Computational chemistry-based
improvement ofbioconjugation reactions
2022/12/22
Literature seminar
M2 Habazaki1

Contents

Contents◆Introduction◆Main➢ Historical example: Bioorthogonal Cycloaddition• Mutual orthogonality & Motif expansion• Reactivity / stability trade-off➢ Recent example: Cysteine Bioconjugation• Reactivity• Modularity◆Summary & Perspective2

Introduction

Features of Bioconjugations
Bioconjugations• The reaction of two molecules and a crosslinking agent that covalently links the components together.
• At least one of the molecules is of biological origin or is a fragment or derivative of a biomolecule.

• Its use is directed toward biological or life science applications.
• Assay and Quantification• Detection, Tracking, and Imaging• Purification, Capture, and Scavenging

Bioorthogonal ligations• Therapeutics and In Vivo Diagnostics• Vaccines and Immune Modulation

Chemoselective modifications• Unnatural functional group• Natural functional group (mainly amino acid)• High site specificity• Immediate accessibility without the need formore specialist techniques• Within the complex milieu of a cell or a cell lysate
Hermanson Bioconjugate Techniques Elsevier Science, 2013

Spicer, Davis Nat. Commun. 2014, 5, 4740

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Introduction

Requirements of Bioconjugations
Fundamental requirements for bioconjugation reactions• Mutual reactivity in a physiological environment(<37 ℃, pH 6–8, aqueous solvent)(a) Mutual orthogonality• Rapid reaction• Near total conversion to generate homogenous constructs• High stability of reagents and adducts• High chemoselectivity• Innocuous (or no) byproducts• Not to disrupt biomoleculars’ architecture and/or function

Requirements for more effective bioconjugation tools(b) Modularity• Mutual orthogonality (a)• Modularity (b)• Metabolic introduction of a unique functional group

Prescher, Bertozzi Nat. Chem. Biol. 2005, 1, 13–21
Shih, Kamber, Prescher Curr. Opin. Chem. Biol. 2014, 21, 103–111
Spicer, Davis Nat. Commun. 2014, 5, 4740
4Bird, Zhou et al. Bioconjugate Chem. 2021, 32, 2457–2479 Baumann, Hackenberger et al. J. Am. Chem. Soc. 2020, 142, 9544–9552

Introduction

Importance of Mechanistic Elucidation and Improvement of Bioconjugations
Challenging points• The prerequisites critically restrict the scope of chemical reactions that can be performed in a biological context.

Key point in development
An in-depth understanding of mechanisms and structural effects is essential for accessing new and improved
versions of bioconjugation chemistry.
• Mechanistic principles• Structure–reactivity trends

One Powerful Strategy (Today’s Topic)
Computational Mechanistic Analysis using DFT Calculation to Improve Bioconjugation Reactions

Deb, Franzini et al. Chem. Rev. 2021, 121, 6850–6914

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Contents

Contents◆Introduction◆Main➢ Historical example: Bioorthogonal Cycloaddition• Mutual orthogonality & Motif expansion• Reactivity / stability trade-off➢ Recent example: Chemoselective Cysteine Bioconjugation• Reactivity• Modularity◆Summary & Perspective

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ContentsContents◆Introduction◆Main➢ Historical example: Bioorthogonal Cycloaddition• Mutual orthogonality & Motif expansion• Reactivity / stability trade-off➢ Recent example: Chemoselective Cysteine Bioconjugation• Reactivity• Modularity◆Summary & Perspective

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Bioorthogonal Cycloaddition

Features of Bioorthogonal Cycloadditions
Bioorthogonal Cycloadditions

Cu-catalysed azide-alkyne cycloaddition (CuAAC)• The most studied bioorthogonal reactions• Fast reactions, Good regioselectivity, Well-established chemistry• The contributions of computations to this field began in the past
decade and have matured.
Strain-promoted azide-alkyne cycloadditions (SPAAC)

Inverse electron-demand Diels-Alder cycloadditions(IEDDA, Tetrazine ligation)

Liu, Liang, Houk Acc. Chem. Res. 2017, 50, 2297–2308

L'Islea, Unciti-Broceta et al. Curr. Opin. Chem. Biol. 2021, 61, 32–428

Bioorthogonal Cycloaddition

Computational Chemistry in Cycloaddition Reactions
Distortion/Interaction (D/I) model• A relatively new tool for chemists to understand reactivities andselectivities(By Prof. Morokuma → Prof. Houk & Prof. Bickelhaupt)• ΔEact is decomposed into two contributions:① The distortion energy associated with the structural distortion (ΔEdist)② The interaction between these increasingly distorted reactants (ΔEint)※ ΔE (Electronic energy) & ΔG (Gibbs free energy)• E: The energy of the electrons in atoms, ions, or molecules, which
are bound by the electric field of the nucleus• G = H – TS = (U + pV) – TS = (E + Etrans + Erot + Eviv) + pV – TS
• Arrhenius equation: k = A exp(–ΔG‡ / kBT)
E → for analysis of reactivity trend, G → for prediction of reaction rate
ΔE‡act = ΔE‡dist + ΔE‡int
Bickelhaupt, Houk et al. Angew. Chem., Int. Ed. 2017, 56, 10070–10086
Liu, Liang, Houk Acc. Chem. Res. 2017, 50, 2297–2308

