supplementary materials for - science advances · 2016. 1. 12. · supplemental figure 1. acid...

advances.sciencemag.org/cgi/content/full/2/1/e1500678/DC1

Supplementary Materials for

Chemical synthesis of erythropoietin glycoforms for insights into the

relationship between glycosylation pattern and bioactivity

Masumi Murakami, Tatsuto Kiuchi, Mika Nishihara, Katsunari Tezuka, Ryo Okamoto, Masayuki Izumi,

Yasuhiro Kajihara

Published 15 January 2016, Sci. Adv. 2, e1500678 (2016)

DOI: 10.1126/sciadv.1500678

The PDF file includes:

Fig. S1. Acid stability of sialyloligosaccharide phenacyl ester.

Fig. S2. General scheme of the synthesis of a sialylglycopeptide-α-thioester by an

improved Boc SPPS method.

Fig. S3. HPLC profile and ESI mass spectrum of H-[Ala1-Gly28]-α-thioester.

Fig. S4. HPLC profile and ESI mass spectrum of H-[Cys29,33 (Acm)-Tyr49]-α-

thioester.

Fig. S5. HPLC profile and ESI mass spectrum of H-[Cys29,33(Acm)-

Asn38(glycan)-Tyr49]-α-thioester.

Fig. S6. HPLC profile and ESI mass spectrum of H-[Cys79(Thz)-Trp88-(formyl)-

Lys97]-α-thioester.

Fig. S7. HPLC profile and ESI mass spectrum of H-[Cys79(Thz)-Asn83(glycan)-

Trp88(formyl)-Lys97]-α-thioester.

Fig. S8. HPLC profile and ESI mass spectrum of H-[Cys98(Thz)-Ala127]-α-

thioester.

Fig. S9. HPLC profile and ESI mass spectrum of H-[Cys50-Ala78]-α-hydrazide.

Fig. S10. HPLC profile and ESI mass spectrum of H-[Ala1-Asn24(glycan)-Gly28]-

α-thioester.

Fig. S11. Monitoring NCL between H-[Cys29, 33(Acm)-Asn38(glycan)-Tyr49]-α-

thioester and H-[Cys50-Ala78]-α-hydrazine.

Fig. S12. Monitoring NCL between H-[Cys29, 33(Acm)-Asn38(glycan)-Ala78]-α-

hydrazide and H-[Cys79-Asn83(glycan)-Arg166]-OH.

Fig. S13. Monitoring the desulfurization reaction of H-[Cys29, 33, 161(Acm)-Cys50,

79, 98, 128-Asn38, 83(glycan)-Arg166]-OH.

Fig. S14. Monitoring of the removal of Acm group of H-[Cys29, 33, 161(Acm)-

Asn38, 83(glycan)2-Arg166]-OH by RP-HPLC and ESI-MS.

Fig. S15. Monitoring the NCL between H-[Ala1-Asn24(glycan)-Gly28]-α-thioester

and H-[Cys29-Asn38, 83(glycan)2-Arg166]-OH.

Fig. S16. The folding reaction of EPON24, N38, N83 (polypeptide form of H-[Ala1-

Asn24, 38, 83(glycan)3-Arg166]-OH.

Fig. S17. The folding reactions of EPON38, N83 (polypeptide form of H-[Ala1-

Asn38, 83(glycan)2-Arg166]-OH) and EPON24, N83 (polypeptide form of H-[Ala1-

Asn24, 83(glycan)2-Arg166]-OH).

Fig. S18. The folding reactions of EPON24, N38 (polypeptide form of H-[Ala1-

Asn24, 38(glycan)2-Arg166]-OH) and EPON83 (polypeptide form of H-[Ala1-

Asn83(glycan)-Arg166]-OH).

Results of folding experiments

Fig. S19. Monitoring of in vitro folding by SDS-PAGE.

Fig. S20. Analysis of disulfide bond positions of EPON24, N38, N83 2 by trypsin

digestion.

Fig. S21. Analysis of disulfide bond positions of EPON38, N83 3 by trypsin

digestion.


digestion.


digestion.

