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IntroDuctIonChemical synthesis of proteins supports biomedical research through the production of proteins (e.g., post-translationally modified proteins, circular proteins, mirror-image proteins) that cannot be easily obtained via recombinant technology13. The most successful strategy for protein chemical synthesis has proven to be the condensation of unprotected peptides in aque-ous buffers by a chemoselective reaction, namely native chemical ligation, which was developed by Kent and co-workers4,5. This method relies on the use of peptide thioesters as key intermediates, which can be successfully prepared through Boc solid-phase peptide synthesis (SPPS)6. Nonetheless, the Boc method is usually not suitable for making protein segments carrying acid-sensitive modifications (e.g., phosphoryl and glyco-syl groups). Furthermore, the use of highly corrosive HF (hydro-gen fluoride) for global deprotection in the Boc method can be an operational hazard79.

To overcome the above problems, extensive studies have been carried out to develop methods for synthesizing peptide thioesters or their equivalents through the Fmoc-SPPS1018. In our recent work, we found that peptide hydrazides constitute good thioester surrogates for the native chemical ligation (Fig. 1)19,20. Through an operationally simple NaNO2 activa-tion and thiolysis process (usually via 4-mercaptophenylacetic acid (MPAA)5, introduced as an external thiol at a ~100 mM concentration), a peptide hydrazide can be cleanly and rapidly converted to a peptide thioester in an epimerization-free manner and then used in the native

chemical ligation without separation of any intermediates. It is important to note that peptide hydrazides with post-translational modifications can be readily prepared through the standard Fmoc chemistry either manually or by using commercially available automated synthesizers. Furthermore, peptide hydrazides are not reactive in native chemical ligation unless they are activated by NaNO2, and thus they can be used as masked thioesters in the convergent synthesis of proteins20.

By using the hydrazide-based native chemical ligation method, we and others have successfully synthesized a number of proteins such as the ribosomal protein S25, backbone-cyclized cyclotides and ubiquitinated histone H2B2124. In these studies, the synthetic strategy usually involves two key steps: first, Fmoc-SPPS of pep-tide hydrazides by using the 2-Cl-(Trt)-NHNH2 resin; second, the native chemical ligation of peptide hydrazides through in situ NaNO2 activation and thiolysis. Here we detail the proce-dure for the use of peptide hydrazides to synthesize -synuclein, a protein with an important role in the development of

Chemical synthesis of proteins using peptide hydrazides as thioester surrogatesJi-Shen Zheng, Shan Tang, Yun-Kun Qi, Zhi-Peng Wang & Lei Liu

Tsinghua-Peking Center for Life Sciences, Key Laboratory of Bioorganic Phosphorus Chemistry & Chemical Biology (Ministry of Education), Department of Chemistry, Tsinghua University, Beijing, China. Correspondence should be addressed to L.L. (lliu@mail.tsinghua.edu.cn).

Published online 14 November 2013; doi:10.1038/nprot.2013.152

this protocol provides a detailed procedure for the chemical synthesis of proteins through native chemical ligation of peptide hydrazides. the two crucial stages of this protocol are (i) the solid-phase synthesis of peptide hydrazides via Fmoc chemistry and (ii) the native chemical ligation of peptide hydrazides through in situ nano2 activation and thiolysis. this protocol may be of help in the synthesis of proteins that are not easily produced by recombinant technology and that include acid-sensitive modifications; it also does not involve the use of hazardous HF. the utility of the protocol is shown for the total synthesis of 140-aa-long a-synuclein, a protein that has an important role in the development of parkinsons disease. the whole synthesis of the target protein a-synuclein in milligram scale takes ~30 working days.

cO

O

ONaNO2 (10 eq), pH 3.0, 15 C, 1520 min

0.2 M phosphate buffer containing 6 M GnHCl

SR

Peptide

PeptidePeptide Peptide

PeptideCys

Cys(1 eq)

Ligation

ActivationNHNH2 N3Peptide

MPAA (100 eq), pH 6.87.0In situ thiolysis

aCl

5% NH2NH2/DMF H2N-NH

2-Cl-(Trt)-Cl resin 2-Cl-(Trt)-NHNH2 resin

b Fmoc-Aa1-OH (4 eq)HCTU (3.8 eq)DIPEA (8 eq)

Fmoc-Aa2-OH (4 eq)HCTU (3.8 eq)DIPEA (8 eq)

DMF

DMF

Fmoc-Aa1

Fmoc-Aa2-Aa1

Aa120% piperidine/DMF

O

OO

O

Aan-Aan-1...-Aa2-Aa1

HN-NH

HN-NH

-Peptide elongation-Final Fmoc deprotion-Cleavage by TFA

Peptide hydrazides

HN-NH

HN-NH2

H2N-NH

Figure 1 | General description for the native chemical ligation of peptide hydrazides. (a) Preparation of 2-Cl-(Trt)-NHNH2 resin from the commercially available 2-Cl-(Trt)-Cl resin. (b) Preparation of peptide hydrazides through Fmoc-SPPS. (c) Native chemical ligation of peptide hydrazides.

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Parkinsons disease2527. Because -synuclein undergoes many post-translational modifications, chemical total synthesis of this protein provides otherwise-difficult-to-access starting materials to study the proteins detailed biology2830.

Experimental designFrom a practical point of view, only peptides containing fewer than about 50 aa can be prepared cost-effectively through the SPPS. Thus, to synthesize -synuclein, which contains 140 aa, we pre-pare separately four peptide segments (Fig. 2 and Supplementary Fig. 1), i.e., -synuclein(129)-NHNH2 (1), -synuclein (3068, A30C)-NHNH2 (2), -synuclein(69106, A69C)-NHNH2 (3) and -synuclein(107140, A107C)-OH (4). These peptides can be efficiently synthe-sized through standard Fmoc-SPPS with good isolated yields (18%, 21%, 30% and

26%, respectively). The four peptide blocks are then assembled in sequence via native chemical ligation of peptide hydrazides to form the complete protein. It is worth mentioning that no Cys resi-dues are present in -synuclein, and thus, to make native chemical ligation reactions possible, we replace three Ala residues (Ala30, Ala69 and Ala107) with Cys. After the entire protein sequence is assembled, these Cys residues can be chemically desulfurized back to Ala residues through a ligation-desulfurization strategy31. We anticipate that the protocol described here can be used for the preparation of other small proteins containing 100200 aa.

To assemble the polypeptide chain of -synuclein, we carry out the N-to-C sequential ligation of the four peptide segments 14. First, peptide 1 is treated with NaNO2 at pH 3.0 in a 15 C ice/salt bath for 15 min. Subsequently, MPAA and peptide 2 are added to the reaction mixture while the pH value is adjusted to 6.87.0. After 5 h, the ligation between 1 and 2 is completed, affording the desired product -synuclein(168, A30C)-NHNH2 5 in 56% isolated yield. Peptide 5 is then reacted with 3 by using the above strategy again to afford -synuclein(1106, A30,69C)-NHNH2 (6) in 49% isolated yield. Finally, 6 is reacted with 4 to afford full-length -synuclein(1140, A30,69,107C) (7) in 54% isolated yield.

