DFT‐Guided Discovery of Ethynyl‐Triazolyl‐Phosphinates as Modular Electrophiles for Chemoselective Cysteine Bioconjugation and Profiling [original]

Bioorthogonal Chemistry Hot Paper

DFT-Guided Discovery of Ethynyl-Triazolyl-Phosphinates as

Modular Electrophiles for Chemoselective Cysteine Bioconjugation

and Profiling

Christian E. Stieger, Yerin Park, Mark A. R. de Geus, Dongju Kim, Christiane Huhn,

J. Sophia Slenczka, Philipp Ochtrop, Judith M. Müchler, Roderich D. Süssmuth,

Johannes Broichhagen, Mu-Hyun Baik,* and Christian P. R. Hackenberger*

In memory of Professor Ulf Diederichsen

Abstract: We report the density functional theory

(DFT) guided discovery of ethynyl-triazolyl-phosphi-

nates (ETPs) as a new class of electrophilic warheads for

cysteine selective bioconjugation. By using CuI-catalysed

azide alkyne cycloaddition (CuAAC) in aqueous buffer,

we were able to access a variety of functional electro-

philic building blocks, including proteins, from diethyn-

yl-phosphinate. ETP-reagents were used to obtain

fluorescent peptide-conjugates for receptor labelling on

live cells and a stable and a biologically active antibody-

drug-conjugate. Moreover, we were able to incorporate

ETP-electrophiles into an azide-containing ubiquitin

under native conditions and demonstrate their potential

in protein–protein conjugation. Finally, we showcase the

excellent cysteine-selectivity of this new class of electro-

phile in mass spectrometry based, proteome-wide cys-

teine profiling, underscoring the applicability in homo-

geneous bioconjugation strategies to connect two

complex biomolecules.

Introduction

The chemical functionalization of proteins allows probing

and altering their biological function and generating new

biotherapeutics, including antibody-drug-conjugates

(ADCs).[1,2] In these applications a homogeneous and well-

defined conjugate is desired.

One approach to achieve this relies on incorporating

unnatural amino acids with distinct bioorthogonal

reactivities.[3–5] Alternatively, one can exploit the inherent

reactivity of canonical amino acid side chains. Chemo-

selective modifications of most amino acids in proteins are

typically carried out by electrophilic reagents targeting

nucleophilic amino acids, especially lysine and cysteine.[2, 6–8]

The sulfhydryl group of cysteine is a particularly attractive

target for bioconjugation due to its low natural abundance

and high nucleophilicity under physiological conditions.[9]

Maleimides are most frequently employed as electrophiles

in cysteine bioconjugation even though the formed thiosuc-

cinimide linkage is susceptible to retro-Michael reaction,

which can be problematic for the conjugation of cytotoxic

payloads.[10,11] To overcome this problem, several alternative

methodologies for cysteine labelling have been developed

including vinylsulfonamides,[12] carbonyl acrylic

derivatives,[13] hypervalent iodine reagents,[14,15] and organo-

metallic reagents.[16,17] Nonetheless, moderate selectivity or

low reactivity under physiological conditions continue to be

problematic.[18]

In addition to selectivity and stability concerns, the

synthetic accessibility and modularity of the electrophile are

important. Whereas peptides can be functionalized with an

[*] C. E. Stieger, Dr. M. A. R. de Geus, C. Huhn, Dr. P. Ochtrop,

J. M. Müchler, Dr. J. Broichhagen, Prof. Dr. C. P. R. Hackenberger

Leibniz-Forschungsinstitut für Molekulare Pharmakologie (FMP)

Robert-Rössle-Strasse 10, 13125 Berlin (Germany)

E-mail: [email protected]

C. E. Stieger, C. Huhn, Dr. P. Ochtrop, J. M. Müchler,

Prof. Dr. C. P. R. Hackenberger

Department of Chemistry, Humboldt Universität zu Berlin

Brook-Taylor-Straße 2, 12489 Berlin (Germany)

Y. Park, D. Kim, Prof. Dr. M.-H. Baik

Department of Chemistry, Korea Advanced Institute of Science and

Technology (KAIST)

Daejeon 34141 (Republic of Korea)

Y. Park, D. Kim, Prof. Dr. M.-H. Baik

Center for Catalytic Hydrocarbon Functionalizations, Institute for

Basic Science (IBS)

Daejeon 34141 (Republic of Korea)

E-mail: [email protected]

J. S. Slenczka, Prof. Dr. R. D. Süssmuth

Institut für Chemie, Technische Universität Berlin

Strasse des 17. Juni 124, 10623 Berlin (Germany)

published by Wiley-VCH GmbH. This is an open access article under

the terms of the Creative Commons Attribution Non-Commercial

NoDerivs License, which permits use and distribution in any med-

ium, provided the original work is properly cited, the use is non-

commercial and no modifications or adaptations are made.