Fig. S24. Analysis of disulfide bond positions of EPON83 6 by trypsin digestion.

Fig. S25. Characterization of misfolded EPON24, N83 (compound 7).

High-resolution mass spectra of EPO glycoforms

Fig. S26. High-resolution mass spectrum of EPON24, N38, N83 2.

Fig. S27. High-resolution mass spectrum of EPON38, N83 3.

Fig. S28. High-resolution mass spectrum of EPON24, N38, 4.

Fig. S29. High-resolution mass spectrum of EPON38, N83 5.

Fig. S30. High-resolution mass spectrum of EPON83 6.

Supplemental Figure 1. Acid stability of sialyloligosaccharide phenacyl ester. (A) The proposed

mechanism of intramolecular catalyst for the acceleration of acid hydrolysis of sialyl linkage. (B)

Hydrolysis yield of sialyl linkage under acid condition (40 mM HCl) and the structure of released sialic

acid (1). Green lozenge indicates the yield of free sialic acid released from Fmoc-Asn-

(sialyloligosaccharide)-OH shown in (B). Blue circle in the panel indicates that phenacyl ester interferes

the hydrolysis of sialyl linkage. The released sialic acid by acid hydrolysis was estimated from the

consumption of Fmoc-Asn-(sialyloligosaccharide)-OH with UV-detected HPLC-mass instrument (1).

(1) M. Murakami, et. al. Angew Chem Int Ed Engl 51, 3567-3572 (2012)

Supplemental Figure 2. General scheme of the synthesis of a sialylglycopeptide-α-thioester by an

improved Boc SPPS method (1).

(1) M. Murakami, et. al. Angew Chem Int Ed Engl 51, 3567-3572 (2012)

Supplemental Figure 3. HPLC profile and ESI mass spectrum of H-[Ala1-Cys7-Gly28]-α-thioester. (A) Analytical RP-HPLC of purified H-[Ala1-Cys7-Gly28]-α-thioester. (B) ESI-Mass spectrum of the

purified H-[Ala1-Cys7-Gly28]-α-thioester (m/z calcd. for C139H233N39O46S3: [M+H]+ 3283.8, found

3282.4 (deconvoluted)). Purification was performed by preparative HPLC (Proteonavi C4 Φ10 × 250

mm, 0.1% TFA: 0.1% TFA in 90% MeCN = 95 : 5 over 5 min then 65 : 35 to 45 : 55 over 30 min at 2.5

mL/min).

Supplemental Figure 4. HPLC profile and ESI mass spectrum of H-[Cys29, 33(Acm)-Tyr49]-α-thioester. (A) Analytical RP-HPLC of purified H-[Cys29, 33(Acm)-Tyr49]-α-thioester. (B) ESI-Mass

spectrum of the purified H-[Cys29, 33(Acm)-Tyr49]-α-thioester (m/z calcd. for C111H171N29O39S4: [M+H]+

2665.0, found 2665.4 (deconvoluted)). Purification was performed by preparative HPLC (Proteonavi C4

Φ10 × 250 mm, 0.1% TFA : 0.1% TFA in 90% MeCN = 95 : 5 over 5 min then 75 : 25 to 50 : 50 over

30 min at 2.5 mL/min).

Supplemental Figure 5. HPLC profile and ESI mass spectrum of H-[Cys29, 33(Acm)-Asn38(glycan)-Tyr49]-α-thioester. (A) Analytical RP-HPLC of purified H-[Cys29, 33(Acm)-Asn38(glycan)-Tyr49]-α-

thioester. (B) ESI-Mass spectrum of the purified H-[Cys29, 33(Acm)-Asn38(glycan)-Tyr49]-α-thioester

(m/z calcd. for C211H319N35O102S4: [M+H]+ 5107.2, found 5106.4 (deconvoluted). Purification was

performed by preparative HPLC (Proteonavi C4 Φ10 × 250 mm, 0.1% TFA : 0.1% TFA in 90% MeCN

= 95 : 5 over 5 min then 75 : 25 to 55 : 45 over 30 min at 2.5 mL/min).

