(a) sds-page/coomassie staining analysis of purified ... filebroadband high mass 8000.0 m/z laser...

Supplementary Figure 1

SDS-PAGE and CD spectra of purified betatrophin 9

(a) SDS-PAGE/Coomassie staining analysis of purified synthetic betatrophin 9. (b) circular dichroism

spectra of refolded synthetic betatrophin 9. ~20μM of betatrophin 9 in PBS buffer pH 7.2 was analyzed in a 1 mm quartz cell.

Nature Protocols: doi:10.1038/nprot.2016.052

Supplementary Data 1: RP-HPLC analysis of betatrophin Fragment 1 H2N-F1(22–59)-Leu-KA running on a C18 column.

H-APNleGGPELAQHEELTLLFHGTLQLGQALNGVYRTTEGR-Leu-KA Calculated M.W: 4229.7 Isolated yield: 7.5–8.5% Calculated E.M: 4227.2 (The linear gradient for analytical HPLC: 5% buffer B in buffer A for 5 min and 5% buffer B to 95% B in A over 20 min at rt with flow rate 1 ml/min) (Isolated gradient: 15% buffer B in buffer A for 7.5 min and 10% buffer B to 90% B in A over 45 min at rt with flow rate 40 ml/min)

(a) RP-HPLC trace of the crude Fragment 1 H2N-F1(22–59)-Leu-KA. (b) RP-HPLC trace of the purified Fragment 1 H2N-F1(22–59)-Leu-KA.

H NH

O

O

OH

1

Me

Me

F1 (22-59)

!" #$%" %" &$%" '!" '#$%" '%" '&$%" #!" ##$%" #%" #&$%" (!" !" #$%" %" &$%" '!" '#$%" '%" '&$%" #!" ##$%" #%" #&$%" (!"

Acquisition Parameter11.06.2013 15:38:43Acquisition Date:High_mass_MALDI_Witt new2Method:Rolf HäfligerOperator:File Name: D:\Data\ESI-MALDI Daten\FT026xx\FT02649_0_D3.d

Dual (MALDI/ESI)Source 1.3 barPositive Polarity Nebulizer Gas303.2 m/z 0.005 secLaser Shot FrequencyBroadband Low Mass Drying Gas Flow Rate 3.7 L/min

16.0 lpLaser Power CapillaryBroadband High Mass 8000.0 m/z 4500.0 V50 No. of Laser Shots1 No. of Cell Fills Drying Gas

Temperature200.0 °C

0.002 secFlight Time to Acq. CellApodization Sine-BellMultiplication

1448.4655

4343.2424

+MS

1000 2000 3000 4000 5000 6000 7000 m/z0.0

0.5

1.0

1.5

2.0

2.5

9x10Intens.

4341.2321

4342.2435

4343.2424

4344.2467

4345.2553

4346.2672

4347.2707

4348.27174349.2669

FT02649_0_D3.d: +MS

0

20

40

60

80

Intens.[%]

4340 4342 4344 4346 4348 4350 m/z

FT02649 Thibault Harmand/Bode - TJH 168 - CCA uncal

ETH - MS-Service LOC - D-CHAB 1 of 2Page Bruker Daltonics SOLARIX






1448.4655

4343.2424

+MS

1000 2000 3000 4000 5000 6000 7000 m/z0.0

0.5

1.0

1.5

2.0

2.5

9x10Intens.

4341.2321

4342.2435

4343.2424

4344.2467

4345.2553

4346.2672

4347.2707

4348.27174349.2669

FT02649_0_D3.d: +MS

0

20

40

60

80

Intens.[%]

4340 4342 4344 4346 4348 4350 m/z








1448.4655

4343.2424

+MS

1000 2000 3000 4000 5000 6000 7000 m/z0.0

0.5

1.0

1.5

2.0

2.5

9x10Intens.

4341.2321

4342.2435

4343.2424

4344.2467

4345.2553

4346.2672

4347.2707

4348.27174349.2669

FT02649_0_D3.d: +MS

0

20

40

60

80

Intens.[%]

4340 4342 4344 4346 4348 4350 m/z



Acquisition Parameter30.06.2014 14:08:39Acquisition Date:High_mass_MALDI_Witt new2Method:Rolf HäfligerOperator:File Name: D:\Data\ESI-MALDI Daten\FT052xx\FT05212a_0_H10_0_H12.d





650.9466

1952.7828

2724.7003

4230.2211

5427.3935

+MS

1000 2000 3000 4000 5000 6000 7000 m/z0

1

2

3

8x10Intens.

