Chemically Induced Vinylphosphonothiolate Electrophiles for Thiol-Thiol Bioconjugations

Article abstract of DOI:10.1021/jacs.0c03426

Herein we introduce vinylphosphonothiolates as a new class of cysteine-selective electrophiles for protein labeling and the formation of stable protein-protein conjugates. We developed a straightforward synthetic route to convert nucleophilic thiols into electrophilic, thiol-selective vinylphosphonothiolates: In this protocol, intermediately formed disulfides can be chemoselectively substituted with vinylphosphonites under acidic conditions to yield the desired vinylphosphonothiolates. Notably, this reaction sequence enables the installation of vinylphosphonothiolate electrophiles directly on cysteine side chains within peptides and proteins. In addition to labeling the monoclonal antibody trastuzumab with excellent cysteine-selectivity, we applied our protocol for the site-specific conjugation of two proteins with unique cysteine residues yielding a nonhydrolyzable phosphonothiolate-linked diubiquitin and an ubiquitin-α-synuclein conjugate. The latter was recognized as a substrate in a subsequent enzymatic ubiquitination reaction.

Full text of DOI:10.1021/jacs.0c03426

Subscriber access provided by UNIV OF NEW ENGLAND ARMIDALE
Article
Chemically induced vinylphosphonothiolate
electrophiles for thiol-thiol bioconjugations
Alice Leonie Baumann, Sergej Schwagerus, Kevin Broi, Kristin Kemnitz-Hassanin,
Christian Ewald Stieger, Nils Trieloff, Peter Schmieder, and Christian P.R. Hackenberger
J. Am. Chem. Soc., Just Accepted Manuscript • Publication Date (Web): 27 Apr 2020
Downloaded from pubs.acs.org on April 27, 2020
Just Accepted
“Just Accepted” manuscripts have been peer-reviewed and accepted for publication. They are posted
online prior to technical editing, formatting for publication and author proofing. The American Chemical
Society provides “Just Accepted” as a service to the research community to expedite the dissemination
of scientific material as soon as possible after acceptance. “Just Accepted” manuscripts appear in
full in PDF format accompanied by an HTML abstract. “Just Accepted” manuscripts have been fully
peer reviewed, but should not be considered the official version of record. They are citable by the
Digital Object Identifier (DOI®). “Just Accepted” is an optional service offered to authors. Therefore,
the “Just Accepted” Web site may not include all articles that will be published in the journal. After
a manuscript is technically edited and formatted, it will be removed from the “Just Accepted” Web
site and published as an ASAP article. Note that technical editing may introduce minor changes
to the manuscript text and/or graphics which could affect content, and all legal disclaimers and
ethical guidelines that apply to the journal pertain. ACS cannot be held responsible for errors or
consequences arising from the use of information contained in these “Just Accepted” manuscripts.
is published by the American Chemical Society. 1155 Sixteenth Street N.W.,
Washington, DC 20036
Published by American Chemical Society. Copyright © American Chemical Society.
However, no copyright claim is made to original U.S. Government works, or works
produced by employees of any Commonwealth realm Crown government in the course
of their duties.

Page 1 of 16
Journal of the American Chemical Society
1
2
3
4
5
6
7
8
9
Chemically induced vinylphosphonothiolate electrophiles for
thiol-thiol bioconjugations
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
Alice L. Baumann‡,§,∥, Sergej Schwagerus‡,§,∥, Kevin Broi∥, Kristin Kemnitz-Hassanin§,
Christian E. Stieger§,∥, Nils Trieloff§, Peter Schmieder§, Christian P. R. Hackenberger§,∥
§
Leibniz-Forschungsinstitut für Molekulare Pharmakologie (FMP), Robert-Rössle-Straße 10, 13125 Berlin, Germany
∥
Department of Chemistry, Humboldt Universität zu Berlin, Brook-Taylor-Strasse 2, 12489 Berlin, Germany
Keywords: Chemical protein modification, cysteine-selective bioconjugation, ubiquitin conjugation, induced reactivity, vinylphosphonothiolates
ABSTRACT: Herein we introduce vinylphosphonothiolates as a new class of cysteine-selective electrophiles for protein labeling and the
formation of stable protein-protein conjugates. We developed a straightforward synthetic route to convert nucleophilic thiols into
electrophilic, thiol-selective vinylphosphonothiolates: In this protocol, intermediately formed disulfides can be chemoselectively substituted
with vinylphosphonites under acidic conditions to yield the desired vinylphosphonothiolates. Notably, this reaction sequence enables the
installation of vinylphosphonothiolate electrophiles directly on cysteine side chains within peptides and proteins. In addition to labeling the
monoclonal antibody trastuzumab with excellent cysteine-selectivity, we applied our protocol for the site-specific conjugation of two proteins
with unique cysteine residues yielding a non-hydrolyzable phosphonothiolate-linked diubiquitin and an ubiquitin-α-synuclein conjugate. The
latter was recognized as a substrate in a subsequent enzymatic ubiquitination reaction.
Introduction
to cysteines and subsequently reacted with various natural or
unnatural nucleophiles.
The development and improvement of chemical methods for the
modification of proteins with synthetic molecules such as
fluorescent probes and drugs, or even other proteins, is crucial for
Alternatively, rather than attaching a preexisting electrophilic
functional group, some approaches are known to directly
transform the cysteine residue itself into an electrophile. Such
approaches are attractive because they allow for a one-pot
conjugation between the formed electrophile and a second thiol
component without problems of homodimer formations
a plethora of applications in life sciences and pharmacology[1-4]
.
Many protein conjugation methods make use of electrophilic
reagents by addressing nucleophilic amino acids like cysteine[5-8]
or lysine[9]. Other strategies involve redox-based reagents for the
modification of methionine[10]
,
radical reactions with
tryptophan[11] and cycloadditions with unnatural amino acids
bearing alkynes, azides[12] or tetrazines[13]
associated with bifunctional linkers. For instance, in a
chemoenzymatic protocol, a formyl-glycine generating enzyme
converts a former cysteine residue into an electrophilic aldehyde-
containing amino acid[23]. In addition, chemical protocols can
deliver electrophiles, that can be reacted with canonical
nucleophilic amino acids in a selective manner.
.
In contrast to exploiting the inherent reactivity of natural amino
acid side chains or making use of laboriously introduced
unnatural functionalities, a different approach is to transform a
given nucleophilic residue into an electrophilic motif (Fig. 1A).
This inversion of reactivity allows for subsequent modifications
with various nucleophiles including nucleophilic amino acid
residues of other proteins. Such approaches enable selective
conjugation between two polypeptides bearing only canonical
amino acids, yielding homogenous conjugates, which is in high
The most well known protocol following this concept is the
formation of electrophilic disulfides with Ellmann’s reagent,
which facilitates the formation of disulfide-linked protein-protein
conjugates. This strategy has been applied to various protein
substrates, especially in the area of chemical ubiquitylation to
construct ubiquitinated histones[24] ubiquitin-PCNA[25] and
,
demand for studying various biochemical processes[4, 14]
.
ubiquitin-α-synuclein conjugates[26]. The applicability of this
approach is notably limited due to the inherent lability of
dislufides in reducing environments[27]. In another approach, a
cysteine residue can be chemoselectively converted into an
electrophilic dehydroalanine (Dha) by means of a bis-alkylation-
elimination protocol using 2,5-dibromohexanediamide[28]. Dha is
susceptible to thiol[29] and aza-Michael-addition[30], as well as
carbon-based free radical chemistry[31]. Brik and co-workers
employed thiol addition of a Cys-peptide to Dha and subsequent
Due to its distinct reactivity among the canonical amino acids,
cysteine is particularly well suited for the selective transformation
into an electrophilic motif. A common approach utilizes bis-
electrophilic molecules or linkers, such as dibromomaleimides[15,
16]
17]
,
dibromopyridazinediones[16,
,
3-bromo-2-bromomethyl-1-propenes[18]
N,N’-ethylene bis(acrylamide)[20]
,
diselenoesters[19]
,
,
chloroacetaldehyde[21] or
palladium oxidative addition complexes[22], which can be attached
1
ACS Paragon Plus Environment

