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protocol

Chemical synthesis of proteins using peptide


hydrazides as thioester surrogates
Ji-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

2013 Nature America, Inc. All rights reserved.

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.

INTRODUCTION
Chemical synthesis of proteins supports biomedical research chemical ligation without separation of any intermediates. It is
through the production of proteins (e.g., post-translationally important to note that peptide hydrazides with post-translational
modified proteins, circular proteins, mirror-image proteins) modifications can be readily prepared through the standard Fmoc
that cannot be easily obtained via recombinant technology13. chemistry either manually or by using commercially available
The most successful strategy for protein chemical synthesis has
automated synthesizers. Furthermore, peptide hydrazides are not
proven to be the condensation of unprotected peptides in aque- reactive in native chemical ligation unless they are activated by
ous buffers by a chemoselective reaction, namely native chemical NaNO2, and thus they can be used as masked thioesters in the
ligation, which was developed by Kent and co-workers4,5. convergent synthesis of proteins20.
This method relies on the use of peptide thioesters as key
By using the hydrazide-based native chemical ligation method,
intermediates, which can be successfully prepared through Boc we and others have successfully synthesized a number of proteins
solid-phase peptide synthesis (SPPS)6. Nonetheless, the Boc such as the ribosomal protein S25, backbone-cyclized cyclotides
method is usually not suitable for making protein segments and ubiquitinated histone H2B2124. In these studies, the synthetic
carrying acid-sensitive modifications (e.g., phosphoryl and glyco- strategy usually involves two key steps: first, Fmoc-SPPS of pepsyl groups). Furthermore, the use of highly corrosive HF (hydro- tide hydrazides by using the 2-Cl-(Trt)-NHNH2 resin; second,
gen fluoride) for global deprotection in the Boc method can be the native chemical ligation of peptide hydrazides through
an operational hazard79.
in situ NaNO2 activation and thiolysis. Here we detail the proceTo overcome the above problems, extensive studies have dure for the use of peptide hydrazides to synthesize -synuclein,
been carried out to develop methods for synthesizing peptide
a protein with an important role in the development of
thioesters or their equivalents through the
Fmoc-SPPS1018. In our recent work, we
found that peptide hydrazides constitute
a
5% NH2NH2/DMF
good thioester surrogates for the native
H2N-NH
Cl
19,20
chemical ligation (Fig. 1)
. Through
2-Cl-(Trt)-Cl resin
2-Cl-(Trt)-NHNH2 resin
an operationally simple NaNO2 activation and thiolysis process (usually via
4-mercaptophenylacetic acid (MPAA) 5,
b
Fmoc-Aa1-OH (4 eq)
O
HCTU (3.8 eq)
O
introduced as an external thiol at a ~100 mM
DIPEA (8 eq)
20% piperidine/DMF
HN-NH
H2N-NH
concentration), a peptide hydrazide can
HN-NH
Fmoc-Aa1
Aa1
DMF
be cleanly and rapidly converted to a
Fmoc-Aa2-OH (4 eq)
peptide thioester in an epimerizationO
O
-Peptide elongation
HCTU (3.8 eq)
-Final Fmoc deprotion
DIPEA (8 eq)
free manner and then used in the native
HN-NH
Aa -Aa ...-Aa -Aa
Fmoc-Aa -Aa
HN-NH
2

DMF

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.

c
NHNH2

MPAA (100 eq), pH 6.87.0


In situ thiolysis

-Cleavage by TFA

NaNO2 (10 eq), pH 3.0, 15 C, 1520 min


0.2 M phosphate buffer containing 6 M GnHCl

O
Peptide

Cys

O
Peptide

SR

O
Peptide

Activation

n-1

Peptide hydrazides

N3

Peptide
(1 eq)
Ligation

Peptide Cys

Peptide

nature protocols | VOL.8 NO.12 | 2013 | 2483

protocol
Cys - 3167 -Gly-NHNH2
2

Cys - 70105 -Gly-NHNH2


3

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 residues 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] dihydrochloride), 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 characterized 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.