4228.2144

4229.2178

4230.2211

4231.2231

4232.2274

4233.2302

4234.23434235.2291 4236.2513

FT05212a_0_H10_0_H12.d: +MS

4228.2149

4229.2182

4230.2206

4231.2247

4232.2281

4233.2315

4234.23504235.2359 4236.2393

FT05212a_0_H10_0_H12.d: C 188 H 300 N 53 O 58 ,4228.220

5

10

15

Intens.[%]

0

20

40

60

80

[%]

4228 4229 4230 4231 4232 4233 4234 4235 4236 4237 m/z

FT05212a Thibault Harmand/Bode - TJH 253 - CCA







650.9466

1952.7828

2724.7003

4230.2211

5427.3935

+MS

1000 2000 3000 4000 5000 6000 7000 m/z0

1

2

3

8x10Intens.

4228.2144

4229.2178

4230.2211

4231.2231

4232.2274

4233.2302

4234.23434235.2291 4236.2513

FT05212a_0_H10_0_H12.d: +MS

4228.2149

4229.2182

4230.2206

4231.2247

4232.2281

4233.2315

4234.23504235.2359 4236.2393

FT05212a_0_H10_0_H12.d: C 188 H 300 N 53 O 58 ,4228.220

5

10

15

Intens.[%]

0

20

40

60

80

[%]

4228 4229 4230 4231 4232 4233 4234 4235 4236 4237 m/z








650.9466

1952.7828

2724.7003

4230.2211

5427.3935

+MS

1000 2000 3000 4000 5000 6000 7000 m/z0

1

2

3

8x10Intens.

4228.2144

4229.2178

4230.2211

4231.2231

4232.2274

4233.2302

4234.23434235.2291 4236.2513

FT05212a_0_H10_0_H12.d: +MS

4228.2149

4229.2182

4230.2206

4231.2247

4232.2281

4233.2315

4234.23504235.2359 4236.2393

FT05212a_0_H10_0_H12.d: C 188 H 300 N 53 O 58 ,4228.220

5

10

15

Intens.[%]

0

20

40

60

80

[%]

4228 4229 4230 4231 4232 4233 4234 4235 4236 4237 m/z



Acquisition Parameter30.06.2014 14:04:18Acquisition Date:High_mass_MALDI_Witt new2Method:Rolf HäfligerOperator:File Name: D:\Data\ESI-MALDI Daten\FT052xx\FT05211a_0_L7_0_H10.d





650.9555

1952.7859

4152.2339

5427.3898 6560.0131

+MS

1000 2000 3000 4000 5000 6000 7000 m/z0.00

0.25

0.50

0.75

1.00

1.25

8x10Intens.

4150.2270

4151.23124152.2339

4153.2363

4154.2382

4155.2412

4156.2497

FT05211a_0_L7_0_H10.d: +MS

4150.2302

4151.2330

4152.2366

4153.2398

4154.2432

4155.2467

4156.25024157.2512 4158.2546

FT05211a_0_L7_0_H10.d: C 178 H 302 N 57 O 55 S 1 ,4150.230

5

10

15

Intens.[%]

0

20

40

60

80

[%]

4150 4151 4152 4153 4154 4155 4156 4157 4158 4159 m/z








650.9466

1952.7828

2724.7003

4230.2211

5427.3935

+MS

1000 2000 3000 4000 5000 6000 7000 m/z0

1

2

3

8x10Intens.

4228.2144

4229.2178

4230.2211

4231.2231

4232.2274

4233.2302

4234.23434235.2291 4236.2513

FT05212a_0_H10_0_H12.d: +MS

4228.2149

4229.2182

4230.2206

4231.2247

4232.2281

4233.2315

4234.23504235.2359 4236.2393

FT05212a_0_H10_0_H12.d: C 188 H 300 N 53 O 58 ,4228.220

5

10

15

Intens.[%]

0

20

40

60

80

[%]

4228 4229 4230 4231 4232 4233 4234 4235 4236 4237 m/z



a b 1 1


Supplementary Data 2: RP-HPLC analysis of betatrophin Fragment 2 Opr-F2(62–95)-Leu-SY running on a C18 column.

