Chemical Lysine Modification at a single site
2018/12/20
M1 Murata

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Contents• Amino acid target for bioconjugation• Chemical Lysine modification
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N-hydroxysuccinimide (NHS)-esterα, β-unsaturated sulfonamide
Sulfotetrafluorophenyl esters
Stilbene
β-lactamSulfonyl acrylate（Lysine activated)• Summary2

Contents• Amino acid target for bioconjugation• Chemical Lysine modification
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•
•
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N-hydroxysuccinimide (NHS)-ester
α,β-unsaturated sulfonamide
Sulfotetrafluorophenyl esters
Stilbene
β-lactamSulfonyl acrylate（Lysine activated)• Summary3

Amino acids for bioconjugation
Conventional(pKa=8.7)

Recent(pKa=10.5)

 Reactive because of nucleophilicity
 Abundant (Lys)(pKa=10.46)
 Less-reactive RareTrp… keto-ABNO
Tyr… reactivity can be efficiently tuned by pH control
Met…only native residue that can be alkylated under acidic conditions

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Biochemistry 2017, 56, 3863−3873

Cys and Lys residues for bioconjugation
The advantage of targeting Cys or Lys residues① abundant (Lys)② Easy to taeget because of high reactivity
Ratio in human protein(%)

The position of residue

Comments

Cys

1.9

within the folded proteinsNot accessible

Lys5.9On the surface of proteins

accessible

Native lysine residues are more convenient targets for protein modification than cysteine residues,

Targeting naturally occurring side chains in a chemo- and region-selective fashion
remains a great and unexplored challenge.
5J. Am. Chem. Soc. 2018, 140, 4004−4017

Problems in Lys-bioconjugation
Problem- Conventional method – Difficulties to target specific single Lys residue
 Require a large excess of often-valuable reagents. (e.g. 10 eq) Require metals(e.g. Ru, Pd…) or other additives that must later be removed from thereaction mixture.

“A general way to selectively target single Lysine is lacking”
6J. Am. Chem. Soc. 2018, 140, 4004−4017

A main literature discussed in this seminar- Sulfonyl acrylate（Lysine activated）Hydrogen bond assisted chemo- and regioselective modification of lysine on native proteins Stoichiometric amount of sulfonyl acrylate reagent
 Proceed to completion rapidly (1~2h)
 Under mild conditions (pH=8.0, rt-37℃)
 Applicable to a range of native protein types
 The products are stable
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 The method is compatible with Cys bioconjugation method Bernardes et al., J. Am. Chem. Soc. 2018, 140, 4004−4017

Contents• Amino acid target for bioconjugation• Chemical Lysine modification
••
•
•
•
•

N-hydroxysuccinimide (NHS)-ester
α,β-unsaturated sulfonamide
Sulfotetrafluorophenyl esters
stilbene
β-lactamSulfonyl acrylate（Lysine activated)• Summary8

Chemical Lysine modification• N-hydroxysuccinimide (NHS)-ester• α,β-unsaturated sulfonamide• Sulfotetrafluorophenyl esters• Stilbene• β-lactam9

Chemical Lysine modification 1- N-hydroxysuccinimide (NHS)-ester type -10Chen et al., Bioconjugate Chem. 2012, 23, 500−508

Chemical Lysine modification 1- N-hydroxysuccinimide (NHS)-ester type -Rnase A the highest solvent accessibility.

Lysozyme C hydrogen bond interaction between K1 and E7
11Chen et al., Bioconjugate Chem. 2012, 23, 500−508

Chemical Lysine modification 1- N-hydroxysuccinimide (NHS)-ester type Advantage
 Fast (2h)
 Low cost
 The risk of misfolding is lowDisadvantage
 the requirement of substoichiometricamounts of reagent➡ imcomplete conversion

Chen et al., Bioconjugate Chem. 2012, 23, 500−508
12Chemical Lysine modification 2- α,β-unsaturated sulfonamide type -Proposed Mechanism of the Conjugation Reaction with HSA

Angew. Chem. Int. Ed. 2014, 53, 11783 –11786
Bioconjugate Chem. 2016, 27, 2271−227513

Chemical Lysine modification 2- α,β-unsaturated sulfonamide type -Screening of the Takeda chemical library was carried out
To develop a new therapeutic agent for sepsis.