By using a free radical desulfurization reaction initiated by VA-044 (2,2-azobis[2-(2-imidazolin-2-yl)propane] dihydrochlo-ride), 7 can be readily reduced to the target protein -synuclein (8) in 72% isolated yield. Thus, the synthetic -synuclein 8 is obtained with an overall isolated yield of 11%, as calculated from the four peptide segments. The identity of 8 is character-ized by ESI-MS and SDS-PAGE. The circular dichroism (CD) spectrum of 8 shows that -synuclein exists as a random coil in the aqueous solution with a strong negative absorption at 198 nm. This observation is consistent with a previous report32. Furthermore, the aggregation of -synuclein 8 can be measured by the thioflavin T (ThT) fluorescence assay (Fig. 3): ThT can form stable fluorescent complexes exclusively with aggregated -synuclein 8 and then undergo a shift in emission frequency.

H- 128 -Ala-NHNH2 Cys - 3167 -Gly-NHNH2 Cys - 70105 -Gly-NHNH2 Cys - 108140 -OH1 2 3 4

5

6

(Desulfurization)(72% isolated yield)

H- 128 -Ala-Cys- 3167 -Gly-NHNH2

NaNO2, 15 C, pH 3.0

MPAA, rt, pH 6.8, 5 h

NaNO2, 15 C, pH 3.0, 15 min

MPAA, rt, pH 6.8, 4 h

NaNO2, 15 C, pH 3.0, 15 min

MPAA, rt, pH 6.8, 4 h

(First ligation)(56% isolated yield)

(Second ligation)(49% isolated yield)

(Third ligation)(54% isolated yield)

TCEP HCl, VA-044,tBu-SH, 37 C, 4 h

7

H- 128 -Ala-Cys- 3167 -Gly-Cys- 70105 -Gly-NHNH2

H- 128 -Ala-Cys- 3167 -Gly-Cys- 70105 -Gly-Cys- 108140 -OH

H- 128 -Ala-Ala- 3167 -Gly-Ala- 70105 -Gly-Ala- 108140 -OH

MDVFMKGLSK10AKEGVVAAAE20KTKQGVAEAA30GKTKEGVLYV40GSKTKEGVVH50GVATVAEKTK60EQVTNVGGAV70VTGVTAVAQK80TVEGAGSIAA90ATGFVKKDQL100GKNEEGAPQE110GILEDMPVDP120DNEAYEMPSE130EGYQDYEPEA140

Synthetic native -synuclein (8)(10.7% overall yield for the ligations and desulfurization)

Figure 2 | Synthetic route for the preparation of -synuclein 8. rt, room temperature.

600 800

15+964.9

14+1033.7

13+1,113.2

16+904.7

17+851.5

18+804.3

12+1,205.9

11+1,315.3

10+1,446.7 9+

1,607.4

19+762.0

20+723.9

21+689.5

1,000 1,200 1,400 1,600m/z

Synthetic -synucleinM.W.14,459.99

0 5 10 15 20 25 30

Synthetic -synucleina

(kDa)170130957255433426

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10

-synuclein 8

c

02468

1012

190 200 210 220 230 240 250Wavelength (nm)

Mea

n el

liptic

ity (m

deg)

260

2

d1,200

1,000

ThT

fluor

esce

nce

800

600

400

200

00 72 120

Thioflavin T assays of-synuclein

240Time (h)

300 360 480

e

b

Figure 3 | Characterization of synthetic -synuclein 8. (a) HPLC profile (detection at 214 nm) of purified -synuclein 8 shows a single peak at 22.04 min. The analysis is run on a C4 column with a gradient of 5% buffer B in buffer A to 95% buffer B in buffer A over 30 min at room temperature. (b) ESI-MS of purified -synuclein 8 (found: 14,458.6 Da, calculated: 14,460.0 Da). (c) SDS-PAGE/Coomassie staining analysis of purified -synuclein 8. (d) CD spectroscopy of purified -synuclein 8. A concentration of 10 M of 8 in 20 mM potassium phosphate (pH 7.4) is analyzed in a 1-mm quartz cell. (e) Thioflavin T assays of purified -synuclein 8. Synthetic -synuclein 8 at 10 M (containing 20 mM Tris, 150 mM NaCl, pH 7.5) is incubated at 37 C. Aliquots are taken and analyzed by ThT fluorescence (450 nm/482 nm) at the indicated time points (expressed in hours). The experiments are performed in triplicate.

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Protein aggregation can be determined by monitoring the inten-sity of the shifted emission frequency. The increase of the ThT signal shows that -synuclein 8 forms mature fibrils after 72 h of incubation at 37 C, an observation that is also consistent with the previous studies28.

If manual Fmoc-SPPS is performed, the preparation of each of the peptide segments 14 requires ~5060 working hours.

Each of the three ligation steps, as well as the final desulfurization step, takes about five working hours for completion of the reac-tion, five working hours for the reverse-phase HPLC (RPHPLC) separation and 12 d for the lyophilizations. Thus, we estimate that the whole synthesis takes ~250 working hours (without counting the time needed for the lyophilizations) to generate the target protein -synuclein on a 110-mg scale.

MaterIalsREAGENTS! cautIon N,N-dimethylformamide, dichloromethane and piperidine are harmful on inhalation, ingestion or skin contact. All the organic solvents are flammable, whereas piperidine and diethyl ether are highly flammable. Trifluoroacetic acid and acetic anhydride are corrosive. Therefore, all organic solvents and chemicals used in this protocol should be handled inside a chemical fume hood, and appropriate personal protective equipment (lab coat, gloves and protective glasses) should be used.

