New semi-synthesis of ubiquitin C-terminal conjugate with 7-amino-4-methylcoumarin

Article abstract of DOI:10.1002/psc.2568

The ligation of peptide hydrazides at a Gly site carrying a removal auxiliary was found to be an efficient process. This technology was successfully used for the synthesis of ubiquitin C-terminal conjugates. Recombinant Ub(1-75)-NHNH₂ was prepared through the hydrozinolysis of the Ub(1-75)-intein fusion protein. It was ligated with a glycine derivative modified with an acid-sensitive thiol auxiliary. The final acid treatment produced the desired bioactive ubiquitin conjugates in practical quantities. Thus, the method described here extends the protocols of expressed protein ligation. Copyright 2013 European Peptide Society and John Wiley & Sons, Ltd. Native chemical ligation of a recombinant peptide hydrazide with a Gly-peptide carrying a removal auxiliary was examined and used to synthesize ubiquitin C-terminal conjugates. Copyright

Full text of DOI:10.1002/psc.2568

Special Issue Article
Received: 21 July 2013
Accepted: 27 August 2013
Published online in Wiley Online Library: 3 October 2013
(wileyonlinelibrary.com) DOI 10.1002/psc.2568
New semi-synthesis of ubiquitin C-terminal
conjugate with 7-amino-4-methylcoumarin‡
Yi-Tong Li,a,b Jun Liang,b Jia-Bin Li,b Ge-Min Fang,b Yong Huanga,b
and Lei Liub*
The ligation of peptide hydrazides at a Gly site carrying a removal auxiliary was found to be an efficient process. This technology
was successfully used for the synthesis of ubiquitin C-terminal conjugates. Recombinant Ub(1–75)-NHNH2 was prepared through
the hydrozinolysis of the Ub(1–75)-intein fusion protein. It was ligated with a glycine derivative modified with an acid-sensitive
thiol auxiliary. The final acid treatment produced the desired bioactive ubiquitin conjugates in practical quantities. Thus, the
method described here extends the protocols of expressed protein ligation. Copyright © 2013 European Peptide Society and John
Wiley & Sons, Ltd.
Keywords: native chemical ligation; peptide hydrazides; expressed protein ligation; ubiquitin; deubiquitinating enzymes
subsequent auxiliary-mediated ligation of the peptide hydrazide
(Scheme 1) [33,34]. Our experiments show that this new proce-
Introduction
Ubiquitination is a reversible post-translational modification
process that plays a highly important role in cell biology [1–3]. It
involves the covalent attachment of a small, functional regulatory
protein, i.e. ubiquitin (Ub) to the target protein molecule. This
process regulates the degradation of cellular proteins by the Ub-
proteasome system and thereby controls a protein’s half-life and
expression levels. The attachment of Ub to a target protein
requires the sequential action of Ub-activating enzymes (E1),
Ub-conjugating enzymes (E2), and Ub ligases (E3). On the other
hand, the removal of Ub from a modified protein is mediated by
the deubiquitinating enzymes (DUBs) [4–6]. The ubiquitinating
and deubiquitinating enzymes function in chorus to preprocess
the proteins degraded by the proteasome.
Many recent studies have revealed the involvement of
malfunctioning DUBs in some human diseases [7–11]. To monitor
the activity of DUBs and to screen small molecule inhibitors
against DUBs, efforts have been directed to the development of
various biochemical tools such as the C-terminal conjugates of
Ub with fluorophores such as 7-amino-4-methylcoumarin (AMC)
[12–15]. Such Ub-fluorophore conjugates exhibit a significant
increase in fluorescence following the DUB-mediated hydrolysis,
resulting in a readily measured signal that is directly proportional
to enzyme turnover and DUB activity [16–20]. Furthermore, the
C-terminal vinyl sulfone conjugate of Ub has also been developed
as a mechanism-based inhibitor that can covalently capture the
DUB active site thiol [21–23]. This activity-based probe has been
used for the identification of active or novel DUBs in crude lysates.
