGalGlcNAcMan)2Man(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

(NH2NH2·1.5 H2O), N,N′-diisopropylcarbodiimide (DIC), 3,6-dioxa-1,8-octane-dithiol (DODT),

formic acid, 1-methyl-2-pyrrolidinone (NMP) and triisopropylsilane (iPr3SiH) were purchased from

Sigma-Aldrich (Sydney, Australia). N,N-Dimethylformamide (DMF) and acetonitrile (MeCN) were

supplied from Scharlau (Barcelona, Spain). Dichloromethane (CH2Cl2) 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

N4-(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 oC at pH 7.0.

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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/iPr3SiH/H2O/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 H2O) 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)

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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, 4.6 × 250 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, 4.6 × 250 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

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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.

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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 335.2

mg of crude 20; Rt 14.88 min; m/z (ESI-MS) 988.6 ([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; Rt 15.75 min; m/z

(ESI-MS) 988.1 ([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); Rt 28.10 min; m/z (ESI-MS) 1317.6 ([M + 3H]3+

requires 1317.5),

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.

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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;

4.6 × 250 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






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; Rt 31.84 min; m/z (ESI-

MS) 1070.2 ([M + 4H]4+

requires 1070.9), (Figure S 5). NH2NH2·1.5 H2O (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; Rt 29.80 min; m/z (ESI-MS)

1038.9 ([M + 4H]4+

requires 1039.4), (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


8




requires 1039.4), Figure

S 7.

Figure S 4 LCMS traces of crude 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 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.



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Figure S 7 RP-HPLC and ESI-MS traces of pure pramlintide analogue 2; Dionex P680, VydacTP Diphenyl (5μ;

4.6 × 250 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







44.8 x 10-3



MS) 1070.3 ([M + 4H]4+

requires 1070.9), (Figure S 9). NH2NH2·1.5 H2O (3.2 mL) was then added

and reaction mixture was further stirred for 2 h during which time formation of the acetate


1038.8 ([M + 4H]4+

requires 1039.4), (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





requires 1039.4), Figure

S 11.

10








11

Figure S 11 LCMS traces of pure pramlintide analogue 3; Hewlett Packard (HP) 1100MSD, Zorbax 300SB–C3 3.5μ;



]pramlintide 4







43.2 x 10-3



MS) 1070.5 ([M + 4H]4+

requires 1070.9), (Figure S 13). NH2NH2·1.5 H2O (3.2 mL) was then

added and reaction mixture was further stirred for 2 h during which time formation of the acetate


1038.9 ([M + 4H]4+


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); Rt 28.33 min; m/z

(ESI-MS) 1039.1 ([M + 4H]4+


12








13





]pramlintide 5






mmol) was suspended in 20 mL mixture of 5% NH2NH2·1.5

H2O, 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; Rt

29.13 min; m/z (ESI-MS) 1038.7 ([M + 4H]4+

requires 1039.4), (Figure S 17). Crude product 5 was

purified by RP-HPLC on a preparative Waters XTerra

Prep. C18 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); Rt 29.36 min; m/z (ESI-MS) 1038.8 ([M + 4H]4+


14








]pramlintide 6







46.2 x 10-3



MS) 1070.0 ([M + 4H]4+




15

1038.6 ([M + 4H]4+


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











16





]pramlintide 7






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; Rt 31.73 min; m/z

(ESI-MS) 1070.4 ([M + 4H]4+




1038.8 ([M + 4H]4+


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




17








18



Synthesis of [Asn(penta)3]pramlintide 8 using Endo-A

Buffer 53.4 L;

Title compound 8 (0.390 mg, 49%); analytical HPLC: Rt = 13.69 min; ESI-MS: calculated for

C205H324N53O78S2 [M+H]+: 4840.2457. Found: 4840.2416 (Figure S 27).

Figure S 27

Synthesis of [Asn(penta)14

]pramlintide 9 using Endo-A

Buffer 46.1 L;



19

Figure S 28



Buffer 53.4 L;



Figure S 29



20

Buffer 54.1 L;



Figure S 30



Buffer 31.0 L;



Figure S 31



21

Buffer 32.4 L;



Figure S 32

Synthesis of [Asn(SG)3]pramlintide 14 using N175Q Endo-M

Buffer 54.9 L;



22

Figure S 33

Synthesis of [Asn(SG)14

]pramlintide 15 using N175Q Endo-M

Buffer 47.5 L;



Figure S 34



23

Buffer 55.2 L;



Figure S 35



Buffer 55.6 L;



24

Figure S 36



Buffer 31.9 L;



Figure S 37



25

Buffer 33.4 L;

Title compound 19 (0.030 mg, 34%); analytical HPLC: Rt = 13.50 min; ESI-MS: calculated for for


Figure S 38

26

[1]. Mitchell, A. R.; Kent, S. B. H.; Engelhard, M.; Merrifield, R. B. J. Org. Chem. 1978, 43,

2845.

[2]. Harris, P. W. R.; Yang, S. H.; Brimble, M. A. Tetrahedron Lett. 2011, 52, 6024.

[3]. Inazu, T.; Kobayashi, K. Synlett 1993, 869.

[4]. Rising, T. W. D. E.; Heidecke, C. D.; Moir, J. W. B.; Ling, Z. L.; Fairbanks, A. J. Chem.

Eur. J. 2008, 14, 6444.

[5]. Seko, A.; Koketsu, M.; Nishizono, M.; Enoki, Y.; Ibrahim, H. R.; Juneja, L. R.; Kim, M.;

Yamamoto, T. Biochim. Biophys. Acta, Gen. Subj. 1997, 1335, 23.

[6]. Umekawa, M.; Higashiyama, T.; Koga, Y.; Tanaka, T.; Noguchi, M.; Kobayashi, A.; Shoda,

S.; Huang, W.; Wang, L. X.; Ashida, H.; Yamamoto, K. Biochim. Biophys. Acta, Gen. Subj. 2010,

1800, 1203.

[7]. Tomabechi, Y.; Krippner, G.; Rendle, P. M.; Squire, M. A.; Fairbanks, A. J. Chem. Eur. J.

2013, 19, 15084.

[8]. Heidecke, C. D.; Ling, Z. L.; Bruce, N. C.; Moir, J. W. B.; Parsons, T. B.; Fairbanks, A. J.

ChemBioChem 2008, 9, 2045.

[9]. Bailey, R. J.; Hay, D. L. Peptides 2006, 27, 1367.

[10]. Gingell, J. J.; Qi, T.; Bailey, R. J.; Hay, D. L. Peptides 2010, 31, 1400.

[11]. Harris, P. W. R.; Kowalczyk, R.; Hay, D. L.; Brimble, M. A. Int. J. Pept. Res. Ther. 2013,

19, 147.

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