Chemical synthesis of UB-AMC via ligation of peptide hydrazides

Article abstract of DOI:10.1007/s11426-013-4885-x

The C-terminal conjugate of ubiquitin with 7-amino-4-methylcoumarin (Ub-AMC) is an important probe for fluorescence-based analysis of deubiquitinating enzyme (DUB) activity. It is important to develop more efficient methods for the preparation of Ub-AMC b

Full text of DOI:10.1007/s11426-013-4885-x

SCIENCE CHINA
Chemistry
• ARTICLES •
doi: 10.1007/s11426-013-4885-x
doi: 10.1007/s11426-013-4885-x
Chemical synthesis of Ub-AMC via ligation of peptide hydrazides
LIANG Jun1, 3, FANG GeMin1, HUANG XiuLiang2, MEI ZiQing2, 3, LI Juan3,
TIAN ChangLin1*, LIU Lei3*
1High Magnetic Field Laboratory, Chinese Academy of Sciences; University of Science and Technology of China,
Hefei 230026, China
2Ministry of Education Protein Science Laboratory, Center for Structural Biology, School of Life Sciences, Tsinghua University, Beijing
100084, China
3Tsinghua-Peking Center for Life Sciences, Department of Chemistry, Tsinghua University, Beijing 100084, China
Received March 10, 2013; accepted April 4, 2013
The C-terminal conjugate of ubiquitin with 7-amino-4-methylcoumarin (Ub-AMC) is an important probe for fluorescence-
based analysis of deubiquitinating enzyme (DUB) activity. It is important to develop more efficient methods for the prepara-
tion of Ub-AMC because the currently available technology is still expensive for scaled-up production. In the present work we
report an efficient strategy for total chemical synthesis of Ub-AMC through ligation of peptide hydrazides. Three peptide seg-
ments are assembled via N-to-C sequential ligation and the resulting product is converted to Ub-AMC via TCEP-mediated
desulfurization. The synthetic Ub-AMC is shown to have expected biological functions through the measurement of its DUB
activity.
ligation of peptide hydrazides, peptide segment condensation, deubiquitinating enzymes, ubiquitin
a thiol-exchange reaction. In the final step, the E3 ligase
1 Introduction
mediates the transfer of Ub to the Lys side chain of the tar-
get protein. Note that the last step necessitates the help of
RING-domain E3 family which brings the Ub thioester-E2
and the ε-amino group of a Lys residue close together to
form an isopeptide bond regio-specifically.
Ubiquitination is one of the most common protein
post-translational modifications. It plays an important role
in cell biology, because ubiquitination not only can direct
the location and degradation of the modified protein, but
also may facilitate the transcription and repair of DNA
[15]. As the key unit of ubiquitination, Ub is a highly con-
served small protein of 76 amino acid residues. Ubiquitina-
tion occurs through the attachment of Ub’s C-terminus to
the lysine residue (or less commonly, the N-terminus) of the
target protein. This process is mediated by three enzymes
named E1, E2 and E3. In the beginning of the process, the
C-terminus of Ub is activated with the assistance of E1 and
ATP to form an Ub-thioester intermediate. Subsequently,
the conjugating E2 enzyme covalently binds to Ub through
The detail process of ubiquitination is fairly complex
because seven lysine residues in Ub (i.e. K6, K11, K27,
K29, K33, K48, K63) can be used to link the consecutive
Ub molecules. As a result, the attachment of Ub to a target
protein may involve a chain of Ubs with various linkage
types and different lengths. Previous studies have mostly
focused on Lys48- and Lys63-linked Ub chains due to the
poor availability of the E2/E3 enzymes systems for prepara-
tion of other Ub linkage types [6]. Moreover, the identifica-
tion, isolation, and characterization of E2/E3 enzymes for a
specific ubiquitination target remain extraordinarily tedious
even with the most advanced technologies. Therefore, it is
important to develop chemical approaches to prepare Ub
*Corresponding authors (email: cltian@ustc.edu.cn; lliu@mail.tsinghua.edu.cn)
© Science China Press and Springer-Verlag Berlin Heidelberg 2013
chem.scichina.com www.springerlink.com