Supplemental Figure 6. HPLC profile and ESI mass spectrum of H-[Cys79(Thz)-Trp88(formyl)-Lys97]-α-thioester. (A) Analytical RP-HPLC of purified H-[Cys79(Thz)-Trp88(formyl)-Lys97]-α-

thioester. (B) ESI-Mass spectrum of the purified H-[Cys79(Thz)-Trp88(formyl)-Lys97]-α-thioester (m/z

calcd. for C103H160N26O32S3: [M+H]+ 2371.7, found 2371.4 (deconvoluted). Purification was performed

by preparative HPLC (Proteonavi C4 Φ10 × 250 mm, 0.1% TFA : 0.1% TFA in 90% MeCN = 95 : 5

over 5 min then 75 : 25 to 50 : 50 over 30 min at 2.5 mL/min). Another peak beside main mass peak in

B is the desired H-[Cys79(Thz)-Trp88(formyl)-Lys97]-α-thioester, but formyl group of tryptophan

removed. This formyl group would be removed during synthesis of EPO-full peptide by NCL and

therefore this segment was used for NCL without further purification.

Supplemental Figure 7. HPLC profile and ESI mass spectrum of H-[Cys79(Thz)-Asn83(glycan)-Trp88(formyl)-Lys97]-α-thioester. (A) Analytical RP-HPLC of purified H-[Cys79(Thz)-Asn83(glycan)-

Trp88(formyl)-Lys97]-α-thioester. (B) ESI-Mass spectrum of the purified H-[Cys79(Thz)-Asn83(glycan)-

Trp88(formyl)-Lys97]-α-thioester (m/z calcd. for C203H308N32O95S3: [M+H]+ 4814.0, found for 4814.1

(deconvoluted). Purification was performed by preparative HPLC (Proteonavi C4 Φ10 × 250 mm, 0.1%

TFA : 0.1% TFA in 90% MeCN = 95 : 5 over 5 min then 75 : 25 to 50 : 50 over 30 min at 2.5 mL/min).

Supplemental Figure 8. HPLC profile and ESI mass spectrum of H-[Cys98(Thz)-Ala127]-α-thioester. (A) Analytical RP-HPLC of purified H-[Cys98(Thz)-Ala127]-α-thioester. (B) ESI-MS spectrum

of the purified H-[Cys98(Thz)-Ala127]-α-thioester (m/z calcd. for C131H226N38O44S3: [M+H]+ 3134.6,

found 3134.8 (deconvoluted)). Purification was performed by preparative HPLC (Proteonavi C4 Φ10 ×

250 mm, 0.1% TFA : 0.1% TFA in 90% MeCN = 95 : 5 over 5 min then 75 : 25 to 50 : 50 over 30 min

at 2.5 mL/min).

Supplemental Figure 9. HPLC profile and ESI mass spectrum of H-[Cys50-Ala78]-α-hydrazide. (A)

Analytical RP-HPLC of purified H-[Cys50-Ala78]-α-hydrazide. (B) ESI-Mass spectrum of the purified

H-[Cys50-Ala78]-α-hydrazide (m/z calcd. for C143H233N43O39S2: [M+H]+ 3243.8, found 3244.1

(deconvoluted). Purification was performed by preparative HPLC (Proteonavi C4 Φ10 × 250 mm, 0.1%

TFA : 0.1% TFA in 90% MeCN = 95 : 5 over 5 min then 75 : 25 to 50 : 50 over 30 min at 2.5 mL/min).

Supplemental Figure 10. HPLC profile and ESI mass spectrum of H-[Ala1-Cys7-Asn24(glycan)-Gly28]-α-thioester. (A) Analytical RP-HPLC of purified H-[Ala1-Cys7-Asn24(glycan)-Gly28]-α-thioester.

(B) ESI-Mass spectrum of the purified H-[Ala1-Cys7-Asn24(glycan)-Gly28]-α-thioester (m/z calcd. for

C239H381N45O109S3: [M+H]+ 5726.0, found 5725.7 (deconvoluted). Purification was performed by using

a linear gradient (Proteonavi C4 Φ10 × 250 mm, 0.1% TFA : 0.1% TFA in 90% MeCN = 95 : 5 over 5

min then 75 : 25 to 50 : 50 over 30 min at 2.5 mL/min).

