Improvement of
Native Chemical Ligation
by Extending from Sulfur to Selenium
2021/06/17
Literature Seminar
M1 Habazaki1

Contents

Contents◆Introduction:

Background and limitations of native chemical ligation (NCL)◆Main: Development of extended NCL using selenium➢Extending NCL to selenocysteine➢Extending NCL to selenoester➢Extending NCL to diselenide-selenoester ligation (DSL)◆Summary & Perspective

2

Contents

Contents◆Introduction:

Background and limitations of native chemical ligation (NCL)◆Main: Development of extended NCL using selenium➢Extending NCL to selenocysteine➢Extending NCL to selenoester➢Extending NCL to diselenide-selenoester ligation (DSL)◆Summary & Perspective

3

Introduction

The need for chemical synthesis of proteins
Significance of access to proteins

How to access to large proteins• Elucidation of protein and PTM functions• Biological expression• Development of peptide/protein drugs• Chemical synthesis
Specific modifications can be introducedsite-selectively and homogeneously!

Monbaliu, Melnyk et. al. Chem. Rev. 2019, 119, 7328–7443

Payne et. al. Nat. Rev. Chem. 2018, 2, 1–17

4

Introduction

Solid phase peptide synthesis (SPPS)• An extremely reliable platform for the

preparation of peptides• Longer syntheses are often plagued by theaccumulation of uncoupled sequences,
unwanted side products, and epimerization.
(limited to 40-50 residues)

Payne et. al. J. Pept. Sci. 2014, 20, 64–77
Sarojini et. al. Chem. Asian J. 2019, 14, 1088–1097

5Introduction

Native Chemical Ligation (NCL)

Reversible, rate-determining step

• A pioneering method for chemical ligation of peptides• Without any other functional or protecting groups
than thioester!
• In purely aqueous media at neutral pH!

• Synthesis of proteins about 200-400 residues in length!
Kent et. al. Science 1994, 266, 776–779
Monbaliu, Melnyk et. al. Chem. Rev. 2019, 119, 7328–7443

Payne et. al. Nat. Rev. Chem. 2018, 2, 1–17

6

introduction

Native Chemical Ligation (NCL)
Typical reaction conditions
Gn･HCldenaturating agent

TCEP
disulfide bond reducing reagent～ 2 eq

1-5 mM

MPAA / PhSH (large excess)catalyst

H2 N
NH2

Buffer (pH 7-7.5)

H2 N
NH2

r.t. 8-24 h (or more)

MPAA

Monbaliu, Melnyk et. al. Chem. Rev. 2019, 119, 7328–7443

Whitesides et. al. J. Org. Chem. 1991, 56, 2648–2650

7

introduction

Limitations of Native Chemical Ligation (1)Typical reaction conditions
Gn･HCldenaturating agent

TCEP
disulfide bond reducing reagent～ 2 eq

1-5 mM

MPAA / PhSH (large excess)catalyst

H2 N

H2 N

Buffer (pH 7-7.5)

NH2

Cys

NH2

r.t. 8-24 h (or more)
Frequency in proteinsis only 1.48%...

Limitation (1)• The need for a Cys residue on the N terminus of one of the peptide fragments
Agouridas, Melnyk et. al. Bioorg. Med. Chem. 2017, 25, 4938–4945

Payne et. al. Nat. Rev. Chem. 2018, 2, 1–17

8

introduction

Limitations of Native Chemical Ligation (2)Typical reaction conditions
Gn･HClCausingdesulfurization

denaturating agent

Hydrolysis

TCEP
disulfide bond reducing reagent～ 2 eq

1-5 mM

MPAA / PhSH (large excess)catalyst

H2 N

H2 N

Buffer (pH 7-7.5)

NH2

NH2

r.t. 8-24 h (or more)

Directaminolysis

High watersolubility is required

Limitation (2)• The need for relatively long reaction time and high concentration of peptide fragments
owing to moderate reactivity
Durek et. al. Angew. Chem. Int. Ed. 2011, 50, 12042–12045