Cys - 108140 -OH


4

NaNO2, 15 C, pH 3.0
(First ligation)
MPAA, rt, pH 6.8, 5 h
(56% isolated yield)
H- 128 -Ala-Cys- 3167 -Gly-NHNH2
5
NaNO2, 15 C, pH 3.0, 15 min
(Second ligation)
(49% isolated yield) MPAA, rt, pH 6.8, 4 h
H- 128 -Ala-Cys- 3167 -Gly-Cys- 70105 -Gly-NHNH2
6
NaNO2, 15 C, pH 3.0, 15 min
(Third ligation)
MPAA, rt, pH 6.8, 4 h
(54% isolated yield)
H- 128 -Ala-Cys- 3167 -Gly-Cys- 70105 -Gly-Cys - 108140 -OH
7
(Desulfurization) TCEP HCl, VA-044,
(72% isolated yield) tBu-SH, 37 C, 4 h
H- 128 -Ala-Ala- 3167 - Gly-Ala- 70105 -Gly-Ala - 108140 -OH

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.

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 design
From 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 prepare 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 synthesized through standard Fmoc-SPPS with
a
good isolated yields (18%, 21%, 30% and

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.
2484 | VOL.8 NO.12 | 2013 | nature protocols

Synthetic -synuclein

15+ 14+
964.9 1033.7
16+
13+
904.7
1,113.2 Synthetic -synuclein
M.W.14,459.99
17+
851.5
12+
18+
1,205.9
804.3
19+
762.0
20+
723.9
21+
689.5

10

(kDa)
170
130
95
72
55
43
34
26
17
10

15

20

25

600

30

d
-synuclein 8

11+
1,315.3
10+
1,446.7

800

1,000

1,400

1,600
m/z

1,000

0
2
4
6
8
10
12
190

1,200

9+
1,607.4

Thioflavin T assays of
-synuclein

1,200

2
Mean ellipticity (mdeg)

2013 Nature America, Inc. All rights reserved.

MDVFMKGLSK10AKEGVVAAAE20 KTKQGVAEA A30GKTKEGVLYV40GSKTKEGV


VH 50 GVATVAEKTK60 EQVTNVGGA V70VTGVTAVAQK80 TVEGAGSIAA90 ATGFVK
KDQL100 GKNEEGAPQE110 GILEDMPVDP120 DNEAYEMPSE130 EGYQDYEPEA140

ThT fluorescence

H - 128 -Ala-NHNH2
1

800
600
400
200

200

210 220 230 240


Wavelength (nm)

250

260

72 120 240 300 360 480


Time (h)

protocol
Protein aggregation can be determined by monitoring the intensity 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 reaction, 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.

2013 Nature America, Inc. All rights reserved.

MATERIALS
REAGENTS
! 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-GlyOH, 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-OH
2-Cl-(Trt)-Cl resin (Tianjin Nankai HECHENG S&T, loading: 0.56 mmol g1)
1-[Bis(dimethylamino)methylene]-1H-benzotriazoliumhexafluorophosphate 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 water
Guanidine 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)
EQUIPMENT
Analytical balance (AL 104; Mettler Toledo)
Polypropylene dropper, 4 ml
Set 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 lids
Magnetic stirrer (e.g., Synthware, cat. no. S360205)
Magnetic stirring apparatus (e.g., Tokyo Rikakikai, EYEL4: RCH-20L)
Thermostat water bath
Thermostat oil bath
Capillary (e.g., Synthware, cat. no. P230001)
Kaiser test micro tubes
Heating and stirring mantles/heater
Water ring vacuum pump
Oil 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 system
Centrifuges (e.g., Eppendorf: microcentrifuge 5418/5418R; centrifuge
5810/5810R)
Vortex mixer
Analytical and semipreparative HPLC system (Shimadzu, SPD-20A UV/VIS
detector; LC-20AT prominence)
HPLC vials
Lyophilizer (e.g., Tokyo Rikakikai, EYEL4: M-1000)
Plastic syringe, 1, 2.5, 5 ml
LC syringe (e.g., Thermo Scientific: 25, 100 and 500 l)
Microporous membrane filter (organic system, 13 mm or 50 mm 0.22 m)
UV detector
CD spectrometer
Electrospray ionizationmass spectrometry (ESI-MS) instrument
Thermometer, 20~110 C (e.g., Synthware, cat. no. T167611)
pH meter (DELTA 320; Mettler Toledo)
Liquid nitrogen container
Rubber hose
Rubber plugs
Measuring cylinder
Ultrasonic oscillators (SB-5200D, Ningbo Scientz Biotechnology)
Conical flask (Synthware: 100 ml, cat. no. F304100; 250 ml, cat. no. F304250;
500 ml, cat. no. F304500)

nature protocols | VOL.8 NO.12 | 2013 | 2485

2013 Nature America, Inc. All rights reserved.

protocol
REAGENT SETUP
Piperidine 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 SETUP
Manual 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 reactions (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

2486 | VOL.8 NO.12 | 2013 | nature protocols

protocol
PROCEDURE
Preparation 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.