Dichloromethane (DCM; Sinopharm Chemical Reagent (SCRC), cat. no. 80047360)N,N-Dimethylformamide (DMF; SCRC, cat. no. 8100771933)Hydrazine hydrate (NH2NH2xH2O; SCRC, cat. no. 80070418)Standard Fmoc-protected amino acids (GL Biochem): Fmoc-Ala-OH, Fmoc-Arg(Pbf)-OH, Fmoc-Asn(Trt)-OH, Fmoc-Asp(OtBu)-OH, Fmoc-Cys(Trt)-OH, Fmoc-Gln(Trt)-OH, Fmoc-Glu(OtBu)-OH, Fmoc-Gly- OH, Fmoc-His(Trt)-OH, Fmoc-Ile-OH, Fmoc-Leu-OH, Fmoc-Lys(Boc)-OH, Fmoc-Met-OH, Fmoc-Phe-OH, Fmoc-Pro-OH, Fmoc-Ser(tBu)-OH, Fmoc-Thr(tBu)-OH, Fmoc-Trp(Boc)-OH, Fmoc-Tyr(tBu)-OH, Fmoc-Val-OH2-Cl-(Trt)-Cl resin (Tianjin Nankai HECHENG S&T, loading: 0.56 mmol g1)1-[Bis(dimethylamino)methylene]-1H-benzotriazoliumhexafluorophos-phate 3-oxide (HBTU; GL Biochem, cat. no. 00702)2-(6-Chloro-1H-benzotriazole-1-yl)-1,1,3,3-tetramethylaminiumhexa-fluorophosphate (HCTU; GL Biochem, cat. no. 00706)1-[Bis(dimethylamino)methylene]-1H-1,2,3-triazolo-[4,5-b]pyridinium hexafluorophosphate 3-oxide (HATU; GL Biochem, cat. no. 00703)N,N-Diisopropylethylamine (DIEA; GL Biochem, cat. no. 90600)1-Hydroxy-7-azabenzotriazole (HOAt; GL Biochem, cat. no. 00601)1-Hydroxybenzotriazole, anhydrous (HOBt; GL Biochem, cat. no. 00602)Piperidine (SCRC, cat. no. 80104216)Methanol (MeOH; SCRC, cat. no. 10014118)Ninhydrin hydrate (SCRC, cat. no. 30130212)Pyridine (SCRC, cat. no. 10018118)Potassium cyanide (KCN; SCRC, cat. no. 10016518)Trifluoroacetic acid (TFA; GL Biochem, cat. no. 10601)Phenol (SCRC, cat. no. 10015318)Triisopropylsilane (TIPS; GL Biochem, cat. no. 91100)Diethyl ether (Et2O; SCRC, cat. no. 10009318)1,1,1,3,3,3-Hexafluoro-2-propanol, hexafluoroisopropanol (HFIP; SCRC, cat. no. 39143370)Acetonitrile (ACN, HPLC grade; J.T. Baker, cat. no. B9017-03)Deionized waterGuanidine hydrochloride (GnHCl; SCRC, cat. no. 30095516)Sodium chloride (NaCl; SCRC, cat. no. 10019318)Sodium nitrite (NaNO2; SCRC, cat. no. 10020018)Sodium hydroxide (NaOH; SCRC, cat. no. 10019762)Hydrochloric acid (HCl; SCRC, cat. no. 10011018)Disodium hydrogen phosphate dodecahydrate (Na2HPO412H2O; SCRC, cat. no. 10020318)Sodium dihydrogen phosphate dihydrate (NaH2PO42H2O; SCRC, cat. no. 20040718)4-mercaptophenylacetic acid (MPAA; Alfa Aesar, cat. no. H27658)Tris(2-carboxyethyl)phosphine hydrochloride (TCEPHCl; Aladdin, cat. no. T107252-5g)

2,2-Azobis[2-(2-imidazolin-2-yl)propane] dihydrochloride (VA-044; J&K Scientific, cat. no. 503236)2-methyl-2-propanethiol (tBuSH; J&K Scientific, cat. no. 533251)

EQUIPMENTAnalytical balance (AL 104; Mettler Toledo)Polypropylene dropper, 4 mlSet of adjustable pipettes, 0.510, 220, 1050, 10100, 1001,000 l (Eppendorf research)Pipette tip, 0.510, 220, 1050, 10100, 1001,000 l (Eppendorf research)Polypropylene centrifuge tubes, 1, 2, 4, 10, 15, 50 ml (safe-lock tubes; Eppendorf) with lidsMagnetic stirrer (e.g., Synthware, cat. no. S360205)Magnetic stirring apparatus (e.g., Tokyo Rikakikai, EYEL4: RCH-20L)Thermostat water bathThermostat oil bathCapillary (e.g., Synthware, cat. no. P230001)Kaiser test micro tubesHeating and stirring mantles/heaterWater ring vacuum pumpOil vacuum pump (e.g., Tokyo Rikakikai, EYEL4: GLD-N051)Rotary evaporators (e.g., Tokyo Rikakikai, EYEL4: N-2100)Peptide synthesis vessels (Synthware, 25 ml, cat. no. P150025M; 50 ml, cat. no. P150050M; 100 ml, cat. no. P150100M)Peptide synthesizer (triple-entries: C S Bio Model, CS136XT; serial no.: 110703; double-entries: C S Bio Model, CS136XT; serial no.: 121204)Dewar flask (Synthware: 100 ml, cat. no. F240100; 500 ml, cat. no. F250500)Constant-temperature shaker (Eppendorf)Iron support system, with iron clamps (Synthware, cat. no. W200002, W192203)Vacuum/Ar gas systemCentrifuges (e.g., Eppendorf: microcentrifuge 5418/5418R; centrifuge 5810/5810R)Vortex mixerAnalytical and semipreparative HPLC system (Shimadzu, SPD-20A UV/VIS detector; LC-20AT prominence)HPLC vialsLyophilizer (e.g., Tokyo Rikakikai, EYEL4: M-1000)Plastic syringe, 1, 2.5, 5 mlLC syringe (e.g., Thermo Scientific: 25, 100 and 500 l)Microporous membrane filter (organic system, 13 mm or 50 mm 0.22 m)UV detectorCD spectrometerElectrospray ionizationmass spectrometry (ESI-MS) instrumentThermometer, 20~110 C (e.g., Synthware, cat. no. T167611)pH meter (DELTA 320; Mettler Toledo)Liquid nitrogen containerRubber hoseRubber plugsMeasuring cylinderUltrasonic oscillators (SB-5200D, Ningbo Scientz Biotechnology)Conical flask (Synthware: 100 ml, cat. no. F304100; 250 ml, cat. no. F304250; 500 ml, cat. no. F304500)

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REAGENT SETUPPiperidine in DMF, 20% (vol/vol) Mix 100 ml of piperidine with 400 ml of DMF. The solution can be stored for up to 1 month at room temperature (~25 C). ! cautIon Pyridine is highly flammable and toxic.TFA cleavage cocktails (TFA/phenol/H2O/TIPS = 88/5/5/2 (vol/wt/vol/vol)) Mix 13.2 ml of TFA, 0.75 ml of phenol, 0.75 ml of deionized H2O and 0.3 ml of TIPS to prepare 15 ml of cleavage cocktails. ! cautIon TFA is strongly corrosive and toxic. Phenol is toxic and corrosive. TIPS is flammable. crItIcal Freshly prepare the solution before use.Kaiser test reagents Kaiser test reagents include reagent A: 80% (wt/vol) phenol in ethanol; reagent B, 5% (wt/vol) ninhydrin in ethanol; and reagent C, 2% (vol/vol) potassium cyanide (KCN) in a 1 mM aqueous solution in pyridine. The reagents can be stored for up to 2 months at room temperature. ! cautIon KCN is highly toxic. Wear a lab coat, gloves and a mask.NH2NH2, 5% (vol/vol) Add 0.2 ml of hydrazine hydrate to 3.80 ml of DMF. Freshly prepare NH2NH2 before use. ! cautIon Hydrazine hydrate is a dangerous substance and may cause cancer. Wear a lab coat, gloves and a mask.ACN (50%) containing 0.1% TFA (vol/vol) Add 0.1 ml of TFA to 100 ml of 50% (vol/vol) aqueous ACN. The solution can be stored for up to 1 week at room temperature.NaOH, 1 M and 6 M Dissolve 40 mg or 240 mg of NaOH in 1 ml of deionized H2O. ! cautIon Solid NaOH and its solution are highly corrosive. Wear a lab coat, gloves and a mask. crItIcal The solutions should be prepared freshly before use, as NaOH will react with CO2 after long-term exposure to air.HCl, 6 M Mix 1 ml of 12 M HCl with 1 ml of deionized H2O. ! cautIon Concentrated HCl solutions are highly corrosive, strong mineral acids. Wear a lab coat, gloves and a mask. crItIcal The solution should be prepared freshly before use, as HCl will evaporate after long-term exposure to air.Two different phosphate solutions (0.2 M) containing 6 M GnHCl (pH 3.03.1 and pH 6.97.0, respectively) For a 10-ml solution, mix 312 mg of NaH2PO42H2O and 5.74 g of GnHCl into a 10-ml volumetric flask and adjust it to the final volume with deionized H2O. Adjust the pH to 3.03.1 and 6.97.0 with 6 M NaOH and 6 M HCl. Filter the solution by using a 13 mm 0.22 m microporous membrane filter. The filtered solution can be stored at 4 C for at least 1 month.NaNO2, 0.5 M Dissolve 17 mg of NaNO2 in 0.5 ml of deionized H2O. crItIcal The solution should be prepared just before use, as NaNO2 may decompose after long-term storage.

