Synthesis of l-and d-Ubiquitin by One-Pot Ligation and Metal-Free Desulfurization

Article abstract of DOI:10.1002/chem.201600101

Native chemical ligation combined with desulfurization has become a powerful strategy for the chemical synthesis of proteins. Here we describe the use of a new thiol additive, methyl thioglycolate, to accomplish one-pot native chemical ligation and metal-free desulfurization for chemical protein synthesis. This one-pot strategy was used to prepare ubiquitin from two or three peptide segments. Circular dichroism spectroscopy and racemic protein X-ray crystallography confirmed the correct folding of ubiquitin. Our results demonstrate that proteins synthesized chemically by streamlined 9-fluorenylmethoxycarbonyl (Fmoc) solid-phase peptide synthesis coupled with a one-pot ligation-desulfurization strategy can supply useful molecules with sufficient purity for crystallographic studies.

Full text of DOI:10.1002/chem.201600101

DOI: 10.1002/chem.201600101
Full Paper
&
Protein Synthesis
Synthesis of l- and d-Ubiquitin by One-Pot Ligation and Metal-
Free Desulfurization
Yi-Chao Huang +,[a, b] Chen-Chen Chen+,[c] Shuai Gao,[b] Ye-Hai Wang,[a] Hua Xiao,[a]
Feng Wang,*[b] Chang-Lin Tian,*[c] and Yi-Ming Li*[a]
Abstract: Native chemical ligation combined with desulfuri-
zation has become a powerful strategy for the chemical syn-
thesis of proteins. Here we describe the use of a new thiol
additive, methyl thioglycolate, to accomplish one-pot native
chemical ligation and metal-free desulfurization for chemical
protein synthesis. This one-pot strategy was used to prepare
ubiquitin from two or three peptide segments. Circular di-
chroism spectroscopy and racemic protein X-ray crystallogra-
phy confirmed the correct folding of ubiquitin. Our results
demonstrate that proteins synthesized chemically by stream-
lined 9-fluorenylmethoxycarbonyl (Fmoc) solid-phase pep-
tide synthesis coupled with a one-pot ligation–desulfuriza-
tion strategy can supply useful molecules with sufficient
purity for crystallographic studies.
ported by Wan and Danishefsky.[8] This metal-free desulfuriza-
tion (MFD) reaction occurs in a buffered solution that contains
the sulfur scavenger tris(2-carboxyethyl) phosphine (TCEP), rad-
ical initiator VA-044, and a hydrogen-donor compound, for ex-
ample, an alkyl thiol species such as tBuSH,[8] glutathione,[6b] or
sodium 2-mercaptoethanethiolate (MESNa).[9] This powerful
stepwise ligation–desulfurization protocol has been widely em-
ployed by the chemical protein synthesis community to pro-
duce several challenging protein targets.[10]
Introduction
Both the total and semi-synthesis of proteins can produce ho-
mogeneous macromolecules with well-defined structures on
the atomic level to pinpoint specific functions of target pro-
teins.[1] Native chemical ligation (NCL) has become one of the
leading methodologies in the field of chemical protein synthe-
sis over the past two decades.[2] NCL entails a mild chemose-
lective Xaa–Cys (Xaa = any amino acid) amide-bond-forming
reaction between a C-terminal Xaa thioester and an N-terminal
Cys moiety in a neutral buffer at room temperature.[3] In 2001,
Yan and Dawson extended the permitted ligation site of NCL
from Xaa-Cys to Xaa-Ala by using a metal-assisted reductive
desulfurization strategy.[4] Since then this ligation–desulfuriza-
tion approach[5] has been expanded through the use of N-ter-
minal residues that contain 1,2-aminothiol/selenol[6] or 1,3-ami-
nothiol[7] units. Another important advancement, a highly effi-
cient homogeneous radical desulfurization reaction, was re-
To streamline sequential NCL and MFD reactions, a straight-
forward one-pot operation would be highly desirable to save
time and labor costs (Figure 1A). Unfortunately, the radical
MFD process can be quenched by the aryl thiol catalyst[11,12]
used for NCL, normally the gold-standard thiol additive 4-mer-
captophenylacetic acid (MPAA, pKa =6.6).[11] Recently several
methods, including ether extraction of the aryl thiol[7f] and
solid-support capture of the aryl thiol[13] or ligation product,[14]
have been used to separate MPAA from the one-pot reaction
mixture. Another approach towards a one-pot NCL-MFD reac-
tion is to use a catalytically potent alkyl thiol to replace MPAA.
