Phosphorus-Fluoride Exchange (PFEx)~Another good reaction for click chemistry~2022/11/10(Thr) M2 Tomoyuki Fukuta

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Table of contents

1. Introduction : Application using other P(V) reagents
2. K. B. Sharpless suggests the utility of “PFEx”
3. Biological application of P(V)-F derivatives

4. Summary & Perspective2

Introducing Phosphorus group provides divergent features.
Phosphate

Phosphinate (P-C bond)✓ Strong tolerance toward hydrolysis

Kanai, M et. al. ACS Cent. Sci. 2020, 6, 2, 283–292

Thiophosphate (P-S bond)

Baran, P. et. Al .Nat. Chem. 2021, 12, 2760.

Org. Biomol. Chem., 2013, 11, 119-129

Phosphoamidate (P-N bond)

ACS Med. Chem. Lett. 2020, 11, 9, 1704–1710

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P(V) reagents are desirable for complex substates

Traditional P(V) reagentsNo OH selectivity

No other nucleophileselectivity

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Recent advances of P(V) reagents for bioconjugation
PSI reagents
Activated by5-membered ringSee Fujiyoshi-san’s Lit 2021.7.29 in detail
Baran, P. et. al. J. Am. Chem. Soc. 2020, 142, 41, 17236–17242

Chlorothiophosphate

High His-selectivity in protein substrates
J. Am. Chem. Soc. 2019, 141, 18, 7294–7301

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K.B. Sharpless suggests the utility of “PFEx”.

K.B. Sharpless6Comparison between P(V)-F and P(V)-ClActivation by CsF

less stablestable

Good leaving group(Cl)

Poor leaving ability(F)

React w/o activation

Activation is required(F- or imidazole)

Application of P(V)-F was limited.
J. Chem. Soc. Chem. Commun., 1981, 101-103
J. Am. Chem. Soc. 1984, 106, 4, 1060–1065

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Feature of P(V)-F compounds
Comparison of Stability/Reactivity : F vs Cl

P(V)-F bond is relatively stable, which means low reactivity.

Synthesis

Synthesis of P(V)-F compound is well-investigated, but the application is limited.

8Selectivity of P(V)-Cl and P(V)-F ; O or N ?

O-selectivePredominant reaction w/ N

Weinberger, W. A. et. al. Can. J. Chem. 1967, 45, 495.

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Mechanistic study for the difference between P(V)-Cl and P(V)-Fπ interaction between p(Y)-d(P)= Electrophilicity of P-center
1<2<3
How selectivity changes?

Weinberger, W. A. et. al. Can. J. Chem. 1970, 48, 1351.

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Mechanistic study for the difference between P(V)-Cl and P(V)-F
π interaction between p(Y)-d(P)= Electrophilicity of P-center
1<2<3
How selectivity changes?

As for P(V)-Cl
More active P produced more P-N product.
1
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As for the leaving group (X)Poorer leaving ability, more P-O product.
Weinberger, W. A. et. al. Can. J. Chem. 1970, 48, 1351.

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Mechanistic study for the difference between P(V)-Cl and P(V)-F

Good leaving ability

Follow the equation𝑣 = 𝑘 𝑁𝑢 [𝑃𝐶𝑙]Weinberger, W. A. et. al. Can. J. Chem. 1970, 48, 1351.

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Mechanistic study for the difference between P(V)-Cl and P(V)-F

Good leaving ability

rate-determining step
Follow the equation𝑣 = 𝑘 𝑁𝑢 [𝑃𝐶𝑙]

P(V)-F 123 kcal/mol
P(V)-Cl 83 kcal/mol
P(V)-N 50-70 kcal/mol
P(V)-O ~100 kcal/mol
Weinberger, W. A. et. al. Can. J. Chem. 1970, 48, 1351.

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Mechanistic study for the difference between P(V)-Cl and P(V)-F

Good leaving ability

rate-determining step
Follow the equation𝑣 = 𝑘 𝑁𝑢 [𝑃𝐶𝑙]

P(V)-F 123 kcal/mol
P(V)-Cl 83 kcal/mol
P(V)-N 50-70 kcal/mol
P(V)-O ~100 kcal/mol

Si(VI)-F 136 kcal/molIs P(V)-F compound applicable to click
chemistry like SuFEx?
Weinberger, W. A. et. al. Can. J. Chem. 1970, 48, 1351.

