Palladium Mediated Rapid Deprotection of N-Terminal Cysteine under Native Chemical Ligation Conditions for the Efficient Preparation of Synthetically Challenging Proteins

Article abstract of DOI:10.1021/jacs.5b13580

Facilitating the process of chemical protein synthesis is an important goal in order to enable the efficient preparation of large and novel protein analogues. Native chemical ligation, which is widely used in the synthesis and semisynthesis of proteins, has been going through several developments to expedite the synthetic process and to obtain the target protein in high yield. A key aspect of this approach is the utilization of protecting groups for the N-terminal Cys in the middle fragments, which bear simultaneously the two reactive groups, i.e., N-terminal Cys and C-terminal thioester. Despite important progress in this area, as has been demonstrated in the use of thiazolidine protecting group in the synthesis of over 100 proteins, finding optimal protecting group(s) remains a challenge. For example, the thiazolidine removal step is very slow (>8 h), and in some cases the applied conditions lead to undesired side reactions. Here we show that water-soluble palladium(II) complexes are excellent reagents for the effective unmasking of thiazolidine, enabling its complete removal within 15 min under native chemical ligation conditions. Moreover, palladium is also able to rapidly remove propargyloxycarbonyl-protecting group from the N-terminal Cys in a similar efficiency. The utility of the new removal conditions for both protecting groups is exemplified in the rapid and efficient synthesis of Lys34-ubiquitinated H2B and for the first time neddlyated peptides derived from cullin1. The current approach expands the use of palladium in protein chemistry and should significantly facilitate the chemical and semisynthesis of synthetically challenging proteins from multiple fragments.

Full text of DOI:10.1021/jacs.5b13580

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Article
Palladium Mediated Rapid Deprotection of N-terminal
Cysteine Under Native Chemical Ligation Conditions for the
Efficient Preparation of Synthetically Challenging Proteins
Muhammad Jbara, Suman Kumar Maity, Mallikanti Seenaiah, and Ashraf Brik
J. Am. Chem. Soc., Just Accepted Manuscript • DOI: 10.1021/jacs.5b13580 • Publication Date (Web): 29 Mar 2016
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Palladium Mediated Rapid Deprotection of N-terminal Cyste-
ine Under Native Chemical Ligation Conditions for the Effi-
cient Preparation of Synthetically Challenging Proteins
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‡
Muhammad Jbara, Suman Kumar Maity, Mallikanti Seenaiah and Ashraf Brik*
‡
Schulich Faculty of Chemistry, Technion-Israel Institute of Technology Haifa, 3200008 (Israel)
ABSTRACT: Facilitating the process of chemical protein synthesis is an important goal in order to enable the efficient
preparation of large and novel protein analogues. Native chemical ligation, which is widely used in the synthesis and
semisynthesis of proteins, has been going through several developments to expedite the synthetic process and to ob-
tain the target protein in high yield. A key aspect of this approach is the utilization of protecting groups for the N-
terminal Cys in the middle fragments, which bear simultaneously the two reactive groups i.e. N-terminal Cys and C-
terminal thioester. Despite important progress in this area, as has been demonstrated in the use of thiazolidine protect-
ing group in the synthesis of over 100 proteins, finding optimal protecting group(s) remains a challenge. For example,
the thiazolidine removal step is very slow (>8 hours) and in some cases the applied conditions lead to undesired side
reactions. Here we show that water-soluble palladium (II) complexes are excellent reagents for the effective unmasking
of thiazolidine, enabling its complete removal within 15 min under native chemical ligation conditions. Moreover, palla-
dium is also able to rapidly remove propargyloxycarbonyl-protecting group from the N-terminal Cys in a similar efficien-
cy. The utility of the new removal conditions for both protecting groups is exemplified in the rapid and efficient synthesis
of Lys34-ubiquitinated H2B and for the first time neddlyated peptides derived from cullin1. The current approach ex-
pands the use of palladium in protein chemistry and should significantly facilitate the chemical and semisynthesis of
synthetically challenging proteins from multiple fragments.
7
Introduction: The chemical protein synthesis field is
witnessing important progresses both at the application
level and method developments. The latter is particularly
important in order to streamline the synthetic process
and enables facile access of large and novel protein
analogues. The most effective peptide ligation methods
are those that based on installing unique reactive func-
tionalities at the C- and N-termini of unprotected pep-
thiazolidine protecting group (Thz). Despite this, protein
chemists continue to search for an optimal protecting
group for the middle fragments and new ones have been
recently developed such as p-boronobenzyloxycarbonyl
8
9
group (Dobz),
(dimethylamino) phenacyloxycarbonyl group (Mapoc)
N-acetoacetyl group (AcA),
4-
1
0
1
1
and azide. Despite these efforts, the protecting groups
suffer either from the use of harsh removal conditions
(e.g. Hg(OAc) for Acm, pH 13-14 for Msc), require-
ment of peptide isolation prior to the removal step (e.g.
Acm) and/or special preparations (e.g. Mapoc).