Payne et. al. Nat. Rev. Chem. 2018, 2, 1–17

9

Introduction

Extending NCL from sulfur to selenium
One of the most succussed ligation• Extended NCL using selenium (Se) instead of sulfur (S) has unexpectedly succeeded!
1994
NCL
2001
NCL
@Selenocysteine
2011
NCL@Selenoester

2015
DSL
@Selenocysteine@Selenoester
10

Contents

Contents◆Introduction:

Background and limitations of native chemical ligation (NCL)◆Main: Development of extended NCL using selenium➢Extending NCL to selenocysteine➢Extending NCL to selenoester➢Extending NCL to diselenide-selenoester ligation (DSL)◆Summary & Perspective

11

Extending NCL to selenocysteine

Extending NCL to selenocysteine
Selenocysteine (Sec)• The 21st proteinogenic amino acid

• Incorporated into natural selenoproteins(25 selenoproteins in human!)

Cysteine(Cys / C)

Selenocysteine(Sec / U)

Selenoproteins
Selenoproteins plays important and unique roles
and has been gaining interest!

Sec

The production of selenocysteine-containing
Scheerer et. al. Biochim. Biophys. Acta, Mol.
Cell. Biol. Lipids 2018, 1863, 1095–1107

Gladyshev et. al. Science 2003, 300, 1439–1443

proteins by biological expression is problematic…

Payne et. al. J. Pept. Sci. 2014, 20, 64–77

12Extending NCL to selenocysteine

Extending NCL to selenocysteine• Sec is the chalcogenic analogue of Cys→ Sec was competent in NCL-like transformations with peptide thioesters!
Raines et. al. J. Am. Chem. Soc. 2001, 123, 5140–5141
Donk et. al. Org. Lett. 2001, 3, 1331–1334

Hilvert et. al. Helv. Chim. Acta 2001, 84, 1197–1206
Payne et. al. J. Pept. Sci. 2014, 20, 64–77

13

Extending NCL to selenocysteine

Differences between Cys and Sec
Cysteine (S)Selenocysteine (Se)Atomic radius

100 pm115 pmElectronegativity

2.58

2.55

Polarizability volume

2.9 Å

3.8 Å

Redox potential-180 mV-381 mV

pKa

8.30

5.24

X-H

367 kJ/mol

310–315 kJ/mol

C-X

309.3 kJ/mol*

257 kJ/mol**

BDE*Value of H C–SH, **Value of H C–SeH
3
3• Sec is the chalcogenic analogue of Cys↔ Sec exhibits some strikingly different physicochemical properties!
Monbaliu, Melnyk et. al. Chem. Rev. 2019, 119, 7328–7443
Raines et. al. J. Am. Chem. Soc. 2001, 123, 5140–5141

Koppenol et. al. J. Org. Chem. 2010, 75, 6696–6699
Payne et. al. J. Pept. Sci. 2014, 20, 64–77

14Extending NCL to selenocysteine

Reactivity of Sec compared to Cys
Sec

TCEP

kCys (pH=pKa=8.30)

kSec (pH=pKa=5.24)

kSec/kCys

3.7 x 102 M-1s-1

9.5 x 102 M-1s-1

2.6

Cys
pKaSecpKaCys• Selenols/selenolate pairs are more nucleophilic
Cys (S)Sec (Se)

Polarizability
volume

2.9 Å

3.8 Å

pKa

8.30

5.24

because of their higher polarizability.

• Sec selenols are significantly more reactive mainly
because of their higher acidity.
Raines et. al. J. Am. Chem. Soc. 2001, 123, 5140–5141

Koppenol et. al. J. Org. Chem. 2010, 75, 6696–6699
Rabenstein et. al. J. Am. Chem. Soc. 1989, 111, 6553–6558 15

Extending NCL to selenocysteine

The rate of Sec NCL compared to Cys NCL
Sec selenols are significantly more reactive

Problem ①than Cys thiol!