Angewandte

Chemie

Research Articles www.angewandte.org

How to cite: Angew. Chem. Int. Ed. 2022, 61, e202205348

International Edition: doi.org/10.1002/anie.202205348

German Edition: doi.org/10.1002/ange.202205348

electrophile with ease in organic solvents, aqueous environ-

ments pose formidable challenges. Genetically encoding

latent bioreactive electrophiles were partially successful,[19]

but encoding more reactive electrophiles often results in

unspecific side reactions with cytosolic thiols.[20] Therefore,

the most common strategy to position a highly reactive

electrophile on proteins is the use of bis-electrophilic linkers

that can be attached to cysteines or other nucleophilic

residues; nevertheless, undesired intramolecular reactions[21]

and dimerization are often observed.[22,23] Alternatively, a

nucleophilic amino acid can be chemically converted into an

electrophile as exemplified by the umpolung of cysteine,

tyrosine, and histidine.[24–27]

Another attractive application of electrophiles is in the

area of chemoproteomics[28] to label a specific proteome for

convenient separation and profiling. For optimal results, the

labelling reagents should offer a high target coverage, a

uniform modification, excellent amino acid selectivity, and

stability of the formed conjugate.[18] To date, this approach is

widely applied in elucidating the proteome-wide redox-

sensitivity[29] and ligandability of cysteines by electrophilic

fragments,[30,31] natural products,[32] or drugs.[33]

As part of our recent effort to establish diethynyl

phosphinates as disulfide rebridging reagents for stable

antibody modifications, we employed these bisfunctional

cysteine-selective electrophiles for the step-wise functionali-

zation of a protein substrate (Scheme 1B).[34] In this proto-

col, an electrophilic group was installed on a former cysteine

sidechain by reaction with diethynyl phosphinate and further

reacted with a thiol-containing small-molecule or peptide to

generate the desired conjugate. Despite these advances, the

incorporation of thiols into small molecules can be syntheti-

cally laborious and is accompanied by the inherent problem

of disulfide formation. Therefore, we wanted to exploit

other modular and synthetically straightforward strategies to

incorporate cysteine-selective electrophiles into small mole-

cules, peptides and proteins.

Our current paper describes the discovery of 1,2,3-

triazolyl-substituted ethynyl phosphinates (ETPs) as readily

accessible, fast, and highly selective thiol-electrophiles

(Scheme 1C), guided by density functional theory-based

computer models. These molecules can be easily accessed

from various azide-containing molecules via copper-cata-

lyzed azide-alkyne cycloaddition (CuAAC) in aqueous

buffer. Upon cycloaddition, the electrophiles show a

remarkably high reactivity towards cysteine compared to

diethynyl-phosphinates and outperform our previously re-

ported phosphonamidate electrophiles (Scheme 1A)[35] in

antibody-labelling experiments. We showcase the utility of

these reagents in the generation of various biologically

relevant peptide-, protein-, and antibody-conjugates. Addi-

tionally, we demonstrate that diethynyl-phosphinates can be

used to functionalize azide-containing proteins with an ETP-

electrophile in aqueous systems and use it for protein-

protein conjugation. Finally, we demonstrate the excellent

cysteine selectivity of ETP-electrophiles by proteome-wide

reactivity profiling.