1
5-6
tides. Despite important advancements being made in
these directions, native chemical ligation (NCL) remains
2
2
1
0
the method of choice when attempting the ligation of
unprotected peptides in aqueous media. NCL utilizes N-
terminal thiol modified amino acid (e.g. Cys) from the
first peptide and C-terminal thioester functionality from a
second peptide, which chemoselectivley react to form an
Since its first demonstration in chemical protein synthe-
7
a
sis by Kent and coworkers, Thz is considered the pro-
tecting group of choice for many laboratories when at-
tempting protein synthesis from three or more fragments.
Remarkably, Thz has been used in the synthesis of over
hundred different proteins, such as HIV-1 protease and
native and modified alpha-synuclein,14
human parathyroid hormone-related protein (hPTHrP),
different forms of erythropoietin (EPO), several ubiq-
uitinated proteins and modified histones. The
3
amide bond at the ligation junction.
The synthesis of medium to large proteins (>120 amino
acids) often requires three or more peptide fragments
wherein the middle fragment(s) must bear both the N-
terminal thiol modified amino acid and the thioester func-
4
c,12
13
its analogues,
4b,15
4
13b,13c,16
17
tionality. As a result, the N-terminal thiol modified amino
acid must be protected to avoid peptide polymerization
and/or cyclization. Several protecting groups have been
used for this goal including methylsulfonylethyloxycar-
wide utility of this protecting group is mainly due to its
removal conditions, which is achieved by adjusting the
pH of the ligation mixture to ~ 4 in presence of a large
excess of methoxylamine. This is in addition to the
5
6
bonyl amine (Msc), acetamidomethyl (Acm), and the
1
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commercial availability of the thiazolidine-protected form
of Cys and its straightforward installation in thiol modified
followed by the addition of 1,4-dithiothreitol (DTT) to
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quench the Pd species as well as to release any bound
Pd to the peptide. This led to a complete conversion of
the Proc-Cys-peptide-1 to Cys-peptide-1, within 1 hour.
Under these conditions no racemization of the N-terminal
Cys was observed (Supporting Information).
1
7c,18
amino acids.
However, the deprotection step is slow
and requires long period of reaction (>8 hours), which
might lead to accumulation of side reactions and slow
down the synthetic process of the final target. In the case
of peptide thioester bearing C-terminal Gly residue,
methoxylamine has been reported to react with the thi-
We then examined if the Proc protecting group can be
removed under NCL conditions in particular in the pres-
ence of the ligation additives (i.e. phosphine and thiols)
as well as the high concentration of guanidine, which is
known to chelate Pd(II) in aqueous media and affect its
1
7b,19
oester peptide when one-pot ligation is employed.
In
addition, it has been reported during the oxidative
switching of hydrazide peptide, that the Thz was found to
be unstable. Hence, a more facile deprotection of the
0
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reactivity. For this, two model peptides were prepared,
where the first having N-terminal Cys, Cys-peptide-1 and
the other have both N-terminal Proc-Cys and C-terminal
thioester functionality Proc-Cys-peptide-1-SR, (R = Me-
thyl 3-mercaptopropionate). The two fragments were
ligated under NCL conditions in presence of 20 equiv of
4-mercaptophenylacetic acid (MPAA) and 10 equiv of
Thz under NCL conditions is desired. In addition, a new
protecting group, which can be orthogonal or comple-
mentary to the Thz and removed efficiently under NCL
conditions in one-pot manner, could also find important
applications in protein synthesis.
Here we report that water-soluble palladium (II) com-
plexes are excellent reagents for the effective unmasking
of Thz, enabling its complete removal within 15 min un-
der NCL conditions. Moreover, these complexes are able
to rapidly remove propargyloxycarbonyl group (Proc)
from the N-terminus Cys of the middle fragment pep-
tides. The utility of these novel conditions for Thz and
Proc removal from the N-terminal Cys of the middle
fragment is exemplified in the rapid and efficient synthe-
sis of Lys34-ubiquitinated H2B and for the first time
neddlyated peptides derived from cullin1.
28
Tris(2-carboxyethyl)phosphine (TCEP). Upon comple-
tion of the ligation reaction and without isolation or
changing the reaction conditions, 20 equiv of
[Pd(allyl)Cl] was added to the reaction mixture. Howev-
2
er, under these conditions the reaction was extremely
slow, presumably due to the presence of MPAA and
TCEP, which could interfere with the metal reactivity.
Notably, with the addition of extra 20 equiv of
[Pd(allyl)Cl] , a quantitative deprotection of the ligation
2
product was observed within 60 min, which was con-
firmed by HPLC-MS analysis (Figure 1).
