Supporting Information

© Wiley-VCH 2008

69451 Weinheim, Germany

SUPPORTING MATERIALS

Synthesis of Protein Mimics with Non-Linear Backbone Topology by Combined Recombinant,

Enzymatic and Chemical Synthesis (CRECS) Strategy

Stephan Pritz, Oliver Kraetke, Annerose Klose, Jana Klose, Sven Rothemund, Klaus Fechner,

Michael Bienert, Michael Beyermann*

General:

The template T, Biotin-K[Z-GGGQK(Mhx-Ahx-GEGK(Dde)GEGKGEG)GEG]-amide, ECD2

(cyclo(CGVQLTVSPEVHQSNVAWSRLG)), ECD3 (cyclo(CGIGKLHYDNEKSWFGKRPGVYT

DYG)), and ECD4 (cyclo(CGVNPGEDEVSRVVFIYFNSFG)) were synthesised using the Fmoc-

strategy and a standard protocol for automated solid-phase synthesis (Applied Biosystems, 433a).

Peptides were purified by preparative RP-HPLC (purity >95% according to analytical RP-HPLC

with 220 nm uv detection) and their identity was confirmed by ESI-mass spectrometry.

Unless otherwise noted, mass accuracy of ESI-mass spectrometry was better than 60 ppm.

Preparation of the receptor loops by SPPS and native chemical ligation (exemplarily for

ECD4):

SPPS was carried out on Trt-Tentagel resin (Rapp-Polymere, Germany, 0.25 mmol/g, 1.5 g) using

Fmoc-strategy (double couplings with 3 equi. of Fmoc-aa/ HBTU/ 6 equi. DIPEA). The N-terminal

cysteine was coupled as BOC-Cys(Trt)-OH. The fully protected peptide was cleaved from the

peptide-resin with 20 mL of HOAc/TFE/DCM (2/2/6) for 1 h at rt. After addition of n-hexane (100

mL), the solution was evaporated to dryness and the product was lyophilised twice from dioxane

(yield: 1.09 g (94%)). Thiol ester was prepared according to a recently published procedure.[12]

Briefly, 1.09 g (0.29 mmol) protected ECD4, 66.5 mg (0.435 mmol) HOBt, 167 mg (1 mmol) p-

acetamidothiophenol (Aatp), and 68 µL (0.435 mmol) diisopropylcarbodiimide (DIPCDI) dissolved

in 50 mL of DCM were reacted for 2 h at rt. After 2 h, 4 h, 6 h, and 8 h same quantities of DIPCDI

and Aatp were again added and the reaction mixture was stirred overnight. The reaction mixture was

then evaporated to dryness, the resulting product was dissolved in 20 mL of a mixture of water(5)/

phenol(5)/ tris-isopropylsilane(2)/ TFA(88) for deprotection (3 h at rt). After evaporation (to 5 mL),

the thiol ester was precipitated with 250 mL of diethylether, washed with diethylether, ACN,

diethylether and dried (yield: 770 mg). 50 mg of crude product gave 12.6 mg of the purified thiol

ester by preparative HPLC (Vydac C-8, 300×25 mm, linear gradient 30-80% B in 70 min, A: 0.1 %

TFA, B: 80% ACN-0.1% TFA). Cyclisation (NCL) was accomplished by dissolving 60 mg of the

thiol ester and 60 mg TCEP in 30 mL of 0.5 M aqueous sodium bicarbonate and stirring for 1 h at rt.

After addition of 300 µL HClconc, the solution was evaporated, the precipitate was separated by

centrifugation, washed with water, ACN and diethylether, and lyophilised from ACN/water (yield:

27 mg of ECD4).

Preparation of the three-loops-template construct T-ECD4-3-2-NH2 (see Fig.2):

Thiol-maleimide ligation, step A: To 33 mg of template T (12.5 µmol) and 37 mg (15 µmol) of

ECD4 dissolved in 0.4 mL DMSO, 1.6 mL water and 0.25 mL of 1 M NaHCO3, pH 8.5, were added.