Supplemental Figure 11. Monitoring NCL between H-[Cys29,33(Acm)-Asn38(glycan)-Tyr49]-α-thioester and H-[Cys50-Ala78]-α-hydrazine. (A) RP-HPLC analysis of reaction mixture at a) starting

point (t

Supplemental Figure 12. Monitoring NCL between H-[Cys29, 33(Acm)-Asn38(glycan)-Ala78]-α-hydrazide and H-[Cys79-Asn83(glycan)-Arg166]-OH. (A) RP-HPLC analysis of reaction mixture a)

after 5 h and b) after purification of the product (H-[Cys29, 33(Acm)-Asn38, 83(glycan)2-Arg166]-OH). (B)

ESI-MS analysis of the product (H-[Cys29, 33(Acm)-Asn38,83(glycan)2-Arg166]-OH). m/z calcd. for

C853H1374N208O322S8: [M+H]+ 19952.8, found 19952.8 (deconvoluted).

Supplemental Figure 13. Monitoring the desulfurization reaction of H-[Cys29, 33, 161(Acm)-Cys50, 79,

98, 128-Asn38, 83(glycan)2-Arg166]-OH (A) RP-HPLC analysis of reaction mixture at a) starting point (t

Supplemental Figure 14. Monitoring of the removal of Acm group of H-[Cys29, 33, 161(Acm)-Asn38,

83(glycan)2-Arg166]-OH by RP-HPLC and ESI-MS. (A) RP-HPLC analysis of reaction mixture at a)

starting point (t

Supplemental Figure 15. Monitoring the NCL between H-[Ala1-Asn24(glycan)-Gly28]--thioester and H-[Cys29-Asn38, 83(glycan)2-Arg166]-OH. (A) RP-HPLC analysis of reaction mixture at a) starting

point (t

Supplemental Figure 16. The folding reaction of EPON24, N38, N83 (polypeptide form of H-[Ala1-

Asn24, 38, 83(glycan)3-Arg166]-OH. (A) Scheme of EPON24, N38, N83 2 folding with redox conditions. (B)

RP-HPLC profiles of folding intermediate; 0.1 mg/mL sample a) in dissolving buffer; b) in buffer A for

20 h; c) in buffer B for 1 h and d) for 20 h; e) in buffer C for 15 h, f) 0.01mg/mL sample in buffer C for 15 h. A large peak at 14.5 min in each HPLC profiles is due to change the gradient of solution. (C) SDS-

PAGE of the folding intermediates. All sample were loaded and analyzed without dithiothreitol (DTT)

treatment.

Supplemental Figure 17. The folding reactions of EPON38, N83 (polypeptide form of H-[Ala1-Asn38,

83(glycan)2-Arg166]-OH) and EPON24, N83 (polypeptide form of H-[Ala1-Asn24, 83(glycan)2-Arg166]-

OH). ESI-Mass spectrum of (A) EPON38, N83 3 and (B) EPON24, N83 4. RP-HPLC profiles of folding

intermediate (C) EPON38, N83 3 and (D) EPON24, N83 4; 0.1 mg/mL sample a) in dissolving buffer; b) in

buffer A for 20 h; c) in buffer B for 1 h and d) for 20 h; e) in buffer C for 15 h, f) 0.01 mg/mL sample in

buffer C for 15 h. A large peak at 14.5 min in each HPLC profiles is due to change the gradient of

solution.

Supplemental Figure 18. The folding reactions of EPON24, N38 (polypeptide form of H-[Ala1-Asn24, 38(glycan)2-Arg166]-OH) and EPON83 (polypeptide form of H-[Ala1-Asn83(glycan)-Arg166]-OH). ESI-

Mass data of (A) EPON24, N38 5 and (B) EPON83 6. RP-HPLC profiles of folding intermediate (C)

EPON24, N38 5 and (D) EPON83 6; 0.1 mg/mL sample a) in dissolving buffer; b) in buffer A for 20 h; c) in

buffer B for 1 h and d) for 20 h; e) in buffer C for 15 h, f) 0.01 mg/mL sample in buffer C for 15 h. A

large peak at 14.5 min in each HPLC profiles is due to change the gradient of solution.