Journal of the American Chemical Society
Page 2 of 16
A) General concept:
native chemical ligation for the construction of protein-ubiquitin
conjugates.[32] Furthermore, they used Dha on ubiquitin for
activity-based probes to catch deubiquitinating enzymes
(DUBs).[33, 34, 35] However, to the best of our knowledge, no direct
protein-protein conjugation with Dha proteins has been reported
for non-affinity induced systems (such as the DUB example). A
disadvantage for the formation of protein-protein conjugates via
Dha can be, that the addition of a nucleophile to Dha results in a
mixture of diastereoisomers in the protein backbone.[32] Finally,
selenocysteine has also been employed as an electrophile for
Nu
1
2
3
El
POI
transformation
POI
POI
POI
POI
POI = peptide or
protein of interest
B) Our strategy in this work:
4
5
Vinyl-
phosphonothiolates
HS
electrophilc
disulfide
1)
2)
Nu
O
O
POI
R
SH
6
7
8
S
P
OEt
S
S
P
OEt
El
EtO
OEt
P
POI= ubiquitin,
α-synuclein,
antibody
R = small molecule,
peptide, ubiquitin
R
R
POI
9
C) Previous work:
EtO
37]
protein conjugation, when activated by means of oxidation.[36,
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
Phosphonamidates
O
OEt
P
HS
O
However, the incorporation of seleno-cysteine into proteins
requires additional genetic engineering and is therefore labor
intensive.
POI
HN
P
HN
P
OEt
S
OEt
R
N3
R = small molecule,
peptide
R
R
POI
POI= antibody,
GFP
With the disadvantages of the forementioned methods in
mind, we set out to develop a method for the conversion of thiols
in cysteine side chains into electrophilic handles based on
unsaturated, electrophilic phosphorous(V) compounds.
Figure 1: A) The general concept of this work is to convert a
nucleophilic, canonical amino acid residue into an electrophilic
moiety, thereby inducing reactivity for the subsequent addition of a
nucleophilic residue of a second protein. B) Our strategy in this work
is to convert an activated cysteine residue (electrophilic disulfide) via
the reaction with diethyl-vinylphosphonite into an electrophilic
vinylphosphonothiolate, thereby inducing reactivity for a subsequent
thiol addition. C) Electrophilic phosphonamidates, generated from
As such, we herein present a straightforward reaction sequence
for the generation of vinylphosphonothiolate electrophiles
originating from thiol-containing substrates (Fig. 1B). In this
approach,
chemoselective
vinylphosphonothiolate.
a
nucleophilic thiol is formally converted in
a
azides and vinyl- or ethynylphosphonites, react selectively with
process into an electrophilic
38,
cysteine residues on proteins[8,
39]. Nu: nucleophile, El:
electrophile, EWG: electron-withdrawing group, R: as specified in
scheme. POI: protein of interest.
We have previously shown that phosphonamidates, another class
of electrophilic phosphorous(V) reagents, can be generated from
vinyl- or alkynyl-phosphonites via Staudinger-phosphonite
reactions with azides (Fig. 1C)[8, 38]. In these reactions, the
electron-rich double or triple bond in the phosphonites is
Results and Discussion
Synthesis
of
small
molecule
and
peptide
transformed into an electrophile, inducing reactivity for
a
vinylphosphonothiolates
subsequent thiol addition. The thiol addition proceeds with
excellent chemoselectivity in aqueous buffer, yielding highly
stable conjugates, as shown for the construction of stapled
At the outset of our studies we developed a synthetic route to
access vinylphosphonothiolates from thiol precursors via
electrophilic disulfides. For this, we activated small molecule
thiols with 2,2’-dithiobis(5-nitropyridine) to obtain the
respective electrophilic mixed disulfides 1-5. To investigate the
reaction with diethyl-vinylphosphonite we used the ethyl-
derivative 1 as a model substrate and employed 31P-NMR as a
read-out for reaction optimization (Fig. 2, Fig. S1). We first
peptides[38] and efficacious antibody-drug conjugates[39]
.
In
order
to
convert
vinylphopshonites
into
vinylphosphonothiolates, we envisioned electrophilic disulfides
as appropriate reagents. This proposal was based on our recent
work, in which we could show that electrophilic disulfides react
selectively with phosphites to deliver phosphorylated cysteine
residues on unprotected peptides.[40] It should be noted that
Trishin and co-workers synthesized vinylphosphonothiolates
from sulfenyl chlorides and vinylphosphonites in the late
1980s;[41] however, we regarded the synthesis of sulfenyl chlorides
as prohibitively challenging on complex substrates like
unprotected peptides and proteins. Furthermore, we envisioned
that upon installation on activated cysteine disulfide residues,
vinylphosphonothiolates could be used as electrophilic handles
for subsequent cysteine-selective bioconjugation. Small molecule
vinylphosphonothiolates could also be used for cysteine-selective
protein labeling, thereby contributing to the toolbox of
electrophilic reagents for cysteine modification.
reacted electrophilic disulfide
1 with one equivalent of
vinylphosphonite in DMSO. Complete conversion of 1 was
observed in less than one minute upon addition of the
vinylphosphonite. The desired vinylphosphonothiolate 6 was
isolated next to the side product 13, identified as the addition
product of the 6-nitropyridine-3-thiolate leaving group to 6, in a
ratio of almost 1:1 (Fig. 2A and Fig. S1A). The fact that 13 is
formed in situ, shows that vinylphosphonothiolates are reactive
towards thiols (Fig. 2B). We reasoned that the formation of the
re-attack by-product could be circumvented under acidic
conditions, since the leaving group would be protonated and thus
not reactive anymore as a nucleophile. Indeed, when the reaction
was performed in the presence of ten equivalents of
trifluoroacetic acid (TFA), the formation of the re-attack product
was inhibited (Fig. 2C and Fig. S1B). In addition, we identified
diethyl-vinylphosphonate in the reaction mixture, which is the
oxidation product of the vinylphosphonite. Due to this partial
oxidation in DMSO containing TFA, 1.5-2 equivalents are
needed to obtain full conversion of disulfide 1.
2
ACS Paragon Plus Environment

