Selenoimidazolium Salts as Supramolecular Reagents for Protein Alkylation

Article abstract of DOI:10.1002/cbic.202000557

Se-benzyl selenoimidazolium salts are characterized by remarkable alkyl-transfer potential under physiological conditions. Structure-activity relationship studies show that selective monoalkylation of primary amines depends on supramolecular interactions

Full text of DOI:10.1002/cbic.202000557

Combining Chemistry and Biology
Accepted Article
Title: Selenoimidazolium salts as supramolecular reagents for protein
alkylation
Authors: David Lim, Xiaojin Wen, and Florian P. Seebeck
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To be cited as: ChemBioChem 10.1002/cbic.202000557
Link to VoR: https://doi.org/10.1002/cbic.202000557
01/2020

10.1002/cbic.202000557
ChemBioChem
Selenoimidazolium salts as supramolecular reagents for protein al-
kylation
David Lim1, Xiaojin Wen1, and Florian P. Seebeck1*
1 Department of Chemistry, University of Basel, Mattenstrasse 24a, Basel 4002, Switzerland
* florian.seebeck@unibas.ch
Abstract: Se-benzyl selenoimidazolium salts are characterized by remarkable alkyl transfer potential under physiological condi-
tions. Structure-activity correlation studies show that selective monoalkylation of primary amines depends on supramolecular inter-
actions between the selenoimidazole leaving group and the target nucleophile. We demonstrate that these reagents can be used for
site-selective and nearly quantitative modification of the model protein lysozyme on lysine 13, bypassing the higher intrinsic reac-
tivities of lysines 1 and 33. These observations introduce selenoimidazolium salts as novel class of electrophiles for selective N-
alkylation of native proteins.
Se
NH3
Lys13
H2N
O
RN
NR
O
37°C, pH 7.4
1
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10.1002/cbic.202000557
ChemBioChem
Introduction
Protein alkylation has emerged as an important strategy to endow proteins with new functions.[1] The most common targets for
protein alkylation are cysteine residues.[2] Thiols are characterized by pKa values near the physiological pH, thiolates are
exceptionally nucleophilic and cysteines residues are sufficiently rare so that most proteins of interest contain no surface accessible
thiols. Recombinant proteins with engineered cysteine residues can be alkylated efficiently with a broad variety of electrophiles.
Surface exposed amino groups in the form of lysine side chains or the N-terminus are less reactive that thiolates, but are much more
abundant.[3] Hence, with the appropriate reagents, N-alkylation may be a more general approach for the modification of native
proteins.[4] The central challenge in N-alkylation lies in singling out one specific amino group.[3, 5] One strategy for site specific
modification with small electrophiles is to target amino groups that are inherently activated by their local environment (Figure 1A).[3-
4, 6] These protocols require careful design of reaction conditions that maximize yield and selectivity via reagent limitation and kinetic
control. A potential problem of this approach may be that reactive lysines are often located at functional sites and their alkylation
may interfere with protein function.[7] This correlation between reactivity and functionality may facilitate the design of lysine-
reactive inhibitors,[8] but it may render labeling of functional proteins difficult.
O
A:
O
MeO2S
O
NH2
HN
O
Figure 1. Selective alkylation of lysine residues on
protein of interest (POI) by targeting intrinsically
reactive residues (A),[3] by S-adenosylmethionine
(SAM) dependent protein methyltransferases
(PMT, B),[9] or by selenoimidazolium salts (C).
Transferred group in blue, alkyl donor in red.
PO
I
H3N
NH3
H3N
HSO2Me
NH3
B:
S
R
R
PMT:SAM
NH
NH2
NH2
PO
I
H3N
H3N
H3N
NH3
NH3
NH3
PMT
+ SAH
C: This study:
NR
N
R
Se
R
imidazyl
selenonium
R
NH
NH3
NH2
PO
I
H3N
H3N
NH3
NH3
H3N
NH3
Se
NR
RN
Enzymes that catalyze protein alkylation, such as protein N-methyltransferases (PMT), do not depend on the inherent reactivity of
the target nucleophile.[10] PMTs in complex with the methyl donor S-adenosylmethionine (SAM) form ternary complexes with
specific lysine residues based on multiple weak interactions with the surface of the client protein (Figure 1B). Formation of this
complex stabilizes the neutral amino group by desolvation and juxtaposition of the activated nucleophile with the electrophilic
sulfonium moiety of SAM. Proper positioning of the reactants in the ground state combined with electrostatic and dynamic features
that stabilize the reactants in the transition state structure both contribute to afford efficient and specific alkyl transfer.[11] Native and
2
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10.1002/cbic.202000557
ChemBioChem
engineered PMTs have been shown to accept SAM-analogs and mediate the transfer of larger alkyl groups, showing that enzymes
can be useful tools for specific protein alkylation.[9] However, enzyme catalysis is limited to client proteins for which specific
modifying enzymes are known or can be obtained by protein engineering.