The preparation of the aforementioned Ub conjugates is
usually performed through direct aminolysis of the Ub-thioester
generated through the intein technology [24–32]. This aminolysis
reaction is a relatively slow transformation and therefore, may
suffer from heavy hydrolysis, difficult product separation, and
low yields. To overcome this problem, we now examine the use of
an alternative approach to prepare the Ub C-terminal conjugates.
The new method involves preparation of Ub(1–75)-NHNH2 through
the hydrozinolysis of the Ub(1–75)-intein fusion protein and
dure can be used to prepare Ub conjugates with good efficiency.
The present study also shows the viability for the ligation of
peptide hydrazides with a non-Cys amino acid carrying a remov-
able thiol auxiliary [35–40].
Experimental
Materials and Methods
7-Amino-4-methylcoumarin was obtained from Alfa Aesar;
anhydrous piperidine was supplied by Acros; and POCl3, DMF,
DCM, DIEA, MeOH, ethyl acetate (EtOAc), and other common
reagents were purchased from Sinopharm Chemical Reagent
Co. Ltd. Analytical HPLC was run on a SHIMADZU (Prominence
LC-20AT; Kyoto, Japan) instrument using an analytical column
(Grace Vydac ‘Protein C18’, 250 × 4.6 mm, 5 μm particle size, flow
rate 1.0 ml/min, rt). Analytical injections were monitored at 214
and 254 nm. Semi-preparative HPLC was run on a SHIMADZU
(Prominence LC-20AT) instrument using a semi preparative column
(Grace Vydac Peptide C18, 250 × 10 mm, 10 μm particle size,
flow rate 4.0ml/min). Solvent A was 0.1% TFA in acetonitrile,
and solvent B was 0.1% TFA in water. The analytical analysis was
*
Correspondence to: Lei Liu, Tsinghua-Peking Center for Life Sciences, Department
of Chemistry, Tsinghua University, Beijing 100084, China. E-mail: lliu@mail.
tsinghua.edu.cn
‡
This article is published in Journal of Peptide Science as part of the Special Issue
devoted to contributions presented at the Chemical Protein Synthesis Meeting,
April 3–6, 2013, Vienna, edited by Christian Becker (University of Vienna, Austria).
a Key Laboratory of Chemical Genomics, School of Chemical Biology and
Biotechnology, Peking University, Shenzhen Graduate School, Shenzhen
518055, China
b Tsinghua-Peking Center for Life Sciences, Department of Chemistry, Tsinghua
University, Beijing 100084, China
J. Pept. Sci. 2014; 20: 102–107
Copyright © 2013 European Peptide Society and John Wiley & Sons, Ltd.

SEMI-SYNTHESIS OF Ub-AMC
Scheme 1. New synthesis of Ub-AMC.
carried out on a C18 analytical column using a gradient of 10–60% sol-
vent A over 30 min. Identification of the synthetic peptides was
performed by using ESI-MS Agilent (Santa Clara, CA, USA). H and
24–72 h. A further 10.0 mmol glycine and 0.7 mmol sodium
cyanoborohydride were added after 24 h, and refluxing was contin-
ued for a further 48 h. The reaction was then evaporated to dryness.
The crude residues were purified by column chromatography over
silica (EtOAc/methanol) to give the products as white or yellow
solids (386 mg, 0.75 mmol, 75%). 1H NMR (400 MHz, CDCl3): δ = 7.36
(5H, m), 7.21 (10H, m), 6.97 (1H, d, J= 8.4 Hz,), 6.38 (2H, m), 3.80
1
13C NMR spectra were recorded on an Oxford 400 MHz spectrometer
(Oxfordshire, UK) in deuteriochloroform (CDCl3), deuterated acetone
((CD3)2CO), or DMSO-d6. Data are reported in the following order:
chemical shifts are given (δ), and multiplicities are indicated as s
(singlet), d (doublet), t (triplet), q (quartet), and m (multiplet).
For the biochemistry experiments, bacterial cells were grown in
Luria–Bertani (LB) broth or on LB agar medium Sigma (St. Louis,
MO, USA). Antibiotics were used at final concentrations of
100 μg/ml for ampicillin (Sigma). Chitin beads for purification of
intein-CBD fusion proteins were obtained from New England
BioLabs (Ipswich, MA, USA). All pictures of protein gels were taken
on a ChemDoxXRS+ Bio-Rad (Hercules, CA, USA).