Page 3 of 16
Journal of the American Chemical Society
vinyl-
phosphonothiolate
A)
O
A)
OEt
P
OEt
P
P
re-attack
product
N
NO2
S
OEt
S
O
P
1
2
EtO
NO2
N
O
P
OEt
crude
+
S
R
S
OEt
crude, 2.0 eq.
S
N
S
13
S
OEt
DMSO
< 1 min
1
S
6
ca. 1:1
R
3
DMSO + 10 eq. TFA,
rt, < 1 min
NO2
6-10
1-5
4
5
6
7
8
9
B) without TFA
C) with TFA
O
P
O
P
O
P
in situ
R
S
OEt
S
OEt
S
S
OEt
S
entry
1
Comp. No.
yield
R
6
13
X
N
re-attack
product
N
HS
N
6
7
80%
55%
NO2
NO2
NO2
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
2
3
Figure 2: A) The reaction of electrophilic disulfide 1 and crude
vinylphopshonite (formed from commercially available diethyl
chlorophosphite and vinylmagnesium bromide as previously
O
O
NH
H
8
9
80%
70%
HN
N
H
reported[38]
) in DMSO gives a mixture of the desired
S
H
vinylphosphonothiolate 6 and re-attack side-product 13. B and C)
The in situ formed re-attack of the thiolate leaving group to the
product can be inhibited in the presence of TFA.
HO
4
O
Using these optimized reaction conditions (two equivalents of
phosphonite in DMSO and ten equivalents TFA) we synthesized
vinylphosphonothiolate derivatives 6-10 in good yields, starting
from the respective electrophilic disulfides 1-4 (Fig. 3A). The
carboxylic acid derivative 9 can be used as a modular building
block in amide couplings as shown for the synthesis of the
fluorescent EDANS-derivative 12 (Fig. 3B). We also installed the
vinylphosphonothiolate moiety on cysteine-residues on model
peptides with the sequence KYRCXK, whereas X are different
amino acids as specified in Fig. 3A. The amino acid X was varied
to probe the chemoselectivity of the reaction. The
vinylphosphonothiolate peptides 10a-10e could be obtained in
29-41% isolated yield after HPLC purification. This result
demonstrates that it is possible to install vinylphosphonothiolates
chemoselectively on a cysteine side chain in an unprotected
peptide and that the reaction proceeds chemoselectively in the
presence of other functional groups. The main side product in
this reaction is reduced cysteine peptide (ca. 10-14% isolated
yield) (Table S2).
5
6
7
KYRCAK
KYRCSK
KYRCHK
10 a
10 b
10 c
41%a
33%a
32%a
H
H
H
NH2
NH2
NH2
8
9
KYRCDK
KYRCMK
10 d
10 e
29%a
35%a
H
H
NH2
NH2
a: isolated via reverse-phase HPLC
O
N
O
P
O
B)
O
P
O
NHS, DIC
dioxane/EtOAc
0 °C to rt, 1h
EtO
S
O
EtO
S
OH
O
11, 84%
9
EDANS
SO3H
DIPEA, DMF
rt, 15 min
Thiol addition to vinylphosphonothiolates
O
O
With the isolated vinylphosphonothiolates in hand, we next set
out to investigate their reactivity towards thiols. Since we aimed
at using vinylphosphonothiolates for the synthesis of protein-
protein conjugates, we were particularly interested in the kinetics
and selectivity of the thiol addition, as well as in the stability of
the vinylphosphonothiolates before and after thiol-addition. In a
first model reaction, we reacted benzyl derivative 7 with reduced
glutathione at pH 8.5, containing 10% DMF to ensure solubility
(Fig. 4). We observed the formation of 14 as the single product,
which could be isolated in 77% yield by HPLC. Similarily, we
reacted purified peptides 10a-10e with reduced glutathione in
aqueous buffer and observed clean conversion to the desired
thiol-addition products and no side products as analyzed by
UPLC-MS and 31P-NMR (Fig. S8).
P
NH
EtO
S
12, 54%
N
H
Figure 3: A) Synthesis of vinylphosphonothiolates from
electrophilic disulfides. In this table, isolated yields after silica gel
chromatography are given, or for peptides after HPLC purification.
B) Carboxylic acid derivative 9 was further functionalized into a
fluorescent EDANS derivative 12 via amide coupling.
3
ACS Paragon Plus Environment

Journal of the American Chemical Society
Page 4 of 16
reduction-alkylation protocol[8] (Fig. 5A). After reduction with
O
P
O
2.0 eq. GSH
S
OEt
S
P
OEt
1
2
3
4
DTT, the antibody was incubated with 8 at 14 °C for 16 hours at
either pH 7.4 or 8.5. Analysis by intact protein MS revealed a
labeling degree of 1.3 and 3.1 biotins per antibody at pH 7.4 and
pH 8.5, respectively using 100 equivalents of biotin derivative 8
at an antibody concentration of 5 mg/ml.
50 mM NH4HCO3, 1 mM EDTA,
pH 8.5, 10% DMF, 25 °C
7
14, 77%
S
glutathione
DMF
14
5
180 min
A)
6
7
8
60 min
0 min
O
7
1) 33 mM DTT or no DTT
pH 8.0, 37 °C, 30 min
S
P
OEt
S
biotin
2) 3.3 mM 8, pH 7.4 or 8.5,
9
0.0
0.5
1.0
1.5
2.0
2.5
3.0
3.5
4.0
4.5
5.0
0
0.5
1
1.5
2
2.5
3
3.5
4
4.5
5
14 °C, o/n
n
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
trastuzumab
5 mg/ml (ca. 33 µM)
Figure 4: Thiol addition of glutathione (GSH) to 7 (10 mM) gives
thiol-conjugate 14. The reaction was monitored by LC-UV (220
nm).
B)
C)
pH 7.4
_
pH 8.5
1.0
_
M
+
+
23441
(LC0
DTT
HC
kDa
70
)
55
40
49996
(HC2)
49574
0.5
0.0
35465
(PNGaseF)
Using the fluorescent EDANS derivative 12 we investigated
the reaction kinetics of the thiol addition by monitoring the
addition of glutathione with fluorescent HPLC. We observed
clean conversion to the thiol addition product and deduced
second order rate constants of 0.0021 M-1s-1 (at pH 7.4) and
0.074 M-1s-1 (at pH 8.5) (Fig. S3). These rates are in the range of
other typically applied protein modification reactions[3] and allow
installation of vinylphosphonothiolates on proteins at dilute
conditions.
(HC1
)
23862
(LC1
49153
(HC0)
50418
(HC3
)
35
25
)
LC
20000
60000
40000
Mass (Da)
WB (anti-biotin)
Figure 5: A) Labeling of IgG antibody trastuzumab with 100 eq.
biotin-vinylphosphonothiolate at pH 7.4 and 8.5 gives a
modification degree of n= 1.3 and 3.1 biotins per antibody,
respectively. B) Western-Blot (anti-biotin). C) Deconvoluted MS
spectra of trastuzumab labelled with 8 at pH 8.5. LC: light chain,
HC: heavy chain, subscripts: number of modification.
8
When using vinylphosphonothiolates for protein modification,
the stability of these reagents in aqueous media under buffered
conditions is of high importance. In aqueous solution at pH 7.4,
more than 90% of vinylphosphonothiolate 12 remained intact
after 90 hours, both at r.t. and at 37 °C (Fig. S2). In contrast, at
pH 8.5 at 37 °C, we observed ca. 40% hydrolysis of the P-S bond
after 90 hours. Notably, vinylphosphonothiolates show excellent
stability under acidic conditions and can be stored at 4 °C for
several months in isolated form and as solutions in DMSO
without any observable decomposition.
In a control reaction without DTT reduction of disulfides, no
antibody labeling was observed, thus demonstrating excellent
cysteine-selectivity of vinylphosphonothiolates (Fig. 5B, Fig. S5
& S6). This finding is in line with previously published results
from our group, where we could show that electrophilic
phosphonamidates show excellent cysteine-selectivity as well[8, 39]
.
Taken
together,
these
results
demonstrate
that
vinylphosphonothiolates are suitable reagents for the cysteine-
selective labeling of biomolecules such as antibodies to generate
stable bioconjugates.
We
also
monitored
the
stability
of
isolated
vinylphosphonothiolate peptides 10a-10e in aqueous buffers at
pH 7.4 or 8.5. Depending on the pH we observed P-S hydrolysis
and in addition a migration of the vinyl-O-ethylphosphate group
to other nucleophilic residues (Fig. S7). As a consequence of
these two processes ca. 50% of peptides 10a-10e are
decomposed after 24 h at neutral pH (Fig. S7C). However, when
the purified vinylphosphonothiolate peptides 10a-10e are
directly reacted with reduced glutathione in aqueous buffer, clean
conversion into the thiol addition product to the
vinylphosphonothiolate is observed (as mentioned above) and
no migration of the P(V) moiety to other residues (Fig. S8).
Vinylphosphonothiolates
conjugation
for
protein-protein
Other than using small molecule vinylphosphonothiolates for
protein labeling, we wanted to apply the chemoselective
induction of electrophilicity on amino acid side chains to the
conjugation of two proteins. Hereby, we focused on the synthesis
of biologically relevant ubiquitin-protein conjugates.
Ubiquitination is one of the largest reversible post-translational
modification of proteins, regulating various cellular processes
from protein degradation and trafficking to DNA repair and
transcription[42]. Ubiquitin chains are usually connected through
the C-terminus of a distal ubiquitin to any of seven available
lysine residues by E1, E2 and E3 enzymes via an isopeptidic bond
at the proximal ubiquitin[42, 43]. Studying the biological function of
different ubiquitin chain linkages as well as probing the impact of
ubiquitination on substrate proteins constitutes a major challenge
in current research. Consequently, there is a great need for
Next we investigated the stability of the thiol-addition
products. For this, we made use of a previously reported
fluorescence-quenching assay[8]. Generally, the conjugates show
very good stability in buffer, in Hela cell lysate and in human
serum, which is crucial for applications in biological systems (Fig.
S4). Furthermore, the conjugates are stable in the presence of
reducing agents and excess free thiols, and in contrast to our
38]
previously reported phosphonamidates[8,
conditions (1 N HCl, pH 0).
also under acidic
homogeneous site-specific ubiquitinated substrates for functional
investigations[44, 45]. Inspired by the work of A. Brik[21,
47], A.
32, 46,
Encouraged by these results, we probed the applicability of
vinylphosphonothiolates for the labeling of cysteine-residues on
proteins in general and antibodies in particular. We reacted the
therapeutically relevant, monoclonal IgG antibody trastuzumab
Marx[48], M. R. Pratt[26] and E. Strieter[14, 49] in the field of chemical
ubiquitination we wanted to apply our vinylphosphonothiolate
conjugation strategy to generate non-hydrolyzable homogenous
ubiquitin-protein conjugates. Within this goal, we particularly
with biotin derivative 8, following
a
previously described
4
ACS Paragon Plus Environment