For example, alkyl thiols MESNa (pKa =9.2),[9] 2,2,2-trifluoroetha-
nethiol (TFET, pKa =7.3),[15] and mercaptopropionylcysteine[16]
have been successfully used to assist NCL. Among them, TFET
was found to be as effective as MPAA and enabled one-pot
NCL-MFD reactions to proceed in a satisfactory manner.[15] Al-
though this thiol additive has been used in several reported
cases,[6l,m,17] TFET is a malodorous compound, which might
limit its usage on a larger synthetic scale.
[a] Y.-C. Huang ,+ Y.-H. Wang, Prof. H. Xiao, Prof. Y.-M. Li
School of Medical Engineering
Hefei University of Technology, Hefei, 230009 (P. R. China)
and
State Key Laboratory of Medicinal Chemical Biology
NanKai University, 94 Weijin Road, Tianjin 300071 (P. R. China)
E-mail: lym2007@mail.ustc.edu.cn
ymli@hfut.edu.cn
[b] Y.-C. Huang ,+ S. Gao, Prof. F. Wang
Department of Chemistry, School of Life Sciences
Tsinghua University, Beijing, 100084 (P. R. China)
E-mail: wfeng@mail.tsinghua.edu.cn
Here we describe a one-pot ligation–desulfurization strategy
assisted by a new thiol additive, methyl thioglycolate (MTG,
pKa =7.9)[18] (Figure 1B). This strategy has four notable fea-
tures: 1) MTG possesses favorable reaction kinetics that are
comparable to MPAA (ꢀ1.8-fold difference), 2) MTG (Sigma Al-
drich, USD $78.8/500 g) is a low-cost reagent, 3) MTG is more
polar and has a lower extinction coefficient at l=214 nm than
[c] C.-C. Chen,+ Prof. C.-L. Tian
High Magnetic Field Laboratory
Chinese Academy of Sciences, Hefei, 230026 (P. R. China)
E-mail: cltian@ustc.edu.cn
[+] These authors contributed equally to this work.
Supporting information for this article is available on the WWW under
http://dx.doi.org/10.1002/chem.201600101.
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reactions (see Figures S1–S4 in the Supporting Information). All
reactions were conducted between the model peptide
1 (2 mm), which had an N-terminal Cys residue, and model
peptides 2–5 (3 mm), which had a C-terminal thioester, in
aqueous guanidine hydrochloride (GnHCl; 6m) and Na2HPO4
(100 mm) buffer (pH 6.5) that contained MPAA or MTG
(100 mm). Peptide MESNa thioesters were prepared by oxida-
tion–thioesterification[19] of peptide hydrazides (see Scheme S1
in the Supporting Information) obtained by Fmoc SPPS.[20] To
our delight, MTG performed almost equally as well as MPAA
for ligations at sterically less-hindered residues (Gly, Ser, and
Leu). All the reactions essentially finished within 6 h for both
MTG and MPAA (see Figure S5 in the Supporting Information).
In the case of kinetically less-favored ligation sites, such as Val,
MTG was less efficient yet comparable to MPAA (ꢀ1.8-fold ki-
netic difference). Reaction of the Val-terminated thioester 5 at
378C overnight completely consumed 1 in the presence of
MPAA or MTG (see Figure S5 in the Supporting Information).
