Supporting Information Aromatic capping surprisingly ... · 1. Fmoc deprotection 2. Coupling FmocHN...

Supporting Information

Aromatic capping surprisingly stabilizes furan moieties in peptides against acidic degradation during resin cleavage

Kurt Hoogewijs, Annelies Deceuninck and Annemieke Madder* Materials and Methods Products Polystyrene PHB (Wang resin, capacity ~0.99 mmol/g, mesh 100-200) and 2-Chlorotrityl chloride resin (capacity ~1.55mmol/g, mesh 100-200) were obtained from Iris Biotech GmbH. Rink Amide AM resin was obtained from Novabiochem. All amino acids and coupling reagents were purchased from Novabiochem. L-amino acids were used throughout the synthesis. 4-dimethylaminopyridine (DMAP) was purchased from Acros. N,N-dimethylformamide (DMF) extra dry with molecular sieves was obtained from Acros. DMF peptide synthesis grade and N-methylpyrrolidon (NMP) were purchased from Biosolve. Dichloromethane (DCM) and N,N-diisopropylethylamine (DIPEA) were obtained from Aldrich. Trifluoroacetic acid (TFA) was obtained from Iris Biotech GmbH. All chemicals were used without further purification. All reagents used for automated peptide synthesis were peptidesynthesis grade. Peptide syntheses For manual peptide synthesis, reactions were performed in a peptide vessel comprising a sintered glass funnel and a 3-way stopcock for easy filtration and washing or in plastic vials equipped with a sintered filter. The solid phase reactions were performed on a shaker (Selecta Vibromatic) at 200 U/min or on a Yellow Line TTS 2 vortexer at 1200 rpm. Peptides were synthesized by standard Fmoc/tBu strategy using PyBOP/DIPEA or DIC/HOBt couplings. Automated peptide syntheses were performed on a 24-reactor block SYRO Multiple Peptide Synthesizer equipped with a vortexing unit (Multisyntech, Witten, Germany). Peptides were synthesized by standard Fmoc/tBu strategy using HBTU/DIPEA couplings. Analyses ESI-MS spectra were recorded using an LCQ ion trap mass spectrometer (Finnigan MAT). RP-HPLC analyses were performed on an Agilent 1100 Series instrument with a Phenomenex Luna C18(2) column (250 x 4.6 mm, 5 μm at 35 °C). A flow rate of 1 ml/min was used with the following solvent systems: 0.1% TFA in H2O (A) and MeCN (B). The column was flushed for 3 min with 100% A, then a gradient from 0 to 100% B over 15 min was used, followed by 5 min of flushing with 100% B. Alternatively, a Phenomenex Clarity column (250 x 4.6mm, 5µm at 50°C) as used with 0,1M TEAA with 5% MeCN (A) and MeCN(B). The column was flushed for 3 min with 100% A, then a gradient from 0 to 100% B over 60 min was used, followed by 5 min of flushing with 100% B. LC-MS data were collected on an Agilent 1100 Series instrument with a Phenomenex Luna C18(2) column (250 x 4.6 mm, 5 μm at 35 °C) connected to an ESMSD type VL mass detector using the following solvent systems: 5 mM NH4OAc in H2O (A) and MeCN (B). The column was flushed with 100% A for 2 min, then a gradient from 0 to 100% B over 15 min was used, followed by 5 min of flushing with 100% B.

Electronic Supplementary Material (ESI) for Organic & Biomolecular ChemistryThis journal is © The Royal Society of Chemistry 2012