(3H, s), 3.65 (3H, s), 3.46 (2H, s), 2.97 (2H, m, CH2), 2.79 (1H, m). 13
C
NMR: δ = 171.11, 161.68, 158.35, 144.36, 129.58, 128.02, 126.77,
125.30, 113.93, 104.80, 98.90, 67.25, 55.43, 50.72, 47.74, 33.34,
21.51. m/z (ESI): 536.20 [MNa]+, C31H31NO4SNa+ requires 536.19.
2-(1-(2,4-Dimethoxyphenyl)-2-mercaptoethylamino)-N-(4-methyl-2-oxo-2H-
chromen-7-yl)acetamide (compound 3)
To a cold solution (À15 °C) of compound 2 (260 mg, 0.5 mmol)
and 7-amino-4-methylcoumarin (87.5 mg, 0.5 mmol) in anhydrous
pyridine (2 ml), phosphoryl chloride (0.055 ml, 0.55 mmol) was
added dropwise. A color change going from yellow to orange
was observed. The mixture was stirred for 2 h at À15 °C and
then allowed to warm to room temperature and stirred for an
additional hour. The reaction mixture was quenched with water.
The solvents were evaporated under reduced pressure. The crude
reaction mixture was purified by chromatography. The product
was then treated with 95% TFA with 2.5% triisopropylsilane and
2.5% water for 2 h. The concentrated residue was dissolved in
water and purified by semi-preparative HPLC. Finally, the dried
product was obtained by lyophilization as a white powder
(41 mg, 0.095 mmol, 19% over two steps). 1H NMR (400 MHz,
DMSO-d6): δ = 10.75 (1H, S), 7.77 (1H, S), 7.72 (1H, S), 7.38 (2H, t),
6.65 (2H, m), 6.32 (1H, s), 4.59 (1H, s), 3.80 (3H, s), 3.78 (3H, s), 3.69
(2H, s), 3.17 (3H, m), 2.41 (3H, s). 13C NMR (400MHz, CD3COCD3):
δ = 163.78, 163.76, 161.19, 160.54, 160.51, 155.44, 153.81, 142.58,
126.90, 117.25, 116.58, 114.32, 107.75, 106.81, 100.15, 100.08,
66.58, 61.02, 56.76, 56.72, 56.30, 18.86. m/z (ESI): 429.0 [M+ 1]+,
C22H24N2O5S requires 428.14.
Experimental Procedure for the Synthesis of Compound 3
2-Keto-2-(2,4-dimethoxyphenyl) (S-trityl) ethanethiol (compound 1)
2-Bromo-2,4-dimethoxyacetophenone (1.0 g, 3.88 mmol) was
dissolved in DMF (20.0 ml). DIEA (808 μl, 4.64 mmol) was added,
followed by trityl thiol (4.64 mmol). The reaction mixture was
stirred at room temperature for 2 h and then evaporated to
dryness. The crude residue was dissolved in EtOAc (100 ml) and
washed with brine (100.0 ml). The aqueous mixture was extracted
with EtOAc (50.0 ml), and the combined organic extracts were
dried over magnesium sulfate and filtered and evaporated under
vacuum to give yellow solids, which were purified by column
chromatography over silica (EtOAc/petroleum ether) to give the
1
products as white or yellow solids (1.67 g, 3.68 mmol, 95%). H
NMR (400 MHz, CDCl3): δ = 7.75 (1H, d), 7.47 (6H, m), 7.30 (6H, m),
7.23 (3H, m) 6.51 (1H, d), 6.35 (1H, s), 3.85 (3H, s), 3.51 (5H, s). 13C
NMR: δ = 45.34, 55.31, 55.55, 66.07, 98.12, 105.30, 119.62, 126.58,
127.87, 129.66, 133.16, 144.80, 160.71, 164.75, 194.51. m/z (ESI):
477.2 [MNa]+, C29H26O3SNa+ requires 477.15.