Lead compound

Yamada et al., J. Med. Chem. 2005, 48, 7457-7467
Barbas et al., Angew. Chem. Int. Ed. 2014, 53, 11783 –11786
Barbas et al., Bioconjugate Chem. 2016, 27, 2271−227514

Chemical Lysine modification 2- α,β-unsaturated sulfonamide type - rapidly modify HSA
 excellent serum stability

Rhodamine-linked cyclohexene sulfonamide compound (cHx-Rho).

Barbas et al., Angew. Chem. Int. Ed. 2014, 53, 11783 –11786
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Barbas et al., Bioconjugate Chem. 2016, 27, 2271−2275

Chemical Lysine modification 2- α, β-unsaturated sulfonamide type Time-course study of trastuzumab, HSAdIC34S-LC,
and Fc-HSAdIC34S labeling with 10 equiv of cHx-Rho.

HER2 for trastuzumab and fusion conjugates.

Advantage Site-selectivity
 Antibody conjugates were also stablein human plasma
Disadvantage
 Imcomplete conversion
 Need large amount of reagent (10 eq)
 Basic condition. (pH=9.0)

Antibody conjugation using HSAdI as a fusion proteinshould be amenable to therapeutic applications.

Barbas et al., Angew. Chem. Int. Ed. 2014, 53, 11783 –11786
Barbas et al., Bioconjugate Chem. 2016, 27, 2271−2275 16

Chemical Lysine modification 3- sulfotetrafluorophenyl esters - broad reactivity
 good selectivity for lysine residues in the human proteome.

Cravatt et al., Nat. Chem., 2017, 9,17

Chemical Lysine modification 3- sulfotetrafluorophenyl esters -Advantage
 more stable in aqueous solution than NHS esters.
Disadvantage
 the localization of hyper-reactive lysines to pockets could
also restrict their access to post-translational machinery,such as ubiquitylation processes
 In order to prevent overreaction it is necessary to wellcontrol the equivalence of reagent and the reaction time.

Cravatt et al., Nat. Chem., 2017, 9,18Chemical Lysine modification 4- Stilbene-

designed stilbenes that selectively and covalently modify the prominent plasma protein transthyretin

Transthyretin(TTR) : One of the causative proteins of amyloidosis.
- Amyloidosis caused by transthyretin - Senile systemic amyloidosis (SSA) Familial amyloid polyneuropathy (FAP)
Figure. Crystal structure of the WT TTRWhen transthyretin becomes unstable, it becomes amyloid fiber and aggregates.

A treatment method to stabilize transthyretin is required.

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Kelly et al., Nat. Chem. Biol., 2010, 6

Chemical Lysine modification 4- Stilbene-

designed stilbenes that selectively and covalently modify the prominent plasma protein transthyretin

Design of chemoselective covalent TTR kinetic stabilizers

Reduce electrostatic repulsion between lysine residues
Stabilization of transthyretin Ester or thioester group→allow amino group of Lys to approach at Burgi-Dunitz angle

Most chemoselective・Percent fibril formation of WT TTR(3.6 μM, black font)and V30M TTR(3.6 μM, blue font)・Maximum modification percent is 50%.

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Kelly et al., Nat. Chem. Biol., 2010, 6

Chemical Lysine modification 4- Stilbene-

designed stilbenes that selectively and covalently modify the prominent plasma protein transthyretin

Design of chemoselective covalent TTR kinetic stabilizers

K15A-TTR subunit modified
by 1, 2, 3 was observed.

Not observed→High chemoselectivity

Most chemoselective
・Percent fibril formation of WT TTR(3.6 μM, black font)and V30M TTR(3.6 μM, blue font)・Maximum modification percent is 50%.

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Kelly et al., Nat. Chem. Biol., 2010, 6

Chemical Lysine modification 4- Stilbene-

designed stilbenes that selectively and covalently modify the prominent plasma protein transthyretin

Transthyretin(TTR) : One of the causative proteins of amyloidosis.

Green: hydrophobic
Purple: polar
Blue: exposedFigure. Crystal structure of the WT TTR

 Bridging hydrogen bonds are formed between the 4-OH of the benzoyl substructure and the Ser117 andSer117′ hydroxyls from adjacent TTR monomers.
 One Lys15 ε-amine group and one Lys15′ ε-ammonium group at pH 7.

22Kelly et al., Nat. Chem. Biol., 2010, 6

Chemical Lysine modification 4- Stilbene-

designed stilbenes that selectively and covalently modify the prominent plasma protein transthyretin

Transthyretin(TTR) : One of the causative proteins of amyloidosis.