General procedure for peptide synthesis on Wang resin

The resin is preswollen in DCM for 30 min and then filtered off. HOBt (5 equiv), DIC (5 equiv) and DMAP (0.3 equiv) are added to a solution of amino acid (5 equiv) in DCM: DMF (2:1). After 30 min of preactivation, this mixture is added to the resin. The reaction is shaken for 3 h. The resin is filtered off and washed with DMF (3 x 30 s), MeOH (3 x 30 s) and DCM (3 x 30 s). The coupling is repeated using the same protocol. After preloading the resin with the first amino acid, peptide synthesis is performed on an automated peptide synthesizer using the following protocols for Fmocdeprotection and coupling. Fmoc deprotection: A solution of 40% piperidine in DMF is added to the resin. The resin is shaken for 3 min and filtered off. Then a solution of 20% piperidine in DMF is added to the resin. The reaction mixture is shaken for 12 min. The resin is filtered off and washed with DMF (6 x 30 s) Coupling: 5 equiv of a 0.5 M solution of amino acid in DMF, 5 equiv of a 0.5 M solution of HBTU in DMF and 10 equiv of a 2.0 M solution of DIPEA in NMP are added to the resin. The reaction mixture is shaken for 40 min. The resin is filtered off and washed with DMF (4 x 30 s). General procedure for peptide synthesis on 2-Chlorotrityl chloride resin

The resin is preswollen in DCM for 30 min and then filtered off. A solution of amino acid (1.2 equiv) and DIPEA (4.8 equiv) in dry DCM is added to the resin. The reaction is shaken for 2 h. The resin is filtered off, treated with a mixture of DCM, DIPEA and MeOH (17:1:2), and washed with DMF (3 x 30 s), MeOH (3 x 30 s) and DCM (3 x 30 s). After preloading the resin with the first amino acid, peptide synthesis is performed on an automated peptide synthesizer using the following protocols for Fmocdeprotection and coupling. Fmoc deprotection: A solution of 40% piperidine in DMF is added to the resin. The resin is shaken for 3 min and filtered off. Then a solution of 20% piperidine in DMF is added to the resin. The reaction mixture is shaken for 12 min. The resin is filtered off and washed with DMF (6 x 30 s) Coupling: 5 equiv of a 0.5 M solution of amino acid in DMF, 5 equiv of a 0.5 M solution of HBTU in DMF and 10 equiv of a 2.0 M solution of DIPEA in NMP are added to the resin. The reaction mixture is shaken for 40 min. The resin is filtered off and washed with DMF (4 x 30 s).


General procedure for peptide synthesis on Rink amide AM resin

Rink amide AM

SYRO1. Fmoc deprotection

2. CouplingPeptideFmocHN

The resin is preswollen in DMF for 30 min and then filtered off. Peptide synthesis is performed on an automated peptide synthesizer using the following protocols for Fmocdeprotection and coupling. Fmoc deprotection: A solution of 40% piperidine in DMF is added to the resin. The resin is shaken for 3 min and filtered off. Then a solution of 20% piperidine in DMF is added to the resin. The reaction mixture is shaken for 12 min. The resin is filtered off and washed with DMF (6 x 30 s) Coupling: 5 equiv of a 0.5 M solution of amino acid in DMF, 5 equiv of a 0.5 M solution of HBTU in DMF and 10 equiv of a 2.0 M solution of DIPEA in NMP are added to the resin. The reaction mixture is shaken for 40 min. The resin is filtered off and washed with DMF (4 x 30 s). Cleavage of the furan modified peptides TFA in combination with the appropriate scavengers is added to the resin. The resin is shaken for 1 h for short peptides (< 8 amino acids) or for 1.5 h for longer peptides (≥ 8 amino acids). The re sin is then filtered off and washed three times with neat TFA. The combined filtrates are evaporated and the peptide is precipitated by adding a 10-20 fold excess of cold MTBE-ether. The mixture is centrifugated at 0°C and the supernatans is carefully removed. The precipitated peptide is then dissolved in water and lyophilized to obtain a powder.