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Short summary
P(V)-F is stable reagent

traditional activation method

J. Am. Chem. Soc. 1984, 106, 4, 1060–1065

PFEx

Activation by Silicon
P(V)-F 123 kcal/mol
P(V)-O ~100 kcal/mol
Si(VI)-F 136 kcal/mol
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Table of contents

1. Introduction : Application using other P(V) reagents
2. K. B. Sharpless suggests the utility of “PFEx”
3. Biological application of P(V)-F derivatives

4. Summary & Perspective16

Initial trial of “PFEx” drawing insight from classical SuFEx
SuFEx✓ Developed by K.B. Sharpless et al in 2014
✓ Si mediates very fast reactions of S-F as electrophiles.
Sharpless, K. B. et. al. Angew. Chem. Int. Ed. 2014,53, 9430 – 9448

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Initial trial of “PFEx” drawing insight from classical SuFEx
SuFEx✓ Developed by K.B. Sharpless et al in 2014
✓ Si mediates very fast reactions of S-F as electrophiles.
Sharpless, K. B. et. al. Angew. Chem. Int. Ed. 2014,53, 9430 – 9448

PFEx (This work)

PFEx successfully occurred in good yields
using various Ar-Si ester.
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Accelerated SuFEx click chemistry
Accelerated SuFEx (ASCC); external Si additives

Moses, et. al. Angew. Chem. Int. Ed. 2022, 1, e202112375.
In situ generation of Silyl ester19

Accelerated SuFEx click chemistry
Accelerated SuFEx (ASCC); external Si additives

Moses, et. al. Angew. Chem. Int. Ed. 2022, 1, e202112375.

Employed ASCC conditions to PFEx

In situ generation of Silyl ester

Demonstrate the superiority of P(V)-F over P(V)-Cl20

Optimization for PFEx
Base screeninganiline

alcoholaldehydeketoneStronger base is necessary for
deprotonation of phenol.
(-)-cholesterolketoneamideester

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Polyfluorinated organophosphorus compounds

Activation by CsF

Inorg. Chem. 1996, 35, 10, 2915–2929

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Polyfluorinated organophosphorus compounds

With substrates comprising multiple P-F bonds anopportunity for diversity orientated clicking is presented.

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The orthogonal reactivity between PFEx and other click reactions
Reason?

FThe striking difference in reactivity between P-F and S-F clickable hubs
creates a window of opportunity for orthogonal connective chemistry.
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Short summary✓ P(V)-F derivatives are stable but once activated, show sharp reactivity.
✓ PFEx is orthogonal over other click reactions.

Angew. Chem. Int. Ed. 2022, 1, e202112375.

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Table of contents

1. Introduction : Application using other P(V) reagents
2. K. B. Sharpless suggests the utility of “PFEx”
3. Biological application of P(V)-F derivatives

4. Summary & Perspective

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P(V)-F shows activated serine-selective reactivity.
Mechanism of action; Irreversible phosphonylation of Ser

http://molecules.a.la9.jp/acetylcholine%20esterase.html

Serine at Catalytic triad…. A set of three coordinated aminoacids in the active site of enzymes

Activated Serine…. Found in Serine hydrolases (SHs)Ex）

Serine esterase
Serine protease
Phospholipase

https://www.chegg.com/homework-help/questions-and-answers/amino-acid-serine-formcatalytic-triad-amino-acid-cysteine-form-catalytic-dyad-q18222430

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Activity-based protein profiling (ABPP) for SHs
LinkerActive-Ser selectiveWarhead
Tag

No reaction w/ denatured proteins

Identification of SH activity enzyme from rat tissues

Soluble fractions of rat testis>10 testicular proteins detected
Preheating ; 80°C, 5 min
Cravatt, B. F. et. al. Proc. Natl. Acad. Sci. U.S.A. 1999, 96, 14694.

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Phospholipase-selective probeTag(sn-1)

WarheadLigand*(sn-2)https://www.mfc.co.jp/product/kouso/phospholipase/index.html

Reactivity of 1 and 2 with poorly-investigated lipase

Good features for identification of CDTA
Red : probe 1
Blue : probe 2

IC50
Probe1 : 3 nM
(Probe2 : 165 nM)
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Cravatt, B. F. et. al. J. Am. Chem. Soc. 2010, 132, 10, 3264–3265.

Further application of ABPP and perspective of P(V)-F

Cell Chem. Biol. 2020, 27, 937.

SuFEx for divergent application✓ React with Ser/ Tyr/ Lys/His✓ Target identification and validation✓ Mapping of enzyme binding site (other than SH)✓ Covalent drugs
https://sites.rutgers.edu/moschitto-lab/research/

P(V)-F could be another CR with high selectivity toward O.
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SummaryPFEx…. is another good reaction for click chemistry…. the transformation proceeds smoothly like SuFEx…. original feature derived from P ; Selectivity toward O…. further development (activation by LA or H network) is desired
Phosphorus-oriented selectivity

Preliminary example of labeling31

Appendix

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Trivalent Phosphoryl reagent ; P(III) reagentsNat. Chem. 2021, 12, 2760.