Results and Discussion

We started our investigation by determining the kinetic

parameters of the second thiol addition to diethynyl-

phosphinates (Scheme 1B). Thus, we reacted a small thiol-

containing fluorophore (EDANS-SH) with an excess of

ethyl diethynyl-phosphinate 1and obtained thiovinyl-

ethynyl phosphinate 2in good yield (Z-isomer, 76%). 2was

exposed to an equimolar amount of reduced glutathione in

an aqueous buffer at pH 8.5[36,37] and we observed smooth

conversion to a mixture of the E- and Z-isomers of the

glutathione adduct. The second-order rate constant

(0.29 M1s1, Figure S5) was found to be slightly lower than

the kinetics we reported earlier for the first thiol-addition of

the diethynyl-phosphinate (0.47 M1s1, Scheme 1B).[34] To

increase the reaction speed, we first envisioned to use

electron-withdrawing (EWG) alcohol-substituents in 1, as

previously demonstrated for unsaturated

Scheme 1. General concept of unsaturated PV-electrophiles in Cys-

selective protein labelling. A) Electrophilic phosphonamidates, gener-

ated via the chemoselective Staudinger-phosphinonite reaction, react

selectively with cysteine residues on proteins. B) Diethynyl-phosphi-

nates can react selectively with two distinct thiol-nucleophiles and

enable protein double-modification and interchain disulfide rebridging

in IgG antibodies. C) CuI-click functionalisation of diethynyl-phosphi-

nates generates ETP-reagents that show superior reaction kinetics in

cysteine selective protein modification.

Angewandte

Chemie

Research Articles

phosphonamidates,[36] however, all attempts to isolate dieth-

ynyl-phosphinates bearing EWG alcohol-substituents failed

due to rapid hydrolysis of the PO bond. Prior work from

our laboratories showed that density functional theory

(DFT) calculations can predict the relative rates of thiol

conjugations to the respective PV-electrophiles.[38] Also,

analysis of the molecular orbitals suggested that the key

factor in enhancing the reactivity is to stabilize the

negatively charged intermediate generated upon the nucleo-

philic attack of thiolate, where delocalization of the anionic

character is achieved through hyperconjugation. The bond

P–XEt (X: S, O, and NH) located in an antiperiplanar

position to the lone-pair played a decisive role in accepting

the electron density. Based on this concept, we interrogated

if we can use the accepting ability of new substituents

featuring π systems accessible from 1(i.e., π-acidity) to

improve the reactivity. We envisioned that several hetero-

cyclic substituents containing electronegative oxygen and/or

nitrogen atoms would be suitable to achieve both, an

electron-withdrawing inductive effect and π-conjugation

with the lone-pair electrons. Therefore, we focused on the

reagents A–Econtaining isoxazole (A), 1,2,3-triazole (Band

C), 1H-pyrazole (D) and 3H-pyrazole (E) substituents and

calculated their reaction profiles using DFT. We modelled

the reactivity of previously reported 2by truncating the

pendant substituent as a methyl group as compound F

(Figure 1).

The general scheme of the reaction energy profile for

the Michael-addition to the electrophiles A–Fis shown in

Figure 1, including ethanethiol (EtSH) as a model nucleo-

philic reaction component.

In aqueous buffer (e.g. pH 6.5–8.5), EtSH and the

corresponding thiolate anion EtSare in equilibrium. Since

the thiolate is the better nucleophile, we assumed the

stepwise deprotonation of EtSH and subsequent nucleo-

philic attack of EtSto form the carbanion intermediate.

The calculated reaction barriers (ΔG�) for respective

electrophiles were used to compare the reaction kinetics

among them. Calculated ΔG�values decrease in the order

of 23.3 (F)>20.0 (C)�19.5 (E)�19.0 (D)>18.1 (A)�

18.1 kcalmol1(B), suggesting that the newly designed

electrophiles A–Eshould be much more reactive than F.

Finally, the intermediate is protonated by water irreversibly

to form the final thiol-addition product. The electrophilicity

of the series of PV-reagents can be explained by DFT-

calculated partial charges of the reactive terminal carbon

atom on the ethynyl group, as atomic charges can be used as

a measurement of the electron-withdrawing ability of

various functional groups.[39–43] Natural population analysis[44]

was conducted to compare the inductive effect posed by the

distinctive substituents in electrophiles A,C, and F(Fig-

ure 2). The atomic charges (qC) are correlated to the

reaction barriers, being most positive for A(0.092),

followed by C(0.108), and most negative for F(0.122).

Along with the partial charges, the frontier molecular

orbitals can be used to account for the electrophilicity or

nucleophilicity,[42,45–48] where the energy levels of the orbitals

affect the efficiency of the bond-forming interaction.[49]

Here, the energy of the reactive π* orbital of the PV-

electrophiles is lowest for A(1.65 eV), followed by C

(0.93 eV), and highest for F(0.33 eV), in line with the

reactivity trend (Figure 2).