Spectral data of cleaved furan modified peptides

HPLC analysis after acidolytic cleavage of Fur-Val-Glu(tBu)-Asp(tBu)-Arg(Pbf)-Thr-Val-Asp(tBu)-Val-His(Trt)-Ile-Arg(Pbf)-Arg(Pbf)-Leu-Arg(Pbf)-Lys(Boc)-Ala-Leu-Glu(tBu)-Pro-Gly-R (R = 2-Cl Trt resin)with TFA, DTT and H2O

HPLC analysis after acidolytic cleavage of Fur-Ala-His(Trt)-Asn(Trt)-Leu-Ala-R (R = Wang resin) with TFA, DTT and H2O

OHVal Glu Asp Arg Thr Val Asp Val His Ile Arg Arg Leu Arg Lys Ala Leu Glu Pro GlyO

O

tBu tBu Pbf tBu tBu Trt Pbf Pbf Pbf Boc tBu

min0 2.5 5 7.5 10 12.5 15 17.5 20 22.5

mAU

0

500

1000

1500

2000

DAD1 A, Sig=214,20 Ref=off (I:\07-01-10\AD000006.D)

OHAla His Asn Leu AlaO

O

Trt Trt

min0 5 10 15 20

mAU

0

500

1000

1500

2000

2500

DAD1 A, Sig=214,20 Ref=off (F:\07-03-05\022-1901.D)

9.56

7

10.16

1

11.88

5 12.68

8

13.54

7

14.25

9

15.11

0 15

.486

15.86

5

17.01

0

17.55

3 17

.933

18.31

3 18

.615

19.85

4

20.54

1 20

.840

21.74

1

λ= 214 nm

λ= 214 nm


HPLC analysis after acidolytic cleavage of Fur-Ala-His(Trt)-Asn(Trt)-Leu-Ala-R (R = Wang resin) with TFA, TIS and H2O

compound 3 Chemical Formula: C29H42N8O9 Exact Mass: 646,31 g mole-1 Molecular Weight: 646,69 g mole-1 MS (m/z): 647.2 [M+H]+, 771,3 [unknown impurity] compound 4 Chemical Formula: C29H46N8O9 Exact Mass: 650,34 g mole-1 Molecular Weight: 650,72 g mole-1 MS (m/z): 326.1 [M+2H]2+, 651.1 [M+H]+

min0 5 10 15 20

mAU

0

500

1000

1500

2000

2500

DAD1 A, Sig=214,20 Ref=off (F:\07-03-05\023-2001.D)

8.48

1 9.53

3

10.04

9

13.91

3

20.55

5

λ= 214 nm 4

3 PG


MS spectra from LC-MS after acidolytic cleavage of Fmoc-Leu-FurAla-Gly-Lys(Boc)-Val-R (R = Wang resin)

compound 6: Chemical Formula: C41H54N6O9 Exact Mass: 774,39523 g mole-1 Molecular Weight: 774,90226 g mole-1 MS (m/z): 775,2 [M+H]+ compound 7: Chemical Formula: C41H56N6O10 Exact Mass: 778,42653 g mole-1 Molecular Weight: 778,93402 g mole-1 MS (m/z): 793,2 [M+H]+


compounds 8/9: Chemical Formula: C41H56N6O9 Exact Mass: 776,41088 g mole-1 Molecular Weight: 776,91814 g mole-1 MS (m/z): 777,2 [M+H]+

compound 10: Chemical Formula: C41H58N6O9 Exact Mass: 778,42653 g mole-1 Molecular Weight: 778,93402 g mole-1 MS (m/z): 779,2 [M+H]+


compounds 11/12: Chemical Formula: C45H64N6O11S2 Exact Mass: 928,40745 g mole-1 Molecular Weight: 929,15326 g mole-1 MS (m/z): 929,4 [M+H]+, 911,3 [M+H-H2O]+ compound 13: is out of range for the LC-MS system, a simple ESI-MS of the crude peptide after acidolytic cleavage with TFA/DTT (95:5) reveals the formed side-products: Chemical Formula: C49H74N6O13S4 Exact Mass: 1082,41967 g mole-1 Molecular Weight: 1083,40426 g mole-1 MS (m/z): 775,4 [6+H]+; 929,4 [11/12+H]+; 1083,3 [13+H]+


