P(V) reagents for stereodivergent
synthesis of phosphothioate

Literature Seminar M2 Fujiyoshi 2021/7/29
Knouse, K. W.; Baran, P.; et al. Science, 2018, 361, 1234

Contents• Introduction of antisense oligonucleotide• Reagents for chiral phosphorothioate synthesis: 𝜓 reagent• The principle of 𝜓 reagent’s reactivity• Stereoselectivity• Chemoselectivity• High reactivity• Summary
2021/7/29

M2 Fujiyoshi

1Contents• Introduction of antisense oligonucleotide• Reagents for chiral phosphorothioate synthesis: 𝜓 reagent• The principle of 𝜓 reagent’s reactivity• Stereoselectivity• Chemoselectivity• High reactivity• Summary
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M2 Fujiyoshi

2Antisense Oligonucleotide (ASO)
ASO binds to RNA

Steric block mechanism

One of the FDA approved ASOs

RNA degradation mechanism

Knouse, K. W.; Baran, P.; et al. Science, 2018, 361, 1234

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Bennett, C. Annu. Rev. Med., 2019, 70, 307

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Common chemical modifications in ASO

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T. Roverts et al., Snat. Rev. Drug. Discov., 2020, 19, 673

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4Why Sulfur? The Thio effectKnouse, K. W.; Baran, P.; et al. Science, 2018, 361, 1234

2021/7/29(See an latest article for mechanism of cellular uptake
Laurent, Q.; et al. Angew. Chem. Int. Ed. 2021, 60, 1)

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5The stereochemistry at phosphorus
The stereochemistry of phosphorothioate affect• the stability against nucleases• the efficiency of degradation of the targeted RNA.
Mipomersen andstereochemically pure counterparts

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Stability in rat liver homogenate

N. Iwamoto et al., Nat. Biotechnol., 2017, 35, 845

Activity of RNase HCon ASO–RNA heteroduplexes

M2 Fujiyoshi

6What is problems of current methods for construction of PS linkage?

• Stepwise-synthesis using P(III) chemistry is operationally cumbersome.
• P(III) reagent is air and moisture sensitive.
• The stereochemistry at phosphorus is often disregarded in scalable preparation.

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Knouse, K. W.; Baran, P.; et al. Science, 2018, 361, 1234

M2 Fujiyoshi

75’-O-DMT-nucleoside 3’-O-(2-thio-1,3,2-oxathiaphospholane)
DMTO
DMTOHOBaseOOO+SPSBaseBaseSOBaseODMTOODBUOOR-SO

Single stereoisomer
Rp or SpPSOOO

BaseSPO

BaseO

O

OR

OR

Diastereoselective coupling
without protection

Problems• 3(4) step synthesis of nucleotide loaded reagent via phosphoramidite P(III) (See appendix)• Separation of the diastereomers of reagents• Scale limited
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Stec, W.; et al. J. Am. Chem. Soc. 1995, 117, 863
Knouse, K. W.; Baran, P.; et al. Science, 2018, 361, 1234

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8Phosphorus–Sulfur Incorporated (PSI, 𝜓) reagent

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Knouse, K. W.; Baran, P.; et al. Science, 2018, 361, 1234

M2 Fujiyoshi

9Contents• Introduction of antisense oligonucleotide• Reagents for chiral phosphorothioate synthesis: 𝜓 reagent• The principle of 𝜓 reagent’s reactivity• Stereoselectivity• Chemoselectivity• High reactivity• Summary
2021/7/29

M2 Fujiyoshi

10Synthesis and Stereochemistry of 𝜓 reagents
Loading condition: alcohol 1.0 eq., 𝜓 1.3 eq., DBU 1.3 eq., MeCN, 25 ºC, 30 min

Coupling condition: alcohol 2.0 eq., Nucleoside P(V) 1.0 eq., DBU 3.0 eq., MeCN, 25 ºC, 30 min

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Knouse, K. W.; Baran, P.; et al. Science, 2018, 361, 1234

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Loading and Coupling of Nucleosides with 𝜓 reagents

P(V)
SS

MePMeOH

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Knouse, K. W.; Baran, P.; et al. Science, 2018, 361, 1234

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CDN Synthesis: Standard approach vs. 𝜓 reagent platform

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Knouse, K. W.; Baran, P.; et al. Science, 2018, 361, 1234

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13Automated synthesis of PS oligonucleotides• 𝜓 reagent can be utilized in stereoselectiveSPOS.

