MS#: JOPC-D-10-00082

Revised on 8/18/2010

Su pplementary information for

Semisynthesis of a protein with cholesterol at the C-

terminal, targeted to the cell membrane of live cells

Kenta Teruya, Keiko Nishizawa and Katsumi Doh-ura*

Department of Neurochemistry

Tohoku University Graduate School of Medicine

2-1 Seiryo-cho, Aoba-ku, Sendai, Miyagi 980-8575, Japan

1 *Corresponding author. E-mail: [email protected]

Telephone: (+81)-22-717-8232, Fax: (+81)-22-717-7656

2 Abbreviations CBD: chitin binding domain Chol: cholesterol CTAB: cetyl trimethyl ammonium bromide DCM: dichloromethane DEG: diethylene glycol DIEPA: N,N-diisopropyl ethylamine DMF: dimethyl formamide DTT: dithiothreitol F: fluorescein FAB: fast atom bomberdment GFP: green fluorescent protein MALDI-TOF: matrix-assisted laser desorption ionization – time of flight MESNa: mercapto ethane sulfonic acid sodium salt MS: mass spectrometry RP-HPLC: reversed phase – high performance liquid chromatography TFA: trifluoroacetic acid TLC: thin layer chromatography Trt: trityl

3 Synthesis of NH2-DEG-Chol and L-Cys-NH-DEG-NHCO-Chol

An aliquote of 4, 7, 10-trioxa-1, 13-tridecane diamine (360 uL, 1.65 mmol, Sigma-

Aldrich, MO, USA) was dissolved in dichloromethane (DCM) (3.0 mL). To the solution, cholesterol chloroformate (500 mg, 1.1 mmol, TCI, Tokyo, Japan) in DCM (2.0 mL) was added drop-wise [2]. In this condensation, the diamine was in an excess molar amount to the cholesterol derivative. After 60 min incubation, the reaction mixture was washed by water three times. The reaction mixture was applied to a silica column, then a product was eluted by

chloroform:methanol:AcOH = 10:2:1. Fractions containing NH2-DEG-NHCO-Chol were combined and evaporated. The residue weighted 200 mg (ca. 0.32 mmol). The coupling product

was detected by ninhydrin for the amino group, followed by CuSO4 in orthophosphoric acid for the cholesterol moiety on a TLC analysis (Figure S1). FAB-MS, 633.5202 (theoretical [M+H]+ =

633.5206) (Figure S2); 1H-NMR (Figure S3);13C-NMR (Figure S4).

An aliquot of NH2-DEG-NHCO-Chol was dissolved in DMF. To the solution, Trt-

Cys(Trt)-OSu (50 mg, 71 umol, Calbiochem-Novabiochem, CA, USA) in DMF(1.0 mL) and

DIEPA (20 uL, 0.12 mmol) was added. Reaction was monitored by TLC developed by 5%

NH4OH aq. in EtOH. After 60 min incubation, desired product was purified on a silica column.

Fractions containing the desired product were combined and evaporated. To the residue, TFA

(1.0 mL), DCM (1.0 mL) and triisopropylsilane (100 uL) were added. The reaction mixture was evaporated under nitrogen, and the residue was subject to purification by a silica column. A

product Cys-DEG-NHCO-Chol was eluted by ethylacetate:H2O:AcOH:EtOH = 4:1:1:1. Fractions containing Cys-DEG-Chol were combined and evaporated. The residue was suspended in water and frozen until use (Figure S5). FAB-MS, 758.5122 (theoretical [M+Na]+ = 758.5118)

(Figure S6).

4 Synthesis of F-NH-DEG-NH2 and F-NH-DEG-NHCO-Chol

Each of 4, 7, 10-trioxa-1, 13-tridecane diamine (200 uL, 0.91 mmol) and NH 2-DEG-

NHCO-Chol (20 mg, ca. 32 umol) was dissolved in methanol (1.0 mL). To the solution, 5- carboxyfluorescein succimidyl ester (2.0 mg, invitrogen, CA, USA) in DMSO (200 uL) and

DIEPA (20 uL, 0.12 mmol) were added. After 60 min incubation, the reaction mixture was

subject to purification by a silica column. A fluorescein-labeled product was eluted by NH4OH

aq.:MeOH = 1:20. Reaction was monitored by TLC developed by NH4OH aq.:MeOH = 1:20.

Fractions containing a target compound were collected and evaporated. The residue was dissolved in water. UV measurement of the solution revealed that 2.4 mg (1.0 mL of 4.2 mM

solution) and 2.5 mg (1.0 mL of 2.5 mM solution) were obtained for F-NH 2 and F-Chol,

+ respectively. FAB-MS, 579.2347 (theoretical [M+H] = 579.2343) for F-NH2 and 1013.5507

(theoretical [M+Na]+ = 1013.5503) for F -Chol, respectively (Figure S7) .