Opr-KARNSLGLYGRTIELLGQEVSRGRDAAQELRASL-Leu-SY Calculated M.W: 4151.7 Isolated yield: 17–19% Calculated E.M: 4149.2 (The linear gradient for analytical HPLC: 5% buffer B in buffer A for 5 min and 5% buffer B to 95% B in A over 20 min at rt with flow rate 1 ml/min) (Isolated gradient: 10% buffer B in buffer A for 7.5 min and 10% buffer B to 90% B in A over 45 min at rt with flow rate 40 ml/min)

(a) RP-HPLC trace of the crude Fragment 2 Opr-F2(62–95)-Leu-SY. (b) RP-HPLC trace of the purified Fragment 2 Opr-F2(62–95)-Leu-SY.

NH

NH

OHN

OO

CN

S

O

2

Me

Me

H2NOC2

F2 (62-95)

!" #$%" %" &$%" '!" '#$%" '%" '&$%" #!" ##$%" #%" #&$%" (!" !" #$%" %" &$%" '!" '#$%" '%" '&$%" #!" ##$%" #%" #&$%" (!"






1448.4655

4343.2424

+MS

1000 2000 3000 4000 5000 6000 7000 m/z0.0

0.5

1.0

1.5

2.0

2.5

9x10Intens.

4341.2321

4342.2435

4343.2424

4344.2467

4345.2553

4346.2672

4347.2707

4348.27174349.2669

FT02649_0_D3.d: +MS

0

20

40

60

80

Intens.[%]

4340 4342 4344 4346 4348 4350 m/z








1448.4655

4343.2424

+MS

1000 2000 3000 4000 5000 6000 7000 m/z0.0

0.5

1.0

1.5

2.0

2.5

9x10Intens.

4341.2321

4342.2435

4343.2424

4344.2467

4345.2553

4346.2672

4347.2707

4348.27174349.2669

FT02649_0_D3.d: +MS

0

20

40

60

80

Intens.[%]

4340 4342 4344 4346 4348 4350 m/z








1448.4655

4343.2424

+MS

1000 2000 3000 4000 5000 6000 7000 m/z0.0

0.5

1.0

1.5

2.0

2.5

9x10Intens.

4341.2321

4342.2435

4343.2424

4344.2467

4345.2553

4346.2672

4347.2707

4348.27174349.2669

FT02649_0_D3.d: +MS

0

20

40

60

80

Intens.[%]

4340 4342 4344 4346 4348 4350 m/z








650.9555

1952.7859

4152.2339

5427.3898 6560.0131

+MS

1000 2000 3000 4000 5000 6000 7000 m/z0.00

0.25

0.50

0.75

1.00

1.25

8x10Intens.

4150.2270

4151.23124152.2339

4153.2363

4154.2382

4155.2412

4156.2497

FT05211a_0_L7_0_H10.d: +MS

4150.2302

4151.2330

4152.2366

4153.2398

4154.2432

4155.2467

4156.25024157.2512 4158.2546

FT05211a_0_L7_0_H10.d: C 178 H 302 N 57 O 55 S 1 ,4150.230

5

10

15

Intens.[%]

0

20

40

60

80

[%]

4150 4151 4152 4153 4154 4155 4156 4157 4158 4159 m/z








650.9555

1952.7859

4152.2339

5427.3898 6560.0131

+MS

1000 2000 3000 4000 5000 6000 7000 m/z0.00

0.25

0.50

0.75

1.00

1.25

8x10Intens.

4150.2270

4151.23124152.2339

4153.2363

4154.2382

4155.2412

4156.2497

FT05211a_0_L7_0_H10.d: +MS

4150.2302

4151.2330

4152.2366

4153.2398

4154.2432

4155.2467

4156.25024157.2512 4158.2546

FT05211a_0_L7_0_H10.d: C 178 H 302 N 57 O 55 S 1 ,4150.230

5

10

15

Intens.[%]

0

20

40

60

80

[%]

4150 4151 4152 4153 4154 4155 4156 4157 4158 4159 m/z








650.9555

1952.7859

4152.2339

5427.3898 6560.0131

+MS

1000 2000 3000 4000 5000 6000 7000 m/z0.00

0.25

0.50

0.75

1.00

1.25

8x10Intens.