HPLC analysis after acidolytic cleavage of Acetyl-Val-Glu(tBu)-Asp(tBu)-Arg(Pbf)-Thr-Val-Asp(tBu)-Val-His(Trt)-Ile-Arg(Pbf)-Arg(Pbf)-Leu-Arg(Pbf)-Lys(Boc)-Ala-Leu-Glu(tBu)-Pro-Gly-FurAla-R (R = Rink Amide AM resin)

Chemical Formula: C110H185N37O32 Exact Mass: 2536,40 g mole-1 Molecular Weight: 2537,87 g mole-1 MS (m/z):2538.0 [M+H]+, 2521.4 [M+H-H2O]+

min0 2.5 5 7.5 10 12.5 15 17.5 20 22.5

mAU

0

200

400

600

800

1000


λ= 214 nm


Oxidation of peptide 14

To 1ml of peptide 14 in MilliQ (394 µM) was added 10 µL of a NBS solution (39,4 mM). The reaction is analyzed after 4 hours by MALDI-TOF. Chemical Formula: C110H185N37O33 Exact Mass: 2552,39 g mole-1 Molecular Weight: 2553.9 g mole-1 MALDI-TOF: 2553.9 [M+H]+, 2536.9 [M+H-H2O]+


HPLC analysis acidolytic cleavage of Acetyl-Val-Glu(tBu)-Asp(tBu)-FurAla-Thr-Val-Asp(tBu)-Val-His(Trt)-Ile-Arg(Pbf)-Arg(Pbf)-Leu-Arg(Pbf)-Lys(Boc)-Ala-Leu-Glu(tBu)-Pro-Gly-R (R = Rink Amide AM resin)

Chemical Formula: C104H173N33O31 Exact Mass: 2380,30 g mole-1 Molecular Weight: 2381,69 g mole-1 MS (m/z): 2382.5 [M+H]+

min0 2.5 5 7.5 10 12.5 15 17.5 20 22.5

mAU

-600

-400

-200

0

200

400


λ= 214 nm

999 .0 1599 .4 2199 .8 2800 .2 3400 .6 4001 .0Mass (m/z )

0

1 .8E+4

0

10

20

30

40

50

60

70

80

90

100

% In

tens

ity

Voyager Spec #1[BP = 2382.6, 17609]

2382 .5211

2365 .8014

2386 .4678

2342 .3437

2326 .72972345 .1100

2360 .45842022 .75301333 .5913 2308 .60291783 .7890 2479 .65361569 .8853



To 1ml of peptide 15 in MilliQ (420 µM) was added 10.1 µL of a NBS solution (41.6 mM). The reaction is analyzed after 4 hours by MALDI-TOF. Chemical Formula: C104H173N33O32 Exact Mass: 2396,29 g mole-1 Molecular Weight: 2397.7 g mole-1 MALDI-TOF: 2398.8 [M+H]+, 2382.9 [15+H]+


HPLC analysis after acidolytic cleavage of Acetyl-Val-Glu(tBu)-Asp(tBu)-Arg(Pbf)-Thr-Val-Asp(tBu)-FurAla-His(Trt)-Ile-Arg(Pbf)-Arg(Pbf)-Leu-Arg(Pbf)-Lys(Boc)-Ala-Leu-Glu(tBu)-Pro-Gly-R (R = Rink Amide AM resin)

Chemical Formula: C105H176N36O31 Exact Mass: 2437,33 g mole-1 Molecular Weight: 2438,74 g mole-1 MS (m/z): 2440,1 [M+H]+

min0 2.5 5 7.5 10 12.5 15 17.5 20 22.5

mAU

0

200

400

600

800

1000

1200

1400


λ= 214 nm

999 .0 1599 .4 2199 .8 2800 .2 3400 .6 4001 .0Mass (m/z )

0

1 .3E+4

0

10

20

30

40

50

60

70

80

90

100

% In

tens

ity

Voyager Spec #1[BP = 2440.2, 13212]