✓ Alcohol-selective phosphorylation over Amine✘ Containing oxidation steps✘

No-selectivity among OH

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P(V) reagents are desirable for protein labeling (w/o oxidation step)36

Warning in case of handing P(V)-F compounds

37Raw data of Mechanistic study

Weinberger, W. A. et. al. Can. J. Chem. 1970, 48, 1351.

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Synthesis of P(V)-F compounds
Phosphoramidic difluoride

Phosphoramidofluoridate39Warning in case of handing P(V)-F compounds

40Phosphorous Fluoride derivatives
Representative P(V)-F derivatives

Diisopropyl fluorophosphate

Sarin

Phosphorofluoridic acid

MipafoxMechanism of action; Irreversible phosphorylation of Ser

http://molecules.a.la9.jp/acetylcholine%20esterase.html

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Serine hydrolases42

PF probeとしての活用法

https://www.sciencedirect.com/science/article/pii/S245194
5619300753

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FP probe for detection of Serine hydrase

Cravatt, B. F. et. al.
Proc. Natl. Acad. Sci. U.S.A.
1999, 96, 14694.

✓ SH selective✓ Broad reactivity
across the SH class

Cravatt, B. F. et. al.
J. Am. Chem. Soc. 2010,
132, 10, 3264–3265.

Li, N. et. al.
Anal. Chem. 2022, 94, 24,8625–8632.

In cell lysate
more selective for reacting withphospholipases (over other SHs)

The broad-spectrum serine hydrolase probe

Serine protases
Phospholipases
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SuFEx45

SuFEx46

SuFEx47

SuFEx for activity-based protein profiling

PNAS, 2019, 116, 18809.

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SuFEX

Dual use of SuFEx and PFEx
https://reader.elsevier.com/reader/sd/pii/S2451945620302816?token=F14E2FBC6E423E1AA1B10EF1491C38F1431A101FB2BA2C1CFE2C3085395BDDB771A028B451B020A53A3B3D
B9C4E36E37&originRegion=us-east-1&originCreation=20221107124339

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アニリンとrefluxするとPFも反応してしまう

PNAS, 2019, 116, 18809.

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Imidazole acts as nucleophilic catalyst

https://pubs.acs.org/doi/10.1021/ja1034733
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Selective-phosphorylation using imidazole as a nucleophilic catalyst

2.7 Å

Miller, S. J. et. al. J. Am. Chem. Soc. 2001,
123, 41, 10125–10126

Miller, S. J. et. al. J. Am. Chem. Soc. 2002,
124, 39, 11653–11656

Miller, S. J. et. al.J. Am. Chem. Soc. 2013,
135, 33, 12414–12421

Kinase mimic catalyst forasymmetric phosphorylationof inositol

Site-selective phosphorylation of
the enantiotopic 1- and 3positions of the inositol ringSite-selective phosphorylation of
three distinct hydroxyl groups within
the complex glycopeptide
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今回の話題内在性のMgを使ってこういうactivation

https://pubs.rsc.org/en/content/articlelanding/2005/cc/b506344b
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Other synthetic method for P(V)-FJ. Chem. Soc., Perkin Trans. 1, 1994, 817-820

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Activating reagent for minimal-protection phosphateJ. Am. Chem. Soc. 2022, 144, 17, 7517–753055リン酸化体を釣ってくる方法
内在性のキナーゼを使ってbiotinを導入

塩基でデヒドロアラニンにしてチオールあなどの1,4-付加（phosphotyrosineには使えない）

https://chemistry-europe.onlinelibrary.wiley.com/doi/full/10.1002/cbic.202200407
https://pubs.acs.org/doi/10.1021/ja066828o

Phosphoimidazole (pyrophosphorylation)

樹脂を使ってリン酸基にアミンを縮合する方法

https://pubs.acs.org/doi/pdf/10.1021/cb6003564
https://chemistry-europe.onlinelibrary.wiley.com/doi/full/10.1002/cbic.202200407

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P-N化合物の特性

レムデシビルなどのプロドラックのphophateのマスキング保護基として使われている
Phosphoryl amidazeで切ってたけど、これにすると酵素非依存で放出されるhttps://onlinelibrary.wiley.com/doi/10.1002/anie.202008665

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Bond dissociation energy

https://www.chegg.com/homework-help/questions-and-answers/using-standard-enthalpyformation-data-show-standard-enthalpy-formation-hf-g-used-determin-q37373914

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