4150.2270

4151.23124152.2339

4153.2363

4154.2382

4155.2412

4156.2497

FT05211a_0_L7_0_H10.d: +MS

4150.2302

4151.2330

4152.2366

4153.2398

4154.2432

4155.2467

4156.25024157.2512 4158.2546

FT05211a_0_L7_0_H10.d: C 178 H 302 N 57 O 55 S 1 ,4150.230

5

10

15

Intens.[%]

0

20

40

60

80

[%]

4150 4151 4152 4153 4154 4155 4156 4157 4158 4159 m/z



b a 2 2


Supplementary Data 3: RP-HPLC analysis of betatrophin Fragment 3 FmocOpr-F3(98–132)-Leu-KA running on a C18 column.

FmocOpr-TQNleEEDILQLQAEATAEVLGEVAQAQKVLRDSVQR-Leu-KA Calculated M.W: 4342.8 Isolated yield: 12–18% Calculated E.M: 4340.2 (The linear gradient for analytical HPLC: 35% buffer B in buffer A for 3 min and 35% buffer B to 80% B in A over 30 min at rt with flow rate 1 ml/min) (Isolated gradient: 35% buffer B in buffer A for 7.5 min and 35% buffer B to 80% B in A over 45 min at rt with flow rate 40 ml/min)

(a) RP-HPLC trace of the crude Fragment 3 FmocOpr-F3(98–132)-Leu-KA. (b) RP-HPLC trace of the purified Fragment 3 FmocOpr-F3(98–132)-Leu-KA.

NH

NH

OFmocN

OO

OH

O

5

Me

Me

F3 (98-132)

!" #$%" %" &$%" '!" '#$%" '%" '&$%" #!" ##$%" #%" #&$%" (!" (#$%" (%" !" #$%" %" &$%" '!" '#$%" '%" '&$%" #!" ##$%" #%" #&$%" (!" (#$%" (%"






1448.4655

4343.2424

+MS

1000 2000 3000 4000 5000 6000 7000 m/z0.0

0.5

1.0

1.5

2.0

2.5

9x10Intens.

4341.2321

4342.2435

4343.2424

4344.2467

4345.2553

4346.2672

4347.2707

4348.27174349.2669

FT02649_0_D3.d: +MS

0

20

40

60

80

Intens.[%]

4340 4342 4344 4346 4348 4350 m/z



b a

5

5


Supplementary Data 4: RP-HPLC analysis of betatrophin Fragment 4 FmocOpr-F4(135–165)-Leu-KA running on a C18 column.

FmocOpr-VQLRSAWLGPAYREFEVLKAHADKQSHILWA-Leu-KA Calculated M.W: 4083.6 Isolated yield: 10–12% Calculated E.M: 4081.1 (The linear gradient for analytical HPLC: 20% buffer B in buffer A for 3 min and 20% buffer B to 95% B in A over 17 min at rt with flow rate 1 ml/min) (Isolated gradient: 30% buffer B in buffer A for 7.5 min and 30% buffer B to 80% B in A over 25 min at rt with flow rate 40 ml/min)

(a) RP-HPLC trace of the crude Fragment 4 FmocOpr-F4(135–165)-COOH. (b) RP-HPLC trace of the purified Fragment 4 Opr-F4(135–165)-COOH.

NH

NH

OFmocN

OO

OH

O

3

Me

Me

F4 (135-165)

!" #$%" %" &$%" '!" '#$%" '%" '&$%" #!" ##$%" #%" !" #$%" %" &$%" '!" '#$%" '%" '&$%" #!" ##$%" #%"

b a 3 3


Supplementary Data 5: RP-HPLC analysis of betatrophin Fragment 5 Opr-F5(168–198)-COOH running on a C18 column.