TCEP, 0.1 M Dissolve 58 mg of TCEPHCl into 1 ml of 0.2 M phosphate solution (pH 3.0) containing 6 M guanidine hydrochloride (GnHCl). Adjust the pH to 6.07.0, and filter the solution by using a 13 mm 0.22 m microporous membrane filter. crItIcal The solution should be prepared freshly before use, as TCEP can be easily oxidized by O2 after exposure to air.TCEP, 1.0 M Dissolve 143 mg of TCEPHCl into a 2-ml Eppendorf reaction tube, and add 0.5 ml of 0.2 M phosphate solution containing 6 M GnHCl (pH 7.0). Filter the solution by using a 13 mm 0.22 m microporous membrane filter. Adjust the pH to about 5.0. crItIcal The solution should be prepared freshly before use, as TCEP can be easily oxidized by O2 after exposure to air.VA-044, 0.1 M Weight 9.7 mg of VA-044 into a 2-ml Eppendorf reaction tube and add 0.3 ml of 0.2 M phosphate solution (pH 6.97.0) containing 6 M GnHCl. Completely dissolve VA-044 by using a vortex and an ultrasonic cleaning bath. crItIcal The solution should be prepared freshly before use, as VA-044 is easily oxidized by exposure to air.EQUIPMENT SETUPManual peptide-synthesis apparatus Place 2-Cl-(Trt)-NHNH2 resin, which is prepared as described in the PROCEDURE (Steps 114), into an SPPS reaction vessel. During the peptide elongating reactions, immobilize the reaction vessel with iron clamps in a constant-temperature shaker at 30 C . During the washing steps, connect the vessel with water ring vacuum pump via rubber hoses to remove the washing solvents.C S Bio automated synthesizer Use a CS136XT automated synthesizer running with a synthesis scale of 0.25 mmol. A standard synthesis program is as follows: 2 DMF, 30 s; 1 20% (vol/vol) piperidine in DMF, 5 min; 1 20% (vol/vol) piperidine in DMF, 20 min; 2 DMF, 30 s; 2 DCM, 30 s; 3 DMF, 30 s; amino acid activation by HCTU in the presence of DIEA in DMF for ~0.51 min; add AA solution to the resin, and couple 0.51 h; 2 DMF, 30 s; 1 DCM, 30 s.Analytical HPLC Use an HPLC gradient system equipped with a UV detector (214 nm) and a reversed-phase C4 column (4.6 mm 150 mm or 250 mm, 5 m). Run a linear gradient as shown in Table 1 for the analysis of crude peptides or ligation products.Semipreparative HPLC Use an HPLC gradient system equipped with a detector (214 nm) and a reversed-phase C4 column (10 mm 250 mm or 22 mm 150 mm, 10 m). Run a linear gradient as shown in Table 1 for the isolation of crude peptides or ligation products.

table 1 | Analytical and semipreparative HPLC conditions.

analytical semipreparative

Column Vydac C4 reverse-phase column (4.6 150 mm or 4.6 250 mm, pore size: 300 , particle size: 5 m)

Vydac C4 reverse-phase column (22 150 mm or 10 250 mm, pore size: 300 , particle size: 10 m)

Solvents A: deionized H2O containing 0.1% (vol/vol) TFA; B: ACN containing 0.08% (vol/vol) TFA

A: deionized H2O containing 0.1% (vol/vol) TFA; B: ACN containing 0.08% (vol/vol) TFA

Flow rate 1.0 ml min1 5.0 ml min1 (for crude peptide) or 3.0 ml min1 (for ligation product)

Run time 30 min 30 min

Gradient Alterable, depending on the peptides or reac-tions (e.g., 595%, 1575% or 2050% of B). The detailed gradients for each peptide and reaction are shown in supplementary Figures 2, 4, 6, 8 and supplementary Data 14.

Alterable, depending on the peptides (e.g., 2050%, or 3045% of B). The detailed gradients for each peptide and reaction are shown in supplementary Figures 2, 4, 6, 8 and supplementary Data 14.

Injection 500 l (maximum) 5.0 ml (maximum)

Wavelength 214 nm and 254 nm 214 nm and 254 nm

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proceDurepreparation of 2-cl-(trt)-nHnH2 resin tIMInG 2 h crItIcal Hydrazination of 2-Cl-(Trt)-Cl resin (Steps 814) should take no more than 2 h. The 2-Cl-(Trt)-NHNH2 resin should be freshly prepared for stability reasons.1| Weigh 450 mg of 2-Cl-(Trt)-Cl resin (0.56 mmol g1) into a peptide synthesis vessel.

2| Add 5 ml of DMF to the resin from the top of the reaction vessel, gently agitate it for 10 s and then drain it.

3| Repeat Step 2 two more times.

4| Add 5 ml of DCM to the resin, gently agitate it for 10 s and then drain it.

5| Repeat Step 4 two more times.

6| Repeat Step 2 three more times.

7| Swell the resin in 4 ml of 50% (vol/vol) DMF/DCM for 30 min, and then drain it.

8| Add 4 ml of 5% (vol/vol) NH2NH2 to the resin for hydrazination. Gently agitate the mixture for 30 min in a constant-temperature shaker at 30 C and then drain the solution by vacuum filtration.? troublesHootInG

9| Add 4 ml of DMF to the resin, gently agitate it for 10 s and then drain it.

10| Repeat Step 8.

11| Wash the resin by repeating Steps 26 twice.

12| Add 4 ml of 5% (vol/vol) MeOH/DMF to the resin, gently agitate it for 10 min and then drain it. crItIcal step This step is crucial, as it ensures that unreacted sites on the resin are capped.

13| Repeat Steps 26 to wash the resin thoroughly.