Results of folding experiments

HPLC-Analytical yield was estimated by the calculation: area of folded EPO / (area of folded EPO +

misfolded EPO).

Folded EPON24, N38, N83 2: The yield of compound 2 was estimated to be 86% by HPLC. ESI-MS: m/z

calcd. for C1065H1718N250O423S5: [M+H]+ 24953.8, found 24953 (deconvoluted).

Folded EPON38, N83 3: The yield of compound 3 was estimated to be 90% by HPLC. ESI-MS: m/z calcd.

for C981H1582N244O362S5: [M+H]+ 22747.9, found 22747 (deconvoluted).





Folded EPON83 6: The yield of compound 6 was estimated to be 63% by HPLC. ESI-MS: m/z calcd. for

C897H1446N238O301S5: [M+H]+ 20541.9, found 20541 (deconvoluted).

Supplemental Figure 19. Monitoring of in vitro folding by SDS-PAGE. Results of (A) EPON38, N83 3,

(B) EPON24, N83 4, (C) EPON24, N38 5, (D) EPON83 6. All sample were loaded and analyzed without DTT

treatment. In terms of EPON24, N38, N83 2 folding is shown in fig. S16.

Supplemental Figure 20. Analysis of disulfide bond positions of EPON24, N38, N83 2 by trypsin

digestion. (A) HPLC data of a) after trypsin digestion (12 h incubation) and b) after trypsin digestion

(12 h incubation) followed by the treatment with TCEP. The blue dotted line indicates a disulfide bond.

(B) ESI-Mass spectra of peak (1) – (6). Based on the mass data, we determined peptide and glycopeptide

sequence as well as the number of disulfide bond. Peptide of peak (1) was found to have a disulfide

bond and this peak (1) disappeared after reduction with TCEP. This observation indicated peptide of

peak (1) had a disulfide bond. The products reduced could not be found in the same analytical

conditions. A peak (4) was newly observed after TCEP treatment and this peak did not include peptide-

products, so we concluded this peak (4) might be derived from reagent. Peak (5) identical with the

structure of the reduced peptide of peak (2). Peak (3) and (6) were found to be an identical peptide. The

asterisk (*) means from an unspecific compound.

Supplemental Figure 21. Analysis of disulfide bond positions of EPON38, N83 3 by trypsin digestion. (A) HPLC data of a) after trypsin digestion (12 h incubation) and b) after trypsin digestion (12 h

incubation) followed by the treatment with TCEP. The blue dotted line indicates a disulfide bond. (B)

ESI-Mass spectra of peak (1) – (6). Analysis of these peptide fragments was performed with the same

protocol in the analysis of EPON24, N38, N83 2 (fig. S20). The asterisk (*) means from an unspecific

compound. A peak (4) was newly observed after TCEP treatment and this peak did not include peptide-

products, so we concluded this peak (4) might be derived from reagent.

Supplemental Figure 22. Analysis of disulfide bond positions of EPON24, N83 4 by trypsin digestion.

(A) HPLC data of a) after trypsin digestion (12 h incubation) and b) after trypsin digestion (12 h






Supplemental Figure 23. Analysis of disulfide bond positions of EPON24, N38 5 by trypsin digestion. (A) HPLC data of a) after trypsin digestion (12 h incubation) and b) after trypsin digestion (12 h






Supplemental Figure 24. Analysis of disulfide bond positions of EPON83 6 by trypsin digestion. (A)

HPLC profile of a) after trypsin digestion (12 h incubation) and b) after trypsin digestion (12 h