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Knouse, K. W.; Baran, P.; et al. Science, 2018, 361, 1234

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14Other applications of 𝜓 reagents
Enantiodivergent formation of C–P bond

Native DNA manipulation

Xu, D.; Baran, P.; et al. J. Am. Chem. Soc. 2020, 142, 5785

Flood, D. T.; Baran, P.; et al., ACS Cent. Sci. 2020, 6, 1789

Serine selective bioconjugation

Total Synthesis of Tagetitoxin
Oligonucleotide synthesis

He, C.; Baran, P.; et al. J. Am. Chem. Soc. 2020, 142, 13683

Vantourout, J.; Baran, P.; et al. J. Am. Chem. Soc. 2020, 142, 17236
Knouse, K. W.; Baran, P.; et al. Science, 2018, 361, 1234

2021/7/29

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Short summary• Redox-economic• Bench-stable reagents• Simplified protocol• Solid and solution phase• Rapid and scale amenable• Highly broad applications
other than ASO synthesis

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Knouse, K. W.; Baran, P.; et al. Science, 2018, 361, 1234

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Contents• Introduction of antisense oligonucleotide• Reagents for chiral phosphorothioate synthesis: 𝜓 reagent• The principle of 𝜓 reagent’s reactivity• Stereoselectivity• Chemoselectivity• High reactivity• Summary
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17The noteworthy points of 𝜓 reagent

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18

Reaction mechanism of phosphoryl transfer
D N + ANNuO
P OR
ROORANDN

AN + DN

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NuO
P
RO OR

ORO
NuP
OR
RO ORO
P OR
Nu
OR

ORO
NuP
OR
RO OR

Pereira et al., Phys. Chem. Chem. Phys. 2016, 18, 18255

ANDN
POSOP transition state

AN + DN
R2PEP intermediate

AN + DN𝜓 reagent intermediate

Domon, K.; Fujiyoshi, K.; Kanai, M.; et al.
ACS Cent. Sci. 2020, 6, 283

Fujiyoshi, K.; Motomu, K.; et al.
Synlett 2021, 32, 1135

Vantourout, J.; Baran, P.; et al.
J. Am. Chem. Soc. 2020, 142, 17236 M2 Fujiyoshi

19Trigonal-bipyramidal (TBP) phosphorane intermediate② Addition and elimination occurs from apical
apical

① Apicophilicity

A
Equatrial EHO• Electronegative

EquatrialEP
E Equatrial
AOOO
PHO OMe

PO

O
OH

MeOH

• Small ligand
F > O > C, N, SO
P
O OMeapicalOOO

OH–OO POMeOH

O P OMe-O OH③pseudorotation④ Configuration at phosphorusLGInversion

BA

P

NuO

A

Retention

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BLG

P

NuO

LG
B P OANuA
B P OLGNu– LG-BA

PO

Nupseudorotation

LGA P ONu
B– LG-ANuPOB
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20Proposed mechanism

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Vantourout, J.; Baran, P.; et al. J. Am. Chem. Soc. 2020, 142, 17236

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The noteworthy points of 𝜓 reagent

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Question: The stereochemistry of 𝜓 reagents

Retention

Each loading and coupling step showsa retention of configuration at phosphorus.

Retention↓Why?

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Knouse, K. W.; Baran, P.; et al. Science, 2018, 361, 1234

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23

Possible pathways for methanolysis of the oxathiaphospholane
The methanolysis of 1b occurrs witha retention of configuration at phosphorus.
↓• To minimize the ring strain, the five-membered ringoccupies one apical and one equatrial position in TBP.
• Anionic S atom derived from P=S is always equatrial.
↓Three pathways A, B and C are possible.

Retention

Inversion

Only path A show a retention of configuration at phosphorus.
↓
However…

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Uchimaru, T.; Stec, W.; Taira K. J. Org. Chem., 1997, 62, 5793

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24

Possible pathways for methanolysis of the oxathiaphospholane
Only path A show a retention of configuration at phosphorus.
↓However…
↓?
?