One approach to emulate enzyme-like recognition of lysine side chains has emerged from supramolecular chemistry in the form of
molecular tweezers.[12] Rigid U-shaped molecules that provide a preorganized aromatic cavity decorated with peripheral anionic
groups were shown to bind lysine residues on protein surfaces via a combination of electrostatic and dispersive attraction.[13] These
interactions are sufficiently specific and strong to alter protein functions under physiological conditions and in vivo.[12a, 14] However,
supramolecular systems that leverage noncovalent recognition for covalent modification – as do PMTs – have not yet been
developed.[15] In the following report we disclose a new class of reagents that may do just that. We found that selenoimidazolium
salts can act as slow but specific alkylation reagents under physiological conditions (Figure 1C). Structure-activity correlation
analysis with model reactions provides evidence that the selenoimidazolium cations bind and activate their nucleophilic substrates
via non-polar interactions. A similar mechanism appears to facilitate specific and near-quantitative alkylation of lysozyme at Lys13,
bypassing the higher intrinsic nucleophilicities of Lys1 or Lys33.
Results and Discussion
Proof of concept. The idea to explore selenoimidazoles as alkyl carriers was inspired by the report that the natural metabolite
ergothioneine participates in the biosynthesis of lincomycin as an alkyl carrier (Figure 2A).[16] We surmised that the involved alkyl
transferase (LmbV) likely binds the mercaptoimidazole ring in protonated form to make ergothioneine an efficient leaving group.
Indeed, mercaptoimidazolium salts with a hypervalent sulfur atom are excellent reagents for alkynylation,[17] chlorocyanation,[18] or
alkylation[19] of carbon, nitrogen, oxygen and sulfur nucleophiles in organic solvents (Figure 2B – 2D). Selenoimidazolium salts
have not yet been examined in this regard.
During our recent efforts to synthesize the selenoimidazole containing natural product selenoneine, we noticed the mobility of the
Se-benzyl group on the model compound 1 under mild conditions (Scheme 1).[20] To examine this reactivity more closely we
synthesized a number of derivatives (3 – 10, 13, 14), [21] and measured their ability to alkylate benzylamine, serine or cysteine in
phosphate buffer (pH 7.4) at 37°C (Scheme 1, Table 1, Figures S1 – S17). Not surprisingly, cysteine reacted most efficiently with
selenoimidazolium salts. At a cysteine concentration of 100 mM, debenzylation of 1 was found completed within 2 min. The reaction
with benzylamine proceeded at a much slower but observable rate (Table 1), whereas products resulting from benzyl transfer to
serine or to hydroxide were not detected.
3
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10.1002/cbic.202000557
ChemBioChem
The common benzylation reagent benzyl bromide reacted 20-fold faster with benzylamine and produced a mixture of dibenzylamine
(93 %) and tribenzylamine (7 %, Table 1). Benzyl bromide also reacted with serine and hydroxide at least 103-fold faster than the
selenoimidazolium salt. Evidently, the selenium reagent 1 is less reactive than benzyl bromide, but is also more nucleophile selective
and more resistant towards hydrolysis. The mercaptoimidazolium compound 2 showed no alkyl transfer activity to benzylamine,
serine or hydroxide under these conditions, highlighting the significant difference in the reactivity of sulfonium and selenonium
functions.[22]
NHR1
OH
NHR1
OH
OH
HO
O
R2
OH
HO
HO
S
S
O
Figure 2. Mercaptoimidazoles as group transfer
reagents. Enzyme-catalyzed (LmbV) transfer of a
glycosyl group from ergothioneine to mycothiol
(A).[16] Imidazyl thione-mediated alkynylation
(B),[17] chlorocyanation (C),[18] and methylation (D)
in organic solvents.[19] Transferred carbon group in
blue.