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Bioorthogonal Cycloaddition

Distortion/Interaction (D/I) Model
Example in SN2 reactions

ΔEdist(ξ)ΔEdist(ξ)

X－ = Nucleophile
Y = Leaving group
ΔE(ξ)
ΔE(ξ)

ΔEint(ξ)

a. A better nucleophile→ A stronger Lewis basicity→ Lowering the SN2 barrier by enhancingthe stabilizing interaction

ΔEint(ξ)

b. A poorer leaving group→ A higher energy penalty to lengthen astronger C−Y bond→ Raising the SN2 barrier because of amore destabilizing distortion curve
Bickelhaupt, Houk et al. Angew. Chem., Int. Ed. 2017, 56, 10070–10086

10Mutual orthogonality & Motif expansion

Elucidation of Mutual Orthogonality in Bioorthogonal Cycloadditions
The mutual orthogonality of azide–DBCO and tetrazine–TCO cycloadditions

QuestionWhy do their selectivities differ dramatically?

AzideDibenzocyclooctyne(DBCO)

Tetrazine (Tz)Trans-cyclooctene(TCO)

DFT-calculation approach• Point out the factors that control thereactivity patterns• Develop a set of design principles fornew orthogonal cycloadditions• Predict that two new bioorthogonal
reagents

Cyclooctyne
Karver, Weissleder, Hilderbrand Angew. Chem., Int. Ed. 2012, 51, 920
Liang, Houk et al. J. Am. Chem. Soc. 2012, 134, 17904–17907

Hu, Schomaker ChemBioChem 2021, 22, 3254–3262

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Mutual orthogonality & Motif expansion

Elucidation of Mutual Orthogonality in Bioorthogonal Cycloadditions
TS1-AzideTCOCyclooctyne

TS1-TzTS2-Azide

TS2-TzTS3-Azide

TS3-TzDBCO

TS1-Azide

TS2-Azide

TS3-Azide

TS1-TzTS2-TzTS3-TzActivation energy (ΔE‡act)
Distortion energy (ΔE‡act, Azide/Tetrazine)
Distortion energy (ΔE‡act, Alkene/Alkyne)
Interaction energy (ΔE‡int)• Trans-cyclooctene (TCO):
The interaction energy of TS1-Tz is much larger than that of TS1-Azide.

• Dibenzocyclooctyne (DBCO):
The extremely sluggish kinetics of the DBCO–tetrazine cycloaddition is mainly due to very high distortion energy.
Liang, Houk et al. J. Am. Chem. Soc. 2012, 134, 17904–17907

Liu, Liang, Houk Acc. Chem. Res. 2017, 50, 2297–2308

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Mutual orthogonality & Motif expansion

Elucidation of Mutual Orthogonality in Bioorthogonal Cycloadditions
Trans-cyclooctene (TCO): ΔEint

Dibenzocyclooctyne (DBCO): ΔEdist
2.38

TS3-Azide

2.19

Close to thesum of van derWaals radii
2.19

TS3-Azide

TS3-TzTS3-Tz• Frontier molecular orbital (FMO) analysis• The smaller orbital energy gap between
TCO and tetrazine makes the favorable
orbital interaction in TS1-Tz stronger thanthat in TS1-Azide.
Liang, Houk et al. J. Am. Chem. Soc. 2012, 134, 17904–17907• The structural analysis of transition states• Because of the great steric hindrance caused by the two arylhydrogen atoms (marked ○), the steric effect overwhelms the electroniceffect, leading to the exclusive azide selectivity.
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Mutual orthogonality & Motif expansion

Calculation-based Design in Bioorthogonal Cycloadditions
Principles for the design of orthogonal reaction pairs• Intrinsically more reactive substances can be made less reactive by increasing the distortion
controlled by steric effects

Prediction of Mutual Orthogonality of Two New Bioorthogonal Reagents
Methylcyclopropene

Tetramethylthia
cycloheptyne(TMTH)

Liang, Houk et al. J. Am. Chem. Soc. 2012, 134, 17904–17907

Liu, Liang, Houk Acc. Chem. Res. 2017, 50, 2297–2308

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Mutual orthogonality & Motif expansion