Having demonstrated the satisfactory reaction kinetics of
MTG-catalyzed NCL reactions, we tested the one-pot ligation–
desulfurization process. First, ligations between N-terminal Cys
peptide 1 (2 mm) and a slight excess of peptide thioesters 6–9
(2.2 mm) were conducted. HPLC analysis confirmed almost
quantitative conversion for all of the ligation steps (see Figur-
es S6–S9 in the Supporting Information). Afterwards, the reac-
tion solution was mixed with an equal volume of GnHCl (6m)
and Na2HPO4 (100 mm) desulfurization buffer (pH 6.5) that con-
tained TCEP (500 mm) and MESNa (200 mm). VA-044 (50 mm)
was then added as the radical initiator. The desulfurization re-
action (pH 6.5) was incubated at 408C overnight to ensure
complete transformation (Table 1). The peptide products were
purified by semipreparative HPLC in good isolated yields (44–
69%).
Figure 1. A) Native chemical ligation (NCL) and metal-free desulfurization
(MFD); B) comparison of new thiol additive MTG with the gold-standard NCL
catalyst MPAA and one-pot ligation–desulfurization additive TFET.
MPAA (it normally shows a small peak at the front of the HPLC
profile (see Figures 4 and 5 below), whereas MPAA exhibits
a broad peak[15] that interferes when MPAA coelutes with the
reaction intermediates),[12a,15] and 4) MTG (b.p. 1528C) is less
volatile and malodorous than TFET (b.p. 378C) and has no neg-
ative effect on the desulfurization. To demonstrate the efficien-
cy and robustness of our protocol, we synthesized monoubi-
quitin, a well-known model protein, in a one-pot ligation–de-
sulfurization process on a 30 mg scale. The chemically synthe-
sized monoubiquitin was characterized by high-resolution rac-
emic X-ray crystallography. This synthetic ubiquitin
demonstrates that both tert-butoxycarbonyl solid-phase pep-
tide synthesis (Boc-SPPS)[21] and 9-fluorenylmethoxycarbonyl
(Fmoc) SPPS combined with an NCL-MFD strategy can furnish
proteins with sufficient homogeneity for X-ray crystallographic
structural biology studies.
Table 1. One-pot ligation–desulfurization of MTG thioester peptides 6–9
and an N-Cys peptide.
Entry
Peptide thioester
Ligation
time [h]
HPLC
conversion [%][a]
Isolated
yield [%][b]
Results and Discussion
Our initial aim was to find an alkyl thiol that might become
a useful alternative to TFET. We reasoned that the ideal thiol
catalyst should: 1) be a good leaving group, 2) have good solu-
bility in aqueous buffer, and 3) show good elution behavior for
HPLC analysis (no significant peak overlapping). Because MTG
is cheap and has a lower pKa (7.9) than most alkyl thiols, we
decided to assess MTG as the thiol additive for standard NCL
1
2
3
4
6, Ac-LYRANG-MTG
7, Ac-LYRANS-MTG
8, Ac-LYRANL-MTG
9, Ac-LYRANV-MTG
1.0
4.0
4.0
8.0
>95
>95
>95
90
66
69
67
44
[a] Conversion estimated by integration of the UV trace. [b] The isolated
yield after one-pot NCL-MFD was determined by the weight of the
lyophilized product.
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Next we used the MTG-assisted one-pot ligation–desulfuriza-
tion sequence to synthesize monoubiquitin, an important
model protein that consists of 76 amino acid residues. Ubiqui-
tin has been successfully synthesized by three-segment liga-
tion,[21] two-segment ligation,[22] and direct Fmoc SPPS.[23] To
test the robustness of our one-pot protocol, we adopted
a three-segment (1–27+28–45+46–76) approach to assemble
full-length ubiquitin. All of the thioester and non-thioester
peptides were prepared by air-bath heated Fmoc SPPS.[24]
Met1 was replaced by an isosteric norleucine (Nle) residue to
prevent methionine oxidation.[10a] Ala28 and Ala46 were mutat-
ed to Cys during Fmoc SPPS and were converted back to Ala
after desulfurization. For the racemization-prone dipeptide
motif Asp52–Gly53,[25] a Dmb (2,4-dimethoxybenzyl) or Hmb
(2-hydroxy-4-methoxybenzyl) backbone protecting group was
introduced. In our first method, Fmoc-(Dmb)Gly-OH and Fmoc-
Asp(OtBu)-OH were coupled as usual (Figure 2A). In another
Figure 3. A new protecting group Tfa-Thz undergoes mild two-step depro-
tection to release Thz and N-terminal Cys.