After stirring for 1 h at rt, the product T-ECD4 was formed quantitatively and purified by RP-HPLC.

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22.5

8323

.500

24.0

92

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8323

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92

Kl 011007 Temp_ECL3 04-Oct-2007

m/z200 300 400 500 600 700 800 900 1000 1100 1200 1300 1400 1500 1600

%

0

100

071004_003 366 (3.171) Sm (SG, 3x5.00); Cm (363:371) 1: TOF MS ES+ 2401263.0352

1262.7803

1262.53491010.8491

1010.4523

842.5497299.9827 391.2533

1011.0349

1011.2545

1011.4487

1018.8704

1263.5354

1263.7810

1683.6633

1264.0359

1683.3474

1264.2815

1683.00951267.0405

1277.2905

m / z m / z calcd. (monoisotopic) found (monoisotopic)

[M+3H]3+ 1683.45 1683.34

[M+4H]4+ 1262.84 1262.78

[M+5H]5+ 1010.47 1010.45

[M+6H]6+ 842.22 842.24

M 5047.34 5047.2 ± 0.3

Figure 6: Characterisation of T-ECD4 by analytical HPLC (top) and ESI-MS (bottom).

Introduction of maleimidohexanoic acid, step B: To 61 mg (12 µmol) of T-ECD4 and 32 mg (0.1

mmol) Mhx-OSu dissolved in 3 mL DMSO, 0.5 mL water and 0.1 mL of 1 M NaHCO3, pH 8.5,

were added. After stirring for 1 h at rt, the product T-ECD4-Mhx was formed quantitatively and

purified by RP-HPLC.

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24.1

67Minutes

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67

Kl 181007 Temp_ECL3_Mhx Endpr. 19-Oct-2007

m/z600 650 700 750 800 850 900 950 1000 1050 1100 1150 1200 1250 1300 1350 1400 1450 1500 1550 1600 1650

%

0

100

071018_051 384 (3.327) Sm (SG, 3x5.00); Cm (376:396) 1: TOF MS ES+ 28.31311.5437

1311.2935

1311.0529

608.35951307.0739

1049.6257674.9371 863.7828840.2413 934.5141 972.90691229.7562

1089.9315

1311.7937

1312.0535

1312.2941

1312.5348

1315.7994

1316.3583

1329.82851333.5900 1540.38221510.3867 1682.0345

m / z m / z calcd. (monoisotopic) found (monoisotopic)

[M+4H]4+ 1311.10 1311.07

M 5240.42 5240.3 ± 0.3

Figure 7: Characterisation of T-ECD4-Mhx by analytical HPLC (top) and ESI-MS (bottom).

Thiol-maleimide ligation, step C: To 40.4 mg (7.8 µmol) of T-ECD4-Mhx and 28.2 mg (12 µmol)

of ECD2 dissolved in 2 mL DMSO, 0.25 mL water and 50 µL of 1 M NaHCO3, pH 8.5, were added.

After stirring for 1.5 h at rt, the product was formed quantitatively and purified by RP-HPLC.

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23.8

33

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92

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33

Kl 221007 Temp_ECL3_ECL1 Endpr. 23-Oct-2007

m/z400 500 600 700 800 900 1000 1100 1200 1300 1400 1500 1600

%

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071023_006 370 (3.206) Sm (SG, 3x5.00); Cm (367:376) 1: TOF MS ES+ 43.31519.3972

1519.1693

1518.9830

1266.4515

389.96401085.8550462.6877 950.0118507.2922 1162.5261

1266.7258

1516.98551268.8635

1469.6093

1519.5525

1519.7595

1519.9771

1522.1841

1522.9821

1531.1836

1542.2330 1688.2909

m / z m / z calcd. (monoisotopic) found (monoisotopic)

[M+5H]5+ 1518.72 1518.80

[M+6H]6+ 1265.76 1265.85

M 7588.59 7589.0 ± 0.5

Figure 8: Characterisation of T-ECD4-2 by analytical HPLC (top) and ESI-MS (bottom).