2013 Nature America, Inc. All rights reserved.

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.
PROCEDURE
1. 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).

nature protocols | VOL.8 NO.12 | 2013 | 2487

2013 Nature America, Inc. All rights reserved.

protocol
Synthesis of Fmoc-protected a-synuclein(129)-NHNH2 TIMING 6090 min per amino acid
15| 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 HATU
HATU 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.
2488 | VOL.8 NO.12 | 2013 | nature protocols

protocol
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 acid
17| 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.

2013 Nature America, Inc. All rights reserved.

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 h
23| 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 h
34| 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.

nature protocols | VOL.8 NO.12 | 2013 | 2489

protocol
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.

2013 Nature America, Inc. All rights reserved.

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 h
39| 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 h
40| 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 h
41| 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 h
51| 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.
2490 | VOL.8 NO.12 | 2013 | nature protocols

protocol
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.

2013 Nature America, Inc. All rights reserved.

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 h
61| Repeat Steps 51 and 52.
nature protocols | VOL.8 NO.12 | 2013 | 2491

protocol
-synuclein(129)-NHNH2 + Cys--synuclein(3168, A30C)-NHNH2
1
2

2013 Nature America, Inc. All rights reserved.

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.

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 (prepared in Step 52) and gently agitate the
solution by magnetic stirring for 15 min.

NaNO2
pH 3.0, 15C
MPAA, pH 6.8, rt
-synuclein(129)-N3
1

Cys--synuclein(168, A30C)-NHNH2
5

2, MPAA, pH6.8, rt
-synuclein(129)-MPAA
1

1
4H+
755.7

3H+
1,007.1

1
+

2H
1510.0

a
5
2

600

1,000
3H+
4H+ 1,049.1
786.9

2H
1,572.5
600

1,400 m/z
6H+
1,164.1
8H 7H+
+
5
6H
1,396.7
9H+
+
10H
6H+
11H+
1,745.7

10

15

20

1,000
+

d
0

1
+

1,400 m/z

25

30 time (min)
600

1,000

1,400 m/z

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 h
67| 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.
2492 | VOL.8 NO.12 | 2013 | nature protocols

protocol
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 h
73| 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

2013 Nature America, Inc. All rights reserved.

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 <95%) should be obtained. The HPLC trace of desulfurization and ESI-MS of purified -synuclein 8
are shown in Supplementary Data 4. The overall yield for three ligations and desulfurization is 10.7%.
? TROUBLESHOOTING
Troubleshooting advice can be found in Table 2.
Table 2 | Troubleshooting table.
Step

Problem

Possible reason

Solution

Low yield of peptide hydrazides

The concentration of NH2NH2 is too low


in the preparation of 2-Cl-(Trt)-NHNH2 resin
The 2-Cl-(Trt)-NHNH2 resin has been stored
too long

Use 5% (vol/vol) NH2NH2 in DMF to


prepare the 2-Cl-(Trt)-NHNH2 resin
Prepare the 2-Cl-(Trt)-NHNH2 resin just
before use

55

Peptide hydrazides have been


reduced, as determined by HPLC
and ESI-MS

The temperature at which the oxidation was


carried out was >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

TIMING
Synthesis of peptide hydrazides
Steps 114, preparation of 2-Cl-(Trt)-NHNH2 resin: 2 h
Steps 1516, synthesis of Fmoc-protected -synuclein(129)-NHNH2: 6090 min per amino acid
Steps 1722, peptide deprotection: 25 min per amino acid
nature protocols | VOL.8 NO.12 | 2013 | 2493

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

2013 Nature America, Inc. All rights reserved.

ANTICIPATED RESULTS
All 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.)

502.4 (502.4)

602.7 (602.7)

753.1 (753.0)

1,003.8 (1,003.6)

1,505.3 (1,504.7)

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)

625.9 (625.9)

750.8 (750.8)

938.3 (938.2)

1,250.7 (1,250.5)

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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