groups have been reported to replace Thz, some of them are
unstable to TCEP (a routine reductant in NCL),[28,29] whereas
others require long multistep syntheses.[30,31] Luckily, we no-
ticed that trifluoroacetyl (Tfa) protected primary or secondary
amines could be mildly deprotected by treatment with aque-
ous base.[32] Therefore, Tfa-Thz (Figure 3) could replace Thz
during the hydrazide oxidation.
First we synthesized Tfa-Thz-OH (3 g scale) in one step (see
Scheme S2 in the Supporting Information). Tfa-Thz-OH was
then incorporated into the middle peptide segment UbM (Tfa-
Thz28~Phe45-NHNH2) by N,N’-diisopropylcarbodiimide (DIC)/
Oxyma coupling.[33] Next, we prepared the N-terminal segment
UbN (H-Nle1~Lys27-NHNH2) and the C-terminal segment UbC
(H-Cys46~Gly76-NH2). The UbN and UbM hydrazides were
transformed into their respective MTG thioesters then purified.
In the first step of the one-pot three-segment ligation, UbM
MTG thioester (2.1 mm) was incubated with the UbC fragment
(2.0 mm) in GnHCl (6m) and Na2HPO4 (100 mm) ligation buffer
(pH 6.5) that contained MTG (20 mm) and TCEP (30 mm). After
4 h, the first ligation was complete and partial Tfa deprotection
was observed by analytical HPLC. Clean Tfa deprotection
(pH 10.0, 30 min) followed by Thz deprotection (pH 4.0, 2 h)
was performed by addition of methyoxyamine (0.2m) to give
the UbM+C peptide. The second ligation was initiated at
pH 6.5 by the addition of the UbN MTG thioester (2.2 mm) and
pH re-adjustment. After 16 h, an equal volume of desulfuriza-
tion buffer that contained TCEP (500 mm), MESNa (200 mm),
and VA-044 (40 mm) was poured into the ligation buffer (final
pH=6.5). The reaction mixture was incubated at 408C for 12 h.
HPLC and ESI-MS analysis indicated complete reaction to
afford full-length ubiquitin (Figure 4). However, in our hands,
semipreparative HPLC purification was complicated by overlap
of the UbN hydrolysis peak (ꢀ10% based on the peak-area
ratio from the analytical HPLC data) with the product peak.
Therefore, unfortunately this three-segment assembly strategy
had some practical difficulties at the purification stage.
Figure 2. The racemization-prone Asp–Gly dipeptide motif was incorporated
into the peptide chain with A) Dmb or B) Hmb backbone protection.
method, Fmoc-Gly-OH was coupled to the resin, then the Hmb
group was introduced by reductive amination of the depro-
tected amine with 2-hydroxy-4-methoxybenzaldehyde. Fmoc-
Asp(OtBu)-OH was then coupled onto the phenol hydroxyl
substituent of Hmb and transferred to the secondary amine by
an on-resin six-membered-ring acyl shift (Figure 2B). Both
methods worked well to overcome the formation of asparti-
mide[25] during Fmoc SPPS according to LC-MS analysis.