Application of Methylcyclopropene-Tetrazine Conjugation in Living Systems
Application as a new bioorthogonal reaction• In-vitro selective protein modification• Metabolic incorporation and labeling of cyclopropenes
onto live cell surfaces• Elucidation of the substituent effects on tetrazine–cyclopropene ligation• Identification of new mutual orthogonal reaction pairs• Demonstration of in-cell protein labeling

Patterson, Prescher et al. J. Am. Chem. Soc. 2012, 134, 18638–18643

Liu, Liang, Houk Acc. Chem. Res. 2017, 50, 2297–2308

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Reactivity / stability trade-off

Uncovering the Key Mechanism of the Reactivity/Stability Trade-Off
The influence of aryl substituents of tetrazine on the click reactivity

Question• Are there any yet overlooked mechanistic
aspects other than FMO interactions that havea crucial effect on the reactivity of tetrazine?

• Revealing the key mechanism of thesubstituent effects on Tz-TCO cycloaddition
using Distortion/Interaction model.

Svatunek, Houk, Mikula et al. J. Am. Chem. Soc. 2022, 144, 8171–8177

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Reactivity / stability trade-off

Relationship of the Orbital Energies and the Reaction Rates
FMO analysis using DFT calculation

Experimental measurement of reaction rate

*in 1,4-dioxane• The reactivity trend for Ph, 3Pyr, and 4Pyr seems to be
governed by FMO interactions.
• As expected, the order of the orbital energies was
Ph > 2Pyr, 3Pyr > 4Pyr• 2Pyr is significantly more reactive (>3-fold) than
expected based on the respective orbital energy.
→ The high IEDDA reactivity of 2Pyr cannot be attributed
to the electron-withdrawing effect.

Svatunek, Houk, Mikula et al. J. Am. Chem. Soc. 2022, 144, 8171–8177

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Reactivity / stability trade-off

Distortion/Interaction Analysis of Tetrazine-TCO IEDDA Reactions• ΔEdist for 2Pyr is about 1.5 kcal/mol lower than that for Ph, 3Pyr, and 4Pyr.
→ The increased reactivity of 2Pyr with TCO is mainly caused by a reduced distortion energy.
Svatunek, Houk, Mikula et al. J. Am. Chem. Soc. 2022, 144, 8171–8177

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Reactivity / stability trade-off

Uncovering the Key Role of Distortion in Tetrazine-TCO IEDDA Reactions
Tz-TCO

Tz-ethylene• In Ph, 3Pyr, and 4Pyr, the aryl moiety istilted only in the reaction with TCO due
to the steric demand of the allylic CH2.
• For 2Pyr, a much stronger tilt wasobserved with TCO, which did notchange with ethylene.

→ An intrinsic property of 2-pyridyl-Tz
rather than forced by steric interactions.

• A nitrogen–nitrogen interaction destabilizes the2-pyridyl-Tz.
→ The key factor of the reactions of 2-pyridyl-Tz withTCOs is the reduced Tz distortion energies
caused by N-N repulsive intramolecularinteractions.

Svatunek, Houk, Mikula et al. J. Am. Chem. Soc. 2022, 144, 8171–8177

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Reactivity / stability trade-off

Repulsive O–N Interaction Increases the Reactivity of Vinyl Ether-Tz• Reduced rotational barrier• Non-electron-withdrawing character• Significantly stronger tilt of the vinyl–Tz bond
• Low distortion energies (ΔEdist)• Low free energy of activation (ΔG⧧)

O–N repulsion plays a crucial role regarding
the potentially increased reactivity of MVE

Svatunek, Houk, Mikula et al. J. Am. Chem. Soc. 2022, 144, 8171–8177

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Reactivity / stability trade-off

Uncovering the Key Role of Distortion in The Reactivity/Stability Trade-Off• Non-electron-withdrawing DHP substituents can be used to significantly increase the IEDDA reactivity of Tz while
maintaining a high compound stability.
Svatunek, Houk, Mikula et al. J. Am. Chem. Soc. 2022, 144, 8171–8177

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Bioorthogonal Cycloaddition

Short SummaryTransition states analysis

Distortion/Interaction (D/I) model

Generalized principles development

New design

Mutual orthogonality

New Mutual orthogonal pair

Reactivity / stability trade-off

New structure with highreactivity & stability
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Contents

Contents◆Introduction◆Main➢ Historical example: Bioorthogonal Cycloaddition• Mutual orthogonality & Motif expansion• Reactivity / stability trade-off➢ Recent example: Chemoselective Cysteine Bioconjugation• Reactivity• Modularity◆Summary & Perspective

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Chemoselective Cysteine Bioconjugation

Chemoselective Cysteine Bioconjugation
Cys selective bioconjugation

Ethynyl phosphorus(V) electrophiles• A low natural abundance on accessible
protein surfaces• The unique nucleophilic properties ofthe sulfhydryl group

The most widely used method

5=Maleimide
reagent• Rapid kinetics
Cys selectivity• Stability(Retro-Michael addition in the presence of
external thiols)
Gunnoo, Madder et al. ChemBioChem 2016,17, 529–553

Modularity
Stability
Kasper, Hackenberger et al. Angew. Chem. Int. Ed. 2019, 58, 11625–11630

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Reactivity

Effects of Substituents around Phosphorus on Thiol Addition Reactivity
R1 (O)

R2 (N)R1 (O)

R2 (N)• Electron-withdrawing substituentsat the N- and the O-residue increased
the speed of the thiol addition.