Dde removal, step D: To 63.5 mg (8.36 µmol) of T-ECD4-2 dissolved in 50 mL of ACN/water

(1/1), 0.25 % aqueous ammonia was added until pH 6.3 was reached, and the solution was

lyophilised. The product was dissolved in 3.92 mL DMF and 80 µL hydrazine was added. After

stirring for 10 min at rt, the deblocking was complete, 200 µL TFA were added and the product T-

ECD4-2-NH2 was purified by RP-HPLC.

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1,00

22.9

08

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08

Kl 051107 Temp_ECL3_ECL1-Dde Endpr. 06-Nov-2007

m/z400 500 600 700 800 900 1000 1100 1200 1300 1400 1500 1600

%

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100

071106_004 351 (3.042) Sm (SG, 3x5.00); Cm (349:357) 1: TOF MS ES+ 93.91486.6700

1486.4755

1486.2706

1486.0759

1239.21191239.04361238.8751

1062.1920

1239.3989

1239.5674

1239.7263

1241.56971480.8983

1486.8544

1487.0697

1487.2540

1487.4794

1487.8484

1490.2679

1498.0726

1512.4825

m / z m / z calcd. (monoisotopic) found (monoisotopic)

[M+5H]5+ 1485.90 1485.87

[M+6H]6+ 1238.42 1238.40

[M+7H]7+ 1061.64 1061.62

M 7424.50 7424.3 ± 0.4

Figure 9: Characterisation of T-ECD4-2-NH2 by analytical HPLC (top) and ESI-MS (bottom).

Introduction of maleimidohexanoic acid, step E: To 51 mg (6.9 µmol) of T-ECD4-2-NH2 and

3.32 mg (0.01 mmol) Mhx-OSu dissolved in 3 mL DMSO, 500 µL water and 100 µL of 1 M

NaHCO3, pH 8.5, were added. After stirring for 90 min at rt, the product was formed quantitatively

and purified by RP-HPLC.

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92

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92

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Kl 061107 Temp_ECL3_ECL1_Mhx Endpr. 07-Nov-2007

m/z400 500 600 700 800 900 1000 1100 1200 1300 1400 1500 1600

%

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071107_005 365 (3.163) Sm (SG, 3x5.00); Cm (362:371) 1: TOF MS ES+ 41.81525.2944

1525.0972

1524.9104

1271.2461

901.4907776.6242652.0960400.3844 483.8033 1089.8999 1202.9917

1271.7385

1371.61841274.2314

1513.8918

1505.5922

1525.4915

1525.6886

1525.9169

1526.2800

1528.5228

1528.78271529.0942

1529.2915

1547.86181613.6870

m / z m / z calcd. (monoisotopic) found (monoisotopic)

[M+5H]5+ 1524.52 1524.66

[M+6H]6+ 1270.60 1270.51

M 7617.58 7617.6 ± 0.5

Figure 10: Characterisation of T-ECD4-2-Mhx by analytical HPLC (top) and ESI-MS (bottom).

Thiol-maleimide ligation, step F: To 41 mg (5.4 µmol) T-ECD4-2-Mhx and 26 mg (8.7 µmol)

ECD3 dissolved in 3 mL DMSO, 0.5 mL water and 100 µL of 1 M NaHCO3, pH 8.5, were added.

After stirring for 1 h at rt, the ligation product was formed quantitatively and purified by RP-HPLC.