Page 5 of 16
Journal of the American Chemical Society
20 (Fig. 6B). The reactions were monitored by SDS-PAGE and
aimed at synthesizing diubiquitin and an ubiquitin-α-synuclein
conjugate.
1
2
intact protein MS, which indicated the formation of covalent
protein-protein conjugates. For both conjugation reactions,
maximal conversion was obtained after 24 hours (Fig S27 & S30).
The conjugates DiUb(K48) 19 and Ub-α-synuclein 21 were
purified by size exclusion chromatography and isolated in 16%
and 27% yields, respectively. For both protein conjugates 19 and
21 the yields are based on pure product-containing fractions;
mixed fractions after size-exclusion chromatography are not
accounted for the isolated yield.
First, we applied the previously optimized protocol for the
installation of the vinylphosphonothiolate moiety to a ubiquitin
mutant, carrying a single cysteine (G76C) (Fig. 6A). We chose
Ellmann’s reagent to activate the cysteine-residue in this case
because 2,2’-dithiobis(5-nitropyridine) is poorly soluble in
aqueous buffer and gave incomplete conversion. Addition of 100
equivalents vinylphosphonite to activated ubiquitin 16 in DMSO
containing 1 % TFA at room temperature resulted in full
conversion and yielded vinylphosphonothiolate ubiquitin 17, as
confirmed by ESI-MS. Crude vinylphosphonothiolate ubiquitin
17 was purified by semipreparative HPLC and could be isolated
in 24% yield. The reaction can also be performed at 60 °C with
only 50 eq. of the vinylphosphonite. In this case, full conversion is
3
4
5
6
7
8
9
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
For both conjugates 19 and 21, the site-selective linkages to
UbG76C-phosphonothiolate were confirmed by LC-MS/MS
analysis after tryptic digest of the purified conjugates (Fig. S9 &
S10). In line with the above described observations for the
vinylphosphonothiolate-peptides (Fig. S8), we also did not
observe P(V)-transfer conjugation products on protein level.
achieved in only
4 hours. As mentioned before, excess
vinylphosphonite oxidizes to vinylphosphonate in DMSO;
however, the latter does not react with 16. In addition, non-
activated disulfide-ubiquitin 15 does not react with the
vinylphosphonite at r.t., as confirmed by ESI-MS. The conversion
of an activated disulfide to vinylphosphonothiolates by means of
the reaction with vinylphosphonite requires dry conditions;
hence the applicability of this method is limited to protein
substrates, which are stable under these conditions (in DMSO +
0.1% TFA) and can subsequently be refolded.
Purified thiol conjugates 19 and 21 showed excellent stability in
aqueous buffer and could be stored for several months at 4 °C
without any sign of decomposition. To further verify the utility of
phosphonothiolate-linked conjugates, we demonstrated that
DiUb(K48) conjugate 19 is stable in the presence of
deubiquitinating enzyme USP2-CD (Fig. S36); that it is
recognized by an α-poly-Ub antibody (Fig. 6B); and that it shows
similar elipticity in CD spectra compared to the wildtype
diubiquitin (K48 amide linkage) (Fig. S11).
Next, we probed protein-protein conjugation in a model
system between vinylphosphonothiolate-Ub 17 and an eGFP
cysteine mutant (C70M S147C) and observed more than 80%
conversion after 24 hours at pH 8.5 (Fig. S24). At pH 8.5 the
reaction is significantly faster than at pH 7.4, which is consistent
with the small molecules studies described above. Furthermore,
Finally, to prove that the non-hydrolyzable phosphonothiolate-
linked ubiquitinated proteins can be recognized by ubiquitin
binding proteins, we mixed α-synuclein-ubiquitin conjugate 21
with the enzymes Ubc1 (E2-25k) and E1 to enable enzymatic
ubiquitination (Fig. 6C). The ubiquitin conjugating enzyme
Ubc1 catalyzes the formation of K48-linked ubiquitin chains from
we
investigated
the
stability
of
unconjugated
[50, 51]
mono ubiquitin
. To prevent polyubiquitination, we
vinylphosphonthiolate-ubiqutin 17 in aqueous buffer and, similar
to the observed behavior of small molecule 12, observed P-S
bond hydrolysis over time at pH 8.5, as well as to some extent at
pH 7.4 (Fig. S23). Because of the P-S bond hydrolysis, the free
thiol ubiquitin reacts with intact 21 to form C-terminal ubiquitin
dimers. Having this observation in mind, we decided to perform
protein conjugations with 17 at pH 8.0, which constitutes a
employed the K48M ubiquitin mutant 22. After 24 h incubation
at 37 °C we could detect the formed di-ubiquitinated α-synuclein
23 by ESI-MS, demonstrating that the ubiquitinating enzymes
indeed recognize our phosphonothiolate-linked ubiquitin
substrate. Using a specific anti-polyUb(K48) antibody, we could
additionally confirm by Western-Blot that conjugate 23 is linked
via the K48 residue.
compromise
of
stability
and
reactivity.
Next,
vinylphosphonothiolate-Ub 17 was subjected to the conjugation
with UbK48C mutant 18 as well as the α-synucleinK6C mutant
5
ACS Paragon Plus Environment