• The rate-determining step of ligation
at Sec might be the reduction of

Sec NCL often does NOT proceed faster

diselenide.

than original Cys NCL…

Disulfide

Redox potential

Cys (S)Sec (Se)-180 eV-381 eV

Donk et. al. Org. Lett. 2001, 3, 1331–1334

More rapidly formedandhighly stable

Diselenide

Payne et. al. J. Pept. Sci. 2014, 20, 64–77

16Extending NCL to selenocysteine

The rate of Sec NCL compared to Cys NCL
Sec selenols are significantly more reactive
than Cys thiol!

Problem ②• The use of TCEP as a reducing agent

can induce problematic
deselenization during ligation.

Sec NCL often does NOT proceed fasterthan original Cys NCL…

Thiol-based additive (PhSH/MPAA)
acts as both a catalyst andas a mild reducing agent.

Donk et. al. Org. Lett. 2001, 3, 1331–1334

Monbaliu, Melnyk et. al. Chem. Rev. 2019, 119, 7328–7443
17
Payne et. al. J. Pept. Sci. 2014, 20, 64–77

Extending NCL to selenocysteine

A landmark discovery in Sec NCL chemistry
Sec NCL often does NOT proceed fasterthan original Cys NCL…

Sec NCL have NOT been extensively
used for the synthesis of peptides andproteins that do not possess Sec
residues in the final product…

A groundbreaking discovery (2010)• Application of the unique chemical properties of Sec to selective cleavage of C–Se
bond of Sec in the presence of unprotected Cys after ligation reaction

Keinan, Dawson et. al. Angew. Chem. Int. Ed. 2010, 122, 7203–7207

Payne et. al. Nat. Rev. Chem. 2018, 2, 1–17
Payne et. al. J. Pept. Sci. 2014, 20, 64–77

18

Extending NCL to selenocysteine

Re: Limitations of Native Chemical Ligation (1)Limitation (1)• The need for a Cys residue on the N terminus of one of the peptide fragments

Solution• A post-ligation desulfurization permits the use of Cys as a surrogate for ligation sites
containing a substantially more abundant amino acid, Ala!

Cys

Frequency is
8.42%!

Frequency is
only 1.48%...

Ala
Dawson et. al. J. Am. Chem. Soc. 2001, 123, 526–533
Agouridas, Melnyk et. al. Bioorg. Med. Chem. 2017, 25, 4938–4945

Payne et. al. Nat. Rev. Chem. 2018, 2, 1–17

19

Extending NCL to selenocysteine

Re: Limitations of Native Chemical Ligation (1)Limitations of post-ligation desulfurization• Any conditions result in global desulfurization of
all thiols in the protein.

Metal reagents(Raney Ni or Pd/Al2O3, H2)
Radical initiator (VA-044), TCEP

Dawson et. al. J. Am. Chem. Soc. 2001, 123, 526–533
Danishefsky et. al. Angew. Chem. Int. Ed. 2007, 46, 9248–9252

Monbaliu, Melnyk et. al. Chem. Rev. 2019, 119, 7328–7443
20

Extending NCL to selenocysteine

Traceless ligation by chemoselective deselenization• Traceless ligation using mild

Step 1Sec-mediatedligation

Step 3TCEP-mediateddeselenization

deselenization of Sec wasachieved using TCEP without
an additional radical initiator!

Step 2
Removal of aromatic thiols(act as radical scavenger)

Redox potential

Keinan, Dawson et. al. Angew. Chem. Int. Ed. 2010, 122, 7203–7207

Cys (S)Sec (Se)-180 eV-381 eV21

Extending NCL to selenocysteine

Traceless ligation by chemoselective deselenization• Selective cleavage of C–Se

bond of Sec in the presenceof unprotected Cys wasdemonstrated!

Only traces of the deselenized–desulfurized peptide!

7
Keinan, Dawson et. al. Angew. Chem. Int. Ed. 2010, 122, 7203–7207

22

Extending NCL to selenocysteine

Proposed mechanism of TCEP-mediated deselenization• The Se–H bond is much
weaker than S–H→ Selective formation ofselenyl radical (Ⅰ)
at room temperature!
• The C–Se bond is much
weaker than C–S→ Faster breakdown ofphosphoranyl radical (Ⅱ)

BDECys (S)Sec (Se)

X-H

367 kJ/mol

310–315 kJ/mol

C-X

309.3 kJ/mol*

257 kJ/mol**High selectivity!