2440 .1142

2423 .0424

2444 .4994

1334 .0465

2425 .91182383 .4511

2447 .60352428 .99761064 .7658

1822 .4141 2392 .06401609 .1973 2080 .5852 2361 .0563

1220 .8739 2542 .72591923 .4200 2088 .9815 2308 .26041765 .33341582 .22721419 .83231080 .8115 2133 .28181954 .1173 2484 .23121261 .6390



To 1ml of peptide 16 in MilliQ (410 µM) was added 9,8 µl of a NBS solution (41.6 mM). The reaction is analyzed after 4 hours by MALDI-TOF. Chemical Formula: C105H176N36O32 Exact Mass: 2453,33 g mole-1 Molecular Weight: 2454.7 g mole-1 MALDI-TOF: 2456.2 [M+H]+, 2439.0 [16+H]+ or [M-H2O+H]+


HPLC analysis after acidolytic cleavage of Acetyl-Val-Glu(tBu)-Asp(tBu)-Arg(Pbf)-Thr-Val-Asp(tBu)-Val-FurAla-Ile-Arg(Pbf)-Arg(Pbf)-Leu-Arg(Pbf)-Lys(Boc)-Ala-Leu-Glu(tBu)-Pro-Gly-R (R = Rink Amide AM resin)

Chemical Formula: C104H178N34O31 Exact Mass: 2399,34 g mole-1 Molecular Weight: 2400,74 g mole-1 MS (m/z): 2402,4 [M+H]+

min0 2.5 5 7.5 10 12.5 15 17.5 20 22.5

mAU

0

250

500

750

1000

1250

1500

1750

2000


λ= 214 nm

999.0 1599.4 2199.8 2800.2 3400.6 4001.0Mass (m /z)

0

2.0E +4

0

10

20

30

40

50

60

70

80

90

100

% In

tens

ity

Voyager Spec #1[B P = 2402.4 , 19712]

2402.3107

2423.9157

2385.2763

2405.2787

2445.72892407.9376

2397.42852359.4022

2448.80751901.46112504.56292063.4164



To 250 µl of peptide 17 in MilliQ (417 µM) was added 8 µl of a NBS solution (20,8 mM). The reaction is analyzed after 4 hours by MALDI-TOF. Chemical Formula: C104H178N34O32 Exact Mass: 2415,33 g mole-1 MW: 2416.7 g mole-1 MALDI-TOF: 2415.5 [M+H]+, 2398.9 [M-H2O+H]+


HPLC analysis after acidolytic cleavage of Acetyl-Val-Glu(tBu)-Asp(tBu)-Arg(Pbf)-Thr-Val-Asp(tBu)-Val-His(Trt)-Ile-Arg(Pbf)-FurAla-Leu-Arg(Pbf)-Lys(Boc)-Ala-Leu-Glu(tBu)-Pro-Gly-R (R = Rink Amide AM resin)


min0 2.5 5 7.5 10 12.5 15 17.5 20 22.5

mAU

0

250

500

750

1000

1250

1500

1750


λ= 214 nm

999 .0 1599 .4 2199 .8 2800 .2 3400 .6 4001 .0Mass (m/z )

0

2 .4E+4

0

10

20

30

40

50

60

70

80

90

100

% In

tens

ity

Voyager Spec #1[BP = 2383.4, 23578]

2383 .3558

2366 .2375

2404 .8740

2342 .5438

2350 .18512380 .61241315 .0889

2362 .09271045 .8351 1867 .53151551 .33682322 .12961337 .3128



To 250 µl of peptide 18 in MilliQ (420 µM) was added 5 µl of a NBS solution (20,8 mM). The reaction is analyzed after 4 hours by MALDI-TOF. Chemical Formula: C104H173N33O32 Exact Mass: 2396,29 g mole-1 MW: 2397.7 g mole-1 MALDI-TOF: 2398.0 [M+H]+, 2381.2 [M-H2O+H]+