Journal of the American Chemical Society
Page 6 of 16
OEt
P
1
2
3
4
EtO
1)
O
P
A)
crude, 100 eq.
1) 10 eq. DTT, PBS,
37 °C, 20 min
Ub
G76C
Ub
G76C
Ub
G76C
S
COOH
NO2
DMSO, 1% TFA, r.t., 16 h
S
OEt
S
S
2
2) 50 eq. Ellmann’s reagent,
PBS, r.t., 1 min
2) PBS pH 7.4
17, 24%
15
16, quantitative
5
6
7
8
9
685.5
8902
742.5
1.0
0.5
1.0
810.0
891.0
989.9
calc.: 8900
obs.: 8902
isolated 17
isolated 17
0.5
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
1113.5
1272.4
1484.3
0.0
0.0
9000
Mass (Da)
11000
7000
600 800 1000 1200 1400
m/z
SH
O
P
Ub
SH
B)
O
P
O
P
O
P
K48C
Ub
G76C
α-SynK6C
20, 500 µM
Ub
G76C
Ub
G76C
Ub
G76C
S
OEt
S
S
OEt
S
S
OEt
S
OEt
18, 500 µM
19, 27%
17, 100 µM
17, 100 µM
21, 16%
PBS, pH 8.0,
37 °C, 24 h
PBS, pH 8.0,
37 °C, 24 h
Ub
K48C
α-SynK6C
kDa
15
M
RM
P
M
RM
P
kDa
20
19
23336
17612
21
1.0
1.0
calc.: 17610
obs.: 17612
calc.: 23335
obs.: 23336
isolated 19
isolated 21
10
15
17, 18
20
17
15
10
0.5
0.0
Coomassie
diUb
(K48)wt
0.5
0.0
M
19
11668
15000
Coomassie
18000
14000
Mass (Da)
22000
25000
Mass (Da)
35000
WB (poly-Ub(K48))
S
C)
S
H
N
H2N
O
P
O
P
G76
OH
Ub
K48M
Ub
K48M
K48
Ub
G76C
Ub
G76C
O
S
OEt
S
S
OEt
K48 linkage
O
22
21
23
S
E1 and Ubc1 enzymes
α-SynK6C
α-SynK6C
1) 2) 3)
kDa
M
100
70
E1
32057
1.0
55
40
23
reaction mixture
Ubc1-Ub(22)
calc.: 32056
obs.: 32057
35
25
Ub2-α-Syn 23
21
23336
0.5
0.0
Ubc1
Ub-α-Syn 21
1) Reaction mixture, 0 h (Silver stain)
2) Reaction mixture, 24 h (Silver stain)
3) Reaction mixture, 24 h (WB, anti-polyUb(K48))
15
10
30000
Mass (Da)
38000
22000
UbK48M 22
Figure 6: Vinylphosphonothiolates as cysteine-selective linkers for protein-protein conjugation. A) Synthesis of vinylphosphonothiolate ubiquitin
17 from cysteine mutant 15 via Ellmann’s activated ubiquitin 16. Shown are raw and deconvoluted MS spectra of isolated product 17. DTT:
dithiothreitol, TFA: trifluoroacetic acid. B) Protein conjugation of vinylphosphonothiolate ubiquitin 17 to UbK48C 18 and α-synucleinK6C 20.
Shown are MS spectra of the isolated products and SDS-gels of the isolated products and the reaction mixtures. WB (anti-polyUb(K48) antibody)
analysis reveals, that the phosphonothiolate-linked diUb 19 is linked via K48. RM: reaction mixture, P: product. C) Enzymatic ubiquitination of
α-synuclein-ubiquitin conjugate 21 at position K48 by E1 and Ubc1 enzymes. UbK48M mutant 22 was used as a dead-end building block to avoid
poly-ubiquitination. Shown are a MS spectrum of the reaction mixture and a SDS-gel (silver stain) of the reaction mixture at time 0 and 24 hours.
Additionally, an anti-polyUb(K48) WB supports that the ubiquitins in 23 are linked via the K48 residue.
6
ACS Paragon Plus Environment