*Value of H C–SH, **Value of H C–SeH
3
3

Keinan, Dawson et. al. Angew. Chem. Int. Ed. 2010, 122, 7203–7207

Metanis et. al. Chem. Sci. 2015, 6, 6207–6212

23

Extending NCL to selenocysteine

Traceless ligation by chemoselective deselenization+• The power of traceless Sec NCL methodology was exemplified in protein synthesis!
Metanis et. al. Angew. Chem. Int. Ed. 2016, 55, 992–995

Payne et. al. Nat. Rev. Chem. 2018, 2, 1–17

24

Extending NCL to selenocysteine

Traceless ligation by chemoselective deselenization
Thiol-derived amino acids

Payne et. al. Nat. Rev. Chem. 2018, 2, 1–17

Selenol-derived amino acids

Ala / Ser

Phe

Leu

Asp

Pro

Glu

Monbaliu, Melnyk et. al. Chem. Rev. 2019, 119, 7328–7443 25

Extending NCL to selenocysteine

Short Summary• Selenium was first introduced to NCL as

selenocysteine (Sec) to synthesize
Transesterification

selenoprotein.
• Despite the enhanced nucleophilicity of
selenols, the reaction rate of Sec NCL is

not faster due to the weak reductive
Se,N-acyl transfer

power of aryl thiols.
• The benefit of Sec NCL was expanded by

the demonstration of traceless ligationby chemoselective deselenization.

Chemoselective
deselenization

Limitation (1)Solved!

• The need for a Cys residue on the N
terminus of one of the peptide fragments
26

Contents

Contents◆Introduction:

Background and limitations of native chemical ligation (NCL)◆Main: Development of extended NCL using selenium➢Extending NCL to selenocysteine➢Extending NCL to selenoester➢Extending NCL to diselenide-selenoester ligation (DSL)◆Summary & Perspective27

Extending NCL to selenoester

Re: Limitations of Native Chemical Ligation (2)Limitation (2)• The need for relatively long reaction time and high concentration of peptide fragments
owing to moderate reactivity

Solution• Converting the starting peptide alkyl thioester into a more powerful acyl donor by
performing the NCL in the presence of thiol additives as nucleophilic catalysts!
The best
catalyst

MPAA
Dawson, Kent et. al. J. Am. Chem. Soc. 1997, 119, 4325–4329
Kent et. al. J. Am. Chem. Soc. 2006, 128, 6640–6646

Monbaliu, Melnyk et. al. Chem. Rev. 2019, 119, 7328–7443
28

Extending NCL to selenoester

Re: Limitations of Native Chemical Ligation (2)Limitation (2)• Peptide thioesters bearing sterically or electronically hindered amino acids at the C
terminus suffer from sluggish reaction rates even with thiol catalysts.

Monbaliu, Melnyk et. al. Chem. Rev. 2019, 119, 7328–7443
Dawson et. al. Proc. Natl. Acad. Sci. USA 1999, 96, 10068–10073

29

Extending NCL to selenoester

Quite low reactivity of peptidyl prolyl thioester• An n→π* electronic donation into
the carbonyl carbon leads toreduced electrophilicity of the
prolyl thioesters→ Pro–Cys junctions aresynthetically intractable…

Reaction rate

>

>

>

Raines et. al. Protein Sci. 2003, 12, 1188–1194
Agouridas, Melnyk et. al. Bioorg. Med. Chem. 2017, 25, 4938–4945
Monbaliu, Melnyk et. al. Chem. Rev. 2019, 119, 7328–7443

30

Extending NCL to selenoester

Extending NCL to selenoester
Strategy• Thioesters can be advantageously substituted by selenoesters in NCL reactions due tohigher reactivity of selenoesters toward Cys peptides!

Superior
leaving group!