Opr- GHVQRQRRENleVAQQHRLRQIQERLHTAALP-COOH Calculated M.W: 3796.3 Isolated yield: 12–14% Calculated E.M: 3794.1 (The linear gradient for analytical HPLC: 20% buffer B in buffer A for 3 min and 20% buffer B to 95% B in A over 17 min at rt with flow rate 1 ml/min) (Isolated gradient: 20% buffer B in buffer A for 7.5 min and 20% buffer B to 75% B in A over 25 min at rt with flow rate 40 ml/min)

(a) RP-HPLC trace of the crude Fragment 5 Opr-F5(168–198)-COOH. (b) RP-HPLC trace of the purified Fragment 5 Opr-F5(168–198)-COOH.

OHNH

OHN

O

4

F5 (168-198)

!" #$%" %" &$%" '!" '#$%" '%" '&$%" #!" ##$%" #%" !" #$%" %" &$%" '!" '#$%" '%" '&$%" #!" ##$%" #%"

a b 4 4


Supplementary Data 6: RP-HPLC trace of betatrophin H2N-F1-F2(22–95)-Leu-KA by KAHA ligation

H-APNleGGPELAQHEELTLLFHGTLQLGQALNGVYRTTEGRL- T§KARNSLGLYGRTIELLGQEVSRGRDAAQELRASL-Leu-KA Calculated M.W: 8155.1 Isolated yield: 41–45% Calculated E.M: 8150.3 (The linear gradient for analytical HPLC: 30% buffer B in buffer A for 3 min and 30% buffer B to 75% B in A over 22 min at rt with flow rate 1 ml/min) (Isolated gradient: 30% buffer B in buffer A for 7.5 min and 30% buffer B to 75% B in A over 40 min at rt with flow rate 40 ml/min) a)

H NH

O

O

OH NH

NH

OHN

OO

CN

S

O

1 2

H NH

O

NH

NH

O

O

OH

O

NH3

O

6

1) KAHA ligation2) Oxidation ONE POT(43% Isolated yield)

Me

Me

Me

Me

Me

Me

Me

Me

H2NOC2

F1 (22-59) F2 (62-95)

F1 (22-59) F2 (62-95)

!" #$%" %" &$%" '!" '#$%" '%" '&$%" #!" ##$%" #%"

1 2


b)

c)

d)

!" #$%" %" &$%" '!" '#$%" '%" '&$%" #!" ##$%" #%"

!" #$%" %" &$%" '!" '#$%" '%" '&$%" #!" ##$%" #%"

!" #$%" %" &$%" '!" '#$%" '%" '&$%" #!" ##$%" #%"

1

6ʼ

6

1

6

F1 (22-59)H3N NH

O

F2 (62-95)NH

NH

O

CN

S

O

NH3

O

6'

Me

Me

Me

Me

OH2NOC

2


e)

a) Ligation H2N-F1(22–59)-Leu-KA and Opr-F2(62–95)-Leu-SY at t=0 b) ligation after 8 hours; 6ʼ is the depsi-ligated product sulfurylide c) One pot oxidation d) Purified H2N-F1-F2(22–96)-Leu-KA e) HRMS of the purified H2N-F1-F2(22–96)-Leu-KA

Acquisition ParameterAcquisition Date: 17.07.2015 12:48:19Method: ETH_HyStar_HPLC_QTOF_POS_ProtMass_Loop-AS.mOperator: Louis BertschiD:\Data\max243xx\MAX24386.dFile Name:

Positive ESI Source Type 1.6 BarIon Polarity Set Nebulizer200 °CFocus Active 4500 VSet Capillary Set Dry Heater

Set Dry Gas 8.0 l/minSet End Plate Offset -500 V100 m/zScan Begin5000 m/zScan End Set Collision Cell RF 2000.0 Vpp Source Set Divert Valve

1360.0731

+MS, 0.06-1.02min #8-150

500 1000 1500 2000 2500 3000 3500 4000 4500 m/z0.0

0.5

1.0

1.5

2.0

4x10Intens.

!""#$"%$

&&!'#%"$'

&(!$#$)(&

&!("#$*&(

"$(%#*($'

+,-. $#$! &#$"/01 2* &'$

!!'(#!)&*

*&''#(%)!

%%'&#%)$! &&!&(#!&*) &"!$%#!*)(

+,-. $#$! &#$"/01. 345617689:4; <,=>?1:. %")#*) &!%@#!'. $#&. ('$$$A

0.00

0.25

0.50

0.75

1.00

4x10Intens.