Results and Discussion: The use of palladium in pro-
tein chemistry has been documented in various elegant
studies such as chemical protein modification based on
2
1
aryl/alkenyl halides and other coupling partners e.g.
alkenes 2(3Mizoroki–Heck),
2
2
boronic 24acids (Suzuki–
Miyaura), and alkynes (Sonogashira). Recently, it has
been shown that 2-dicyclohexylphosphino-29,69-
diisopropoxybiphenyl
aryl
(RuPhos)Pd(Ar)X), (X=counter ion) complexes can be
palladium(II)
(
used for selective and rapid Cys modification in pro-
25
teins. In addition, we were intrigued by the recent use
of Pd(0) and Pd(II) complexes to convert chemically
caged Lys analogue to free ε-amine from the Proc-
protected moiety, which was used for masking the cellu-
2
6
lar activity of a Lys residue of an intact protein. We
wondered if such a protecting group can be also used to
mask the N-terminal Cys in the middle fragments during
protein synthesis.
To begin examine the utility of the Proc-protecting group,
we initially prepared eleven-mer peptide, Proc-Cys-
LYRAGLYRAG (peptide-1 = LYRAGLYRAG), wherein
the Proc-Cys was prepared in solution and coupled to
the N-terminus of the peptide by using standard Fmoc-
SPPS (Supporting Information). Alternatively, the Proc
protecting group could be also incorporated on solid
support by reacting the free N-terminus with propargyl
chlorofromate. Peptides with excellent crude purities
were obtained demonstrating that the Proc protecting
group is stable to Fmoc-SPPS. Next, we examined the
Proc removal from the N-terminal Cys by exposing the
peptide to 20 equiv allylpalladium chloride dimer
Figure 1. A) Analytical HPLC and mass analysis of ligat-
ed proc-peptide-2 with the observed mass 2548.1 Da ±
0.6 Da, calcd 2548.9 Da, (average isotopes). B) Analyti-
cal HPLC and mass analysis of deptotected peptide-2
[
Pd(allyl)Cl] reagent (in 6 M Gnd.HCl, pH ~7.2, 37 °C)
2
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with the observed mass 2465.6 Da ± 0.1 Da, calcd
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2
465.9
LYRAGLYRAG,
Da,
(average
isotopes).
Peptide-1:
Peptide-2:
Methyl 3-
CLYRAGLYRAGCLYRAGLYRAG.
mercaptopropionate.
R
=
Encouraged by these results, we then aimed to test the
applicability of this protected group in protein synthesis
and examined it in the total chemical synthesis of ubiqui-
tinated H2B at Lys 34 (H2BK34Ub). This mark has been
proposed to stimulate H3K79 and K4 methylation in vivo
and in vitro through trans-tail regulation, however, the
effect of H2BK34Ub on the regulation of chromatin struc-
ture and the interacting proteins still unclear. The syn-
thesis of this challenging target was recently reported by
our group and was used to learn about the interacting
proteins with ubiquitinated nucleosome.
our approach suffered from multiple purification steps,
which prolonged the time of synthesis and negatively
affected the total yield.
this ubiquitinated histone will assist in dissecting the role
of ubiquitination at this site in chromatin context.
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7a,30
However,
17a,17b
More efficient synthesis of
The previous synthesis of H2BK34Ub depends on using
Acm for the N-terminal Cys as an orthogonal protecting
group of δ-mercaptolysine in fragment 5 (Figure 2).
17a,17b
Figure 2. A) Synthesis of HA-H2BK34Ub: A) Analytical
HPLC and mass analysis of ligated product Proc-
H2B(22-125)K34Ub with the observed mass 20709.5 Da
±
3.2 Da, calcd 20714.5 Da, (average isotopes). B) Ana-
lytical HPLC and mass analysis of deptotected H2B(22-
125)K34Ub with the observed mass 20631.0 ± 3.0 Da,
calcd 20631.5 Da, (average isotopes). Nbz = N-acyl-
benzimidazolinone.
After ubiquitination of Lys34, the ligation product 6 had
to be isolated to enable Acm removal using Ag(OAc) in
2
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0% acetic acid (Figure 2). New synthesis based on terminal Cys was observed (Supporting Information).
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Proc-Cys as an orthogonal protecting group should in
principle overcome this additional purification step where
the removal of the Proc can be achieved directly after
ligation. The key synthetic intermediate 6 was achieved
from one-pot ligation of three fragments employed also
methoxylamine treatment. Notably the Proc protecting
group was stable during the removal of Thz protecting
group of the thiolysine using methoxylamine. Purified 6b
was ligated with Ub-thioester in presence of MPAA and
Notably, when the Thz protected peptide-1 was exposed
to the same Pd complex, however without MPAA and
TCEP, the removal was extremely slow and showed only
40% removal after 4 hours. When adding MPAA or
TCEP, separately, 50-70% of the deprotected peptide
was obtained within 30 min, compared to a complete
removal within 15 min when both TCEP and MPPA are
present. This indicates that both ligation additives, which
are essential part of the NCL conditions, are important
TCEP for
[
3
Pd(allyl)Cl] for 1 hour. The desired product 7 was ob-
hours followed by the addition of
for more efficient removal probably by chelating/reducing
to
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32
2
Pd(II)
Pd(0).
served, however, along with unidentified product (~50%)
having the additional mass of +32 Da, which did not re-
act further in the ligation reaction presumably due to
capping of the free Cys via unclear mechanism associ-
ated with the Pd complex. To try to overcome this, we
retuned back to our model peptides and examined vari-
ous Pd(II) complexes such as PdCl and Pd(OAc) . In
2
2
this case, PdCl gave much better results with a quanti-
2
tative conversion of the ligated product Proc-peptide-2
(peptide-2 = CLYRAGLYRAGCLYRAGLYRAG) to the
free form (NH -peptide-2) within 5 min (Supporting In-
2
formation), prompting us to test these conditions in the
H2BK34Ub synthesis. We were very pleased to find that
with PdCl the removal of the Proc was very rapid (5-10
2
min) and no +32 Da adduct was observed to give
(H2B(22-125)K34Ub) 7 in 40%. Ligation with fragment 4
followed by desulfurization, as we have previously re-
ported, gave the desired product 8, H2BK34Ub, in ~5%
overall yield, which is ~25% increase in the yield com-
pared to the previous synthesis, (Figure 2).
The previous observations in which the Thz group was
not stable during the oxidative switching of Thz-peptide-
NHNH to Thz-peptide-thioester via the acyl azide inter-
2
2
0
mediate prompted us to examine the stability of Proc-
Cys under these conditions. For this, we prepared five-
model peptides based on Proc-Cys-peptide-1-NHNH2
with different C-terminal amino acids. The Proc group in
all these peptides exhibited high stability under the
switching conditions of the C-terminal hydrazide to thioe-
ster. Further, we validated that such a thioester reacts
quantitatively in NCL. These results make the Proc
group a better choice than the Thz for some synthetic
applications involving NCL (Supporting Information).
Figure 3. A) Analytical HPLC and mass analysis of ligat-
ed Thz-peptide-2 with the observed mass 2477.6 Da ±
0
.6 Da, calcd 2477.9 Da, (average isotopes). B) Analyti-
During our work with Pd complexes in one of the model
systems we observed also removal of the Thz protecting
group, which promoted us to study further this reaction
using various Pd complexes, realizing the potential use
cal HPLC and mass analysis of deptotected peptide 2
with the observed mass 2465.9 Da ± 0.3 Da, calcd
2
465.9
LYRAGLYRAG,
Da,
(average
isotopes).
Peptide-1:
Peptide-2:
Methyl 3-
3
1
of this approach in protein synthesis. For this, two
eleven-mer peptides were prepared; the Cys-peptide-1
and the other having both N-terminal Thz and C-terminal
thioester, Thz-peptide-1-SR. The two peptides were li-
gated under NCL conditions in presence of MPAA and
TCEP. Upon completion of the ligation reaction and
CLYRAGLYRAGCLYRAGLYRAG.
mercaptopropionate.
R
=
To further shed light on this, we carried out the removal
without TCEP and MPAA but in presence of sodium
ascorbate, which is known to reduce Pd(II) to Pd(0).
2
6
without isolation, [Pd(allyl)Cl] was added to the ligation
2
We observed significant improvement in the Thz removal
where ~45 % of the deprotected peptide was obtained
within 30 min, supporting that Pd(0) might be the more
reactive species in the removal step. Efforts to directly
work with Pd(0) complexes e.g. Pd(dba) (dba = diben-
2
zylideneacetone), were not successful due to low solu-
mixture followed by DTT to give within 15 min, quantita-
tively the ligated product with the free Cys, (Figure 3).
When using the PdCl complex for the Thz removal the
2
reaction was slower and took around 60 min for comple-
tion. Under these conditions no racemization of the N-
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To further explore the utility of the new removal condi-
bility of this complex in the aqueous media and under
different conditions. Interestingly, in the case of the Proc
removal via PdCl we did not observe difference in the
2
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tions of Thz in the synthesis of challenging proteins, we
undertook the synthesis of neddlyated peptides derived
3
6
removal, with or without the ligation additive i.e. MPAA
and TCEP. This probably due to the different mecha-
nisms involved in the removal of the two different pro-
from cullin1. Neddlyation by the small Ub like protein
NEDD8 (neural precursor cell expressed developmental-
ly downregulated protein 8) has been mainly character-
ized as activating the largest E3-ligases family, known as
the cullin-Ring ligases. In this process, the target of
neddlyation is the cullin1 from the cullins family having
the conserved cullin1 domain. Despite its large se-
quence homology to Ub, NEDD8 expression is known to
be very low and chemical biology tools based on NEDD8
still lacking. This is in contrary to Ub where various con-
jugates and analogues are available and have become
extremely useful in studying ubiquitination and deubiqui-
tecting groups.
One has to note that in the described
conditions for the removal of the two protecting groups
with the different Pd complexes, it is unclear yet what is
the composition of the reactive Pd complex(s) due to
various possibilities of chelation of the ligation additives
to the Pd species(s). The depropargylation mechanism
was proposed to proceed via Pd(0) or/and Pd(II) com-
plexes involving Pd(0)allenylpalladium intermediate or
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26
Pd(II) hydration mechanism. On the other hand, we
propose a mechanism for Thz removal where the Pd
complex activates the sulfur atom of the Thz (a),
37
tination. Hence generating a toolbox based on NEDD8
could be highly valuable for the research community to
study neddylation and denddylation processes. To begin
this journey, we tested whether neddlyated peptides can
be synthesized where Pd can facilitate the rapid assem-
bly of these conjugates. To do so, we decided to
neddlyate 26-mer derived from cullin1 using a similar
approach for ubiquitinated peptides based on orthogonal
(Scheme 1), which initiates the ring opening by the nu-
cleophilic attack of a water molecule to form carbinola-
mine intermediate (b). This intermediate spontaneously
collapses to release free Cys and formaldehyde (c). The
7
b
latter can be trapped by DTT, which is added in large
excess to quench the reaction (Scheme 1).
3
8
protection of the neddlyated Lys (Lys720 of cullin) from
the peptide target, (Scheme 2).
Scheme 1. Proposed mechanism for Pd-assisted Thz
conversion to a free Cys:
Scheme 2. A) Sequence of NEDD8 (1-76) with high-
lighted ligation site. B) Sequence of Cullin (703-728)
with highlighted ligation site. C) Synthesis of
neddlyated cullin (703-728) and neddylated cullin
(
=
714-728). R = Methyl 3-mercaptopropionate. MeNbz
N-acyl-N-methylacylurea.
3
3
Recent efforts from different labs expanded NCL to
3
selenocysteine (Sec) in particular the use of Thz pro-
4
3
5
tected Sec to form selenazolidine (Sez). To examine if
our new approach could convert Sez to a free Sec, we
prepared peptide-1, having N-terminal Sez and C-
terminal thioester functionality, Sez-peptide-1-SR. This
peptide was ligated under NCL conditions in presence of
MPAA with, Cys-peptide-1. Upon completion of the liga-
tion reaction, [Pd(allyl)Cl] or PdCl2 was added to the
2
ligation mixture followed by DTT. Interestingly, despite
rapid disappearance of the starting material we did not
observe the desired product or the dimer oxidized prod-
uct, which is often observed in Sec based ligation. Ra-
ther unidentified products were obtained despite varia-
tions in the removal conditions. This might be due to
differences in the reactivity of Sec compared to Cys,
where it could after removal behave differently with Pd
under these conditions.
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For this synthesis we noted the presence of Ala57 in
NEDD8, which can be used as a ligation junction after
the synthesis of the cullin1 peptide having the isopeptide
bond linked to the C-terminal 19 amino acids of NEDD8.
On the other hand, the complementary part of NEDD8
can be prepared as a thioester. We also decided to
switch Ala714 from the cullin peptide to Thz, which after
full assembly of the neddlyated peptide can be opened
with [Pd(allyl)Cl] to enable further elongation of the cul-
2
lin1 peptide or modification by other groups such as a
fluorescence tag for uses in monitoring the reversible
reaction. Hence, peptide 10 was prepared directly on
solid support to give, after purification, the purified pep-
tide in 50 % yield, (Supporting Information). The com-
plementary part of NEDD8 peptide 9, and peptide 11
Figure 4. A) Analytical HPLC and mass analysis of ligat-
ed product of 9 & 10 with the observed mass 10449.5
Da ± 0.6 Da, calcd 10450.4 Da, (average isotopes). B)
Analytical HPLC and mass analysis of deptotected pep-
tide 12 with the observed mass 10437.8 ± 1.1 Da, calcd
were
(
prepared3
MeDbz) linker recently developed by the Dawson
using the o-amino(methyl)aniline
9
1
0438.4 Da, (average isotopes). C) Analytical HPLC
group to give these peptides in 40-50% yield, (Support-
ing Information). Having these peptides in hand, NCL
was applied between 9 and 10 in presence of MPAA and
and mass analysis of final ligated product with the ob-
served mass 11747.7 Da ± 0.4 Da, calcd 11748.0 Da,
(average isotopes). D) Analytical HPLC and mass analy-
sis of final desulfurized product 13 with the observed
mass 11682.9 Da ± 0.4 Da, calcd 11683.7 Da, (average
isotopes).
TCEP for
[
2 hours, followed by the addition of
Pd(allyl)Cl] for 15 min. The reaction was quenched by
2
DTT to give, after purification, peptide 12 in 45% isolated
yield. Peptide 12 was ligated with fragment 11, followed
4
0
by radical desulfurization to give, after purification, the
neddlyated cullin (703-728) peptide 13 in 30% yield,
(Figure 4).
We also studied the hydrolysis of neddylated cullin (714-
7
28) using ubiquitin C-terminal hydrolase isozyme 3
UCH-L3). This enzyme is known to process both Ub
(
and NEDD8 from their precursors, where the initial gene
products made from Ub or NEDD8 has a carboxyl-
terminal extension and the removal of these extensions
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is necessary before Ub or NEDD8 becomes a substrate
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for conjugation. Yet, it is still unclear if UCH-L3 can
remove NEDD from neddlyated proteins linked via an
ASSOCIATED CONTENT
3
6a
isopeptide bond. In our study UCH-L3 was also able to
remove NEDD8 from the 15-mer peptide, where ~42%
hydrolysis was obtained after 30 min incubation with the
enzyme, (Figure 5). These results suggest that longer
substrates linked to NEDD8 via an isopeptide bond
might be processed by UCH-L3.
Supporting Information. All experimental procedures, ana-
lytical data of synthetic small molecule peptides and pro-
teins, enzymatic hydrolysis studies. This material is availa-
ble free of charge via the Internet at http://pubs.acs.org.
AUTHOR INFORMATION
Corresponding Author
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*
E-mail: abrik@technion.ac.il
Author Contributions
These authors contributed equally.
‡
ACKNOWLEDGMENT
A. Brik is a Neubauer Professor and a Taub Fellow-
Supported by the Taub Foundations. SKM thanks the Israel
Council of Higher Education for a fellowship under the PBC
program. We thank Mr. Shay Laps for assisting in the syn-
thesis of some of the peptides.
REFERENCES
(
1) Schnolzer, M.; Kent, S. B. H. Science 1992, 256, 221.
(2) (a) Bode, J. W.; Fox, R. M.; Baucom, K. D. Angew.
Chem., Int. Ed. 2006, 45, 1248. (b) Zhang, Y.; Xu, C.; Lam, H.
Y.; Lee, C. L.; Li, X. Proc. Natl. Acad. Sci. U. S. A. 2013, 110,
6
657. (c) Raj, M.; Wu, H.; Blosser, S. L.; Vittoria, M. A.; Arora,
P. S. J. Am. Chem. Soc. 2015, 137, 6932.
3) Dawson, P. E.; Muir, T. W.; Clark-Lewis, I.; Kent, S. B.
(
Science 1994, 266, 776.
(4) (a) Weinstock, M. T.; Jacobsen, M. T.; Kay, M. S. Proc.
Natl. Acad. Sci. U. S. A. 2014, 111, 11679. (b) Wang, P.; Dong,
S.; Shieh, J. H.; Peguero, E.; Hendrickson, R.; Moore, M. A.;
Danishefsky, S. J. Science 2013, 342, 1357. (c) Torbeev, V. Y.;
Kent, S. B. Angew. Chem., Int. Ed. 2007, 46, 1667. (d) Sato, K.;
Shigenaga, A.; Kitakaze, K.; Sakamoto, K.; Tsuji, D.; Itoh, K.;
Otaka, A. Angew. Chem., Int. Ed. 2013, 52, 7855. (e) Kumar, K.
S.; Bavikar, S. N.; Spasser, L.; Moyal, T.; Ohayon, S.; Brik, A.
Angew. Chem., Int. Ed. 2011, 50, 6137.
Figure 5. Analytical HPLC and mass data of (A) purified
protein 14 with the observed mass 10373.8±0.2, calcd
0374.3 (average isotopes) and CD spectra of 14; (B) after
UCHL3 treatment where peak b corresponds to the cleaved
peptide fragment from cullin sequence with the observed
mass 1849.6±0.1, calcd 1850.4 (average isotopes), peak c
corresponds to cleaved NEDD8 with the observed mass
8541.8±0.2, calcd 8540.9 (average isotopes). # is bosine
sirum albumin (BSA).
1
(
5) Hackeng, T. M.; Griffin, J. H.; Dawson, P. E. Proc. Natl.
Acad. Sci. U. S. A. 1999, 96, 10068.
6) Canne, L. E.; Botti, P.; Simon, R. J.; Chen, Y. J.; Dennis,
(
E. A.; Kent, S. B. H. J. Am. Chem. Soc. 1999, 121, 8720.
(7) (a) Bang, D.; Kent, S. B. H. Angew. Chem., Int. Ed.
2004, 43, 2534. (b) Villain, M.; Vizzavona, J.; Rose, K. Chem.
Biol. 2001, 8, 673.
(8) Li, J. B.; Li, Y. Y.; He, Q. Q.; Li, Y. M.; Li, H. T.; Liu, L.
Org. Biomol. Chem. 2014, 12, 5435.
(9) Boll, E.; Ebran, J. P.; Drobecq, H.; El-Mahdi, O.; Raibaut,
L.; Ollivier, N.; Melnyk, O. Org. Lett. 2015, 17, 130.
(10) Ueda, S.; Fujita, M.; Tamamura, H.; Fujii, N.; Otaka, A.
Chembiochem 2005, 6, 1983.
(11) Pan, M.; He, Y.; Wen, M.; Wu, F. M.; Sun, D. M.; Li, S.
J.; Zhang, L. H.; Li, Y. M.; Tian, C. L. Chem. Commun. 2014,
50, 5837.
(12) (a) Torbeev, V. Y.; Raghuraman, H.; Hamelberg, D.;
Tonelli, M.; Westler, W. M.; Perozo, E.; Kent, S. B. H. Proc.
Natl. Acad. Sci. U. S. A. 2011, 108, 20982. (b) Torbeev, V. Y.;
Kent, S. B. H. Org. Biomol. Chem. 2012, 10, 5887.
Summary: We have discovered new deprotection meth-
od for the well-utilized Thz protecting group based on Pd
complexes. Moreover, for the first time we extended the
use of the Proc protecting group in chemical protein syn-
thesis and showed its compatibility to peptide synthesis
of the middle fragments. Importantly, the removal of both
protecting groups can be achieved under NCL conditions
without prior peptide isolation and is compatible with the
2
0 natural amino acids. The deprotection step for both
groups is rapid and can be achieved within minutes.
These conditions can be applied in the synthesis of
complex proteins as exemplified in the case of ubiqui-
tinated H2B and for the first synthesis of neddlyated pep-
tides. The ability to prepare neddylated peptides in high
efficiency opens new opportunities in studying this sys-
tem, which we are currently exploring. We believe that
the current approach will find important applications in
protein synthesis and facilitate the efficient chemical
preparation of complex proteins.
(
13) (a) Fauvet, B.; Butterfield, S. M.; Fuks, J.; Brik, A.;
Lashuel, H. A. Chem. Commun. 2013, 49, 9254. (b) Haj-Yahya,
M.; Fauvet, B.; Herman-Bachinsky, Y.; Hejjaoui, M.; Bavikar, S.
N.; Karthikeyan, S. V.; Ciechanover, A.; Lashuel, H. A.; Brik, A.
Proc. Natl. Acad. Sci. U. S. A. 2013, 110, 17726. (c) Hejjaoui,
7
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Page 8 of 9
M.; Haj-Yahya, M.; Kumar, K. S. A.; Brik, A.; Lashuel, H. A.
Angew. Chem., Int. Ed. 2011, 50, 405.
14) Shang, S. Y.; Tan, Z. P.; Danishefsky, S. J. Proc. Natl.
Acad. Sci. U. S. A. 2011, 108, 5986.
15) (a) Liu, S. H.; Pentelute, B. L.; Kent, S. B. H. Angew.
Chem., Int. Ed. 2012, 51, 993. (b) Murakami, M.; Okamoto, R.;
Izumi, M.; Kajihara, Y. Angew. Chem., Int. Ed. 2012, 51, 3567.
(16) Hemantha, H. P.; Bavikar, S. N.; Herman-Bachinsky,
Y.; Haj-Yahya, N.; Bondalapati, S.; Ciechanover, A.; Brik, A. J.
Am. Chem. Soc. 2014, 136, 2665.
(27) Li, S. H.; Lin, Y. J.; Cao, J. G.; Zhang, S. B. J. Org.
Chem. 2007, 72, 4067.
(28) Johnson, E. C. B.; Kent, S. B. H. J. Am. Chem. Soc.
2006, 128, 6640.
(29) Wu, L. P.; Zee, B. M.; Wang, Y. M.; Garcia, B. A.; Dou,
Y. L. Mol. Cell. 2011, 43, 132.
(30) Shema-Yaacoby, E.; Nikolov, M.; Haj-Yahya, M.;
Siman, P.; Allemand, E.; Yamaguchi, Y.; Muchardt, C.; Urlaub,
H.; Brik, A.; Oren, M.; Fischle, W. Cell. Rep. 2013, 4, 601.
(31) Kent and coworkers reported that during the synthesis
of neoglycopeptides applying click chemistry reaction in pres-
ence of Cu(I), the Thz was removed. The authors did not inves-
tigate this reaction further probably due to the difficulties in
working with Cu(I) in the context proteins, for more details see
this reference. Lee, D. J.; Mandal, K.; Harris, P. W. R.; Brimble,
M. A.; Kent, S. B. H. Org. Lett. 2009, 11, 5270.
1
2
3
4
5
6
7
8
9
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
(
(
(
17) (a) Siman, P.; Karthikeyan, S. V.; Nikolov, M.; Fischle,
W.; Brik, A. Angew. Chem., Int. Ed. 2013, 52, 8059. (b)
Seenaiah, M.; Jbara, M.; Mali, S. M.; Brik, A. Angew. Chem.,
Int. Ed. 2015, 54, 12374. (c) Fierz, B.; Kilic, S.; Hieb, A. R.;
Luger, K.; Muir, T. W. J. Am. Chem. Soc. 2012, 134, 19548.
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
(
18) (a) Haj-Yahya, M.; Kumar, K. S. A.; Erlich, L. A.; Brik, A.
Biopolymers 2010, 94, 504. (b) Sayers, J.; Thompson, R. E.;
Perry, K. J.; Malins, L. R.; Payne, R. J. Org. Lett. 2015, 17,
(32) Amatore, C.; Jutand, A.; Thuilliez, A. Organometallics
2001, 20, 3241.
4
902. (c) Creech, G. S.; Paresi, C.; Li, Y. M.; Danishefsky, S. J.
Proc. Natl. Acad. Sci. U. S. A. 2014, 111, 2891.
19) (a) Johnson, E. C. B.; Malito, E.; Shen, Y. Q.; Rich, D.;
Tang, W. J.; Kent, S. B. H. J. Am. Chem. Soc. 2007, 129,
1480. (b) Piontek, C.; Silva, D. V.; Heinlein, C.; Pohner, C.;
(33) (a) Metanis, N.; Keinan, E.; Dawson, P. E. Angew.
Chem., Int. Ed. 2010, 49, 7049. (b) Dery, S.; Reddy, P. S.;
Dery, L.; Mousa, R.; Dardashti, R. N.; Metanis, N. Chem. Sci.
2015, 6, 6207. (c) Malins, L. R.; Mitchell, N. J.; McGowan, S.;
Payne, R. J. Angew. Chem., Int. Ed. 2015, 54, 12716. (d)
Mitchell, N. J.; Malins, L. R.; Liu, X. Y.; Thompson, R. E.; Chan,
B.; Radom, L.; Payne, R. J. J. Am. Chem. Soc. 2015, 137,
14011.
(34) Malins, L. R.; Mitchell, N. J.; Payne, R. J. J. Pept. Sci.
2014, 20, 64.
(35) Reddy, P. S.; Dery, S.; Metanis, N. Angew. Chem., Int.
Ed. 2016, 55, 992.
(36) (a) Enchev, R. I.; Schulman, B. A.; Peter, M. Nat. Rev.
Mo.l Cel.l Biol. 2015, 16, 30. (b) Pan, Z. Q.; Kentsis, A.; Dias,
D. C.; Yamoah, K.; Wu, K. Oncogene 2004, 23, 1985.
(37) (a) Spasser, L.; Brik, A. Angew. Chem., Int. Ed. 2012,
51, 6840. (b) Weller, C. E.; Pilkerton, M. E.; Chatterjee, C. Bi-
opolymers 2014, 101, 144. (c) Abeywardana, T.; Pratt, M. R.
Chembiochem 2014, 15, 1547. (d) Pham, G. H.; Strieter, E. R.
Curr. Opin. Chem. Biol. 2015, 28, 57.
(38) Kumar, K. S. A.; Spasser, L.; Ohayon, S.; Erlich, L. A.;
Brik, A. Bioconjugate Chem. 2011, 22, 137.
(39) Blanco-Canosa, J. B.; Nardone, B.; Albericio, F.; Daw-
son, P. E. J. Am. Chem. Soc. 2015, 137, 7197.
(
1
Mezzato, S.; Ring, P.; Martin, A.; Schmid, F. X.; Unverzagt, C.
Angew. Chem., Int. Ed. 2009, 48, 1941.
(20) Fang, G. M.; Wang, J. X.; Liu, L. Angew. Chem., Int.
Ed. 2012, 51, 10347.
(
21) (a) Tilley, S. D.; Francis, M. B. J. Am. Chem. Soc. 2006,
128, 1080. (b) Li, J.; Chen, P. R. Chembiochem 2012, 13,
1728. (c) Spicer, C. D.; Davis, B. G. Nat. Commun. 2014, 5,
4740.
(22) Kodama, K.; Fukuzawa, S.; Nakayama, H.; Kigawa, T.;
Sakamoto, K.; Yabuki, T.; Matsuda, N.; Shirouzu, M.; Takio, K.;
Tachibana, K.; Yokoyama, S. Chembiochem 2006, 7, 134.
23) (a) Chalker, J. M.; Wood, C. S.; Davis, B. G. J. Am.
(
Chem. Soc. 2009, 131, 16346. (b) Ojida, A.; Tsutsumi, H.;
Kasagi, N.; Hamachi, I. Tetrahedron Lett. 2005, 46, 3301.
(24) (a) Kodama, K.; Fukuzawa, S.; Nakayama, H.; Sa-
kamoto, K.; Kigawa, T.; Yabuki, T.; Matsuda, N.; Shirouzu, M.;
Takio, K.; Yokoyama, S.; Tachibana, K. Chembiochem 2007, 8,
2
32. (b) Li, N.; Lim, R. K. V.; Edwardraja, S.; Lin, Q. J. Am.
Chem. Soc. 2011, 133, 15316.
25) Vinogradova, E. V.; Zhang, C.; Spokoyny, A. M.; Pen-
(
(40) Wan, Q.; Danishefsky, S. J. Angew. Chem., Int. Ed.
2007, 46, 9248.
(41) (a) Wada, H.; Kito, K.; Caskey, L. S.; Yeh, E. T. H.;
Kamitani, T. Biochem. Bioph. Res. Co. 1998, 251, 688. (b)
Linghu, B.; Callis, J.; Goebl, M. G. Eukaryot. Cell. 2002, 1, 491.
telute, B. L.; Buchwald, S. L. Nature 2015, 526, 687.
(26) Li, J.; Yu, J. T.; Zhao, J. Y.; Wang, J.; Zheng, S. Q.; Lin,
S. X.; Chen, L.; Yang, M. Y.; Jia, S.; Zhang, X. Y.; Chen, P. R.
Nat. Chem. 2014, 6, 352.
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