Page 7 of 16
Journal of the American Chemical Society
Stephen Byrne, Jonas Helma-Smets, Heinrich Leonhardt, Tina
Stoscheck, Marcus Gerlach and Dominik Schumacher.
Conclusions
1
2
3
4
5
6
7
8
9
Herein, we presented vinylphosphonothiolates as a new class
of cysteine-reactive reagents for protein labeling and protein-
protein conjugation. Vinylphosphonothiolates can be generated
ACKNOWLEDGMENT
The authors thank Marc-André Kasper for valuable experimental
input and M.-A. Kasper and Dr. Philipp Ochtrop for proofreading the
manuscript. For LC-MS/MS measurements we thank Michal
Nadler-Holly (AG Fan Liu, FMP).
in good yields even on unprotected peptides following
a
straightforward protocol from vinylphosphonites and thiol-
containing molecules via intermediately formed electrophilic
disulfides, thereby inducing reactivity for
a thiol addition.
REFERENCES
Importantly, performing the phosphonite-disulfide reaction
under acidic conditions circumvents the in situ re-attack of the
thiolate leaving group. Excellent cysteine-selectivity of
vinylphosphonothiolates could be demonstrated by labeling the
monoclonal antibody trastuzumab. Importantly, we showed that
vinylphosphonothiolate electrophiles could be installed site-
selectively on a cysteine residue in a single cysteine-containing
ubiquitin mutant. The resulting electrophilic ubiquitin-
vinylphosphonothiolate was conjugated to Cys-containing
mutants of α-synuclein and ubiquitin, generating
nonhydrolyzable conjugates, which were accepted as substrates
for subsequent enzymatic ubiquitynation. Taken together, this
method enables the conjugation of two biomolecules containing
(1): Hoyt E. A.; Cal P. M. S. D.; Oliveira B. L.; Bernardes G. J. L.
Contemporary approaches to site-selective protein modification. Nat.
Rev. Chem. 2019, 3, 147-171.
(2) Spicer C. D.; Davis B. G. Selective chemical protein modification.
Nat. Comm. 2014, 5, article number: 4740.
(3) Row R. D.; Prescher J. A. Constructing new bioorthogonal
reagents and reactions. Acc. Chem. Res. 2018, 51, 1073-1081.
(4): Beck A.; Goetsch L.; Dumontet C.; Corvaia N. Strategies and
challenges for the next generation of antibody-drug conjugates. Nat. Rev.
2017, 16, 315-337.
(5): Gunnoo S. B.; Madder A. Chemical protein modification
through cysteine. ChemBioChem 2016, 17, 529-553.
(6): Zhang C.; Welborn M.; Zhu T.; Yang N. J.; Santos M. S.; Van
Voorhis T.; Pentelute B. L. Π-clamp-mediated cysteine conjugation. Nat.
Chem. 2016, 8, 120-128.
(7): Luo, Q.; Tao Y.; Sheng W.; Lu J.; Wang H. Dinitroimidazoles as
bifunctional bioconjuation reagents for protein functionalization and
peptide macrocyclization. Nat. Comm. 2019, 10, article number: 142.
(8): Kasper M.-A.; Glanz M.; Stengl A.; Penkert M.; Klenk S.; Sauer
T.; Schumacher D.; Helma J.; Krause E.; Cardoso C.; Leonhardt H.;
Hackenberger C. P. R. Cysteine-selective phosphonamidate electrophiles
for modular protein bioconjugations. Angew. Chem. Int. Ed. 2019, 58,
11625-11630.
(9) deGruyter J. N.; Malins L. R.; Baran P. S. Residue-specific peptide
modification: A chemist’s guide. Biochemistry 2017, 56, 3863-3873.
(10): Lin S.; Yang X.; Jia S.; Weeks A. M.; Hornsby M.; Lee P. S.;
Nichiporuk R. V.; Iavarone A. T.; Wells J. A.; Toste F. D.; Chang C. J.
Redox-based reagents for chemoselective methionine bioconjugation.
Science 2017, 355, 597-602.
(11): Seki Y.; Ishiyama T.; Sasaki D.; Abe J.; Sohma Y.; Oisaki K.;
Kanai M. Transition metal-free tryptophan-selective bioconjugation of
proteins. J. Am. Chem. Soc. 2016, 138, 10798-10801.
(12): Nguyen D. P.; Lusic H.; Neumann H.; Kapadnis P. B.; Deiters
A.; Chin J. W. Genetic encoding and labeling of aliphatic azides and
alkynes in recombinant proteins via
synthetase/tRNA(CUA) pair and click chemistry. J. Am. Chem. Soc.
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
single free cysteines in
a chemoselective and site-selective
manner. We believe that the herein presented formal attachment
of electrophilic moieties to a former Cys-residue will find use for
the generation of homogenous peptide and protein conjugates
containing only canonical amino acids and in particular for the
formation of non-hydrolyzable protein-ubiquitin conjugates.
Supporting Information
The Supporting Information is available free of charge on the ACS
Publications website.
Detailed experimental procedures, organic synthesis, compound
characterizations, protein chemistry (PDF).
AUTHOR INFORMATION
Corresponding Author
Prof. C. P. R. Hackenberger, Chemical Biology Department, Leibniz-
Forschungsinstitut für Molekulare Pharmakologie (FMP), Robert-
Rössle-Strasse 10, 13125 Berlin (Germany),
E-Mail: hackenbe@fmp-berlin.de
a
pyrrolysyl-tRNA
Author Contributions
2009, 131, 8720-8721.
A. L. B., S. S. and C. P. R. H. have designed the research and have
written the manuscript. A. L. B. investigated small molecule and
peptide vinylphosphonothiolates. S. S. has conducted experiments
involving ubiquitin.
All authors have given approval to the final version of the manuscript.
/ ‡These authors contributed equally.
(13): Seitchik J. L.; Peeler J. C.; Taylor M. T.; Blackman M. L.;
Rhoads T. W.; Cooley R. B.; Refakis C.; Fox J. M.; Mehl R. A. Genetically
encoded tetrazine amino acid directs rapid site-specific in vivo
bioorthogonal ligation with trans-cyclooctenes. J. Am. Chem. Soc. 2012,
134, 2898-2901.
(14): Valkevich E. M.; Guenette R. G.; Sanchez N. A.; Chen Y. C.; Ge
Y.; Strieter E. R. 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.
(15): Smith M. E. B.; Schumacher F. F.; Ryan C. P.; Tedaldi L. M.;
Papaioannou D.; Waksman G.; Caddick S.; Baker J. R. Protein
modification, bioconjugation, and disulfide bridging using
bromomaleimides. J. Am. Chem. Soc. 2010, 132, 1960-1965.
(16): Morgan R. E.; Chudasama V.; Moody P.; Smith M. E. B.;
Caddick S. A novel synthetic chemistry approach to linkage-specific
ubiquitin conjugation. Org. Biomol. Chem. 2015, 13, 4165-4168.
(17): Chudasama V.; Smith M. E. B.; Schumacher F. F.; Papaioannou
D.; Waksman G.; Baker J. R.; Caddick S. Bromopyridazinedione-
mediated protein and peptide bioconjugation. Chem. Commun. 2011,
47, 8781-8783.
Funding Sources
We are very grateful for continuous support by the Deutsche
Forschungsgemeinschaft within the SPP1623 and the SFB765, the
Einstein Foundation Berlin (Leibniz-Humboldt Professorship), the
Boehringer Ingelheim Fonds (Plus 3 award), the Fonds der
Chemischen Industrie, the Leibniz Society (SAW-2018-FMP-4-
P5label, T18/2017) and the Studienstiftung des Deutschen Volkes
(doctoral
fellowship
to
A.L.B.).
Notes
The technology described in the manuscript is part of a pending
patent application by C.P.R. H., A.-L. B., Marc-André Kasper,
7
ACS Paragon Plus Environment