0

1

2

3

4

4x10

500 750 1000 1250 1500 1750 2000 2250 2500 2750 m/z

5000 6000 7000 8000 9000 10000 11000 12000 13000 Mass [Da]

MAX24386 Thibault Harmand/Bode - TJH-Betratro-F1,F2KA - ACN/H2O

Bruker Daltonics maXis ESI-QTOF Page 1 of 3ETH - MS-Service LOC - D-CHAB


Supplementary Data 7: RP-HPLC trace of betatrophin Opr-F4-F5(135–198)-COOH by KAHA ligation

Opr-VQLRSAWLGPAYREFEVLKAHADKQSHILWAL-T§GHVQRQRRENleVAQQHRLRQIQERLHTAALP -COOH Calculated M.W: 7613.6 Isolated yield: 48–53% Calculated E.M: 7609.1 (The linear gradient for analytical HPLC: 20% buffer B in buffer A for 3 min and 20% buffer B to 95% B in A over 22 min at rt with flow rate 1 ml/min) (Isolated gradient: 25% buffer B in buffer A for 7.5 min and 25% buffer B to 85% B in A over 23 min at rt with flow rate 40 ml/min) a)

NH

NH

OFmocN

OO

OH

O

OHNH

OHN

O

3 4

NH

OHN

O

7

Me

Me

1) KAHA ligation2) O-N shift, deprotection ONE POT (52% Isolated yield)

NH

O

NH

OHN

OH

Me

Me

F4 (135-165) F5 (168-198)

OHF5 (168-198)F4 (135-165)

!" #$%" %" &$%" '!" '#$%" '%" '&$%" #!" ##$%" #%"

4

3


b)

c)

d)

!" #$%" %" &$%" '!" '#$%" '%" '&$%" #!" ##$%" #%"

!" #$%" %" &$%" '!" '#$%" '%" '&$%" #!" ##$%" #%"

!" #$%" %" &$%" '!" '#$%" '%" '&$%" #!" ##$%" #%"

3

7ʼ

7

3ʼ

NH

OFmocN

O F4 (135-165) COOHF5 (168-198)F4 (135-165) NH

MeMe

O

NH

O

NH3

O

7'

3'

NH

NH

OHN

OO

F4 (135-165) OH

O

Me

Me

7


e)

a) Ligation FmocOpr-F4(135–165)-Leu-KA and Opr-F5(168–198)-COOH at t=0 b) ligation after 8 hours; 7ʼ is the depsi-ligated product c) One pot Fmoc deprotection and rearrangement; 3ʼ is the deprotected Opr-F4(135–165)-Leu-KA d) Purified Opr-F4-F5(135–198)-COOH e) HRMS of the purified Opr-F4-F5(135–198)-COOH

Acquisition ParameterAcquisition Date: 15.07.2015 14:49:33Method: ETH_HyStar_HPLC_QTOF_POS_ProtMass_Loop-AS.mOperator: Louis BertschiD:\Data\max243xx\MAX24344.dFile Name:



1088.6022

1523.6379

+MS, 0.08-1.09min #11-161

500 1000 1500 2000 2500 3000 3500 4000 4500 m/z0

1

2

3

4

4x10Intens.

!"##$%"&&!&%'$#%%(

!)&*$%*('

!'"+$)+)*&!&!$'**& &%!)$*((%

,-./ "$"# !$"'012 3!! !%!

+)(*$)'+')(%#$!)''

(%!*$!((*

'!+($"%!' !"'##$#&#( !*'"*$)+)#

,-./ "$"# !$"'012/ 45672879:;5< =->?@2;/ %&'$'' !#!%$"!/ "$!/ *)"""A

0.0

0.5

1.0

1.5

2.0

4x10Intens.