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Liang J, et al. Sci China Chem January (2013) Vol.56 No.1
derivatives required for the biomedical researches.
vide suitable substrates for protein chemical synthesis. Pre-
viously we have applied this method to the synthesis of
several proteins including trifolitoxin, CssІІ, circular pro-
teins, and ribosomal protein S25 [1521]. In the present
work we report the use of peptide hydrazide ligation for the
total synthesis of an Ub derivative Ub-AMC. The molecule
contains 7-amino-4-methylcoumarin at its C-terminus and is
a widely used probe for the monitoring of deubiquitinating
enzymes (DUBs) as well as for the identification of inhibi-
tors against DUBs.
In this context several studies have been reported for the
chemical synthesis of Ub derivatives (Figure 1). Examples
include the synthesis of Ub derivatives via native chemical
ligaiton [7], total chemical synthesis of ubiquitinated pep-
tides bearing various ubiquitin chains by δ-mercaptolysine
assisted ligation [811], dual native chemical ligation at
lysine for synthesis of ubiquitinated peptides [12], and
Fmoc solid-phase peptide synthesis of Ub probes by the use
of pseudoproline building blocks and dimethoxybenzyl di-
peptides [13]. Despite these advances, there are still tech-
nological problems limiting the application of chemical
synthesis in the study of ubiquitination. For instance, the
preparation of the thioester intermediates via mild Fmoc
chemistry remains difficult in the previous synthetic studies.
The use of expensive dipeptide building blocks in some of
the previous studies also prevents the scaled-up production
of Ub derivatives.
Recently, we have developed a new ligation procedure
through the chemoselective reaction between a Cys-peptide
and a C-terminal peptide hydrazide [14]. In this method, the
peptide hydrazide was activated by NaNO2 and an external
thiol in situ to become the peptide thioester. Compared to
the previous methods for the preparation of peptide thioe-
sters, peptide hydrazides can be easily obtained through
Fmoc-based solid-phase peptide synthesis and thereby, pro-
2 Experimental
2.1 Materials
Fmoc-protected amino acids were purchased from Beijing
BoMaiJie Technology Company. HBTU, DIC, 7-amino-4-
methylcoumarin, and POCl3 were obtained from Alfa Aesar.
Et2O, THF, DMF, DCM, DIEA, EtOAc, piperidine, and
other common reagent were gained from Sinopharm Chem-
ical Reagent Co. Ltd. Purification of crude peptides was
carried out by using HPLC (LC-20AT, SHIMADZU) with
dual wavelength (215 and 254 nm). Identification of syn-
thetic peptides was performed by using MALDI-TOF and
ESI-MS (Agilent).
Figure 1 Previous methods for chemical synthesis of Ub derivatives.