Removal of the Z-group, step G: 42.1 mg (3.98 µmol) T-ECD4-3-2 were dissolved at 0°C in the

deblocking mixture (1.8 mL thioanisol, 0.9 mL ethanedithiol, 18 mL TFA, 1.8 mL TFMSA), and the

reaction mixture was stirred for 10 min at 0°C. After evaporation, the product T-ECD4-3-2-NH2 was

precipitated with diethylether and subsequently purified by RP-HPLC.

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92

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Kl 121107 Temp_ECL3_ECL1_ECL2-Z Endpr. 13-Nov-2007

m/z400 500 600 700 800 900 1000 1100 1200 1300 1400 1500 1600

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071113_004 328 (2.844) Sm (SG, 3x50.00); Cm (325:332) 1: TOF MS ES+ 9.911511.7908

1495.4624

1308.7249

1306.49601163.3153

427.3687 1175.7898

1322.9393 1492.8026

1324.6694

1527.9794

1530.1090

1546.4160 1646.1814

m / z m / z calcd. (monoisotopic) found (monoisotopic)

[M+7H]7+ 1495.50 1495.46

[M+7H+TFA]7+ 1511.79 1511.79

[M+7H+2TFA]7+ 1528.07 1527.98

[M+8H]8+ 1308.68 1308.72

[M+8H+TFA]8+ 1322.94 1322.94

[M+9H]9+ 1163.39 1163.32

M 10461.47 10461.2 ± 0.6

Figure 11: Characterisation of T-ECD4-3-2-NH2 by analytical HPLC (top) and ESI-MS (bottom).

Recombinant synthesis of ECD1-LPKTGGRR, step X:

As described previously by Klose et al.[4], E. coli BL21 (DE3), transformed with the plasmid pET-

CRF1-N-terminus with sortase A recognition motif-LPKTGGRR, was grown in LB medium with

antibiotics at 37°C until an optical density of OD600nm= 0.5 – 0.7 was reached. The expression was

started by addition of IPTG. After incubation for 3-4 h at 37°C, an expression yield of approximately

30-35 mg ECD1-LPKTGGRR per liter of medium was obtained. The cells were then harvested by

centrifugation and subjected to lysis. Inclusion bodies, containing ECD1-LPKTGGRR, were

separated by centrifugation and stored at –20°C.

Folding & purification of ECD1-LPKTGGRR, step Y:

The inclusion bodies were resuspended in 5 M GuHCl, 20 mM Tris, pH 7.5 and sonicated. After

centrifugation, ECD1-LPKTGGRR was reduced with tris(2-carboxyethyl) phosphine for 2 h at rt and

the solution was then dialysed against 5 M GuHCl/ 1 mM EDTA, pH 3, overnight at 8°C. Insoluble

material was removed by centrifugation. The supernatant was diluted to a final protein concentration

of 0.5 mg/ml and the pH readjusted to 7.5. Folding of ECD1-LPKTGGRR was achieved by dialysis

against a buffer containing 1 M arginine, 100 mM TrizmaBase, 1 mM EDTA, 1 mM gluthation-

reduced (GSH), and 1 mM GSSG, pH 7.5 at 10°C. The folded protein was purified by preparative

RP-HPLC (Vydac C4, 250×10 mm, 5 µm, 300 Å, with a linear gradient 20-60% B within 70 min, A:

0.1 % TFA, B: 80% aqueous ACN-0.1% TFA, 10 ml/min) and lyophilised.

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18.4

5018

.683

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18.4

5018

.683

MALDI-MS m / z m / z calcd. (monoisotopic) found (monoisotopic)

[M+H]+ 13006.6 13006.1

[M+2H]2+ 6503.8 6506.0

M 13005.6 13007 ± 4

Figure 12: Characterisation of ECD1-LPKTGGRR by analytical HPLC (top) and MALDI-MS

(bottom).

Treatment of the product with α-chymotrypsin and LC-MS analysis of the peptide fragments showed

the identity and the correct disulfide pattern of the ECD1-LPKTGGRR.