14| Add 4 ml of DCM to the resin, agitate it gently for 10 s and then drain it. Directly use the resin for the next coupling step. Please note that the formation of 2-Cl-(Trt)-NHNH2 cannot be monitored by the Kaiser test, as described in box 1 (as 2-Cl-(Trt)-NHNH2 resin is yellow under the Kaiser test), but the resin becomes yellow or light green when the substitution is successful. pause poInt For storage, dry the resin under high vacuum for 2 h and store dried 2-Cl-(Trt)-NHNH2 resin at 20 C under argon gas for up to 2 weeks. The substitution of the resin is noticeably reduced after storage for over 4 weeks.

Box 1 | Qualitative monitoring of peptide synthesis by the Kaiser test tIMInG 5 min The Kaiser test35 is a qualitative test for the completion of a coupling step. This test is based on the reaction of free primary amino groups with ninhydrin, which yields a characteristic blue color.

proceDure1. Transfer a few washed resin beads to a small glass tube.2. Add one drop of each of the Kaiser test reagents A, B and C (as mentioned in Reagent Setup) to the tube.3. Mix the contents of the tube well and heat the tube in a preheated oven (100 C) for 25 min. crItIcal step The test is not suitable for N-terminal Pro (a secondary amine).

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synthesis of Fmoc-protected a-synuclein(129)-nHnH2 tIMInG 6090 min per amino acid15| Couple amino acids according to option A if the amino acids are either sterically hindered or meant to be coupled immediately after a proline residue, or use option B for all other amino acids. crItIcal step Choose the proper reaction conditions for each amino acid, including the number of coupling reactions (e.g., single or double coupling), reaction time (e.g., 6090 min) and the type of coupling reagent (e.g., HCTU or HATUHATU is a more efficient activator than HCTU, and it is used for sterically hindered amino acids). A single coupling reaction using HCTU and a 60-min reaction time is enough for most amino acids. However, a double coupling strategy is needed for sterically hindered amino acid derivatives (e.g., Fmoc-Cys(Trt)-OH, Fmoc-His(Trt)-OH, Fmoc-Ile-OH, Fmoc-Asn(Trt)-OH, Fmoc-Gln(Trt)-OH, Fmoc-Arg(Pbf)-OH, Fmoc-Thr(tBu)-OH, Fmoc-Val-OH and the amino acids to be coupled right after a Pro residue), especially when the stretch of peptide already assembled is longer than 20 amino acids. HTBU/HOBt/DIEA can be used to couple the amino acids Fmoc-Cys(Trt)-OH, Fmoc-His(Trt)-OH and Fmoc-Phe-OH to reduce the racemizations.(a) amino acid double-coupling tIMInG 90 min (i) Dissolve 1 mmol of the desired Fmoc-protected amino acid and 0.95 mmol of HATU (360 mg) with 3 ml of DMF in a 5-ml tube.

crItIcal step HATU is a uranium-based coupling reagent that can cause undesired termination of peptide elongation. Ensure that the concentration of HATU is slightly lower than that of amino acid building blocks. ! cautIon Exposure to HATU can cause an allergic reaction. Wear a lab coat, gloves and a mask.

(ii) Add 2 mmol of DIEA (0.36 ml) to the dissolved solution. Activate the amino acid by vortexing the solution for 0.51 min at room temperature. crItIcal step The activation of amino acids usually involves the formation of a yellow color accompanied by heat release, but this phenomenon is not always observed. crItIcal step The activation should take no more than 5 min, as the activated amino acids isomerize from the more active O-forms to the less active N-forms over time33.

(iii) Add the activated amino acid to the resin; agitate it gently for 20 min at 30 C in a constant-temperature shaker, and then drain the solution by vacuum filtration.

(iv) Add 4 ml of DMF to wash the resin, shake it for 10 s and then drain it. (v) Dissolve 1 mmol of the desired Fmoc-protected amino acid and 0.95 mmol of HCTU (390 mg) with 3 ml of DMF in a 5-ml tube.

! cautIon Exposure to HCTU can cause an allergic reaction. Wear protective clothing, gloves and a mask. crItIcal step HCTU is a uranium-based coupling reagent that can cause undesired termination of peptide elongation. Ensure that the concentration of HCTU is slightly lower than that of the amino acid building blocks.

(vi) Add 2 mmol of DIEA (0.36 ml) to the dissolved solution. Activate the amino acid by vortexing the solution for 0.51 min at room temperature. crItIcal step The activation of amino acids usually involves the formation of a yellow coloration accompanied by heat release, but this phenomenon is not always observed. crItIcal step The activation should take no more than 5 min, as the activated amino acids isomerize from the more active O-forms to the less active N-forms over time33.

(vii) Add the activated amino acid to the resin, agitate it gently for 4060 min at 30 C in a constant-temperature shaker and then drain the solution by vacuum filtration.

(viii) Repeat Steps 26 to thoroughly wash the resin. pause poInt At the end of every coupling cycle, the synthesis can be interrupted and the reaction mixture can be stored in DMF overnight at room temperature, as long as the Fmoc protecting group has not been removed.

(b) amino acid single-coupling tIMInG 60 min (i) Dissolve 1 mmol of the desired Fmoc-protected amino acid and 0.95 mmol of HCTU (390 mg) in 3 ml of DMF in a vial.

! cautIon Exposure to HCTU can cause an allergic reaction. Wear protective clothing, gloves and a mask. (ii) Add 2 mmol DIEA (0.36 ml) to the dissolved solution. Activate the amino acid by vortexing the solution for 0.51 min

at room temperature. crItIcal step The activation of amino acids usually involves the formation of a yellow color accompanied by heat release, but this phenomenon is not always observed. crItIcal step The activation should take no more than 5 min, as the activated amino acids isomerize from the more active O-forms to the less active N-forms over time33. crItIcal step When HATU is used for Cys coupling instead of HCTU, the activation with HATU, DIEA and 1 mmol of HOAt (136 mg) as a coupling additive should take no more than 30 s to reduce the racemization.

(iii) Add the activated amino acid to the resin, gently agitate it for 4060 min at 30 C in a constant-temperature shaker and then drain the solution by vacuum filtration.

(iv) Repeat Steps 26 to thoroughly wash the resin. pause poInt At the end of every coupling cycle, the synthesis can be interrupted and the reaction mixture can be stored in DMF overnight at room temperature, as long as the Fmoc protecting group has not been removed.

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16| Couple Fmoc-Ala-OH to the 2-Cl-(Trt)-NHNH2 resin as described in Step 15A(iviii).

peptide deprotection tIMInG 25 min per amino acid17| Add 4 ml of 20% (vol/vol) piperidine in DMF to the resin, gently agitate it for 5 min at room temperature and then drain the solution.

18| Add 4 ml of 20% (vol/vol) piperidine in DMF to the resin, gently agitate it for 15 min and then drain it.

19| Repeat the coupling and deprotection cycle from Steps 1518 for the subsequent amino acids. Choose proper coupling strategies for different amino acid derivatives until all of the 29 amino acid residues are assembled.

20| (Optional) Completeness of amino acid coupling can be monitored by performing the Kaiser test as described in box 1.

21| Repeat Steps 26 to thoroughly wash the resin.