2-Glycine-2-(2,4-dimethoxyphenyl) (S-trityl) ethanethiol (compound 2)
Overexpression and Purification of Ub(1–75)-NHNH2
Compound 1 (1.0 mmol) was dissolved in MeOH (10.0 ml). Glycine
(10.0 mmol) was added, followed by sodium cyanoborohydride
(44 mg, 0.7 mmol). The reaction mixtures were heated to reflux for
Escherichia coli BL21(DE3) cells (Beijing Transgen Biotech Co., Ltd,
Beijing, China) transformed with the plasmid pTYB2-Ub(1–75)
J. Pept. Sci. 2014; 20: 102–107 Copyright © 2013 European Peptide Society and John Wiley & Sons, Ltd. wileyonlinelibrary.com/journal/jpepsci

LI ET AL.
were grown in 1 l LB medium (100 μg/ml ampicillin) at 37 °C with
shaking at 220 rpm until an OD600 0.6–0.8. Overexpression of Ub
was induced by the 0.2 mM isopropyl β-D-1-thiogalactopyranoside,
and the cells were grown at 16 °C overnight. The cells were
harvested by centrifugation at 5000 rpm for 10 min and were
re-suspended in lysis buffer (20 mM Hepes, 500 mM NaCl, and
1 mM EDTA, pH 7.4), and the cells were lysised by ultrasonification.
The soluble fraction separated from insoluble cellular debris by
centrifugation at 13 000 rpm for 30 min. The supernatants were
filtrated through a 0.22 μm filter and were loaded to a 15 ml chitin
column pre-equilibrated with lysis buffer for 1 h at room temper-
ature. The column was washed with 20 bed volumes of lysis
buffer. The column was incubated for 3 h with cleavage buffer
(20 mM Hepes, 500 mM NaCl, 1 mM EDTA, 5–8% hydrazine hydrate,
pH 7.4). The eluted Ub(1–75)-NHNH2 was purified by C18 semi-
preparative RP-HPLC. The yield of Ub(1–75)-NHNH2 was approxi-
mately 2–3 mg for per 1 l LB medium.
To design the synthetic route for Ub-AMC, we chose the
junction at Gly75-Gly76 to be the ligation site. Thus, we first
prepared the two peptide segments needed for the ligation. Using
our previously developed protocol [33], we used the intein-based
technology to prepare Ub(1–75)-NHNH2 (Figure 1). We employed
the E. coli BL21(DE3) cells transformed with the plasmid pTYB2-Ub
(1–75), which were harvested and lysised to yield soluble
supernatants. The supernatants were then loaded to a chitin
column, which was extensively washed to remove all the other
proteins. Finally, the column was incubated with the cleavage
buffer containing approximately 5–8% hydrazine hydrate at
pH 7.4. The desired product, i.e. Ub(1–75)-NHNH2, was eluted
and purified by semi-preparative RP-HPLC. This protocol can
produce 2–3 mg Ub(1–75)-NHNH2 per 1 l LB medium. In
comparison with the traditional intein technology that typically
uses 2-mercaptoethanesulfonic acid to cleave the fusion protein,
the hydrazine cleavage method is relatively faster (3 h versus
24–48 h). The product of this process (i.e. a protein hydrazide) is
also more stable towards hydrolysis as compared with a protein
thioester.
Auxiliary-mediated Expressed Protein Ligation
Ub(1–75)-NHNH2 (3.9 mg, 0.46 μmol) was dissolved in 0.2 ml of
ligation buffer (6.0 M Gn•HCl, 0.2M Na2HPO4). At a low pH (3.0) and
À10 °C, 20 μl of NaNO2 (0.2 M) was added to the mixture. After
20 min, the mixture was treated by a 0.2 M 4-mercaptophenylacetic
acid solution (0.2 ml), and its pH was slowly adjusted to 7.0 with
2.0 M NaOH solution. Then, the ligation reaction was treated with
compound 3 (2mg, 4.6 μmol) at room temperature. The reaction
was monitored by HPLC at different times. The ligation product
was isolated by semi-preparative HPLC after reduction of the
mixture with tris(2-chloroethyl) phosphate (TCEP). The product
was obtained as a white powder (35%). ESI-MS: m/z = 8918.0
(M + 1+), calc. 8916.8.