We further interrogated how the substituents influence

the π-system to enhance the reactivity. Distortion of the

PC=C bond in the geometry of the transition state (TS)

and carbanion intermediate (Int) is characterized by the

preference for the sp2-hybridised lone-pair to be antiperipla-

nar to the PR bond (Figure 3a). The optimized TS and

intermediate structures of C(C-TS and C-Int) are illustrated

in Figure 3a. This structural feature is also found in the

previous system, where PRσ-bonds (R: S, O, and NH)

accept electron density via hyperconjugation. Similarly,

delocalization of electrons into vacant orbitals related to the

PR bonds is feasible as conceptualized in Figure 3b. The

mixing between π-systems of the ethynyl moiety and PR

bond is clearly captured in the LUMO of the electrophiles.

The conjugation renders the π*yorbital more reactive

compared to π*zorbital, except in the case of F, and

ultimately favors the lone-pair electrons to form in that

direction. Following the guideline provided by the theoret-

Figure 1. Reaction energy profile of the thiol addition of the proposed

reagents A–Eand a model compound F. Energies in kcalmol1,

calculated with B3LYP-D3/cc-pVTZ(-f)//B3LYP-D3/6-31G** level of

theory.

Figure 2. Comparison between electrophiles A,C, and F.qC: Natural

atomic charge at the reactive carbon, E(π*): Energy of the reactive π*

orbital of the electrophile. (For more details see Figure S3 and S4).

Angewandte

Chemie

Research Articles

ical study, we envisioned that 1,2,3-triazole-substituted

phosphinate electrophiles could be easily accessed via CuI-

catalyzed azide-alkyne cycloaddition.

Therefore, we investigated the synthesis of ETP-electro-

philes starting from EDANS-N3and phosphinate 1. While

1–3 equiv phosphinate in the presence of 10 mol% CuBr

resulted mainly in the di-functionalized phosphinate, 5 equiv

phosphinate allowed us to obtain the desired mono-

functionalized molecule 3in 85% yield (Scheme 2a). To

validate the computational results, we performed kinetic

analysis using glutathione as a model thiol as described

before. Phosphinate 3showed accelerated reaction kinetics

of 4.61 M1s1(Figure S5), thus outperforming phosphinate 2

by roughly 15-times, which is in agreement with the DFT-

calculated reaction barriers (Scheme 2a). These findings

inspired us to synthesize various functionalized ETP-electro-

philes (Scheme 2b). Using the described procedure, we were

able to generate ETP-reagents bearing fluorophores (3,4

Figure 3. a) The general conformation of transition states and intermediates viewed from the top (left). Geometry of C-TS and C-Int as

representatives (right). b) Conceptualized orbital mixing between two fragments for the LUMO of electrophiles and the HOMO of intermediates.

(For the actual molecular orbitals, see Figure S1 and S2).

Scheme 2. a) Schematic representation of the experimental thiol addition kinetics of phosphinates 1–3at pH 8.5. b) Synthetic procedure for the

generation of functionalized ETP-electrophiles. c) Functional electrophiles obtained via the synthetic procedure depicted in b (values represent

isolated yield).

Angewandte

Chemie

Research Articles

and 5), affinity-tags (6and 9) or bioorthogonal-click-handles

(7and 8) in moderate to good yield. Moreover, we could

show that this strategy allows for incorporating an ETP-

electrophile into an azide-containing cell-penetrating R10-

peptide (10) (Scheme 2c).

To probe the applicability of ETP-electrophiles for Cys-

selective protein and antibody labelling, we choose the

Her2-targeting therapeutic antibody Trastuzumab as a

model system. At first, we compared thiovinyl-phosphinate

2and triazolyl-phosphinate 3. In brief, Trastuzumab

(5 mgmL1in Tris-buffer pH 8.3) was reduced with 8 equiv

TCEP (37°C, 30 min). Subsequently, 8 equiv of the corre-

sponding phosphinate were added, and the reaction was

allowed to proceed overnight at room temperature (Fig-

ure 4a). While compound 2achieved an average labelling of

4.2 fluorophores per antibody, compound 3reached almost

stoichiometric labelling of all free cysteine thiols in the

antibody corresponding to a fluorophore-to-antibody-ratio

(FAR) of 7.4 (Figure 4b and S6). In a control experiment, in

which the antibody was not reduced prior to the reaction

with the two phosphinates, no modification could be

observed by SDS-PAGE and intact-protein mass spectrom-

etry (MS) (Figure S4b). Also, a larger excess of 3

(100 equiv) did not lead to any antibody-labelling in the

absence of a reducing agent (Figure S7).