22| Wash the resin with DCM as described in Steps 4 and 5 and then vacuum-dry the resin for 5 min. crItIcal step Thoroughly wash and dry the resin to avoid undesired side reactions before the cleavage step.

cleavage of the crude peptide tIMInG 4 h23| Add 15 ml of TFA cleavage cocktails (23 ml per 100 mg of resin) to the dried peptide-containing resin. Note that 0.1 M HCl in HFIP solution may be used as a surrogate for the TFA cocktails34.! cautIon TFA is highly corrosive. Wear a lab coat, gloves and a mask, and work in an efficient fume hood.

24| Gently agitate the reaction in a constant-temperature shaker at 30 C for 23 h, and then filter the cleavage mixture into a 50-ml centrifuge tube.

25| Wash the reaction vessel and resin by using 1 ml of TFA cleavage cocktails and collect the filtrate.

26| Repeat Step 25 two more times.

27| Remove most of the TFA (the volume of the concentrated cleavage mixture is less than 5 ml) by blowing nitrogen over the mixture in an efficient fume hood.

28| Cool diethyl ether to 0 C for 10 min (the volume of diethyl ether should be at least 10 times that of the concentrated cleavage mixture). crItIcal step The anhydrous ether should be precooled to 0 C to avoid violent heat release, which may cause side reactions of the crude peptide.

29| Add the precooled diethyl ether to precipitate crude peptides.

30| Triturate the product and then centrifuge it at 5,000g for 1 min at room temperature.

31| Decant the solvent from the centrifuge tube carefully.

32| Repeat Steps 2931 two more times.

33| Air-dry the peptide product in the open centrifuge tube for about 10 min. Store the dried crude peptides at 4 C for further analysis. pause poInt Dried crude peptides can be stored at 4 C under argon gas for at least 2 months.

analysis, purification and characterization of the crude peptide tIMInG 10 h34| Dissolve 100 mg of crude peptide in 4 ml of 50% ACN containing 0.1% (vol/vol) TFA. Filter the solution by using a microporous membrane filter (13 mm 0.22 m). crItIcal step Filter the solution of crude peptide with a hydrophobic membrane filter before injection into the HPLC for analysis or purification to remove the insoluble impurities, which would clog the HPLC lines.

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35| Dilute 5 l of the crude sample with 95 l of 50% ACN containing 0.1% (vol/vol) TFA.

36| Analyze 20 l of the diluted sample via analytic HPLC according to the conditions described in table 1. Collect the fractions corresponding to the main peak and confirm the target peptide mass by ESI-MS.

37| Purify the crude peptide products by semipreparative HPLC according to the conditions listed in table 1. Collect the pure product in a round-bottom flask.

38| Remove ACN by evaporation under reduced pressure (in a rotary evaporator with the temperature kept below 40 C). Freeze the concentrated solution with liquid nitrogen and then lyophilize it overnight to obtain purified -synuclein(129)- NHNH2: 135 mg (isolated yield: 18%, purity >95% ). RP-HPLC analyses of crude and purified -synuclein(129)-NHNH2 are shown in supplementary Figure 2. The identity of the peptide hydrazide is confirmed by ESI-MS, as shown in supplementary Figure 3.

synthesis of a-synuclein(3068, a30c)-nHnH2 tIMInG 65 h39| Repeat Steps 138 to obtain the purified -synuclein(3068, A30C)-NHNH2: 210 mg (isolated yield: 21%, purity >95% ). RP-HPLC analyses of crude and purified -synuclein(3068, A30C)-NHNH2 are shown in supplementary Figure 4. The identity of the peptide hydrazide is confirmed by ESI-MS, as shown in supplementary Figure 5.

synthesis of a-synuclein(69106 a69c)-nHnH2 tIMInG 62 h40| Repeat Steps 138 to obtain the purified -synuclein(69106, A69C)-NHNH2: 280 mg (isolated yield: 30%, purity >95%). RP-HPLC analyses of crude and purified -synuclein(69106, A69C)-NHNH2 are shown in supplementary Figure 6. The identity of the peptide hydrazide is confirmed by ESI-MS, as shown in supplementary Figure 7.

synthesis of a-synuclein(107140, a107c)-oH tIMInG 60 h41| Repeat Steps 17 to swell the resin, and then drain it.

42| Weigh 1 mmol (311 mg) Fmoc-Ala-OH in a vial, and add 3 ml of DCM and 2 mmol DIEA (0.36 ml) to the vial. Dissolve the amino acids by vortexing for 1 min at room temperature. crItIcal step The solubility of Fmoc-Ala-OH in DCM is relatively low, but this compound is completely dissolved after the addition of 0.36 ml of DIEA.

43| Add the solution prepared in Step 42 to the resin, agitate it gently for 2 h in a constant-temperature shaker at 30 C and then drain it.

44| Repeat Steps 42 and 43 once.

45| Repeat Steps 26 to wash the resin.

46| Add 4 ml of 5% MeOH/DMF (vol/vol), agitate the mixture for 10 min at 30 C in a constant-temperature shaker and then drain it.

47| Repeat Steps 26 to wash the resin. pause poInt After Fmoc-Ala-OH is attached to the 2-Cl-(Trt)-Cl resin, the dried resin can be stored for up to 6 months under argon gas.

48| Repeat Steps 17 and 18 to remove the Fmoc group.

49| Repeat Steps 26 to wash the resin.

50| Repeat Steps 1538 to obtain the purified -synuclein(107140, A107C)-OH: 252 mg (isolated yield: 26%, purity >95%). RP-HPLC analyses of crude and purified -synuclein(107140, A107C)-OH are shown in supplementary Figure 8. The identity of the peptide is confirmed by ESI-MS, as shown in supplementary Figure 9.

synthesis of a-synuclein(168, a30c)-nHnH2 by native chemical ligation of peptide hydrazides tIMInG 10 h51| Prepare 1 ml of 1 M and 6 M aqueous NaOH solution, 2 ml of 6 M aqueous HCl solution, 10 ml of 0.2 M sodium phosphate acidic solution containing 6 M GnHCl, 0.5 ml of 0.5 M aqueous NaNO2 solution and 1 ml of 0.1 M TCEP neutral solution containing 6 M GnHCl as described in Reagent Setup.

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52| To prepare a 15 C ice/salt bath, mix 12.5 g of sodium chloride and 50 g of crushed ice with a stirring rod in a 100-ml Dewar flask. crItIcal step It is important to keep the temperature of the bath at about 15 C. If ice melts into water, suck the water out with a pipe, and then add crushed ice and salt into the Dewar flask. It is also a good idea to use the cryogenic reactor to reach the 15 C condition.

53| Weigh 6.0 mg of -synuclein(129)-NHNH2 into a 2-ml Eppendorf reaction tube. Add 0.4 ml of 0.2 M phosphate solution containing 6 M GnHCl (pH 3.03.1) prepared in Step 51 and completely dissolve the peptide hydrazide by using a vortex and an ultrasonic cleaning bath. Centrifuge the tube at 7,200g for 1 min at room temperature. Place the reaction tube in the 15 C ice-salt bath, and gently agitate the solution by magnetic stirring for 15 min. crItIcal step Centrifuging the tube is necessary to effectively recover the solution that sticks to the tube wall.