For the three-segment iterative NCL-MFD approach, the key
operation was the release of protected N-terminal Cys in the
middle segment. Thz (Figure 3), the most-popular N-terminal
Cys protecting group, comes from the commercially available
Boc- or Fmoc-protected 1,3-thiazolidine-4-carboxylic acid.[26]
Unfortunately, we and others found that Thz was unstable
under the hydrazide oxidation conditions, and Pentelute’s
group reported that an unproductive nitric oxide adduct
might form.[27] Although several N-terminal Cys protecting
To overcome this problem, we tested a two-segment ap-
proach with UbN+M hydrazide (H-Nle1~Phe45-NHNH2) and
UbC (H-Cys46~Gly76-NH2) as the starting materials. The
UbN+M hydrazide was first converted to the UbN+M MTG
thioester. The purified thioester (1.0 mm) was then treated
with UbC (1.0 mm) in the ligation buffer. After 12 h the reac-
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Figure 5. A) A one-pot two-segment ligation–desulfurization approach to
ubiquitin (RSH = MTG); B) monitoring the reaction by HLPC.
Figure 4. A) A one-pot three-segment ligation–desulfurization approach to
ubiquitin (RSH = MTG); B) monitoring the reaction by HPLC.
tion mixture was subjected to the desulfurization protocol de-
scribed above (Figure 5). Semipreparative HPLC purification af-
forded full-length ubiquitin in an excellent isolated yield of
85% (30 mg scale).
By using the same one-pot NCL-MFD protocol, we complet-
ed the one-pot total synthesis of d-ubiquitin. Synthetic l- and
d-ubiquitin were both characterized by HPLC and direct-injec-
tion ESI-MS (Figure 6A). Spontaneous folding was conducted
by directly dissolving lyophilized ubiquitin in aqueous buffer or
double-distilled water (ddH2O). The circular dichroism (CD)
spectra of l- and d-ubiquitin were compared to that of ex-
pressed l-ubiquitin and confirmed the well-folded secondary
structure in ddH2O (0.8 mgmLÀ1) (Figure 6B). To further charac-
terize the homogeneity of our synthetic products, we em-
ployed racemic X-ray crystallography, which was pioneered by
Yeates and Kent.[34] Lyophilized l- and d-ubiquitin were both
dissolved in Tris buffer (10 mm; pH 8.0) to obtain the protein
solution (8 mgmLÀ1) as determined by UV A280 (Nanodrop).
Crystallization trials with l- or d-ubiquitin alone were unsuc-
cessful at low protein concentration (<20 mgmLÀ1), with or
without CdCl2 as a precipitating additive, for one month.[21]
This was consistent with previous observations that direct crys-
tallization of monoubiquitin was difficult without seeding.[21]
Crystals were obtained overnight at 188C under almost half of
the conditions when l- and d-ubiquitin were mixed and under-
went crystallization screening with commercially available
Hampton Research PEG/Ion Screen in the presence of CdCl2.
Figure 6. A) Characterization of the synthetic ubiquitins by analytical HPLC
and direct-injection ESI-MS; B) CD spectra of the synthetic and expressed
ubiquitins; C) representation of racemic l-ubiquitin and d-ubiqutin (see the
Supporting Information (Figure S46) for a colored version of the image).
Diffraction-quality single crystals could be reproducibly gath-
ered by mixing the racemic protein solution (1 mL; l-ubiquitin
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(4 mgmLÀ1)/d-ubiquitin (4 mgmLÀ1)/Tris buffer (10 mm)/CdCl2
(20 mm); pH 8.0) and well solution (1 mL; lithium acetate dihy-
drate (0.2m)/polyethylene glycol 3350 (20% w/v); pH 7.9). X-
ray diffraction data were collected on the Tsinghua Protein X-
ray Crystallography Platform. The structure of synthetic ubiqui-
tin was solved to 1.95 Š by using a molecular replacement
method with the structure of extracted ubiquitin (PDB:1UBQ)
as the search model. As anticipated, the synthetic ubiquitin
had the same molecular shape as previously reported, which
includes an a-helix and five b-strands (Figure 6C).[35]
was characterized by analytical HPLC and ESI-MS. The peptide was
purified, if necessary, by semipreparative HPLC then lyophilized.