0.0

0.2

0.4

0.6

0.8

1.0

1.25x10

500 750 1000 1250 1500 1750 2000 2250 2500 m/z

4000 6000 8000 10000 12000 Mass [Da]

MAX24344 Claudia Murar/Bode - CM-6-BF4F5 - ACN/H2O



Supplementary Data 8: RP-HPLC trace of betatrophin Opr-F3-F4-F5(98–198)-COOH by KAHA ligation

Opr-TQNleEEDILQLQAEATAEVLGEVAQAQKVLRDSVQRL- T§VQLRSAWLGPAYREFEVLKAHADKQSHILWAL- T§GHVQRQRRENleVAQQHRLRQIQERLHTAALP -COOH Calculated M.W: 11690.2 Isolated yield: 47–52% Calculated E.M: 11683.3 (The linear gradient for analytical HPLC: 30% buffer B in buffer A for 3 min and 30% buffer B to 80% B in A over 22 min at rt with flow rate 1 ml/min) (Isolated gradient: 30% buffer B in buffer A for 7.5 min and 30% buffer B to 80% B in A over 25 min at rt with flow rate 40 ml/min) a)

NH

NH

OFmocN

OO

OH

O

5

NH

NH

OHN

OO

NH

OHN

OH8

NH

OHN

O

7

Me

Me

Me

Me

NH

O

NH

OHN

OH

Me

Me

1) KAHA ligation2) O-N shift, deprotection ONE POT (50% Isolated yield)

F3 (98-132) OHF5 (168-198)F4 (135-165)

F4 (135-165) OHF5 (168-198)F3 (98-132)

!" #$%" %" &$%" '!" '#$%" '%" '&$%" #!" ##$%" #%"

5

7


b)

c)

d)

!" #$%" %" &$%" '!" '#$%" '%" '&$%" #!" ##$%" #%"

!" #$%" %" &$%" '!" '#$%" '%" '&$%" #!" ##$%" #%"

!" #$%" %" &$%" '!" '#$%" '%" '&$%" #!" ##$%" #%"

8

8ʼ

8

NH

OFmocN

O F3 (98-132) COOHF5 (168-198)F4 (135-165)NH

O

NH

O

NH3

O

Me

Me

8'


Acquisition ParameterAcquisition Date: 19.09.2014 14:43:30Method: ETH_HyStar_HPLC_QTOF_POS_ProtMass_Loop-AS.mOperator: Oswald GreterD:\Data\max206xx\MAX20624.dFile Name:



1170.0375

1671.0520

+MS, 1.10-1.21min #161-178

500 1000 1500 2000 2500 3000 3500 4000 4500 m/z0.0

0.5

1.0

1.5

4x10Intens.

!"#!$%&'% ()*+ !$!% !$'!,-. /!"! !#0

()*+ !$!% !$'!,-.+ 1234.5467829 :);<=.8+ &>#$?@ !#">$?#+ %$!+ ?&%%%A

0.0

0.2

0.4

0.6

0.8

4x10Intens.

0

2

4

6

4x10

1000 2000 3000 4000 5000 6000 7000 8000 9000 m/z

10000 12000 14000 16000 18000 20000 22000 24000 Mass [Da]

MAX20624 Claudia Murar/Bode - CM4-125-2 - ACN/H2O


e)

a) Ligation FmocOpr-F3(98–132)-Leu-KA and Opr-F4-F5(135–198)-COOH at t=0 b) ligation after 15 hours; 8ʼ is the depsi-ligated product c) One pot Fmoc deprotection and rearrangement d) Purified Opr-F3-F4-F5(98–198)-COOH e) HRMS of the purified Opr-F3-F4-F5(98–198)-COOH

11690.9473


Supplementary Data 9: RP-HPLC trace of betatrophin H2N-F1-F2 F3-F4-F5(22–198)-COOH by KAHA ligation

H-APNleGGPELAQHEELTLLFHGTLQLGQALNGVYRTTEGRL- T§KARNSLGLYGRTIELLGQEVSRGRDAAQELRASLL- T§TQNleEEDILQLQAEATAEVLGEVAQAQKVLRDSVQRL- T§VQLRSAWLGPAYREFEVLKAHADKQSHILWAL- T§GHVQRQRRENleVAQQHRLRQIQERLHTAALP -COOH Calculated M.W: 19801.3 Isolated yield: 22–33% Calculated E.M: 19789.7 (The linear gradient for analytical HPLC: 30% buffer B in buffer A for 5 min and 30% buffer B to 80% B in A over 30 min at rt with flow rate 1 ml/min) (Isolated gradient: 30% buffer B in buffer A for 5 min and 30% buffer B to 80% B in A over 40 min at rt with flow rate 10 ml/min) a)

H NH

O

NH

NH

O

O

OH

O

NH3

O

6

NH

O

NH

OHN

OH

NH

O

NH

OHN

OH

F3 (98-132)NH

O

NH

OHN

OH

NH

O

NH

OHN

OH

9

NH

NH

OHN

OO

NH

OHN

OH8

1) KAHA ligation2) O-N shift ONE POT(33% Isolated yield)

Me

Me

Me

Me

Me

Me

Me

Me

Me

Me

Me

Me

Me

Me

F4 (135-165) OHF5 (168-198)F3 (98-132)F1 (22-59) F2 (62-95)

OHF5 (168-198)F4 (135-165)F3 (98-132)H F1 (22-59) F2 (62-95)

!" #$%" %" &$%" '!" '#$%" '%" '&$%" #!" ##$%" #%" #&$%" (!"

6 8


b)

c)

d)

!" #$%" %" &$%" '!" '#$%" '%" '&$%" #!" ##$%" #%" #&$%" (!"

!" #$%" %" &$%" '!" '#$%" '%" '&$%" #!" ##$%" #%" #&$%" (!"

!" #$%" %" &$%" '!" '#$%" '%" '&$%" #!" ##$%" #%" #&$%" (!"

6 9ʼ

8

9

8

6ʼ

9

COOHF3F4F5 (98-198)F1 (22-59)H3N F2 (62-95) NH

MeMe

O

NH

O

NH3

ONH

MeMe

O

O

9'

6'

F1 (22-59)H3N NH

O

NH

O

NH

OHN

OH

F2 (62-95)

Me

Me

Me

Me

OH

O


e)

a) Final ligation at t=0 b) ligation after 24 hours; 9ʼ is the depsi-Betatrophin c) One pot rearrangement; 6ʼ is the H2N-F1-F2(22–95)-Leu-KA rearranged d) Purified Betatrophin e) HRMS of the purified Betatrophin




622.0289

1061.21571667.0504

+MS, 1.04-1.18min #152-174

500 1000 1500 2000 2500 3000 3500 4000 4500 m/z0

2000

4000

6000

8000

Intens.

!""#$"%&

'$!'#"'()'!!)#$($*

'&**#)"!&

+,-. '#$* '#'%/01 2'(" ')*

19801.6000

+,-. '#$* '#'%/01. 345617689:4; <,=>?1:. (!%#@! ')!@#*(. $#'. @($$$A

0

1000

2000

3000

4000

5000Intens.

0.0

0.5

1.0

1.5

2.0

2.5

4x10

500 1000 1500 2000 2500 3000 m/z

12000 14000 16000 18000 20000 22000 24000 Mass [Da]






622.0289

1061.21571667.0504

+MS, 1.04-1.18min #152-174

500 1000 1500 2000 2500 3000 3500 4000 4500 m/z0

2000

4000

6000

8000

Intens.

!""#$"%&

'$!'#"'()'!!)#$($*

'&**#)"!&

+,-. '#$* '#'%/01 2'(" ')*

19801.6000

+,-. '#$* '#'%/01. 345617689:4; <,=>?1:. (!%#@! ')!@#*(. $#'. @($$$A

0

1000

2000

3000

4000

5000Intens.

0.0

0.5

1.0

1.5

2.0

2.5

4x10

500 1000 1500 2000 2500 3000 m/z

12000 14000 16000 18000 20000 22000 24000 Mass [Da]






622.0289

1061.21571667.0504

+MS, 1.04-1.18min #152-174

500 1000 1500 2000 2500 3000 3500 4000 4500 m/z0

2000

4000

6000

8000

Intens.

!""#$"%&

'$!'#"'()'!!)#$($*

'&**#)"!&

+,-. '#$* '#'%/01 2'(" ')*

19801.6000

+,-. '#$* '#'%/01. 345617689:4; <,=>?1:. (!%#@! ')!@#*(. $#'. @($$$A

0

1000

2000

3000

4000

5000Intens.

0.0

0.5

1.0

1.5

2.0

2.5

4x10

500 1000 1500 2000 2500 3000 m/z

12000 14000 16000 18000 20000 22000 24000 Mass [Da]




(a) sds-page/coomassie staining analysis of purified ... filebroadband high mass 8000.0 m/z laser...

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