54| Weigh 8.0 mg of -synuclein(3068, A30C)-NHNH2 and 13.6 mg of MPAA into a 2-ml Eppendorf reaction tube. Add 0.4 ml of 0.2 M phosphate solution containing 6 M GnHCl (pH 3.03.1) prepared in Step 51. Adjust the pH to 6.5 with 6 M NaOH and mix by using a vortex and an ultrasound ultrasonic cleaning bath until all solids have dissolved. Centrifuge the tube at 7,200g for 1 min at room temperature to recover the solution that sticks to the tube wall.

55| To oxidize the peptide hydrazide to the corresponding azide, pipette 40 l of 0.5 M NaNO2 prepared in Step 51 into the solution from Step 53. Gently agitate the solution for 15 min at 15 C. crItIcal step During the oxidation, make sure that the temperature drops to about 15 C. To prevent side reactions, the oxidation step should proceed for no longer than 20 min.? troublesHootInG

56| To convert the peptide azide to the thioester, pipette the buffer prepared in Step 54 into the oxidation solution from Step 55. Remove the tube from the ice/salt bath and allow it to warm to room temperature. Monitor the pH of the ligation reaction with a micro pH probe and adjust the pH to 6.87.0 with 6 M NaOH. crItIcal step Do not use TCEP to quench the excess NaNO2 in this step, as TCEP will reduce the peptide azide to the corresponding peptide amide. crItIcal step The addition of MPAA converts the peptide azide to peptide thioester, and it also eliminates excess NaNO2 in the reaction system. crItIcal step At pH >6.87.0, the peptide thioester will be hydrolyzed, so avoid over-titration when adding NaOH. It is better to use 1 M NaOH instead of 6 M NaOH once the pH is above 6.0.? troublesHootInG

57| To monitor the ligation reaction, remove 1 l of the reaction mixture and quench it by adding 10 l of 0.2 M phosphate solution containing 6 M GnHCl (pH 3.03.1). We also recommend adding 3 l of 0.1 M TCEP neutral solution. Let the resulting solution incubate for 5 min to reduce the sample. Analyze the sample by analytical HPLC and ESI-MS.

58| Repeat Step 57 once every hour, until the initial reactants completely convert to the ligation product. pause poInt The ligation reaction can be stirred overnight at room temperature.

59| After the peptide ligation has proceeded for ~5 h, reduce the ligation buffer from Step 56 by adding to it 0.4 ml of 0.1 M TCEP neutral solution and letting the solution incubate for 20 min. Analyze the sample by analytical HPLC and ESI-MS of the objective product to confirm that the starting material has been completely consumed and the ligation system has been sufficiently reduced. crItIcal step The reduction with TCEP is a necessary step before HPLC purification of the ligation product in order to reduce the disulfide by-products.? troublesHootInG

60| Purify the product, -synuclein(168, A30C)-NHNH2, by RP-HPLC according to the conditions described in table 1. Please note that before RP-HPLC, the reduced ligation mixture should be diluted tenfold with deionized H2O to bring down the concentration of the salts and additives so that they do not clog the HPLC lines. Collect the target fractions, freeze the combined product solution with liquid nitrogen and then lyophilize it overnight. A measure of 7.8 mg of purified -synuclein(168, A30C)-NHNH2 will be obtained. The HPLC trace of the second ligation and ESI-MS of purified 5 are shown in Figure 4 and supplementary Data 1.

synthesis of a-synuclein(1106, a30,69c)-nHnH2 by native chemical ligation of peptide hydrazides tIMInG 10 h61| Repeat Steps 51 and 52.

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62| Transfer 7.0 mg of -synuclein(168, A30C)-NHNH2 to a 2-ml Eppendorf reaction tube. Add to it 0.2 ml of 0.2 M phosphate solution containing 6 M GnHCl (pH 3.03.1) prepared in Step 51. Completely dissolve the peptide hydrazide by using a vortex and an ultrasonic cleaning bath. Centrifuge the tube at 7,200g for 1 min at room temperature. Place the reaction tube into the 15 C ice/salt bath (pre-pared in Step 52) and gently agitate the solution by magnetic stirring for 15 min.

63| Mix 4.6 mg of -synuclein(69106, A69C)-NHNH2 and 6.8 mg of MPAA and add 0.2 ml of 0.2 M phosphate solution containing 6 M GnHCl (pH 3.03.1) prepared in Step 51. Adjust the pH to 6.5 with 6 M NaOH and mix it by using a vortex and an ultrasonic cleaning bath until everything is dissolved. Centrifuge the tube at 7,200g for 1 min at room temperature to recover the solution that sticks to the tube wall.

64| To oxidize the peptide hydrazide to the corresponding azide, pipette 20 l of 0.5 M NaNO2 into the solution prepared in Step 62. Gently agitate the solution for 15 min at 15 C in the ice/salt bath.

65| To convert the peptide azide to the thioester, pipette the phosphate buffer prepared in Step 63 into the oxidation solution from Step 64. Remove the tube from the ice/salt bath and let it warm to room temperature. Adjust the pH of the ligation reaction to 6.87.0 with 6 M NaOH.

66| Repeat Steps 5760. 5.2 mg of purified -synuclein(1107, A30,69C)-NHNH2 will be obtained. The HPLC trace of the second ligation and ESI-MS of purified 6 are shown in supplementary Data 2.

synthesis of a-synuclein(1140, a30,69,107c)-oH by native chemical ligation of peptide hydrazides tIMInG 10 h67| Repeat Steps 51 and 52.

68| Weigh 4.3 mg of -synuclein(1106, A30,69C)-NHNH2 in a 2-ml Eppendorf reaction tube. Add to it 80 l of 0.2 M phosphate solution containing 6 M GnHCl (pH 3.03.1) prepared in Step 51. Completely dissolve the peptide hydrazide by using a vortex and an ultrasonic cleaning bath. Centrifuge the tube at 7,200g for 1 min at room temperature. Place the reaction tube in the 15 C ice/salt bath and gently agitate the solution by magnetic stirring for 15 min.

69| Mix 3.2 mg of -synuclein(107140, A107C)-NHNH2 and 2.8 mg of MPAA. Add 80 l of 0.2 M phosphate solution containing 6 M GnHCl (pH 3.03.1) prepared in Step 51. Adjust the pH to 6.5 with 6 M NaOH and mix it by using a vortex and an ultrasound cleaning bath until everything is dissolved. Centrifuge the tube at 7,200g for 1 min at room temperature to recover the solution that sticks to the tube wall.

70| To oxidize the peptide hydrazide to the corresponding azide, pipette 8 l of 0.5 M NaNO2 into the solution prepared in Step 68. Gently agitate the solution for 15 min at 15 C in the ice/salt bath.

71| Convert the peptide azide to the thioester for native chemical ligation. Pipette the buffer prepared in Step 69 into the oxidation solution from Step 70. Remove the tube from the ice/salt bath and let it warm to room temperature. Adjust the pH of the ligation reaction to 6.87.0 with 6 M NaOH.