One-pot ligation-desulfurization protocol
Ac-LYRANX-MTG (2.2 mm; X=G, S, L, V) and H-CSPGY-NH2 (2 mm)
were dissolved in GnHCl (6m) and Na2HPO4 ligation buffer
(100 mm) that contained MTG (20 mm) and TCEP (30 mm). The
mixture was adjusted to pH 6.5 and incubated at RT. When the li-
gation was complete, the reaction mixture was combined with an
equal volume of ligation buffer that contained TCEP (500 mm) and
MESNa (200 mm). The mixture was adjusted to pH 6.5. After blend-
ing VA-044 (50 mm) was added to the mixture as radical initiator.
The desulfurization reaction was incubated at 408C overnight to
ensure complete transformation. The desulfurization products were
purified by semipreparative HPLC then lyophilized.
Conclusion
We have developed an efficient strategy for protein synthesis
that relies on one-pot NCL-MFD. Key to this technical advance
is the use of MTG as the thiol additive for the ligation process.
MTG is easily accessible and displays sufficient reactivity. Our
streamlined synthesis of ubiquitin (85% isolated yield, 30mg
scale) demonstrates that this strategy has the potential to effi-
ciently generate large-scale high-quality proteins. Racemic X-
ray crystallography was employed to characterize the synthetic
ubiquitin. We hope that the utility of this one-pot NCL-MFD
approach can be demonstrated in the chemical synthesis of
post-translational modified proteins[30,36] (e.g., glycosylated pro-
teins,[37] lipidated proteins,[38] ubiquitinated proteins,[39] and his-
tone derivatives)[40] in future studies.
Acknowledgements
This work was supported by the NSFC (Grant numbers
21372058, 21572043, and 21302034) and the State Key Labora-
tory of Medicinal Chemical Biology.
Keywords: desulfurization
chemical ligation · proteins · X-ray diffraction
· methyl thioglycolate · native
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Experimental Section
Air-bath heated SPPS
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Screw-cap glass SPPS reaction vessels were obtained from com-
mercial sources. The SPPS glassware was immobilized on a rotary
shaker (130 rpm) during the reaction.
Fmoc-protected Rink amide AM or hydrazide resins were swollen
in 1:1 CH2Cl2/DMF for 10 min before use.
Fmoc deprotection was conducted by treatment with 20% piperi-
dine in DMF (containing 0.1m Oxyma) twice (1 min + 9 min). The
resin was washed with DMF (”5), CH2Cl2 (”5), and then DMF (”5).
Arg coupling was initiated by pouring a preactivated (30 s at RT)
solution of Fmoc-Arg(Pbf)-OH (0.1m in DMF; 4 equiv), O-(6-chloro-
benzotriazol-1-yl)-N,N,N’,N’-tetramethyluronium
hexafluorophos-
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phate (HCTU; 3.6 equiv), and N,N-diisopropylethylamine (DIEA;
8 equiv) onto the resin. The reaction mixture was kept at RT for
45 min. The coupling of amino acids other than Arg was initiated
by pouring a preactivated (5 min at RT) solution of protected
amino acid (0.1m in DMF; 4 equiv) and N,N’-diisopropylcarbodii-
mide (DIC; 4 equiv) in Oxyma (4 equiv) onto the resin. The His(Trt)
coupling was kept at RT for 45 min. The Cys(Trt) coupling was kept
at 508C (air-bath heating) for 20 min. Other residue couplings were
kept at 758C (air-bath heating) for 20 min.
At the end of the SPPS, the peptide was cleaved from the resin
with a modified Reagent K: H2O (5% v/v), thioanisole (5% v/v), and
1,2-ethanedithiol (2.5% v/v) in trifluoroacetic acid (TFA). The com-
bined TFA eluents were concentrated under a flow of nitrogen gas.
The crude peptides were obtained by precipitation with ice-cold
ether and centrifugation (5000 rpm, 48C, 2 min). The peptide pellet
was dissolved in 1:1 CH3CN/H2O that contained TFA (0.1%) and
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Received: January 10, 2016
Published online on April 14, 2016
Chem. Eur. J. 2016, 22, 7623 – 7628
www.chemeurj.org
7628
ꢀ 2016 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim