Selenolysine: A New Tool for Traceless Isopeptide Bond Formation

Article abstract of DOI:10.1002/chem.202000310

Despite their biological importance, post-translationally modified proteins are notoriously difficult to produce in a homogeneous fashion by using conventional expression systems. Chemical protein synthesis or semisynthesis offers a solution to this problem; however, traditional strategies often rely on sulfur-based chemistry that is incompatible with the presence of any cysteine residues in the target protein. To overcome these limitations, we present the design and synthesis of γ-selenolysine, a selenol-containing form of the commonly modified proteinogenic amino acid, lysine. The utility of γ-selenolysine is demonstrated with the traceless ligation of the small ubiquitin-like modifier protein, SUMO-1, to a peptide segment of human glucokinase. The resulting polypeptide is poised for native chemical ligation and chemoselective deselenization in the presence of unprotected cysteine residues. Selenolysine's straightforward synthesis and incorporation into synthetic peptides marks it as a universal handle for conjugating any ubiquitin-like modifying protein to its target.

Full text of DOI:10.1002/chem.202000310

A Journal of
Accepted Article
Title: Selenolysine: a new tool for traceless isopeptide bond formation
Authors: Rebecca Notis Dardashti, Shailesh Kumar, Shawn Sternisha,
Sai Reddy Post, Brian G. Miller, and Norman Metanis
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To be cited as: Chem. Eur. J. 10.1002/chem.202000310
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Selenolysine: a new tool for traceless isopeptide bond formation
Rebecca Notis Dardashti,[a] Shailesh Kumar,[a] Shawn M. Sternisha,[b] Post Sai Reddy,[a] Brian G.
Miller,[b] and Norman Metanis* [a]
[a]
Rebecca N. Dardashti, Dr. Shailesh Kumar, Dr. Post Sai Reddy, Prof. Norman Metanis
The Institute of Chemistry, The Hebrew University of Jerusalem
Edmond J. Safra Campus, Givat Ram, Jerusalem 91904, Israel
E-mail: metanis@mail.huji.ac.il
[b]
Dr. Shawn M. Sternisha, Prof. Brian G. Miller
Department of Chemistry and Biochemistry
Florida State University
Tallahassee, FL, USA 32306-4390
Supporting information for this article is given via a link at the end of the document.((Please delete this text if not appropriate))
Abstract: Despite their biological importance, post-translationally
modified proteins are notoriously difficult to produce in
sites of different PTMs.[4]
a
Additionally, Lys-mediated chemical ligation to form an isopeptide
bond, typically referred to as isopeptide chemical ligation (ICL),
has been used as a tool to conjugate post-translationally
modifying proteins to their targets. Previously, Muir and
coworkers reported ubiquitylated protein synthesis using a
modified Lys with an auxiliary that was removed post-ligation;[13]
more recently, the Liu group employed an auxiliary to produce di-
and tri-ubiquitin chains.[14] Other studies utilized orthogonal
protecting groups to incorporate isopeptide bonds on-resin,[15] or
installed dipeptides at desired modification sites to allow for
downstream ligation.[16] Brik and coworkers introduced δ-
thiolysine in the conjugation of ubiquitin and synthesis of
ubiquitylated proteins or ubiquitin chains.[17] Another Lys
derivative, γ-thiolysine, has been used as a handle in dual NCL
reactions,[18] in which the γ-thiolysine plays a role in both main
chain and isopeptide ligation. Global desulfurization of the final
products gives a native Lys at the ligation site (Scheme 1).
We sought to develop selenium-containing analogs of modified
Lys residues in order to facilitate traceless isopeptide bond
homogeneous fashion using conventional expression systems.
Chemical protein synthesis or semi-synthesis offers a solution to this
problem; however, traditional strategies often rely on sulfur-based
chemistry that is incompatible with the presence of any cysteine
residues in the target protein. To overcome these limitations, we
present the design and synthesis of γ-selenolysine, a selenol-
containing form of the commonly modified proteinogenic amino acid,
lysine. The utility of γ-selenolysine is demonstrated with the traceless
ligation of the small ubiquitin-like modifier protein, SUMO-1, to a
peptide segment of human glucokinase. The resulting polypeptide is
poised for native chemical ligation and chemoselective deselenization
in the presence of unprotected cysteine residues. Selenolysine’s
straightforward synthesis and incorporation into synthetic peptides
marks it as a universal handle for conjugating any ubiquitin-like
modifying protein to its target.
Following ribosomal translation and folding, expressed proteins
can undergo a great number of post-translational modifications
(PTMs) before fulfilling their biological functions.[1] These PTMs
instruct the cell to carry out a variety of regulatory activities
including transportation and degradation, to name a few.[2]
Additionally, Lys, which is one of the more reactive amino acids
under biological conditions, can be modified with post-
translationally modifying proteins such as ubiquitin and other
ubiquitin-like modifiers (Ubl).[3]
formation through ICL.
deselenization reaction in the presence of unprotected Cys
residues (Scheme 1) would give the native Lys.
A
subsequent chemoselective
O
NH2
NH
NH2
Ubl
SH
1) dual NCL & ICL
2) desulfurization
δ
1) ICL
2) desulfurization
γ
HS
Techniques for controlling PTMs in cellular expression are not yet
sufficiently well-developed to allow recombinant expression of
homogenously modified proteins.[4] Alternatively, some methods
for semi-selective modification of expressed proteins have been
developed, most employing Cys chemistry. For example,
dehydroalanine (Dha), a suitable electrophile for Michael addition
reactions, can be generated from Cys,[5] Ser,[6] thioethers,[7] and
several selenium-containing unnatural amino acids.[8] Michael
addition to the resulting Dha has been used to introduce many
small-molecule PTMs and entire PTM proteins, as in the case of
ubiquitylated α-globin.[9] However, a stereoselective method for
addition to Dha has yet to be developed,[9] a drawback in the chiral
world of biology.
N
H
N
H2N
H
O
O
O
γ-thiolysine
Yang et al. 2009
δ-thiolysine
Brik et al. 2009
1) NCL and/or ICL
2) deselenization
NH2
NH2
SeH
δ
γ
HSe
N
H
N
H
O
O
γ- or δ-selenolysine
this work
Chemical protein synthesis (CPS) and semi-synthesis, on the
other hand, offers a direct route to selectively modified proteins.[10]
Based on solid-phase peptide synthesis (SPPS)[11] and native
chemical ligation (NCL)[12], this approach allows the preparation
of modified proteins, including proteins with multiple
Scheme 1. Previously developed δ-thiolysine, by Brik,16, 17 and γ-thiolysine, by
Yang,19, 20 used for NCL and ICL followed by desulfurization for the conjugation
of Ubl proteins to Lys residues. Our designed γ/δ-selenolysines for NCL and/or
ICL followed by chemoselective deselenization for traceless conjugation of Ubl
proteins to Lys residues.
1
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O
O
H
N
O
The aforementioned γ/δ-thiolysines used in ICL/desulfurization
approaches are not compatible in the presence of proteins with
natural Cys residues in their sequences, unless orthogonal Cys
protecting groups are installed. This presents a true challenge in
expressed protein ligation (EPL),15 where installation of protected
Cys residues in Ubl proteins would require specialized expression
methods. In contrast, thanks to selenium’s unique chemical
properties, γ/δ-selenolysine (Se-Lys, Sek) could be used in an
ICL/deselenization approach in the target protein, without the risk
of desulfurizing natural Cys residues.[19]
Here we report the synthesis and use of γ-Se-Lys as a new tool
for traceless isopeptide bond formation. The design and synthesis
of δ-Se-Lys, which could not be completed due to spontaneous
Se-elimination, is described in the SI (Scheme S1).
To obtain a protected form of γ-Se-Lys suitable for SPPS, we
turned to work by Merkx et al., where an Fmoc-Lys[Sγ(StBu)-
Nε(Boc)]-OH was successfully synthesized from γ-chlorolysine.[20]
We opted to follow the previous protocol until selenium
incorporation, protecting the α-amino and carboxylate groups of
γ-chlorolysine with 9-BBN and then the ε-NH2 group with Boc to
give 2 (Scheme 2).
H2
N
NCL
Peptide 2
Peptide 1
Peptide 1
+
SR
Peptide 2
HX
O
XH
X = S or Se
NH-PG
NH-PG
O
O
desulfurization/
deselenization
H
N
H
N
1. deprotection
Peptide 2
Peptide 2
Peptide 1
Peptide 3
Peptide 1
O
2. ICL
O
O
XH
SR
Peptide 3
If X = S; Y = H
If X = Se; Y = SH
HN
Peptide 3
HN
SH
O
O
SH
Y
Scheme 3. γ-thiolysine[18] and γ-selenolysine as tools for dual NCL and ICL at
Lys. Selenolysine has the advantage of allowing chemoselective deselenization
in the presence of unprotected Cys residues.
The less sterically hindered ester in 12 was selectively converted
o
to an aldehyde with DIBAL-H in dry ether at -78 C, which was
treated with NaBH4 in EtOH to give alcohol 13 (68% for 2 steps).
The hydroxyl group in 13 was converted to mesylate using MsCl
and Et3N in CH2Cl2 followed by treatment with NaN3 in DMF at 80
oC, which afforded the azide compound 14 in 88% yield.
Reduction of azide 14 with Ph3P in MeOH, followed by treatment
with Alloc-Cl and Et3N, gave an Alloc-protected amine 15 (71%).
Conversion of di-Boc (15) to mono-Boc (16) with LiBr[25] was
carried out in CH3CN at 65 oC for 24 h in 98% yield. Finally,
hydrolysis of methyl ester with LiOH[17a] in aqueous THF gave the
carboxylic acid 17 in 97% yield (17 can be used in Boc-SPPS).
Compound 17 was treated with TFA in CH2Cl2 to give an amine-
salt, which was finally converted to Fmoc-protected amino acid 18
using Fmoc-OSu and aqueous Na2CO3 in 90% yield. The final
product, Fmoc-Lys[Seγ(Mob)-Nε(Alloc)]-OH, was achieved in 17%
overall yield for 13 steps of synthesis (Scheme 4).
Following successful main-chain NCL, the Alloc protecting group
can be removed off-resin with a ruthenium catalyst[26] and the
resulting peptide can be subjected to isopeptide ligation. Studies
showing the utility of Fmoc-Lys[Nε(Alloc)-Seγ(Mob)]-OH in dual
chemical ligation are currently underway.
Scheme 2. Synthesis of Fmoc-Lys[Seɤ(Mob)-Nɛ(Boc)]-OH
Nucleophilic substitution of Cl to Se in DMF was performed using
Na2Se2, generated in situ from Se powder, hydrazine
monohydrate, and NaOH.[21] The reaction gave the γ-diselenide
product 3 in 42% yield. A reduction with NaBH4 and protection
with p-methoxybenzyl chloride (Mob-Cl or PMB-Cl) gave 4 in 60%
yield. Finally, removal of 9-BBN and subsequent Fmoc-protection
gave the desired Fmoc-Lys[Seγ(Mob)-Nε(Boc)]-OH, 6, in 77%
yield (Scheme 2).
To assess the utility of g-Se-Lys in protein semi-synthesis, we
incorporated our first-generation Se-Lys 6 (Scheme 2) into post-
translationally modified human glucokinase (GCK). Prior studies
demonstrated that the pancreatic isoform of this key glucose
Following our successful synthesis of a protected Se-Lys building
block for SPPS, we set out to design an analog with an orthogonal
protecting group at the ε-NH2 site to allow for dual chemical
ligation. In the proposed application, Se-Lys with an orthogonal
protection at ε-NH2 would first be used in NCL along the main
peptide chain. After NCL, the protecting group of ε-NH2 would be
removed to allow for a second ligation, forming an isopeptide
bond with the desired PTM protein-thioester. Final deselenization
would give native Lys at the PTM site (Scheme 3). Disappointingly,
using γ-chlorolysine, we were unsuccessful in incorporating an
orthogonal protecting group on the ε-NH2. Instead, an alternative
route was pursued from known aldehyde 10, which can be
obtained from L-aspartic acid precursor in 3 steps (Scheme 4).[22]
In this synthesis, the aldehyde 10 was treated with methyl
(triphenylphosphoranylidene)acetate to
afford
the
α,β-
Scheme 4. Synthesis of Fmoc-Lys[Seɤ(Mob)-Nɛ(Alloc)]-OH.
unsaturated ester 11 (85% for 2 steps).[23] Michael addition on 11
with Mob-selenolate (generated in situ from (Mob- Se-)2 and
NaBH4)[24] gave compound 12 in 90% yield.
2
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homeostatic enzyme is modified by the small ubiquitin-like
modifier protein, SUMO-1, at one or more lysine residues near the
N-terminus.[27] Unfortunately, the structural and functional impact
of site-specific GCK SUMOylation has been difficult to assess due
to an inability to produce homogenously SUMOylated GCK. We
viewed the SUMO-1-GCK conjugate as an attractive target, since
both proteins contain unprotected cysteine residues, and thus
require the unique chemical properties of g-Se-Lys.
We devised a three-segment strategy to obtain semisynthetic
SUMOylated human GCK (Scheme 5). First, SPPS was used to
incorporate our Fmoc-protected g-Se-Lys 6 at residue 15*,[28]
within a peptide consisting of the first 18 residues of GCK, GCK(2-
19)-NHNH2 (Scheme 5, Figure S1). This peptide also contained a
C-terminal masked thioester for downstream use in NCL.[29] Next,
SUMO-1 was produced as a C-terminal fusion with the MxeGyrA
C-terminal intein (Figures S2 and S3),[30] which was subsequently
converted to its 2-mercaptoethane sulfonic acid thioester (SUMO-
1-Mes) for ICL (Figure S4, details and method in SI). Following
Scheme 5. Proposed synthesis of the semisynthetic SUMOylated GCK.
ICL,
the
product
underwent
a
one-pot
deselenization/thioesterification (Figure S5), giving an overall
yield of 12.3% of the GCK(2-19) segment linked to SUMO-1 via
an isopeptide bond at Lys15 (Scheme 5, Figure 1).
We expected the ligation step to proceed efficiently, as the size of
the amino acid adjacent to the thioester typically dictates the rate
of the ligation reaction.[31] SUMO-1 contains a desirable Gly-Gly
sequence adjacent to the thioester. However, the ligation step
was surprisingly slow. SUMO-1-Mes was converted to the more
reactive SUMO-1-MPAA thioester within 1 hour. Yet, ligation of
this adduct with the small GCK peptide took up to 22 hours to
complete despite a large excess of the peptide and the presence
of TCEP and sodium ascorbate, which are known to accelerate
the selenium-mediated NCL reaction.[19b, 32] It is unlikely that the
γ-position of the selenol contributes to a slower rate of ligation,
based on a previous report indicating that this has no impact on
ligation rate.[33] Instead, a direct comparison of isopeptide ligation
in the synthesis of diubiquitin with γ- and δ-thiolysine also showed
a slower-than-expected rate of ligation.[20] In fact, the pKa of the
peptide’s amino groups is likely to blame, as the N-terminal α-
amino group of a peptide has an average pKa of 7.7[34], while the
e-amino group of Lys has a pKa of 10.5[35], making it significantly
less reactive at pH 7, the optimized pH for NCL. Together, these
observations suggest that the inefficiency lies in the nature of
isopeptide bond formation, as the first, typically rate-determining
nucleophilic attack in ICL is performed by the less reactive e-
amine.
Figure 1. A. HPLC traces of one-pot SUMO-1-GCK deselenization and
thioesterification over time. 1 is SUMO-1-GCK(2-19)(K15Sek)-NHNH2, 2 is
SUMO-1-GCK(2-19)-NHNH2, 3 shows two peaks, both with the mass of the first
intermediate in thioesterification (Schiff base),[29b] 4 is the final SUMO-GCK(2-
Due to the slow rate of ligation, a significant portion of SUMO-1-
MPAA underwent hydrolysis to inactive SUMO-1-OH rather than
to the desired ligation. As a result, the maximum yield we
observed was 22% for the ligation step, which severely limited the
expected yield of the overall strategy. Notably, for the subsequent
deselenization reaction, the unprotected, native Cys52 of SUMO-
1 was unaffected by this procedure[16a] and unmasking of the
19)-MPAA, and
5 is the second intermediate in thioesterification (Knorr
pyrazole).[29b] * shows a mass that most likely is truncated protein from
expression of the intein, which coeluted with all intermediate products. The slow
rate of thioesterification is attributed to a C-terminal Ile[31]; B. HPLC trace for the
* Using online analysis programs such as SUMOplotTM or SUMOsp 2.0, which
predict possible SUMOylation sites, K12 and K15 are predicted as likely
SUMOylation sites in the N-terminus of pancreatic GCK.[27b, 28a] The first site,
K12, is part of SUMO consensus motif ψ-K-x-D/E, where ψ is a large
hydrophobic amino acid, K is the lysine at which the SUMO is linked, x is any
amino acid and D/E is an acidic amino acid (Asp or Glu, respectively).[28c] The
second SUMOylation site is K15, which is not part of a typical SUMO consensus
motif. Both analysis programs did not predict K13 as a possible SUMOylation
site. In early crystal structures of GCK (PDB entries 1V4S, 1V4T)[28b] the N-
terminus was truncated to facilitate crystallization. In these structures the N-
terminal region of GCK, which begins with Lys12, adopts an α-helix.[28d]
Although K12, K13 and K15 are adjacent residues, each residue would face a
different environment as part of the α-helix, with some inaccessible to the
SUMOylation system. Indeed, based on a more recent crystal structure (PDB
entry 3IDH),[28d] only K15 is solvent exposed, while K12 and K13 are on the
opposite face of the helix and make electrostatic and hydrogen bonding
interactions with the rest of the protein. Based on these observations, we initially
focused on Lys15 as the SUMOylation site in pancreatic GCK; however, our
method should be applicable to Lys12 or any Lys residue in a protein sequence.
3
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final product SUMO-GCK(2-19)-MPAA, and C. Overlay of the simulated HR-MS
(above) and deconvoluted HR-MS (below).
[8]
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peptide’s cryptic thioester provided a segment poised for NCL.
As a final step in the preparation of SUMOylated human GCK, we
recombinantly produced a human GCK variant containing a
Factor Xa recognition sequence directly upstream of a Cys at
position 20 (Scheme 5, and Figure S6). Following purification and
Factor Xa digestion, this procedure yielded a truncated, N-
terminal Cys containing GCK variant, Δ20CysGCK, that displayed
wild-type kinetic characteristics (Figure S7). Unfortunately, efforts
to perform NCL between Δ20CysGCK and the SUMO-1-GCK(1-
19)-COSR segment proved unsuccessful. We observed no
ligation after prolonged incubation of the reaction components in
the absence of denaturants, whereas inclusion of guanidium
hydrochloride in the ligation buffer caused extensive precipitation
of the protein, as has been reported earlier.[36] Nonetheless, we
expect that the methods presented here will provide a route
towards Cys-rich Ubl-protein conjugates. Further exploration of
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using the orthogonal tRNA synthetase/tRNA pairs, as has been
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Wang, Q. Zheng, Y. Huang, J. Wang, L. Liu, J Am Chem Soc 2016, 138,
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In summary, lysine, often a site of ubiquitylation, SUMOylation,
and other Ubl protein PTM conjugations, was designed with an
artificial γ-selenol moiety. The resulting amino acid was
successfully used for traceless isopeptide ligation of SUMO-1 on
a peptide of its natural target protein, human glucokinase.
Additionally,
a selenolysine analog, possessing orthogonal
protecting groups to be used in traceless dual chemical ligation,
was designed and synthesized. Selenolysine presents an
attractive building block in the synthesis of post-translationally
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We thank the Dr. Tsafi Danieli, Dr. Mario Lebendiker, and Dr. Yael
Keren for their assistance in SUMO-1-COSR expression and
purification in the Wolfson Centre for Applied Structural Biology at
HUJI. Thanks to Dr. Bill Breuer of the Mass Spectrometry Unit at
the Institute of Life Sciences, HUJI for assistance with HR-MS.
We wish to thank Prof. Dr. Huib Ovaa (Leiden University Medical
Center, The Netherlands) for the generous gift of chlorolysine.
RND was supported by the Kaete Klausner Ph.D. scholarship, SK
was supported by PBC fellowship for postdocs, NM
acknowledges the support of ISF (783/18) and the US-Israel
Binational Science Foundation (BSF) (2014167), and BGM
acknowledges the support of the NIH (GM115388).
Keywords: Chemical protein synthesis • post-translational modification •
deselenization • expressed protein ligation • SUMOylation
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COMMUNICATION
WILEY-VCH
Entry for the Table of Contents
Insert graphic for Table of Contents here. ((Please ensure your graphic is in one of following formats))
SH
O
SH
O
NH2
NH
Ubl
SR
Ubl
γ
1) NCL and/or ICL
2) Deselenization
HSe
N
H
N
H
O
O
Ubl-protein
conjugate
γ-selenolysine
Insert text for Table of Contents here. The design and synthesis of γ-selenolysine, a selenol-containing form of the commonly
modified proteinogenic amino acid, lysine is reported. The utility of γ-selenolysine is demonstrated with the traceless ligation of the
small ubiquitin-like modifier protein, SUMO-1, to a peptide segment of human glucokinase.
6
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