Supplemental Figure 25. Characterization of misfolded EPON24, N83 (compound 7). (A) Structure of

the resultant glycopeptide and peptide fragments (1), (2) and (3) after trypsin digestion. These structures

of (1)-(3) were determined based on mass analysis. The blue dotted line between Cys 29 and Cys 33

indicate disulfide bond. HPLC profile of a) after trypsin digestion (12 h incubation) and b) after trypsin

digestion (12 h incubation) followed by the treatment with TCEP. Peak (2) is the reduced

sialylglycopeptide fragment of peak (1). (B) SDS-PAGE; line a) without treatment of TCEP; b) treated

with 10 mM TCEP and c) with 40 mM TCEP; d) treated with DTT, e) folded EPON24, N83 (compound 3)

treated with DTT. Line a) shows several smear bands from 50 KDa to ca. 70 KDa (See fig. S19 B). (C)

CD spectrum of compound 7.

High-resolution mass spectra of EPO glycoforms

All measurements were performed with Bruker SolariX (9.4 Tesla). Samples were dissolved into a

solution of 50% MeOH containing 1% acetic acid and subjected into an instrument as direct infusion.

An internal standard (ESI tuning mix: Agilent Technology®) was used (1521.971475 and

2121.933152). Ionization condition employed electrospray ionization under the 200°C and therefore the

small amount of the terminal sialic acid was removed during ionization, although conventional ESI mass

instrument did not show the considerable ion peaks derived from desialylation (Bruker Amazon ETD

shown Fig. 4, and fig. S17 and S18). The amounts of desialylated ion peaks were automatically

increased dependent on the number of sialyloligosaccharides in EPO molecules. This increasing in the

intensity of ion peaks should achieve to ca 20% amount in the case of EPO having three glycans, even

though an individual sialyloligosaccharide includes 3-5% desialylated oligosaccharide. The amount of

population of desialylated oligosaccharides can be estimated by a simple equation (a glycoform having a

desialylated oligosaccharide at the 83 position = 100%N24+100%N38+95%N83. Another example: a

glycoform having two desialylated oligosaccharide at the 24 and 83 position =

95%N24+100%N38+95%N83. Total population of desialylated oligosaccharide in high-resolution mass

spectrometry was observed according to the addition of these total possible calculations. The marks

shown (* and ) in fig. S26-30 are desialylated EPO glycoforms and internal standard ion peak position,

respectively. The large ion peaks () of the internal standard were erased by Photoshop CS3 and other

ion peaks observed are shown as it is.

The most largest monoisotopic ion peak was selected for the determination of correct mass and

these values are shown in figure comparing computational simulation pattern.

Supplemental Figure 26. High-resolution mass spectrum of EPON24, N38, N83 2. The marks shown (*

and ) in Figure are desialylated EPO glycoforms and internal standard ion peak position, respectively.

The observed monoisotopic high resolution mass was 1664.4700 (the most potent monoisotopic peak:

calcd 1664.4712). The estimation (deconvolution) of mass for C1065H1718N250O423S5 is 24952.0500 (based on the most potent monoisotopic peak: calcd 24952.0680).

Supplemental Figure 27. High-resolution mass spectrum of EPON38, N83 3. The marks shown (* and

) in Figure are desialylated EPO glycoforms and internal standard ion peak position, respectively. The

observed monoisotopic high resolution mass was 1517.4114 (the most potent monoisotopic peak: calcd

1517.4195). The estimation (deconvolution) of mass for C981H1582N244O362S5 is 22746.1710 (based on the most potent monoisotopic peak: calcd 22746.2925).

Supplemental Figure 28. High-resolution mass spectrum of EPON24, N38, 4. The marks shown (* and




Supplemental Figure 29. High-resolution mass spectrum of EPON38, N83 5. The marks shown (* and



1517.4195). The estimation (deconvolution) of mass for C981H1582N244O362S5 is 22746.2733 (based on the most potent monoisotopic peak: calcd 22746.2928). A slight disorder of observed peak pattern

between found and simulation might be due to measurement with slight high concentration of

glycoprotein 5 sample.

Supplemental Figure 30. High-resolution mass spectrum of EPON83 6. The marks shown (* and )

in Figure are desialylated EPO glycoforms and internal standard ion peak position, respectively. The



supplementary materials for - science advances · 2016. 1. 12. · supplemental figure 1. acid...

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