To synthesize the other ligation partner, we started with
commercially available 2-bromo-1-(2,4-dimethoxyphenyl)ethanone
(Scheme 2). Through the treatment with triphenylmethanethiol,
we obtained compound 1 in 95% yield. Compound 1 was then
condensed with glycine through reductive amination to generate
compound 2 in 75% yield. Subsequently, compound 2 was
condensed with 7-amino-4-methyl-coumarin in the presence
of POCl3. After the trityl group was removed, we used semi-
preparative RP-HPLC to isolate the final target compound 3 with
an overall yield of approximately 14%.
With both Ub(1–75)-NHNH2 and 3 in hand, we conducted
the ligation (Figure 2). Ub(1–75)-NHNH2 was first treated with
NaNO2 at pH 3 and À10 °C for 20 min. Subsequently, excessive
4-mercaptophenylacetic acid was added, and the pH value was
adjusted to 7.0. Compound 3 was then added into the ligation
mixture at room temperature. The ligation reaction was completed
smoothly in approximately 2 h. The desired product 4 was obtained
in approximately 85% HPLC yield and 35% isolated yield.
Measurement of the Bioactivity of Synthetic Ub-AMC
For 15 min, 0.124 μl ubiquitin carboxyl-terminal hydrolase isoen-
zyme 3 (UCH-L3; 16.4 μM) was incubated at 30 °C in 120 μl of
50 mM Tris7.5 solution containing 1 mM EDTA, 1 mM ovalbumin,
and 1 mM 1,4-dithiothreitol (DTT). After addition of 4 μl Ub-AMC
(0.2 mM), the fluorescence emission was determined at an excita-
tion wavelength of 355 nm and an emission wavelength of
460 nm. Each experiment included three independent reactions
and was repeated three times.
(A)
(B)
Results and Discussion
The native chemical ligation between a peptide thioester and a
Cys-peptide represents one of the most efficient methods for
the condensation of protein segments. A limitation of this
method is the requirement of a Cys residue at the ligation site,
which may sometimes be overcome through the attachment of
a removable thiol functionality at the N-terminal amino group.
Recently, we have shown that peptide hydrazides can be acti-
vated in situ by NaNO2 and then concerted to peptide thioesters.
This transformation enables the use of peptide hydrazides as
thioester surrogates in the native chemical ligation reactions. In
our previous experiments, we have only tested the use of Cys-
peptides in the native chemical ligation of peptide hydrazides.
Thus, the present synthesis of Ub-AMC presents a good opportu-
nity to examine the viability of the hydrazide-based method in
the auxiliary-assisted ligation because Ub-AMC contains no Cys
residue in the protein sequence.
(C)
Figure 1. Recombinant synthesis of Ub(1–75)-NHNH2. (A) SDS-PAGE of the
recombinant Ub(1–75)-NHNH2. Note: ‘*’ corresponds to Ub(1–75)-intein-
CBD. (B) ESI-MS mass of Ub(1–75)-NHNH2. (C) Purified Ub(1–75)-NHNH2.
wileyonlinelibrary.com/journal/jpepsci Copyright © 2013 European Peptide Society and John Wiley & Sons, Ltd. J. Pept. Sci. 2014; 20: 102–107

SEMI-SYNTHESIS OF Ub-AMC
Scheme 2. Synthesis of the auxiliary-modified glycine derivative.
(A)
(B)
(C)
Figure 2. Analytical HPLC traces of the ligation reaction between Ub(1–75)-NHNH2 and 3. (A) At 0 h. Peak i corresponds to 3; ii corresponds to Ub
(1–75)-MPAA thioester; iii corresponds to hydrolysis by-product Ub(1–75)-COOH; (B) After 2 h. Peak iv corresponds to the ligation product 4; (C)
Purified 4 and its ESI-MS.
The final step of the synthesis is the removal of the
auxiliary group. Thus, we treated compound 4 with the
standard cocktail reagent (95% TFA, 2.5% H2O, and 2.5% TIPS)
for 2 h. The crude peptides were collected after the cold ether
precipitation and subsequently purified by semi-preparative
HPLC. The target compound Ub-AMC was obtained as a
white powder in approximately 50% yield. The synthetic
Ub-AMC was successfully characterized by both SDS-PAGE
and ESI-MS (Figure 3).