Liang J, et al. Sci China Chem January (2013) Vol.56 No.1
3
2.2 Fmoc solid-phase peptide synthesis (SPPS)
2-Cl-Trt(Cl) resin (2.0 g, 0.5 mol/g) was added to a flask.
At 0 °C, a solution of hydrazine in DMF (N2H4·H2O :
DIEA : DMF = 0.4 g : 1.33mL : 4.0 mL) was added drop-
wise to the flask. After 90 min, the reaction was quenched
by the addition of methanol (0.2 mL) for 20 min (Eq. (2)).
The hydrazine-Trt(2-Cl) resin was washed with DMF, H2O,
DMF, MeOH, and Et2O in sequence, and dried in a vacuum.
Resin for SPPS was initially swelled with DMF/DCM (1:1)
for 2 h in the screw-cap glass peptide synthesis apparatus,
followed by washing of the resin with DMF, DCM, and
DMF. Fmoc-amino acids were coupled with the DMF solu-
tion containing HBTU (3.6 equiv.), HOBt (4.0 equiv.),
DIEA (8.0 equiv.), and Fmoc-amino acid (4.0 equiv.). Note
that the activation step initiated by DIEA was performed in
less than 5 min, and the condensation was performed for 90
min followed by K-Test. The Fmoc group was removed by
dual treatment (5 and 10 min) with a solution of piperidine
in DMF (20%, v/v). After the assembly of the target peptide,
the resin was washed with DMF and DCM, and dried at
room temperature. A cocktail reagent (87.5% TFA, 5% H2O,
5% PhOH, 2.5% TIPS) was then added to the resin for 2.5 h.
After the concentration of the combined cocktail reagent by
a N2 blowing, the crude peptides were obtained by cold
ether precipitation and centrifugation. The crude peptides
were dissolved in water, identified by ESI-MS and purified
by semi-preparative HPLC. Finally, the dried peptides were
obtained by lyophilization as white powders.
2.5 Preparation of Ub1 and Ub2
The hydrazine-Trt(2-Cl) resin (0.4 g) was swelled with
DMF/DCM (1 : 1) for 1 h in the screw-cap glass peptide
apparatus. The coupling of Fmoc-amino acids was carried
out according to the standard Fmoc SPPS. After dried at
room temperature, the peptide bound resin was treated by
the cocktail reagent for 2.5 h (87.5% TFA, 5% H2O, 5%
PhOH, 2.5% TIPS). The combined reagent was concentrat-
ed by a N2 blowing. The crude peptide hydrazides were
obtained by cold ether precipitation and centrifugation as
white powders. The crude peptides were characterized by
ESI-MS, and purified by HPLC. They were lyophilized as
white powders. ESI-MS: Ub1 m/z = 3048.4 (M + 1), calc.:
3047.6; Ub2 m/z = 2142.0 (M + 1), calc.: 2141.1.
2.3 Synthesis of H-Gly-AMC
2.6 Two step synthesis of Ub3
The 2-Cl-Trt-Cl resin (0.4 g) was swelled with DMF/DCM
(1 : 1) for 15 min in the screw-cap glass peptide apparatus.
The first amino acid was coupled with the DMF solution
containing DIEA (8.0 equiv.) and Fmoc-Gly-OH (4.0
equiv.). Note that the last amino acid attachment was per-
formed with Boc-Cys-OH. After the assembly of the target
peptide, the full protected peptide was cleaved from resin
with HFIP/DCM (1 : 4) at room temperature. After 0.5 h,
the resin was washed with DCM and then the combined
reagent was concentrated in a vacuum. The crude full pro-
tected peptide was obtained by cold ether precipitation and
centrifugation as a white powder. The fully protected pep-
tide was condensed with H-Gly-AMC in DCM by using
PyBOP (5.0 equiv.) initiated with DIEA at room tempera-
ture. After the reaction was stirred overnight, the mixture
was concentrated in vacuum. The protecting group was re-
moved by using a mixed solution (TFA : H2O : TIPS = 95:
2.5: 2.5, v/v) for 3 h. The identification and isolation step
was carried out as described above. ESI-MS: Ub3 m/z =
3657.3 (M+H), calc.: 3654.9.
Eq. (1)
7-Amino-4-methylcoumarin (0.87 g, 5.0 mM) and Boc-Gly-
OH (0.87 g, 5.0 mM) were dissolved in dried pyridine (15
mL). At 15 °C, 0.51 mL of POCl3 was added to the mix-
ture dropwise. After 2 h, the ice-salt bath was removed, and
the mixture was stirred at room temperature. The reaction
was stirred overnight and quenched by treatment with water.
The organic reagents were removed by evaporation in vac-
uum. Boc-Gly-AMC was isolated by column chromatog-
raphy with a yield of 53% (0.87 g, 2.7 mM). Finally, the
removal of Boc group was achieved by treatment with a
solution of TFA/DCM (4:1) for 1 h. The product H-Gly-
AMC was obtained by concentration as solid with a yield of
1
95% (0.49 g) (Eq. (1)). H NMR (400 MHz, DMSO-d6): δ
10.97 (1H, s), 7.78 (2H, d, J = 8.1 Hz), 7.47 (1H, d, J = 8.6
Hz), 6.31 (1H, s), 3.87 (2H, s), 2.41 (3H, s) ppm. 13C NMR
(400 MHz, DMSO-d6): δ 166.14, 160.37, 154.12, 153.53,
141.83, 126.76, 116.04, 115.64, 113.12, 106.26, 41.73,
18.44 ppm. ESI-MS: m/z = 233.2 (M + H), calc.: 232.1.
2.4 Preparation of NH2-NH-Trt(2-Cl) resin
2.7 The ligation of Ub1 with Ub2
Ub1 (6.5 mg, 2.1 μmol) was dissolved in 0.7 mL of ligation
buffer (6.0 M Gn·HCl, 0.2 M Na2HPO4). At a low pH (3.0)
and 10 °C, 70 μL of NaNO2 (0.2M) was added to the mix-
ture. After 20 min, the mixture was treated by a 0.2 M
Eq. (2)