Green: hydrophobic
Purple: polar
Blue: exposedAdvantage
 High-selectivity(chemo-, site-)(Even when 5 eq. of stilbene derivative wereadded, other lysine residues did not react)
Figure. Crystal structure of the WT TTR
 Small molecule
Disadvantage

 Bridging hydrogen bonds are formed between the 4-OH of the benzoyl substructure and the Ser117 and Applicable range is limited to TTR
Ser117′ hydroxyls from adjacent TTR monomers.
 One Lys15 ε-amine group and one Lys15′ ε-ammonium group at pH 7.

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Kelly et al., Nat. Chem. Biol., 2010, 6

Chemical Lysine modification 5- β-lactam- The first site-specific ADC to be generated using a natural Lys for conjugation.
ADC : useful for cancer treatment.

Conventional Site-Selective reaction
 unnatural amino acid by a genetic engineering technique introduce a sequence suitable for an enzymatic reaction

h38C2 :(humanized anti-hapten monoclonal antibody) Having a nucleophilic Lys residue (pKa=~6)in the hydrophobic pocket

The DVD is composed of• variable domains of trastuzumab (blue),• h38C2 (green) with reactive Lys (yellow circle),• constant domains (gray).

It becomes a selective modified target at in physiological pH.

Rader, Roush et al., Nat. Com, 2017, .8, 1112

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Chemical Lysine modification 5- β-lactam- The first site-specific ADC to be generated using a natural Lys for conjugation.
Monomethyl auristatin F (MMAF)…To reduce cytotoxicity

Advantage
 Mutation free
 React irreversibly with Lys.
→prevent premature drug release ADCs to be highly homogeneous conjugation does not eliminate any positive charges
on the antibody→preserve electrostatic properties of the antibody
Disadvantage
 The possibility that other Lys react with reagent
when the reaction time is longer. Rader, Roush et al., Nat. Com, 2017, .8,251112

Contents• Amino acid for bioconjugation• Chemical Lysine modification•••••

N-hydroxysuccinimide(NHS)-esterα, β-unsaturated sulfonamide
Sulfotetrafluorophenyl esters
β-lactam conjugation
Sulfonyl acrylate（Lysine activated)• Summary26

Sulfonyl acrylate-mediated lysine modification- Sulfonyl acrylate（Lysine activated）Hydrogen bond assisted chemo- and regioselective modification of lysine on native proteins Stoichiometric amount of sulfonyl acrylate reagent
 Proceed to completion rapidly (1~2h)
 Under mild conditions (pH=8.0, rt-37℃)
 Applicable to a range of native protein types
 The products are stable.
 The method is compatible with Cys bioconjugation.
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Bernardes et al., J. Am. Chem. Soc. 2018, 140, 4004−4017

pKa & reactivity of amino acids- The reactivity of Lys and Cys residue at various pH conditions -pH 5.5-6.0 Lysine residues are mostly protonated and unreactive.
pH 7.5-.8.0 Lysine residues compete with cysteine as Michael donors.
pH 8.5-The more nucleophilic thiolate of cysteine residues
usually dominate

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Bernardes et al., J. Am. Chem. Soc. 2018, 140, 4004−4017

Screening of Michael acceptors- Computational screening of acrylic acid derivatives-Activation barriers (ΔG‡) were calculated with PCM(H2O)/M06-2X/6-31+G(d,p)

Sulfonylmethyl acrylate 1c was predicted to have a superior reactivity compared to its amide analogues

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Bernardes et al., J. Am. Chem. Soc. 2018, 140, 4004−4017

Detailed analysis of transition states- Sulfonyl acrylate（Lysine activated）Acrylate electrophile derivatives 1a−d used in this study and transition states

This hydrogen bond interaction between the lysine model and the sulfone lowers the energy barrier by 10−16 kcal mol−130
Bernardes et al., J. Am. Chem. Soc. 2018, 140, 4004−4017

Comparison between Cys & Lys- Sulfonylmethyl acrylate（Lysine activated）・Hydrogen-bonding
Cys ＜ Lys・Cys … less polar character of the S−H bond, The positive charge developed at the amino group of
the lysine model is efficiently dissipated by the sulfone

A means to selectively modify lysine even in the presence of free cysteine residues at near neutral pH.
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Bernardes et al., J. Am. Chem. Soc. 2018, 140, 4004−4017

This conjugation is a two step reaction- Sulfonylmethyl acrylate（Lysine activated）-