To confirm the bioactivity of the synthetic Ub-AMC, we moni-
tored its fluorescence change upon the hydrolysis mediated by
a DUB enzyme named UCH-L3. Our measurements indicated that
in the absence of UCH-L3, Ub-AMC is a very stable compound in
the aqueous buffer that does not cause any fluorescence change
over time (Figure 4). On the other hand, when UCH-L3 was added
to the solution of Ub-AMC, we observed a rapid and dramatic
fluorescence increase. As a result, the synthetic Ub-AMC is an
active fluorescent probe for the DUB enzymes.
J. Pept. Sci. 2014; 20: 102–107 Copyright © 2013 European Peptide Society and John Wiley & Sons, Ltd. wileyonlinelibrary.com/journal/jpepsci

LI ET AL.
(A)
(B)
(C)
Science Fund for Distinguished Young Scholars, and the Specialized
Research Fund for the Doctoral Program of Higher Education (Grant
No. 20120002130004).
References
1 Hershko A, Ciechanover A. The ubiquitin system. Annu. Rev. Biochem.
1998; 67: 425–479.
2 Weissman AM, Shabek N, Ciechanover A. The predator becomes the
prey: regulating the ubiquitin system by ubiquitylation and degradation.
Nat. Rev. Mol. Cell Biol. 2011; 12: 605–620.
3 Komander D, Rape M. The ubiquitin code. Annu. Rev. Biochem. 2012;
81: 203–229.
4 Love KR, Catic A, Schlieke C, Ploegh HL. Mechanisms, biology and inhib-
itors of deubiquitinating enzymes. Nat. Chem. Biol. 2007; 3: 697–705.
5 Reyes-Turcu, FE, Wilkinson K. Polyubiquitin binding and disassembly
by deubiquitinating enzymes. Chem. Rev. 2009; 109: 1495–1508.
6 Spasser L, Brik A. Chemistry and biology of the ubiquitin signal.
Angew. Chem. Int. Ed. 2012; 51: 6840–6862.
7 Nijman SMB, Luna-Vargas MPA, Velds A, Brummelkamp TR, Dirac MG,
Sixma TK, Bermards R. A genomic and functional inventory of
deubiquitinating enzymes. Cell 2005; 123: 773–786.
Figure 3. (A) SDS-PAGE of the synthetic Ub-AMC. (B) The observed
ESI-MS mass for Ub-AMC. (C) Purified Ub-AMC after semi-preparative
reverse-phase HPLC.
8 Huang TT, Andrea AD. Regulation of DNA repair by ubiquitylation.
Nat. Rev. Mol. Cell Biol. 2006; 7: 323–334.
9 Conaway RC, Brower CS, Conaway JW. Emerging roles of ubiquitin in
transcription regulation. Science 2002; 296: 1254–1258.
10 Komander D, Clague MJ, Urbe S. Breaking the chains: structure and
function of the deubiquitinases. Nat. Rev. Mol. Cell Biol. 2009; 10:
550–563.
11 McGinty RK, Kim J, Chatterjee C, Roeder RG, Muir TW. Chemically
ubiquitylated
histone
H2B
stimulates
hDot1L-mediated
intranucleosomal methylation. Nature 2009; 453: 812–816.
12 Ekkebus R, Kasteren SIV, Kulathu Y, Scholten A, Berlin I, Geurink PP,
Jong A, Goerdayal S, Neefjes J, Heck AJR, Komander D, Ovva H. On
terminal alkynes that can react with active-site cysteine nucleophiles
in proteases. J. Am. Chem. Soc. 2013; 135: 2867–2870.
13 Oualid FE, Merkx R, Ekkebus R, Hameed DS, Smit JJ, Jone A, Hilkmann
H, Sixma TK, Ovva H. Chemical synthesis of ubiquitin, ubiquitin-based
probes, and diubiquitin. Angew. Chem. Int. Ed. 2010; 49: 10149–10153.
14 Ye Y, Blaser G, Horrocks MH, Ruedas-Rama MJ, Ibrahim S, Zhukov AA,
Orte A, Klenerman D, Jackson SE, Komander D. Ubiquitin chain con-
formation regulates recognition and activity of interacting proteins.