Polarizability volume

Cys (S)Sec (Se)

2.9 Å

3.8 Å

Durek et. al. Angew. Chem. Int. Ed. 2011, 50, 12042–12045 Monbaliu, Melnyk et. al. Chem. Rev. 2019, 119, 7328–7443 31

Extending NCL to selenoester

Rapid ligation at interactable site using selenoester
1 mM

X=F, Y=Se

1 mM

X=F, Y=S
6M Gn･HCl
0.2M Na3PO4
100 mM TCEP

X=P, Y=Se

50 mM MPAA
X=P, Y=S

pH 6.8• Peptide selenoesters reacted significantly

faster than the corresponding thioesters.
• The rate-determining step is likely to beXYk [M-1s-1]

FactorP

S

0.00057 ± 0.00066

1

F

S

0.437 ± 0.032

766

P

Se

0.019 ± 0.002

33

F

Se

0.827 ± 0.039

1450trans-thioesterification of the rapidly formed

MPAA-derived thioester with Cys…
Durek et. al. Angew. Chem. Int. Ed. 2011, 50, 12042–12045

32

Extending NCL to selenoester

Rapid ligation at interactable site using selenoester
1 mM

X=F, Y=Se

1 mM

X=P, Y=Se
6M Gn･HCl
0.2M Na3PO4
100 mM TCEP

28 mM DPDS

X=F, Y=S

pH 6.2

X=P, Y=SX

Yk [M-1s-1]

Factor

P

S

n.d.

n.d.

• Ligation at prolyl selenoester (with selenol

F

S

n.d.

n.d.

additive) were nearly 350 times faster thanPSe

0.198 ± 0.007

347

F

Se

7.7 ± 0.113500

• Peptide selenoesters rapidly and quantitativelytrans-selenoesterified with PhSeH.

traditional NCL (with thiol additive)!
Durek et. al. Angew. Chem. Int. Ed. 2011, 50, 12042–12045

33

Extending NCL to selenoester

Short Summary• Selenoester was introduce to NCL due to higher reactivity toward Cys peptides.
• Selenoester even allowed for rapid ligation even at proline (up to 350-fold faster reaction).

Limitation (2)Solved!

• Peptide thioesters bearing sterically or electronically hindered amino acids at the C

terminus suffer from sluggish reaction rates even with thiol catalysts.
34

Contents

Contents◆Introduction:

Background and limitations of native chemical ligation (NCL)◆Main: Development of extended NCL using selenium➢Extending NCL to selenocysteine➢Extending NCL to selenoester➢Extending NCL to diselenide-selenoester ligation (DSL)◆Summary & Perspective

35

Extending NCL to DSL

Extending NCL to diselenide-selenoester ligation (DSL)
2001
NCL@Selenocysteine
2011
NCL@Selenoester

Concept• If the increased nucleophilicity of Sec could be effectively harnessed and combinedwith the enhanced electrophilicity of a selenoester, the ligation rate should increase.

2015
DSL
@Selenocysteine@Selenoester
Payne et. al. J. Am. Chem. Soc. 2015, 137, 14011–14014

36

Extending NCL to DSL

Rapid additive-free diselenide-selenoester ligation (DSL)
6 M Gn·HCl
0.1 M Na2HPO4
pH 6.2, 25 °C

Additive-free ligation!
(without any reducing reagent!)

Ligation completes
within 60 sec!

• A control experiment involving only diselenide

peptide dimer and peptide phenylselenoester
in denaturing buffer represents unprecedented
reactivity!

Payne et. al. J. Am. Chem. Soc. 2015, 137, 14011–14014

37

Extending NCL to DSL

Insights into dramatic reactivity of DSL• An important factor for the dramatic
Precipitateof DPDS

Payne et. al. J. Am. Chem. Soc. 2015, 137, 14011–14014

ligation rate seems to be removal of DPDS
from solution by precipitation.

Selenoester (R=)

Water solubility

T1/2

H

Insoluble

24 s

CH2COOMe

Sparingly soluble

48 s

CH2COO–soluble> 1h38Extending NCL to DSL

Proposed mechanism; Ligation redox cycle
Diselenide
ligation product

Reducedligation product• Native chemical ligation-type mechanism is proposed.
• The reaction is an equilibrium process, whereby the forward reaction is promoted byremoval of DPDS from solution by precipitation.
Payne et. al. J. Am. Chem. Soc. 2015, 137, 14011–14014

39

Extending NCL to DSL

Proposed mechanism; “Initiation” event

Ea = 111.8 kJ/mol

Ea = 118.3 kJ/mol• In the absence of additives or external reductants, the propagation cycle requires
initiation through an internal reduction event to generate any of the intermediates.