Journal of the American Chemical Society
Page 8 of 16
(18): Lee B.; Sun S.; Jiménez-Moreno E.; Neves A. A.; Bernardes G. J.
strategy to expressed proteins reveal a potential α-globin-modulating
L. Site-selective installation of an electrophilic handle on proteins for
bioconjugation. Bioorg. Med. Chem. 2018, 15, 3060-3064.
(19): Yin X.-G.; Gao X.-F.; Du J.-J.; Zhang X.-K.; Chen X.-Z.; Wang
J.; Xin L.-M.; Lei Z.; Liu Z.; Guo J. Preparation of protein conjugates via
homobifunctional diselenoester cross-linker. Org. Lett. 2016, 18, 5796-
5799.
(20): Holm L.; Moody P.; Howarth M. Electrophilic affibodies
forming covalent bonds to protein targets. J. Biol. Chem. 2009, 284,
32906-32913.
(21): Singh S. K.; Sahu I.; Mali S. M.; Hemantha H. P.; Kleifeld O.;
Glickman M. H.; Brik A. Synthetic uncleavable ubiquitinated proteins
dissect proteasome deubiquitination and degradation, and highlight
distinctive fate of tetraubiquitin. J. Am. Chem. Soc. 2016, 138, 16004-
16015.
(22): Kubota K.; Dai P.; Pentelute B. L.; Buchwald S. L. Palladium
oxidative addition complexes for peptide and protein cross-linking. J. Am.
Chem. Soc. 2018, 140, 3128-3133.
(23): Hudak J. E.; Barfield R. M.; de Hart G. W.; Grob P.; Nogales E.;
Bertozzi C. R.; Rabuka D. Synthesis of heterobifunctional protein fusions
using copper-free click chemistry and the aldehyde tag. Angew. Chem.
Int. Ed. 2012, 51, 4161-4165.
(24): Chatterjee C.; McGinty R. K.; Fierz B.; Muir T. W. Disulfide-
directed histone ubiquitylation reveals plasticity in hDot1L activation.
Nat. Chem. Biol. 2010, 6, 267-269.
(25): Chen J.; Ai Y.; Wang J.; Haracska L.; Zhuang Z. Chemically
ubiquitylated PCNA as a probe for eukaryotic translesion DNA synthesis.
Nat. Chem. Biol. 2010, 6, 270-272.
(26): Meier F.; Abeywardana T.; Dhall A.; Marotta N. P.; Varkey J.;
Langen R.; Chatterjee C.; Pratt M. R. Semisynthetic, site-specific
ubiquitin modification of α-synuclein reveals differential effects on
aggregation. J. Am. Chem. Soc. 2012, 134, 5468-5471.
(27): Powis G, Montfort W. R. Properties and biological activities of
thioredoxins. Annu. Rev. Pharmacol. Toxicol. 2001, 41, 261-295.
(28): Chalker J. M.; Gunnoo S. B.; Boutureira O.; Gerstberger S. C.;
Fernández-González M.; Bernardes G. J. L.; Griffin L.; Hailu H.;
Schofield C. J.; Davis B. G. Methods for converting cysteine to
dehydroalanine on peptides and proteins. Chem. Sci. 2011, 2, 1666-
1676.
(29): Chalker J. M.; Lercher L.; Rose N. R.; Schofield C. J.; Davis B.
G. Conversion of cysteine into dehydroalanine enables access to
synthetic histones bearing diverse post-translational modifications.
Angew. Chem. Int. Ed. 2012, 51, 1835-1839.
(30): Freedy A. M.; Matos M. J.; Boutureira O.; Corzana F.; Guerreiro
A.; Akkapeddi P.; Somovilla V. J.; Rodrigues T.; Nicholls K.; Xie B.;
Jiménez-Osés G.; Brindle K. M.; Neves A. A.; Bernardes G. J. L.
Chemoselective installation of amine bonds on proteins through aza-
Michael ligation. J. Am. Chem. Soc. 2017, 139, 18365-18375.
(31): Wright T. H.; Bower B. J.; Chalker J. M.; Bernardes G. J. L.;
Wiewiora R.; Ng W.-L.; Raj R.; Faulkner S.; Vallée M. R. J.;
Phanumartwiwath A.; Coleman O. D.; Thézénas M.-L.; Khan M.; Galan
S. R. G.; Lercher L.; Schombs M. W.; Gerstberger S.; Palm-Espling M. E.;
Baldwin A. J.; Kessler B. M.; Claridge T. D. W.; Mohammed S.; Davis B.
G. Posttranslational mutagenesis: A chemical strategy for exploring
protein side-chain diversity. Science 2016, 354, 597 and aag1465.
(32): Meledin R.; Mali S. M.; Singh S. K.; Brik A. Protein
ubiquitination via dehydroalanine: development and insights into the
diastereoselective 1,4-addition step. Org. Biomol. Chem. 2016, 14,
4817-4823.
1
2
3
4
5
6
7
8
deubiquitinase. Angew. Chem. Int. Ed. 2018, 57, 5645-5649.
(36): Cohen D. T.; Zhang C.; Pentelute B. L.; Buchwald S. L. An
umpolung approach for the chemoselective arylation of selenocysteine in
unprotected peptides. J. Am. Chem. Soc. 2015, 137, 9784-9787.
(37): Cohen D. T.; Zhang C.; Fadzen C. M.; Mijalis A. J.; Hie L.;
Johnson K. D.; Shriver Z.; Plante O.; Miller S. J.; Buchwald S. L.;
Pentelute B. L. A chemoselective strategy for late-stage functionalization
of complex small molecules with polypeptides and proteins. Nat. Chem.
2019, 11, 78-85.
(38): Kasper M.-A.; Glanz M.; Oder A.; Schmieder P.; von Kries J. P.;
Hackenberger C. P. R. Vinylphosphonites for staudinger-induced
chemoselective peptide cyclization and functionalization. Chem. Sci.
2019, 10, 6322-6329.
(39): Kasper M.-A.; Stengl A.; Ochtrop P.; Gerlach M.; Stoschek T.;
Schumacher D.; Helma J.; Penkert M.; Krause E.; Leonhardt H.;
Hackenberger C. P. R. Ethynylphosphonamidates for rapid and cysteine-
selective generation of efficacious antibody-drug conjugates. Angew.
Chem. Int. Ed. 2019, 58, 11631-11636.
(40): Bertran-Vicente J.; Penkert M.; Nieto-Garcia O.; Jeckelmann J.-
M.; Schmieder P., Krause E., Hackenberger C. P. R. Chemoselective
synthesis and analysis of naturally occurring phosphorylated cysteine
peptides. Nat. Comm. 2016, 7, Article Number: 12703.
9
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
(41): Trishin Y. G.; Bondarenko E. A.; Stepanov I. A.; Chistokletov V.
N. Chemoselectivity of reactions of dialkyl α,β-unsaturated
phosphonites with p-nitrophenylsulfenyl chloride. Zhurnal Obshchei
Khimii 1989, 59, 230-231.
(42): Komander D.; Rape M. The ubiquitin code. Annu. Rev.
Biochem. 2012, 81, 203-229.
(43): Komander D. The emerging complexity of protein
ubiquitination. Biochem. Soc. Trans. 2009, 37, 937-953.
(44): Isasa M.; Katz E. J.; Kim W., Yugo V.; González S.; Kirkpatrick
D. S.; Thomson T. M.; Finley D.; Gygi S. P.; Crosas B.
Monoubiquitination of RPN10 regulates substrate recruitment to the
proteasome. Mol. Cell. 2010, 38, 733-745.
(45): Baker R.; Lewis S. M.; Sasaki A. T.; Wilkerson E. M.; Locasale J.
W.; Cantley L. C.; Kuhlman B.; Dohlman H. G.; Campbell S. L. Site-
specific monoubiquitination activates Ras by impending GTPase
activating protein function. Nat. Struct. Mol. Biol. 2013, 20, 46-52.
(46): Haj-Yahya M.; Fauvet B.; Herman-Bachinsky Y.; Hejjaoui M.;
Bavikar S. N.; Karthikeyan S. V.; Ciechanover A.; Lashuel H. A.; Brik A.
Synthetic polyubiquitinated α-synuclein reveals important insights into
the roles of the ubiquitin chain in regulating its pathophysiology. PNAS
2013, 110, 17726-17731.
(47): Hemantha H. P.; Bavikar S. N.; Herman-Bachinsky Y.; Haj-
Yahya N.; Bondalapati S.; Ciechanover A.; Brik A. Nonenzymatic
polyubiquitination of expressed proteins. J. Am. Chem. Soc. 2014, 136,
2665-2673.
(48): Schneider T.; Schneider D.; Rösner D.; Malhotra S.; Mortensen
F.; Mayer T. U.; Scheffner M.; Marx A. Dissecting ubiquitin signaling
with linkage-defined and protease resistant ubiquitin chains. Angew.
Chem. Int. Ed. 2014, 53, 12925-12929.
(49): Trang V. H.; Rodgers M. L.; Boyle K. J.; Hoskins A. A.; Strieter
E. R. Chemoenzymatic synthesis of bifunctional polyubiquitin substrates
for monitoring ubiquitin chain remodeling. ChemBioChem 2014, 15,
1563-1568.
(50): Rodrigo-Brenni M. C.; Foster S. A.; Morgan D. O. Catalysis of
lysine 48-specific ubiquitin chain assembly by residues in E2 and
ubiquitin. Mol. Cell. 2010, 39, 548-559.
(51): Haldeman M. T.; Xia G.; Kasperek E. M.; Pickart C. M.
Structure and function of ubiquitin conjugating enzyme E2-25K: the tail
is a core-dependent activity element. Biochemistry 1997, 36, 10526-
10537.
(33): Haj-Yahya N.; Hemantha H. P.; Meledin R.; Bondalapati S.;
Seenaiah M.; Brik A. Dehydroalanine-based diubiquitin activity probes.
Org. Lett. 2014, 16, 540-543.
(34): Jbara M.; Laps S.; Morgan M.; Kamnesky G.; Mann G.;
Wolberger C.; Brik A. Palladium prompted on-demand cysteine
chemistry for the synthesis of challenging and uniquely modified
proteins. Nat. Commun. 2018, 9, 3154.
(35): Meledin R.; Mali S. M.; Kleifeld O.; Brik A. Activity-based
probes developed by applying a sequential dehydroalanine formation
8
ACS Paragon Plus Environment