Nature 2012; 492: 266–270.
Figure 4. Enzymatic activity of Ub-AMC. The black spots represent the
change of fluorescence in the absence of UCH-L3. The red spots represent
the change of fluorescence in the presence of UCH-L3.
15 Eger S, Scheffner M, Marx A, Rubini M. Synthesis of defined ubiquitin
dimers. J. Am. Chem. Soc. 2010; 132: 16337–16339.
16 Virdee S, Kapadnis PB, Elliott T, Lang K, Madrzak J, Nguyen DP,
Riechmann L, Chin JW. Traceless and site-specific ubiquitination of
recombinant proteins. J. Am. Chem. Soc. 2011; 133: 10708–10711.
17 Ohayon S, Spasser L, Aharoni A, Brik A. Targeting deubiquitinases
enabled by chemical synthesis of proteins. J. Am. Chem. Soc. 2012;
134: 3281–3289.
Summary
The C-terminal conjugates of ubiquitin are useful biochemical
probes for the study of the DUBs. In the present study, we
described a new synthesis of a representative Ub C-terminal
conjugate, i.e. Ub-AMC. Our synthesis involved the use of
recombinant Ub(1–75)-NHNH2 that is produced through the
hydrozinolysis of the Ub(1–75)-intein fusion protein. It was found
that Ub(1–75)-NHNH2 can be efficiently ligated with an auxiliary-
modified glycine derivative through an in situ NaNO2 activation
and thiolysis process. Overall, our experiments showed that the
new approach can generate bioactive Ub-AMC (and presumably,
other Ub C-terminal conjugates) in practical quantities [41]. Our
work also provided evidence that the ligation of peptide
hydrazides can be smoothly extended to non-Cys amino acid
residues that carry a removable thiol auxiliary.
18 Chen JJ, Ai YX, Wang JL, Haracska L, Zhuang ZH. Chemically
ubiquitylated PCNA as a probe for eukaryotic translesion DNA synthesis.
Nat. Chem. Biol. 2010; 6: 270–272.
19 Virdee S, Ye Y, Nguyen DP, Komander D, Chin JW. Engeineered
diubiquitin synthesis reveals Lys29-isopeptide specificity of an OTU
deubiquitinase. Nat. Chem. Biol. 2010; 6: 750–757.
20 Valkevich EM, Guenette RG, Sanchez NA, Chen YC, Ge Y, Strieter ER.
Forging isopeptide bonds using thiol-ene chemistry: site-specific coupling
of ubiquitin molecules for studying the activity of isopeptidases. J. Am.
Chem. Soc. 2012; 134: 6916–6919.
21 Love KR, Pandya RK, Spooner E, Ploegh HL. Ubiquitin C-terminal elec-
trophiles are activity-based probes for identification and mechanistic
study of ubiquitin conjugating machinery. ACS Chem. Biol. 2009; 4:
275–287.
22 Lu XQ, Olsen SK, Capili AD, Cisar JS, Lima CD, Tan DS. Designed
semisynthetic protein inhibitors of Ub/Ub1 E1 activating enzymes.
J. Am. Chem. Soc. 2010; 132: 1748–1749.
23 McGinty RK, Kohn M, Chatterjee C, Chiang KP, Pratt MP, Muir TW.
Structure-activity analysis of semisynthetic nucleosomes: mechanistic
insights into the stimulation of Dot1L by ubiquitylated histone H2B.
ACS Chem. Biol. 2009; 4: 958–968.
Acknowledgements
This study was supported by the National Basic Research Program
of China (973 program, No. 2013CB932800), the National Natural
Science Foundation of China (Grants No. 91013007), the National
24 Dawson PE, Muir TW, Lewis IC, Kent SBH. Synthesis of protein by
native chemical ligation. Science 1994; 266: 776–779.
wileyonlinelibrary.com/journal/jpepsci Copyright © 2013 European Peptide Society and John Wiley & Sons, Ltd. J. Pept. Sci. 2014; 20: 102–107

SEMI-SYNTHESIS OF Ub-AMC
25 Liu CF, Tam JP. Peptide segment ligation strategy without use of
protecting groups. Proc. Natl. Acad. Sci. U. S. A. 1994; 91: 6584–6588.