Kasper, Hackenberger et al. Chem. Sci. 2019,10, 6322–6329

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Reactivity

The Mechanism behind Enhanced Reactivity of Ethynyl P(V) Electrophiles• The heteroatoms bound to the central
phosphorus atom have a notable influence on
the overall thiol addition reactivity.
• Not consistent with simple electronegativity
considerations (S: 2.5, O: 3.5, N: 3.0).

Pursue a mechanistic rationaleusing DFT calculation

Park, Sun, Baik, Hackenberger et al. Chem. Sci. 2021, 12, 8141–8148

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Reactivity

The Mechanism behind Enhanced Reactivity of Ethynyl P(V) Electrophiles
Possible timings for deprotonation

Computationally examined mechanistic pathway

13• The thiol cannot form an addition product• The thiolate addition is feasible
Park, Sun, Baik, Hackenberger et al. Chem. Sci. 2021, 12, 8141–8148

1415• Calculated barriers correlate
well with the trends observed in
the experimental rates.
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Reactivity

The Mechanism behind Enhanced Reactivity of Ethynyl P(V) Electrophiles• The X–P–C1–C2 dihedral angles are within 50°→ Stereoelectronic interaction• The P–X antibonding orbital accepts the electron density through hyperconjugation.
Park, Sun, Baik, Hackenberger et al. Chem. Sci. 2021, 12, 8141–8148

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Modularity

DFT-Guided Discovery of Ethynyl-Triazolyl-Phosphinates as Modular Electrophiles
Previous modular ethynyl P(V) electrophiles

Discovery of new modular ethynyl P(V) electrophiles• Ethynyl-Triazolyl-Phosphinates (ETPs) as readily
accessible, fast, and highly selective thiol-electrophiles,guided by DFT-based computer models.
Stieger, Baik, Hackenberger et al. Angew. Chem. Int. Ed. 2022, 61, e202205348

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Modularity

DFT-Guided Discovery of Ethynyl-Triazolyl-Phosphinates as Modular Electrophiles
Design to increase the reaction speed of ethynyl P(V) electrophiles• The bond P–XEt located in an antiperiplanar position
to the lone-pair played a decisive role in accepting
the electron density.

Easily accessed via
CuI-catalyzed
azide-alkyne cycloaddition→ Use heterocyclic substituents to achieve bothan electron-withdrawing inductive effect andπ-conjugation with the lone-pair electrons.

Stieger, Baik, Hackenberger et al. Angew. Chem. Int. Ed. 2022, 61, e202205348

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Modularity

DFT-Guided Discovery of Ethynyl-Triazolyl-Phosphinates as Modular Electrophiles
The substituents influence of the π-system to enhance the reactivity

HOMO• Delocalization of electrons into vacant orbitals related to the P−R bonds is feasible.

Stieger, Baik, Hackenberger et al. Angew. Chem. Int. Ed. 2022, 61, e202205348

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Modularity

Application of Ethynyl-Triazolyl-Phosphinates as Modular Electrophiles
The experimental thiol addition kinetics

Cysteine-selective peptide modification• ETP electrophile (3) showed accelerated reaction
kinetics, which is in agreement with the DFT-calculated
reaction barriers• P5-LUXendin645 specifically labelled CHO-K1 cells stably
expressing SNAP-GLP1R.

Stieger, Baik, Hackenberger et al. Angew. Chem. Int. Ed. 2022, 61, e202205348

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Contents

Contents◆Introduction◆Main➢ Historical example: Bioorthogonal Cycloaddition• Mutual orthogonality & Motif expansion• Reactivity / stability trade-off➢ Recent example: Chemoselective Cysteine Bioconjugation• Reactivity• Modularity◆Summary & Perspective

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Summary

Summary & Perspective
Challenges of Bioconjugations• The critically restricted scope of chemical reactions that can be performed in a biological context.

Computational Mechanistic Analysis
In-depth insights and understanding about structure–reactivity trends from transition state analysis→ Constitute a solid foundation for extremely fast, stable reactions with high utility

Combination with Chemical Ingenuity
Improve and expand the bioconjugations, and open new capabilities in the life sciences

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