mycothiol
HO
HN
HN
LmbV
NH
N
O
S
AcN
A:
N
N
NHR3
ergothioneine
O
O
O
O
CO2Me
S
S
CO2Me
S
B:
S
DCM
iPr
iPr
N
+
+
N
iPr
iPr
N
N
S
CF3
CF3
Cl
CN
N
C:
S
H
Cl
1,2 DCE
CN
iPr
iPr
N
+
iPr
iPr
+
N
N
H
D:
Me
N
I
S
S
DMSO
+
+
N
N
N
N
Me
N
I
The non-innocent role of the N-benzyl side chains. Compared to benzyl bromide, reagent 1 has a rather large and hydrophobic
leaving group. Hence, it is possible that limited accessibility reduces the reactivity of the Se-benzyl group. However, the N,N-
dimethylated derivative (6) was found to be only two-fold more reactive towards benzylamine than 1 (Table 1), suggesting that steric
hinderance plays a subordinate role. Instead, reactivity towards serine (> 102-fold) and hydroxide (> 50-fold) increased much more.
This observation suggests that the N-benzyl rings of 1 protect the electrophile against hydrolysis and at the same time increase the
specificity for the nucleophile. In addition, the electronic structure of the N-benzyl groups also influences the reactivity. A
selenoimidazolium derivative armed with electron rich p-methoxy N-benzyl side chains (4), is more reactive than 1, a derivative
with with electron poor p-nitro N-benzyl side chains (3) is less reactive than 1. A similar trend emerges from the observed relative
activities of compounds 7, 13 and 14. This structure-activity correlation indicates that the aromatic side chains may play an active
role in facilitating benzyl transfer.
4
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10.1002/cbic.202000557
ChemBioChem
R
Se
RN
NR
R
H2N
R
HO
HN
OH
HO
HS
NH
S
CO2H
H2N
CO2H
H2N
CO2H
pH 7.4, 37°C
H2N
CO2H
R
R
Se
X
Se
N
N
N
N
N
N
R
R
3: R = NO2
4: R = OMe
5: R = CO2H
1: X = Se
2: X = S
6: R = H
11: R = OMe
R
O
Scheme 1. Nucleophiles and
electrophiles examined in this
study.
R
Se
Se
Se
N
N
N
N
N
N
HO2C
CO2H
HO2C
CO2H
CO2H
9: R = methyl
10: R = proparyl
7: R = OMe
8: R = F
12
O
O
O
Se
Se
Se
N
N
N
N
N
N
R
CO2H
R
R
CO2H
CO2H
15: R = OMe
16: R = CO2H
17: R = OMe
18: R = CO2H
13: R = OMe
14: R = Br
Table 1. Initial rates of benzyl transfer [M-1s-1]
a
b
Alkylation
Reagent
(Scheme 1)
BnNH2
BnNH2
Serine a
Hydrolysis a,c
[x 10-10 M-1 s-1]
pH 7.4
Lysozyme (24 h)d
monobenzylated
lysozyme [%]
[x 10-10 M-1s-1] [x 10-10 M-1s-1]
[x 10-10 M-1 s-1]
pH 7.4
pH 7.4
7.9
< 0.1
3.2
13
pH 10
17
-
5.0
-
6.2
-
-
-
-
-
-
-
-
-
_-
-
-
-
-
1
2
3
4
5
6
7
8
9
10
11
12
13
14
15
16
17
18
< 0.1
< 0.1
< 0.1
0.13
< 0.1
14
2.7
< 0.1
< 0.01
< 0.01
< 0.01
< 0.01
< 0.01
0.45
0.014
< 0.01
< 0.01
< 0.01
-
30
-
< 2
-
28
-
93
< 2
< 2
< 2
< 2
14
90
68
22
39
1.4
18
4.5
< 0.1
< 0.1
< 0.1
-
< 0.1
< 0.1
-
-
-
-
-
-
-
-
-
-
5.1
0.32
-
-
-
0.02
0.001
-
-
-
80
89
49
-
-
BnBr
200
120
> 43
38 (polybenzylated)
a
Reactions contained 100 mM phosphate buffer (pH 7.4), 100 mM benzylamine or serine, and 2 mM selenoimidazolium halide.