26 Muir TW, Sondhi D, Cole PA. Expressed protein ligation: a general
method for protein engineering. Proc. Natl. Acad. Sci. U. S. A. 1998;
95: 6705–6710.
27 Bang D, Makhatadze GI, Tereshko V, Kossiakoff AA, Kent SB. Total
chemical synthesis and X-ray crystal structure of a protein diastereo-
mer: [D-Gln 35] ubiquitin. Angew. Chem. Int. Ed. 2005; 44: 3852–3856.
28 Yang RL, Pasunooti KK, Li FP, Liu XW, Liu CF. Synthesis of K48-linked
diubiquitin using dual native chemical ligation at lysine. Chem.
Commun. 2010; 46: 7199–7201.
33 Fang GM, Li YM, Shen F, Huang YC, Li JB, Lin Y, Cui HK, Liu L. Protein
chemical synthesis by ligation of peptide hydrazides. Angew. Chem.
Int. Ed. 2011; 50: 7645–7649.
34 Li YM, Yang MY, Huang YC, Li YT, Chen PR, Liu L. Ligation of expressed
protein α-hydrazides via genetic incorporation of an α-hydroxy acid.
ACS Chem. Biol. 2012; 7: 1015–1022.
35 Canne LE, Bark SJ, Kent SBH. Extending the applicability of native
chemical ligation. J. Am. Chem. Soc. 1996; 118: 5891–5896.
36 Low DW, Hill MG, Carrasco MR, Kent SBH, Botti P. Total synthesis of
cytochrome b562 by native chemical ligation using a removable
auxiliary. Proc. Natl. Acad. Sci. U. S. A. 2001; 98: 6554–6559.
37 Botti P, Michale RC, Kent SBH. Native chemical ligation using removable
Na-(1-phenyl-2-mercaptoethyl) auxiliaries. Tetrahedron Lett. 2001; 42:
1831–1833.
29 Li X, Fekner T, Ottesen JJ, Chan MK. A pyrrolysine analogue for site-
specific protein ubiquitination. Angew. Chem. Int. Ed. 2009; 48:
9184–9187.
30 Sommer S, Weikart ND, Brockmeyer A, Janning P, Mootz HD.
Expanded click conjugation of recombinant proteins with ubiquitin-
like modifiers reveals altered substrate preference of SUMO2-
modified Ubc9. Angew. Chem. Int. Ed. 2011; 50: 9888–9892.
31 Castaneda CA, Spasser L, Bavikar SN, Brik A, Fushmann D. Segmental
isotopic labeling of ubiquitin chains to unravel monomer-specific
molecular behavior. Angew. Chem. Int. Ed. 2011; 50: 11210–11214.
32 Bavikar SN, Spasser L, Haj-Yahya M, Karthikeyan SV, Kumar KSA, Brik A.
Chemical synthesis of ubiquitinated peptides with varying lengths
and types of ubiquitin chains to explore the activity of
deubiquitinases. Angew. Chem. Int. Ed. 2012; 51: 758–763.
38 Offer J, Boddy CNC, Dawson PE. Extending synthetic access to pro-
teins with a removable acyl transfer auxiliary. J. Am. Chem. Soc.
2002; 124: 4642–4646.
39 Brik A, Yang YY, Wong CH. Sugar-assisted glycopeptides ligation.
J. Am. Chem. Soc. 2006; 128: 5626–5627.
40 Chatterjee C, McGinty RK, Pellois JP, Muir TW. Auxiliary-mediated
site-specific peptide ubiquitylation. Angew. Chem. Int. Ed. 2007;
46: 2814–2818.
41 Liang J, Fang GM, Huang XL, Mei ZQ, Li J, Tian CL, Liu L. Chemical
synthesis of Ub-AMC via ligation of peptide hydrazides. Sci. China
Chem. 2013; 56: 1301–1306.
J. Pept. Sci. 2014; 20: 102–107 Copyright © 2013 European Peptide Society and John Wiley & Sons, Ltd. wileyonlinelibrary.com/journal/jpepsci