• The exact mechanism of “Initiation” event has not yet been elucidated.

Payne et. al. J. Am. Chem. Soc. 2015, 137, 14011–14014

40

Extending NCL to DSL

One-pot ligation-deselenization

5 mM

2.5 mM

63%

Problem of aryl thiol additives• The radical quenching activity of aryl thiols prohibits in situ radical dechalcogenation.
→ Purification and lyophilization steps must be carried out…

Merit of additive-free DSL• The insoluble DPDS (= radical scavenger) can be removed by hexane extraction.
→ One-pot ligation-deselenization can be conducted!
Payne et. al. J. Am. Chem. Soc. 2015, 137, 14011–14014

41

Extending NCL to DSL

Peptide ligation at high dilution
Limitation (2)• The need for high concentration of peptide fragments remained to make challenging
synthesis of lipidated polypeptides and integral membrane proteins.

Solution using DSL• DSL at extremely low concentrations has been achieved and provides application in thesynthesis of a large range of hydrophobic and lipidated targets!

Payne et. al. J. Am. Chem. Soc. 2020, 142, 1090–1100

42

Extending NCL to DSL

Concentration Limit of additive-free DSL

pKa

Cys (S)Sec (Se)

8.30

5.24

Ligation occurs following
the “initiation period”.

• As the concentration of the peptide decreases, the “initiation period” becomes longer.
• No ligation product was observed at 50 μM (= the reaction did not initiate)…
Payne et. al. J. Am. Chem. Soc. 2020, 142, 1090–1100

43

Extending NCL to DSLDevelopment of the Reductive DSL
Hypothesis• The “initiation step” of DSL is proposed to proceed through a bimolecular reaction.
→ Phenylselenoate is needed at high concentrations to enable highly diluted ligation.
• TCEP can be used with DPDS as a radical scavenger to suppress the deselenization.

The lowest
concentration
at to date!

Payne et. al. J. Am. Chem. Soc. 2020, 142, 1090–1100

44

Extending NCL to DSL

Synthesis of FXYD1 by reductive DSL at high dilution• The power of high-dilution DSL technology was exemplified through the high-yielding
synthesis of stable analogues of the membrane protein FXYD1.
Payne et. al. J. Am. Chem. Soc. 2020, 142, 1090–1100

Fuller et. al. J. Biol. Chem. 2011, 286, 36020–36031

45

Extending NCL to DSL

Assay of the effect of palmitoylation using synthesized FXYD1Monopalmitylated FXYD1

Dipalmitylated FXYD1• Mono/di-palmitylated FXYD1
displayed inhibition of Na+/K+ATPase activity.
• Interestingly, dipalmitylated
FXYD1 was less effective than themonopalmitylated variant.

• The inhibitory activity observed inthis study will aid inunderstanding how posttranslational palmitoylation of
FXYD1 regulates the Na+/K+ pump!
Payne et. al. J. Am. Chem. Soc. 2020, 142, 1090–1100

Fuller et. al. J. Biol. Chem. 2011, 286, 36020–36031

46

Contents

Contents◆Introduction:

Background and limitations of native chemical ligation (NCL)◆Main: Development of extended NCL using selenium➢Extending NCL to selenocysteine➢Extending NCL to selenoester➢Extending NCL to diselenide-selenoester ligation (DSL)◆Summary & Perspective

47

Summary & Perspective

Summary & Perspective
Native Chemical Ligation (NCL)

Selenocysteine

Selenoester

Extending the
N-terminal residueof ligation site!

Extending the
C-terminal residueof ligation site!

Diselenide-Selenoester Ligation (DSL)

The unprecedented reactivity of DSL is opening up new
possibilities for protein chemical synthesis!

48