The benzylation products dibenzylamine, S-benzyl cysteine, N-benzyl serine and benzyl alcohol were identified by HPLC
b
comparison with authentic compounds (not shown). At pH 10 benzylamine rather than phosphate is the dominant buffering
5
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10.1002/cbic.202000557
ChemBioChem
molecule. c Bimolecular rate constants for hydrolysis was determined assuming hydroxide ([OH] = 250 nM) as the nucleophile. d 1
mM lysozyme from chicken egg was reacted with 5 mM selenoimidazolium halide or 5 mM benzyl halide in 100 mM phosphate
buffer (pH 7.4) at 37°C. After 24 h the reactions were analyzed by high-resolution electrospray ionization mass spectrometry (HR-
ESI-MS, Figure 3 and S18 – S28). Signal intensities were used to estimate the fraction of monobenzylated lysozyme. Given values
correspond to averages of two independent measurements. Polybenzylated lysozyme accounted for less than 2 % unless stated
otherwise.
Previous studies on host-guest chemistry in aqueous media showed that cyclophanes can bind quinoline in an aromatic environment
and accelerate N-alkylation of this guest by methyl iodide. Desolvation of the nucleophilic guest combined with cation-p interactions
that stabilize the reactants in the cationic transition state were identified as key features that facilitate methyl transfer.[23] Similar
interactions may promote benzyl transfer from N,N-dibenzyl selenoimidazolium salts to benzylamine. The conjugated acid of the
nucleophile (benzylammonium) is characterized by a pKa of 9.3. Consequently, at pH 7.4 the reaction contains about 0.5 mM of the
active nucleophile and 99.5 mM of the conjugated acid. If the reaction rate is limited by the effective concentration of the nucleophile,
one would expect a strong pH dependence. Contrary to this expectation we found that increasing the reaction pH to 10 increased the
reactivities of compounds 1 and 3 by only two-fold. This shallow pH dependence indicates that the selenonium reagent decreases
the effective pKa of the nucleophile. One way this may happen is that the selenoimidazolium cation selectively binds to the
nucleophile in neutral form.
The correlation between the benzyl transfer activity of selenoimidazolium salts and the p-donating abilities of their N-benzyl side
chains (compounds 1, 3 and 4; 7, 13 and 14) indicate that cation-p interactions may also contribute to the observed N-benzylation
reactivity of selenoimidazolium salts. In a way, these reagents provide rudimentary models of methyltransferases that leverage
binding energy to activate their substrate, and accelerate alkyl transfer by transition state stabilization (Figure 1C).
Transferrable alkyl groups. Even though the compound with two p-carboxy N-benzyl side chains (5) proved less reactive than
compounds 1, 3 and 4, we continued with this derivative for further development because the carboxylates improved solubility in
water (> 50 mM). Testing selenoimidazolium salts with different transferrable groups showed that electron rich benzyl and p-
methoxy benzyl groups (5 and 7) are transferred more efficiently than p-fluoro benzyl, methyl or propargyl groups (8, 9 and 10). A
convincing explanation for this effect suggests that the alkyl transfer occurs via a loose transition state with considerable
development of cationic charge on the transferred carbon atom.[24] Conjugative interactions between the cationic carbon and adjacent
donating p-electrons can stabilize this transition state.[9c, 9d]
6
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10.1002/cbic.202000557
ChemBioChem
Application to protein modification. In a next step we examined as to whether selenoimidazolium salts could be used to alkylate
proteins under physiological condition. To our delight, we found that treatment of 1 mM hen egg-white lysozyme with 5 mM 1 or 5
for 24 h at 37°C produced up to 30 % monobenzylated and less than 2 % dibenzylated protein, as inferred by high-resolution
electrospray ionization mass spectrometry (HR-ESI-MS, Figure 3A and 3B). The more reactive derivative 7 converted more than
90 % of lysozyme to the monobenzylated derivative. Tryptic digestion of this product, followed by liquid chromatography–mass
spectrometry analysis (LC-MS) identified lysine 13 (K13) as the exclusive benzylation site (Figure S29, Table S1). This result is
remarkable because lysozyme contains six lysines plus the free amine at the N-terminus. K33 has been shown to be particularly
reactive towards alkylation because its pKa is 0.5 units lower than that of the other lysines.[3] An earlier study identified K1 is the
preferred target of acylation.[6] Hence compound 7 is able to alkylate a specific lysine independent of intrinsic nucleophile reactivity
(Figure 3C). This target specificity is accentuated by the finding that treatment of bovine serum albumin (BSA, 59 lysines) or RNase
A (10 lysines) with 7 under the same conditions produced less than 2 % alkylated protein (Figure S30).