The observation of almost stoichiometric labelling when

using 8 equiv EDANS-ETP led us to explore if this is a

general phenomenon. We titrated Trastuzumab with in-

creasing amounts of phosphinates 2and 3(Figure 4a). To

our delight, we observed full antibody labelling with up to

5 equiv and close to stoichiometric labelling with 6 and

8 equiv for phosphinate 3. In contrast, compound 2reached

only approximately 50% labelling for all equivalents after

the same time (Figure 4b).

Finally, we also compared ethynyl-triazolyl-phosphinates

with our previously reported ethynyl-phosphonamidates

(PA) in antibody labelling.[35] We performed a time-course

experiment, in which reduced Trastuzumab was incubated

with 10 equiv of the corresponding PV-electrophile, monitor-

ing the reaction over 16 h using intact-protein MS. We

observed that both the PA- and ETP-reagents reached close

to full conversion (FAR 7.5 and 7.9, respectively) after

overnight reaction; however, after shorter reaction times

phosphinate 3resulted in a higher degree of functionaliza-

tion (Figure 4c).

Apart from fast reaction kinetics and cysteine-selectivity,

also serum-stability of the linkage is a prerequisite for the

successful application in antibody-drug-conjugates to pre-

vent hazardous off-target effects. To test this, we generated

an antibody-fluorescein conjugate using phosphinate 4(see

Supporting Information 3.5) and incubated it in human

serum for 14 days at 37°C. Gratifyingly, no significant

transfer onto other serum proteins was observed (Fig-

ure S8). In addition, we made use of a fluorescence-

quenching assay to validate the serum stability of thiol-thiol

conjugates formed with diethynyl-phosphinates (see Sup-

porting Information 3.6).[34,35] The quenched FRET-pair F1

was synthesized from phosphinate 3and DABCYL-peptide

P2 (38%, Figure S9a). Incubation of F1 in a physiologic

buffer in the presence of excess small thiols and in human

serum did not show any increase in fluorescence signal,

indicating excellent stability (Figure S9b).

Encouraged by the straightforward accessibility, high

reactivity and stability as cysteine-conjugates we used ETP-

electrophiles in the generation of ADCs.

As a payload, we selected Monomethyl-auristatin E

(MMAE), a potent anti-mitotic drug commonly used as a

cytotoxic payload in the generation of ADCs, which we

previously used in thegeneration of phosphonamidate-linked

ADCs.[50] After HPLC-purification, the ETP-functionalized

drug (11) was obtained in 59% yield after HPLC. (Fig-

ure 5a) With the electrophile modified toxin in hand, we

started to examine its applicability in antibody labelling.

Since the experiments using EDANS-ETP 3showed that

already after 4 hours the majority of the reagent has reacted

(Figure 4c), the reactions were terminated and checked after

that time. Intact-protein MS revealed that already 5 equiv of

the ETP-drug was sufficient to reach an average drug-to-

antibody-ratio (DAR) of approximately 4 after the relatively

short reaction time. Hence, we did a scale-up of the reaction

using 1 mg of Trastuzumab to allow further biological

testing. After purification via size-exclusion-chromatography

(SEC), the functionalized antibody was obtained in 80%

yield (0.8 mg). Analysis via hydrophobic-interaction-chro-

matography (HIC) revealed that most of the antibody-

molecules are functionalized with 3–6 drug molecules

resulting in an average DAR of 4.3 (Figure 5b). The cellular

Figure 4. Generation of antibody-fluorophore-conjugates (AFC) using

different PV-electrophiles a) Reaction principle of the simultaneous

reduction alkylation reaction using phosphinates 2and 3and

phosphonamidate S1 b) Equivalent-screen of the two phosphinates

shows close to quantitative labelling with ETP 3c) Time-course of the

antibody labelling reaction using 10 equiv of the corresponding electro-

phile shows superior reaction kinetics for 3compared to phosphona-

midate S1.

Angewandte

Chemie

Research Articles

Loading more pages...