 Hydrogen bonding between the nucleophilic amino group and the sulfone moiety promotes
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both the aza-Michael addition and the elimination of methanesulfinic acid.
Bernardes et al., J. Am. Chem. Soc. 2018, 140, 4004−4017

rHSA (MS analysis)- Sulfonylmethyl acrylate（Lysine activated）-Total ion chromatogram

A single modification was produced in >95%

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Bernardes et al., J. Am. Chem. Soc. 2018, 140, 4004−4017

rHSA (LC-MS/MS , CD)- Sulfonylmethyl acrylate（Lysine activated）-

LC-MS/MS analysis

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Bernardes et al., J. Am. Chem. Soc. 2018, 140, 4004−4017

Reaction with thiol specific Ellman’s reagent- Sulfonylmethyl acrylate（Lysine activated）-

Ellman’s reagent

 Fast Full conversion
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Bernardes et al., J. Am. Chem. Soc. 2018, 140, 4004−4017

Substrate scope- Application to various type of native protein1 eq

Conversion (%) native protein–1c (pH8, 37 ºC) = 100

Conjugates are stable → Application to ADC is expected(Trastuzumab)

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Regioselective lysine modification is applicable to a wide-range of native protein scaffolds.
Bernardes et al., J. Am. Chem. Soc. 2018, 140, 4004−4017

Relationships between pKa & reactive site- Sulfonyl acrylate（Lysine activated）Theoretical calculation of the most reactive lysine residue
Predicted site of modification: K100
Observed site of modification: K100pKamodification

K100

10.1observed

C95

10.3

Not observed

The selectivity of Lysine residue was observed
because of Hydrogen bond.
LysCys脱プロトンされてる37
Bernardes et al., J. Am. Chem. Soc. 2018, 140, 4004−4017

Site-specific labeling- The ability to precisely conjugate fluorophores and cytotoxic drugs to antibodies -Conventional antibody labeling… relied on labeling using disulfide bonds with genetically engineered freelysine residues or non-natural amino acids.
→Produce heterogeneous compounds
There are possibility that affinity with the target antigen may be compromised.

This method can be applied directly to a therapeutic antibody in its native form.
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Bernardes et al., J. Am. Chem. Soc. 2018, 140, 4004−4017

Site-specific labeling- The ability to precisely conjugate fluorophores and cytotoxic drugs to antibodies -Flow cytometry←Heavy chain←Light chain

SDS-PAGE

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Bernardes et al., J. Am. Chem. Soc. 2018, 140, 4004−4017

Contents• Amino acid target for bioconjugation• Chemical Lysine modification
••
•
•
•

N-hydroxysuccinimide (NHS)-esterα, β-unsaturated sulfonamide
Sulfotetrafluorophenyl esters
β-lactamSulfonyl acrylate（Lysine activated)• Summary40

Summary• Lysine residues are abundant and easy to taeget because of their highreactivity.
• A general way to selectively target single Lysine is lacking.
• This method can provide a single lysine one step modification withcomplete chemo- and region-selectivity.
• Direct applicability to wild-type protein sequence bode well forroutinely accessing site-selectively modified proteins for basic biology
and therapeutic applications.

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Appendix

43Various types of modification• Ligation• Genetic Code expansion• Ligand – directed type• Chemical conjugation using Dha44

Various types of modification• Genetic code expansion

Current Opinion in Chemical Biology 2018, 45:35–47

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Various types of modification• Ligand-directed type46

Various types of modification• Ligation

Chem. Rev. 2018, 118, 6656−6705
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Various types of modification• Chemical conjugation using Dha

Chemical Biology, 2018, 45:35–47

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Chemical Lysine modification 4- Stilbene-

designed stilbenes that selectively and covalently modify the prominent plasma protein transthyretin Noncovalent TTR kinetic stabilizers
 It is known to prevent amyloid formation–
associated cytotoxicity, whereas structurallyrelated compounds with poor TTR binding
capacity do not inhibit cytotoxicity

Resveratrol49

Chemical Lysine modification 6- Sulfonyl acrylate（Lysine activated）-

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Bernardes et al., J. Am. Chem. Soc. 2018, 140, 4004−4017

Chemical Lysine modification 6
Optimisation of reaction conditions between rHSA and 1c with respect to pH, buffer and time

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Bernardes et al., J. Am. Chem. Soc. 2018, 140, 4004−4017

Chemical Lysine modification 352
Bernardes et al., J. Am. Chem. Soc. 2018, 140, 4004−4017