HPLC analysis after acidolytic cleavage of Acr-FurAla-Thr(tBu)-Pro-Lys(Boc)-Arg(Pbf)-Pro-Arg(Pbf)-Gly-Arg(Pbf)-Pro-Lys(Boc)-Lys(Boc)-R (R = Rink Amide AM resin)


min2.5 5 7.5 10 12.5 15 17.5 20 22.5

mAU

0

100

200

300

400

500

DAD1 A, Sig=214,20 Ref=off (E:\09-04-03\KH000007.D)

min2.5 5 7.5 10 12.5 15 17.5 20 22.5

mAU

0

20

40

60

80

DAD1 E, Sig=360,20 Ref=off (E:\09-04-03\KH000007.D)

λ= 214 nm

λ= 360 nm

999.0 1599.4 2199.8 2800.2 3400.6 4001.0Mass (m /z)

0

1.7E +4

0

10

20

30

40

50

60

70

80

90

100

% In

tens

ity

Voyager Spec #1[B P = 1662.6 , 17260]

1662.5895

1665.6040

1668.5139

1621.5091

1577.40251624.6698

1688.6440 1853.53601004.2928 1319.5713 1508.2972


HPLC analysis after acidolytic cleavage of Napht-FurAla-Trp(Boc)-Ser(tBu)-His(Trt)-Pro-Gln(Trt)-Phe-Glu(OtBu)-Lys(Boc)-R (R = 2-Cl Trt resin)

Chemical Formula: C68H80N14O16 Exact Mass: 1348,59 g mole-1 Molecular Weight: 1349,45 g mole-1 LC-MS: tR=15,073min (1349,4=[M+H]+, 675,3=[M+2H]2+/2)

λ= 214 nm


HPLC analysis after acidolytic cleavage of Acr-Gly-FurAla-R (R = Rink Amide AM resin)

Chemical Formula: C23H20N4O4 Exact Mass: 416,15 g mole-1 Molecular Weight: 416,43 g mole-1 RPHPLC: tR=35,1 min (Clarity) LC-MS: tR=12,307 (417,1=[M+H]+) RPHPLC

LC-MS

min0 5 10 15 20

mAU

0

200

400

600

800

1000

DAD1 E, Sig=360,20 Ref=off (063-1601.D)

11.

541

12.

307

min0 10 20 30 40

mAU

-25

0

25

50

75

100

125

150

175

DAD1 E, Sig=360,16 Ref=off (E:\09-03-02\AD000006.D)


MS

m/z100 200 300 400 500 600 700 800 900

0

20

40

60

80

100

*MSD1 SPC, time=12.330 of 063-1601.D API-ES, Pos, Scan, Frag: 70

Max: 441204 417

.1 4

18.1


Oxidation of peptide 23 into 24

To 1 ml of peptide 23 in MilliQ (240 µM) was added 4,3 µl of a NBS solution (55,6 mM). The reaction is analyzed after 1 hours by RPHPLC and ESI-MS.

Chemical Formula: C23H20N4O5

Exact Mass: 432,14 g mole-1 MW: 432,43 g mole-1 RPHPLC: tR=28,6 min; 28,8 min ESI-MS: 452,3 [M+H2O+H]+, 434,4 [M+H]+

min0 10 20 30 40

mAU

-40

-20

0

20

40

60

80

100

120

S#: 49-57 RT: 0,77-0,87 AV: 9 NL: 1,19E7 T: + c ms [ 150,00 - 1000,00]

300 320 340 360 380 400 420 440 460 480 500 520 540 560 580 m/z

0 5

10 15 20 25 30 35 40 45 50 55 60 65 70 75 80 85 90 95

100

Relative Abundance

452,3

434,4

453,4

435,5

474,4 420,4 466,3 416,5 451,6 512,4 475,4 456,3 421,5 336,2 389,4 406,4 488,2 532,5 360,1 518,1 373,2 576,3 584,0 307,1 540,6 553,6 332,1 354,3 568,0 596,7 498,1


Supporting Information Aromatic capping surprisingly ... · 1. Fmoc deprotection 2. Coupling FmocHN...

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Transcript of Supporting Information Aromatic capping surprisingly ... · 1. Fmoc deprotection 2. Coupling FmocHN...