Synthesis of Cys-lantinide binding peptide [1]

Amino acid derivatives for fluorenylmethyloxycarbonyl (Fmoc)-based solid phase peptide synthesis were purchased from Calbiochem-Novabiochem (CA, USA). Starting form

Fmoc-Gly-Wang resin (0.63 mmol/g, 171.4 mg, 0.108 mmol), FIDTNNDGWIEGDELLLEEGG- resin was synthesized on ABI 433A (Applied. Biosystems, CA, USA) utilizing the 2-(1H- benzotriazole-1-yl)-1,1,3,3-tetramethyluronium hexafluorophosphate as a coupling reagent. To this peptide resin, tBoc-Cys(Trt) (1mmol, 463 mg, Watanabe Chemical Industries, Hiroshima,

Japan) was incorporated manually. Protected peptide resin (446 mg) was obtained. An aliquot

(158 mg) of the resin was treated with reagent K+ [3] for 8 h at room temperature. The peptides were precipitated by addition of diethylether. The crude peptide (85 mg) was subject to RP-

HPLC purification (Figure S8A). A fraction containing the peptide gave 2.3 mg of purified peptide as lyophilized powder (Figure S8B). MALDI-TOF MS, 2440.58 (theoretical [M+H]+ = 2440.31)

5 (Figure S9).

Native chemical ligation of GFP-C-thioester with Cys-derivatives

GFP-C-thioester was successfully prepared in good purity (Figure S10A).

Comparative data of GFP-intein-CBD fusion protein and GFP-Met-intein-CBD fusion protein in the cleavage reaction were shown in Figure S10B. Even without the cleavage reaction, cleavage products were observed in GFP-Met-intein CBD at the step of affinity purification with chitin beads.

In a typical ligation, GFP-C-thioester solution (55uM, 40uL, 2.2 nmol, 59 ug),

NaHCO3 aq. (pH 8.5, 1 M, 10 uL), CTAB (200mM, 10 uL) and tris(hydroxypropyl)phosphine (100 mM, 10 uL) were mixed. To an aliquot of the solution, Cys-lantanide binding peptide (0.10 mg,

40 nmol), Cys-NH-DEG-NHCO-Chol (0.10 mg, 20 uL) or DTT (10 uL of 1.0 M, 10 umol) was added. The final volume was adjusted with water to 100 uL. The coupling mixture was incubated for 24 h at room temperature under nitrogen. The reaction buffer was exchanged to phosphate- buffered saline by ultracentrifugation. The concentration of GFP moiety in recovered samples was respectively 26 uM, 17 uM, or 8.5 uM for the coupling with Cys-lantanide binding peptide, with Cys-NH-DEG-NHCO-Chol, or with DTT. No significant difference was observed in the fluorescence spectra between non-modified GFP and modified GFP (FigureS11). The progress of the coupling reactions were monitored by SDS-PAGE.

Each aliquot of the coupling reaction mixtures was subject to chloroform-methanol protein precipitation in order to remove salts and detergents. Precipitated proteins were dissolved in 20% aqueous acetonitrile containing 0.1 % TFA. Coupling reactions were monitored

by RP-HPLC. RP-HPLC analyses were performed with LC10A (Shimadzu) on a C4 column (5C4-

AR300, 4.6 X 150 mm, Nacalai Tesque Inc., Kyoto, Japan) using a water-acetonitrile-0.1% TFA

6 gradient. MALDI-TOF MS, 26857.9 corresponding to GFP (theoretical [M+H]+ = 26997.9), and

27571.1 corresponding to GFP-Chol (theoretical [M+H]+ = 27716.9). The fractionated GFP-Chol was analyzed by RP-HPLC again (Figure S12).

Density gradient fractionation analysis

Technical adequacy for density gradient fractionation analysis of the cell membrane components was tested by analyzing endogenous cellular prion protein distribution in untreated

N2a cells. Density gradient fractionation was performed as described in the 2.8 section of

MATERIALS AND METHODS, except the step of labeling the cells with GFP-Chol or F-Chol.

Immunoblotting detection of prion protein in each fraction was performed by using mouse monoclonal anti-prion protein antibody SAF83 (1:5000; SPI-Bio, Massy, France) as a primary antibody and rabbit anti-mouse IgG as a secondary antibody. The results shown in Figure S13 were consistent with those of a previous report [4].

7 References [1] Franz KJ, Nitz M, Imperiali B. (2003) Lanthanide-binding tags as versatile protein coexpression probes. Chembiochem. 4, 265-271.

[2] Geall AJ, Taylor RJ, Earll ME, Eaton MA, Blagbrough IS. (2000) Synthesis of cholesteryl polyamine carbamates: pK(a) studies and condensation of calf thymus DNA. Bioconjug Chem.

11, 314-326.

[3] King DS, Fields CG, Fields GB. (1990) A cleavage method which minimized side reactions following Fmoc solid phase peptide synthesis. Int. J. Peptide Protein Res. 36, 255–266.

[4] Naslavsky N, Stein R, Yanai A, Friedlander G, Taraboulos A. (1997) Characterization of detergent-insoluble complexes containing the cellular prion protein and its scrapie isoform. J.

Biol. Chem. 272, 6324 – 6331.

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