MRGSHHHHHHGSLQDQRCENLSLTSNVSGLQCNASVDLIGTCWPRSPAGQLVVRPCPAFF 1 ---------+---------+---------+---------+---------+---------+ 60

YGVRYNTTNNGYRECLANGSWAARVNYSECQEILNEEKKSKVHYHVAVLPKTGGRR 61 ---------+---------+---------+---------+---------+------ 116

Figure 13: Sequence of ECD1-LPKTGGRR with N-terminal His6-Tag and C-terminal sortase-

recognition motif. Cysteine residues are underlined, cleavage sites found by

chymotryptic digest are marked in bold (cleavages occurred C-terminally of marked

amino acids).

Table 1: Linear fragments found for the chymotryptic digest of in vitro folded ECD1-

LPKTGGRR.

fragment sequence monoisotopic

mass calcd.

monoisotopic

mass found

deviation

in ppm

2-13 RGSHHHHHHGSL 1397.656 1397.645 -8

22-23 SL 218.127 218.128 6

22-30 SLTSNVSGL 876.455 876.455 -1

24-30 TSNVSGL 676.339 676.336 -4

61-64 YGVR 493.265 493.265 0

61-65 YGVRY 656.328 656.325 -5

62-65 GVRY 493.265 493.264 -2

66-72 NTTNNGY 782.320 782.318 -2

77-81 ANGSW 533.223 533.224 2

82-87 AARVNY 692.361 692.356 -7

95-104 NEEKKSKVHY 1260.646 1260.644 -2

105-111 HVAVLPK 762.475 762.469 -8

105-116 HVAVLPKTGGRR 1289.768 1289.759 -7

112-116 TGGRR 545.303 545.301 -4

All expected cysteine-free fragments being larger than one amino acid were found. Additionally,

some fragments with a missed cleavage site could be identified (e.g. fragment 22-30). The protein

was also cleaved at two residues nontypical for chymotrypsin (Arg64 and Lys111), possibly due to

contamination of the enzyme with trypsin. No linear fragment containing cysteine was found, i.e. all

cysteines were involved in disulfide bonding.

For determination of the disulfide pattern, the masses of all possible combinations of disulfides

(homodimers included) were calculated and the respective masses searched for in the LC-MS run.

Only three disulfide-bridged fragments were found corresponding to the disulfide pattern (C1-C3,

C2-C5, C4-C6) reported for the soluble CRF1-NT.[4]

Table 2. Disulfide-bridged fragments found for the chymotryptic digest of in vitro folded ECD1-

LPKTGGRR.

Fragment Cysteine pattern Monoisotopic

mass calcd.

Monoisotopic

mass found

Deviation

in ppm

14-21 and 39-51 Cys18-Cys42 (C1-C3) 2387.111 2387.075 -15

31-38 and 73-76 Cys32-Cys75 (C2-C5) 1365.601 1365.596 -4

52-59 and 88-94 Cys56-Cys90 (C4-C6) 1705.816 1705.802 -9

Sortase-mediated ligation of ECD1-LPKTGGRR and T-ECD4-3-2-NH2:

A mixture of ECD1-LPKTGGRR (238 µM), T-ECD4-3-2-NH2 (238 µM) and sortase A (5.7 µM)

dissolved in Tris-buffer (50 mM Tris-HCl, 150 mM NaCl, 5 mM CaCl2, 0.95 M urea, pH 7.5) was

shaken at 23°C. After two days a product yield of ~ 60% (as determined by HPLC) was attained

which did not further increase. The crude product was purified by RP-HPLC (Prontosil C-18, 250×8

mm, C18, 300 Å, 5 µm, eluent A: 0.1% TFA, eluent B: 80% ACN-0.1% TFA, linear gradient 30-

80% B in 70 min, 2 ml/min). Lyophilisation of the product fractions yielded 1.678 mg of the desired

product (36.4% yield) which was characterised by HPLC and MS (Fig. 4).