The N-benzyl side chains of 7 proved crucial for alkylation of lysine at K13. Derivatives missing one or both aromatic side chains
alkylated lysozyme much less efficiently (12 and 11). This result shows that selectivity for K13 is not the result of steric exclusion
of other targets. In contrast, the N-benzyl side chains seem to play an active role in promoting benzylation in a similar way as
observed with the model reactions (Figure 3A). The yield of benzylated lysozyme decreased to 78, 33 and 16 % when the reaction
solution contained 10, 20 or 30 % methanol as co-solvent (Figure S31). This sharp decline in alkylation efficiency suggests that
hydrophobic interactions between 7 and K13 are important for efficient benzyl transfer (Figure 1A). Similar hydrophobic
interactions, combined with dispersive forces, have been found to drive association between lysine side chains and the aromatic
scaffold of molecular tweezers.[12a]
The substituents on the N-benzyl side chains also affec the efficiency of lysozyme benzylation, but to a lesser extent that observed
in the model reactions with benzylamine. Derivatives of 7 with one p-carboxy N-benzyl side chain replaced with a p-methoxy benzyl
chain (13) also benzylated lysozyme nearly quantitatively, whereas the derivative with one p-bromo benzyl group (14) proofed
significantly less reactive (Table 1). Nevertheless, given this shallow correlation we anticipate that combinatorial variation of the
substitution pattern on the N-benzyl rings will provide a strategy to develop tailor made alkylation reagents to target specified protein
surfaces.
Finally, we tested as to whether selenoimidazolium salts can also transfer chemical functions that are amenable to bioorthogonal
conjugation.[25] Popular functionalities for this purpose are terminal alkynes which can be conjugated via click reactions.[1c, 9c, 26]
7
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10.1002/cbic.202000557
ChemBioChem
Compound 10 which would deliver a minimal proparyl group did not react with lysozyme under the standard conditions. Compounds
15 and 16 did transfer p-propargyloxy benzyl groups, but not efficiently. The reduced transfer activity is likely related to the electron
withdrawing nature of the alkyne functionality either adjacent to (10) or electronically coupled to (15 and 16) the electrophilic carbon
atom. Consistently, reagents such as 17 and 18, in which the terminal alkyne function is electronically isolated from the benzyl
portion, alkylated lysozyme almost quantitatively under standard conditions (Figure 3D).
CO2
R
R
N
CO2
Se
N
K13
N
A:
5, 7, 18
37°C, pH 7.4
native lysozyme
B:
C:
D:
O
O
native
Lysozyme
Lysozyme
Lysozyme
native
native
Figure 3. Site-specific alkylation of lysozyme at Lys13 by compounds 5, 7 and 18 (A). Reaction products were analyzed by HR-
ESI-MS. Insets: the combined ion series (envelope). Signal intensities in the deconvoluted mass spectrum were used to estimate the
relative fraction of native and modified lysozyme.
Conclusions
In this study we introduce selenoimidazolium salts as a new type of reagents that leverage supramolecular interactions for selective
covalent modification of native proteins. In contrast to leaving groups of conventional alkylation agents such as bromide in benzyl
bromide, the selenoimidazole moiety plays a non-innocent role in promoting specific alkylation. Structure-activity correlation show
that interactions with the N-benzyl side chains of the electrophile are important to recognize and activate the specific nucleophilic
substrate. The same aromatic environment also seems to protect the electrophile against hydrolysis and may accelerate benzyl
transfer via transition state stabilization through cation-p interactions. The modular structure of the described selenoimidazoles and
8
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10.1002/cbic.202000557
ChemBioChem
the simplicity of their synthesis bode well for the development of second-generation supramolecular reagents that may alkylate
specific residues in other proteins of interest.
Acknowledgements. This project was supported by the NCCR for Molecular Systems Engineering and by the “Professur für
Molekulare Bionik”. X.W. is a recipient of the Camille and Henry Dreyfus Foundation PhD Scholarship.
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