Page 9 of 16
Journal of the American Chemical Society
1
2
3
4
5
6
7
8
Insert Table of Contents artwork here
9
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
9
ACS Paragon Plus Environment

Journal of the American Chemical Society
Page 10 of 16
1
2
3
4
5
6
7
8
9
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
ACS Paragon Plus Environment

Page 11 of 16
Journal of the American Chemical Society
1
2
3
4
5
6
7
8
9
10
11
12
13
14
15
16
ACS Paragon Plus Environment

A)
OEt
Journal of the American Chemical Society
Page 12 of 16
P
NO2
N
O
OEt
crude, 2.0 eq.
R
P
S
S
OEt
1
2
3
S
R
DMSO + 10 eq. TFA,
rt, < 1 min
6-10
1-5
4
5
6
7
8
9
entry
1
Comp. No.
yield
80%
R
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
6
7
2
3
55%
80%
O
O
NH
H
8
9
HN
N
H
S
H
HO
4
70%
O
5
6
7
10 a
10 b
10 c
41%a
33%a
32%a
KYRCAK
KYRCSK
KYRCHK
H
H
H
NH2
NH2
NH2
8
9
10 d
10 e
29%a
35%a
KYRCDK
KYRCMK
H
H
NH2
NH2
a: isolated via reverse-phase HPLC
O
N
O
P
O
B)
O
P
O
NHS, DIC
EtO
S
O
dioxane/EtOAc
0 °C to rt, 1h
EtO
S
OH
O
11, 84%
9
EDANS
DIPEA, DMF
rt, 15 min
SO3H
O
O
ACS Paragon Plus Environment
P
NH
EtO
S
N
H
12, 54%

Page 13 of 16
Journal of the American Chemical Society
1
2
3
4
5
6
7
8
9
10
11
12
13
14
15
ACS Paragon Plus Environment

A)
Journal of the American Chemical Society
Page 14 of 16
O
P
1) 33 mM DTT or no DTT
pH 8.0, 37 °C, 30 min
S
1
2
3
4
5
6
7
OEt
S
biotin
2) 3.3 mM 8, pH 7.4 or 8.5,
14 °C, o/n
n
trastuzumab
5 mg/ml (ca. 33 µM)
8
9
B)
C)
pH 7.4
_
pH 8.5
_
1.0
23441
(LC0)
M
+
+
DTT
HC
kDa
70
10
11
12
13
14
15
16
17
18
19
55
40
49996
(HC2)
49574
(HC1)
0.5
35465
(PNGaseF)
23862
(LC1)
49153
(HC0)
50418
(HC3)
35
25
ACS Paragon Plus Environment
LC
20000
0.0
60000
40000
Mass (Da)
WB (anti-biotin)

OEt
P
Page 15 of 16
Journal of the American Chemical Society
EtO
1)
O
P
A)
crude, 100 eq.
1) 10 eq. DTT, PBS,
37 °C, 20 min
1
Ub
G76C
Ub
G76C
Ub
G76C
S
COOH
NO2
DMSO, 1% TFA, r.t., 16 h
2
S
OEt
S
S
3
4
2
2) 50 eq. Ellmann’s reagent,
PBS, r.t., 1 min
2) PBS pH 7.4
17, 24%
15
16, quantitative
5
6
7
8
9
10
11
12
13
14
15
16
17
18
685.5
8902
742.5
1.0
0.5
1.0
810.0
891.0
989.9
calc.: 8900
obs.: 8902
isolated 17
isolated 17
0.5
1113.5
1272.4
1484.3
0.0
0.0
9000
11000
7000
600 800 1000 1200 1400
m/z
Mass (Da)
SH
19
O
P
Ub
K48C
B)
O
P
O
P
O
P
SH
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
Ub
G76C
α-SynK6C
20, 500 µM
Ub
S
Ub
G76C
Ub
S
G76C
S
OEt
S
OEt
S
S
OEt
OEt
G76C
18, 500 µM
19, 27%
21, 16%
17, 100 µM
17, 100 µM
PBS, pH 8.0,
37 °C, 24 h
PBS, pH 8.0,
37 °C, 24 h
Ub
K48C
α-SynK6C
kDa
15
M
RM
P
M
RM
P
kDa
20
19
23336
17612
21
1.0
1.0
calc.: 17610
obs.: 17612
calc.: 23335
obs.: 23336
isolated 19
isolated 21
10
15
17, 18
20
17
15
10
0.5
0.0
Coomassie
diUb
(K48)wt
0.5
0.0
M
19
11668
15000
Coomassie
18000
Mass (Da)
22000
14000
25000
Mass (Da)
35000
WB (poly-Ub(K48))
S
C)
S
H
N
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
H2N
O
P
O
P
G76
OH
Ub
K48M
Ub
K48M
K48
Ub
G76C
Ub
G76C
O
S
OEt
S
S
OEt
K48 linkage
O
22
21
23
S
E1 and Ubc1 enzymes
α-SynK6C
α-SynK6C
1) 2) 3)
kDa
M
100
70
E1
32057
1.0
55
40
23
reaction mixture
Ubc1-Ub(22)
calc.: 32056
obs.: 32057
35
25
Ub2-α-Syn 23
21
23336
0.5
0.0
Ubc1
Ub-α-Syn 21
1) Reaction mixture, 0 h (Silver stain)
2) Reaction mixture, 24 h (Silver stain)
3) Reaction mixture, 24 h (WB, anti-polyUb(K48))
15
30000
Mass (Da)
38000
22000
ACS Paragon Plus Environment
10
UbK48M 22

Journal of the American Chemical Society
Page 16 of 16
R
SH
Nucleophile
1
2
3
4
5
6
7
8
9
10
11
1) Disulfide formation
2) Vinylphosphonite, H+
Electrophile
Protein
labeling
Protein-protein
conjugation
O
O
O
Ubi-
quitin
R
Label S P
S P
S P
δ
POI
S
S
Cysteine-selective
Cysteine-selective
ACS Paragon Plus Environment
OEt
OEt
OEt
Vinyl-
phosphonothiolates