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Liang J, et al. Sci China Chem January (2013) Vol.56 No.1
MPAA solution (0.7 mL) and its pH was slowly adjusted to
7.0 with 2.0 M NaOH solution. Then, the ligation reaction
was treated with Ub2 (6.1 mg, 2.8 μmol) at room tempera-
ture. The reaction was monitored by HPLC at different
times. The ligation product was isolated by semi-preparative
HPLC after reduction of the mixture with TCEP. The
Ub[Met1-Phe45] was obtained as a white powder (30%).
ESI-MS: m/z = 5159.5 (M + H), calc.: 5158.7.
peptide synthesis. However, Ub-AMC consists of 76 amino
acid residues, so that we cannot reliably prepare it through a
single SPPS. Instead, we had to use the method of multiple
peptide segment condensation (Figure 2). Because Ub does
not have any Cys residue, we mutated Ala located at the
positions of 28 and 46 to Cys. After the assembly of three
peptide segments, the two Cys residues were converted
back to Ala by using the metal-free desulfurization method.
It is noteworthy that the three peptide segments (i.e. Ub1,
Ub2, and Ub3) contained 27, 18, and 31 amino acid resi-
dues, respectively. They are therefore, possible to prepare in
large scales. Moreover, Ub1 and Ub2 are peptide hydra-
zides and therefore, can be obtained easily by using the
Fmoc SPPS method with the hydrazine-Trt(2-Cl) resin. On
the other hand, Ub3 contains an AMC group at its
C-terminus and therefore, cannot be synthesized directly by
SPPS. We developed a two-step method to solve the prob-
lem. Specifically, we used the 2-Cl-trityl resin to prepare
the fully protected peptide that contains the first 30 residues.
This intermediate was then condensed with H-Gly-AMC in
DCM by using PyBOP assisted by DIEA. Subsequently, the
protecting group was removed by using the standard TFA
cocktail reagent, and Ub3 was obtained by cold ether pre-
cipitation. The crude product was purified by semi-prepara-
tive HPLC.
2.8 Ligation with Ub3
Ub[Met1-Phe45] (5.6 mg, 1.1 μmol) was dissolved in 0.35
mL of ligation buffer (6.0 M Gn·HCl, 0.2 M Na2HPO4). At
a low pH (3.0) and 10 °C, 35 μL of NaNO2 (0.2M) was
added to the mixture. After 20 min, the mixture was treated
by a 0.2 M MPAA solution (0.35 mL) and its pH was slow-
ly adjusted to 7.0 with 2.0 M NaOH solution. Then, the li-
gation reaction was triggered by addition of Ub3 (5.5 mg,
1.5 μmol) at room temperature. The reaction was monitored
by HPLC at different times. The ligation product was iso-
lated by semi-preparative HPLC after reduction of the mix-
ture with TCEP. The Ub-AMC[Ala28,46Cys] was obtained
as a white powder (35%). ESI-MS: m/z = 8780.5 (M + H),
calc.: 8779.6.
2.9 Free-radical desulfurization
Ub-AMC[Ala28, 46Cys] (1.5 mg, 0.17 μmol) was dissolved
in the buffer solution (6.0 M Gn·HCl，0.2 M Na2HPO4, pH
7.0). The mixture was treated by addition of t-BuSH (0.15
mL), a 0.1 M VA-044 solution, and a 0.5 M TCEP solution
(0.2 mL). Then its pH was adjusted to 7.0 by 2.0 M NaOH
solution. The desulfurization reaction was performed for 12
h at 35 °C. The purified Ub-AMC was obtained by HPLC as
a white powder with a yield of 80%. ESI-MS: m/z = 8716.6
(M + H), calc.: 8715.7.
2.10 Measurement of Ub-AMC activity
1 μL Ubp6 (0.1 mM) was incubated for 15 minutes at 30 °C
in 60 μL of 50 mM Tris 7.5 solution containing 1 mM
EDTA, 1 mM ovalbumin, 5 mM ATP/MgCl2 and 1 mM
DTT. After addition of 4 μL of 0.2 mM Ub-AMC, the fluo-
rescence emission was determined at an excitation wave-
length of 355 nm and an emission wavelength of 460 nm.
Each experiment included three independent reactions and
was repeated three times.
3 Results and discussion
3.1 Design of synthetic route for Ub-AMC
Generally speaking, peptide fragments containing less than
50 residues can be made directly by standard solid-phase
Figure 2 The sequence of Ub-AMC and its synthetic route.