600 1,000 1,400 m/z

600 1,000 1,400 m/z

4H+755.7

3H+1,007.1

2H+1510.0

4H+786.9

3H+1,049.1

2H+1,572.5

6H+1,164.1

6H+1,396.7

6H+1,745.7

7H+8H+

9H+

10H+11H+

1

1

5

600 1,000 1,400 m/z

1

12

2

5

5

5

a

b

c

d0 5 10 15 20 25 30 time (min)

NaNO2pH 3.0, 15C

-synuclein(129)-NHNH2 + Cys--synuclein(3168, A30C)-NHNH21

-synuclein(129)-N31

-synuclein(129)-MPAA1

MPAA, pH 6.8, rt

Cys--synuclein(168, A30C)-NHNH252

2, MPAA, pH6.8, rt

Figure 4 | RP-HPLC traces for the native chemical ligation of -synuclein(129)-NHNH2 1 with -synuclein(3068, A30C)-NHNH2 2. (a) Oxidation of 1 by NaNO2 for 15 min in pH 3 at 15 C. (b) Native chemical ligation with 2 for 15 min. (c) Native chemical ligation with 2 for 5 h. (d) Purified -synuclein(168, A30C)-NHNH2.

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72| Repeat Steps 5760. 3.1 mg of purified ligation product -synuclein(1140, A30,69,107C)-OH will be obtained. The HPLC trace of the third ligation and ESI-MS of purified 7 are shown in supplementary Data 3.

Free radical peptide desulfurization tIMInG 10 h73| Prepare 10 ml of 0.2 M sodium phosphate neutral buffer (pH 6.97.0) containing 6 M GnHCl, 0.5 ml of 1.0 M TCEP solution and 0.3 ml of 0.1 M VA-044 solution as described in Reagent Setup.

74| Dissolve 2.6 mg of -synuclein(1140, A30,69,107C)-OH in 0.2 ml of phosphate neutral buffer (prepared in Step 73) in a 2-ml Eppendorf reaction tube. Add 0.2 ml of 1.0 M TCEP, 40 l of tBuSH and 20 l of 0.1 M VA-044 solution (prepared in Step 73) to the -synuclein(1140, A30,69,107C)-OH solution. Place the solution on a stirrer at 37 C.! cautIon tBuSH is highly malodorous and it must be handled inside an efficient fume hood.? troublesHootInG

75| Monitor the reaction by analytical RP-HPLC: remove 1 l of the reaction mixture and analyze the sample by analytical HPLC and ESI-MS. Complete the desulfurization reaction within 4 h at 37 C.

76| Purify the product -synuclein by RP-HPLC by using the conditions detailed in table 1. Collect the pure target fractions, freeze the combined product solution with liquid nitrogen and then lyophilize it overnight. 1.8 mg of purified synthetic protein -synuclein (purity 10 C The pH of the ligation step is too high

Perform oxidation at 15 C Adjust pH to 6.87.0 for the ligation

56 Low yield of native chemical ligation of peptide hydrazides

The concentrations of peptide reactants were too low

Increase the initial concentration of the reactant peptides to at least 1 mM for native chemical ligation

Peptide hydrazides have been hydrolyzed, as determined by HPLC and ESI-MS

TCEP was added to the reaction mixture too early, during the first hour of the ligation

Add TCEP to the reaction only after the ligation is complete (as monitored by analytic HPLC)

59 Mass observed equals to peptide +166 Da

Failure to reduce the disulfide bond before HPLC purification

Reduction with TCEP should be performed at pH 6.87.0 for at least 10 min

74 Incomplete desulfurization, as determined by HPLC and ESI-MS

TCEP precipitated because of high pH Deterioration of VA-044 Reaction temperature is too low

Adjust the pH to fully dissolve 1 M TCEP VA-044 should be stored at 28 C away from light and moisture Perform desulfurization at 37 C

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Steps 2333, cleavage of the crude peptide: 4 hSteps 3438, analysis, purification and characterization of the crude peptide: 10 hStep 39, synthesis of -synuclein(3068, A30C)-NHNH2: 65 hStep 40, synthesis of -synuclein(69106, A69C)-NHNH2: 62 hSteps 4150, synthesis of -synuclein(107140, A107C)-OH: 60 hsynthesis of a-synuclein by native chemical ligation of peptide hydrazidesSteps 5160, synthesis of -synuclein(168, A30C)-NHNH2: 10 h plus overnight lyophilizationSteps 6166, synthesis of -synuclein(1106, A30,69C)-NHNH2: 10 h plus overnight lyophilizationSteps 6772, synthesis of -synuclein(1140, A30,69,107C)-OH: 10 h plus overnight lyophilizationSteps 7376, free radical peptide desulfurization: 10 h plus overnight lyophilization

antIcIpateD resultsAll the peptide and peptide hydrazide intermediates can be smoothly prepared by using the Fmoc-SPPS protocol detailed here. These peptides can be readily purified by RP-HPLC (>95% purity) and characterized by ESI-MS (m/z):

peptide[M/6 + H]+

calcd. (exp.)[M/5 + H]+

calcd. (exp.)[M/4 + H]+

calcd. (exp.)[M/3 + H]+

calcd. (exp.)[M/2 + H]+

calcd. (exp.)

1 502.4 (502.4) 602.7 (602.7) 753.1 (753.0) 1,003.8 (1,003.6) 1,505.3 (1,504.7)

2 668.3 (668.4) 801.7 (801.7) 1,001.9 (1,001.8) 1,335.5 (1,335.3) 2,002.8 (2,002.3)

3 625.9 (625.9) 750.8 (750.8) 938.3 (938.2) 1,250.7 (1,250.5)

4 779.2 (779.0) 973.8 (973.6) 1,298.0 (1,297.7) 1,946.5 (1,946.4)

The full-length -synuclein 8 can be successfully synthesized through the N-to-C sequential native chemical ligations of the peptide hydrazides and subsequent desulfurization with a 10.7% overall isolated yield (including four purification steps). As an illustrative example, Figure 4 shows the RP-HPLC traces for the native chemical ligation of peptide hydrazides 1 and 2. Synthetic -synuclein 8 can be characterized by ESI-MS, SDS-PAGE, CD and the ThT fluorescence assay (Fig. 3, supplementary Data 4 and supplementary Figs. 10 and 11).

Note: Any Supplementary Information and Source Data files are available in the online version of the paper.

acknowleDGMents This study was supported by the National Basic Research Program of China (973 program, no. 2013CB932800), the 863 Program of the Ministry of Science and Technology (grant no. 2012AA02A700), the National Natural Science Foundation of China (grants no. 20932006), National Science Fund for Distinguished Young Scholars and the Specialized Research Fund for the Doctoral Program of Higher Education (grant no. 20120002130004). We thank T. Chu and Y. Chen for technical assistance in the biophysical characterization of synthetic -synuclein 8.

autHor contrIbutIons L.L. conceived and led the project. J.-S.Z., S.T., Y.-K.Q. and Z.-P.W. conducted the experiments and co-wrote the manuscript with L.L.

coMpetInG FInancIal Interests The authors declare no competing financial interests.

Reprints and permissions information is available online at http://www.nature.com/reprints/index.html.

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