Liang J, et al. Sci China Chem January (2013) Vol.56 No.1
5
3.2 Total synthesis of Ub-AMC
shown in Figure 4. Besides, we found a significant precipi-
tation of the final ligation product. To solve this solubility
problem, we added acetonitrile to the ligation media but this
method did not improve the synthesis.
The final step was the conversion of two cys residues in
the Ub-AMC[Ala28, 46Cys] to two Ala residues to generate
Ub-AMC by using the metal-free desulfurization reaction
(Figure 5). This reaction was performed at pH 7.0 and 35 °C
upon the addition of VA-044, t-BuSH, and TCEP. Ub-AMC
was obtained by HPLC with a yield of 80%.
With Ub1, Ub2, and Ub3 in hand, we synthesized Ub-AMC
by using the N-to-C sequential ligation of three peptide
segments.
Synthesis of Ub-AMC was initiated with the ligation
between Ub1 and Ub2 (Figure 3). At a low pH (3.0) and
10 °C, Ub1 was converted into the peptide azide interme-
diate by the assistance of NaNO2 (6.0 equiv.). After 20 min,
MPAA (65.0 equiv.) was added and the pH values was ad-
justed to 7.0 to facilitate the formation of the thioester in-
termediate Ub[Met1-Lys27]-S-aryl. Then, Ub2 (1.3 equiv.)
was added and this step was allowed to proceed until the
consumption of the thioester peptide. Before purification by
HPLC, TCEP was added to reduce any oxidized thiols. The
yield of the isolated product in this step was 30% and its
product was Ub[Met1-Phe45]. Note that Ub[Met1-Lys27]-S-
aryl was found to produce a circular peptide byproduct
probably through cyclization reaction between the side
chain of Lys27 and the thioester moiety. This side reaction,
which can be slightly suppressed by reducing the acidity of
the mixture, led to the relatively low yield in this step.
Our next step was the second ligation step with Ub3,
which was carried out in a way similar to the first step as
described above (Figure 4). Ub[Met1-Phe45] was oxidized
by NaNO2 to the peptide azide in less than 20 min at pH 3.0.
After the addition of MPAA, Ub[Met1-Phe45]-S-aryl was
formed. After adjusting pH of the mixture to 7.0, Ub3 was
added and triggered the final ligation. The ligation product
Ub-AMC[Ala28,46Cys] was purified from the mixture with
yield of 35%. Fortunately, the impurity contained in the
reagent of Ub3 does not ligate with the peptide thioester as
3.3 DUBs activity assays of Ub-AMC
DUBs can promote the release of strongly fluorescent AMC
moiety from Ub-AMC. The fluorescence of AMC has been
exploited to monitor DUBs. To test the biological activity of
the synthetic Ub-AMC, we observed the intensity change of
fluorescence provided by AMC upon the addition of DUBs
(Figure 6). Furthermore, we carried out the same assay with
Ub-AMC[Ala28, 46Cys] to study the effect of two Cys res-
Figure 4 HPLC monitoring of the ligation between Ub[Met1-Phe45] and
Ub3. (a) After the activation of Ub[Met1-Phe45] followed by addition of
MPAA; (b) after overnight.
Figure 3 HPLC monitoring of the ligation between Ub1 and Ub2. (a)
After the activation of Ub1 followed by addition of MPAA; (b) after over-
night.
Figure 5 Desulfurization of Ub-AMC[Ala28, 46Cys].

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Figure 6 Enzymatic activity with Ub-AMC. The gray spots represent the
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4 Conclusions
In summary, we have developed an efficient method for
chemical synthesis of Ub-AMC by ligation of peptide hy-
drazides. We confirmed that the synthetic Ub-AMC can be
used for the detection of DUBs. The peptide hydrazides
used in Ub synthesis can be readily prepared by Fmoc-SPPS,
and all of the peptide fragments can be prepared readily.
Therefore, this method may be extended to the synthesis of
other Ub derivatives.
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This study was supported by National Basic Research Program of China
(973 program, 2013CB932800) and the National Natural Science Founda-
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