• ITBPA is unstable compared with ITBPB…
(S and N are less apicophilic than O)• The elimination of equatrial ligand
doesn’t occur directly…

Retention

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Inversion↓
Why does the substitution occur in such a way?
↓Ab initio caliculation
Uchimaru, T.; Stec, W.; Taira K. J. Org. Chem., 1997, 62, 5793

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25

Ab initio calculation
1. Nucleophilic attack of methoxide
collinear to the endocyclic P-Sbond should occur in concert withbreaking of the P-S bond. (Path C)
2. Pseudorotation in ITBPB will occur
concomitant with cleavage of theendocyclic P–S bond via thetransition state TSnP–S to
immediately give ITHA, without
forming ITBPA. (Path A)
3. Path A will be preffered over either
path B or C.
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Uchimaru, T.; Stec, W.; Taira K. J. Org. Chem., 1997, 62, 5793

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Answer: The stereochemistry of 𝜓 reagents
TS2MeMe-TS1HMe

X = SC6F5DBU

MeMeS S R OHO P
XHS S
HOO PRXH

H
MeH

MeMeS SRO P OH
SC6F5HMeS SO P
ORH-H
MeS SOO P
RXHMeMeX = OR'

S SO P

H
MeH

O
OR'R

H

SO SP
H
ORMeR'O•

Nucleophilic attack of alcohol will occur from the opposite side of endocyclic oxygen via TS1.

•Pseudorotation will occur accompanied by the cleavage of the endocyclic P–S bond via TS2.

•These processes lead to the diastereoselective reaction with a retention of configuration at phosphorus.
Uchimaru, T.; Stec, W.; Taira K. J. Org. Chem., 1997, 62, 5793

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Knouse, K. W.; Baran, P.; et al. Science, 2018, 361, 1234
Vantourout, J.; Baran, P.; et al. J. Am. Chem. Soc. 2020, 142, 17236

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The noteworthy points of 𝜓 reagent

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28Serine selective bioconjugation

P(V)SSMePMeOH

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Vantourout, J.; Baran, P.; et al. J. Am. Chem. Soc. 2020, 142, 17236

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29

Question: The chemoselectivity of 𝜓 reagent

𝜓 reagent selectively react with serine-OH in the presence of other nucleophiles.
↓How does 𝜓 reagents distinguish nucleophiles?
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Vantourout, J.; Baran, P.; et al. J. Am. Chem. Soc. 2020, 142, 17236

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DFT calculations to rationalize the serine selectivity

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Vantourout, J.; Baran, P.; et al. J. Am. Chem. Soc. 2020, 142, 17236

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31Answer: The chemoselectivity of 𝜓 reagent
ThrTyrLys

𝜓MeH

S SO P
ORH

MeSerOHOH

MeH

NH2MeOH

S S
HXO P
OR RH

MeTS1H
MeSH

H
MeDBU

S S
HOO P
OR SerH
MeCys

DBU

TBP
H
Me-S S
O
O P
Ser
ORH

S SO P

H
MeH

MeO

Ser

ORH

SO SPH
O Ser
MeRO

MeS-H

Me

Me

TS2

MeDBU

S S
H
PS
O
OR CysH

MeSOO PCys
ORH

S SO PH
MeH

SCysOR• Barriers in the formation of TBP intermediate (TS1)
Cys << Ser < Thr << Tyr (limitted reactivity) << Lys (lower acidity of primary amine)• For Cys, the collapse of the TBP intermediate is late limitting step.
TS2 𝛥G‡ (Cys) > TS1 𝛥G‡ (Ser)• These results rationalize the serine selectivity.
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Vantourout, J.; Baran, P.; et al. J. Am. Chem. Soc. 2020, 142, 17236

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The noteworthy points of 𝜓 reagent

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Question: The best reactivity of 𝜓 reagent
Bench stable
HOMeSPSMeSC6F5HODBU

1.3 eq.
1.3 eq.

HOMePMeCNr.t., 30 min

SSO

MeHODBU

2.0 eq.
3.0 eq.

MeCNSHOPOOr.t., 30 min

Knouse, K. W.; Baran, P.; et al. Science, 2018, 361, 1234

𝜓 reagent has great stability for air and moisture and is highly reactive under mild condition.
↓How does 𝜓 reagent realize the balance of stability and reactivity?
DBU is one of the most effective base for activation of alcohol.
However, is the effect of base only a reason for the high reactivity?
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34

What is the key structure for high reactivity?
ParaoxonParathionOO
P
SC6F5S

S
EtO P
EtO O

1.3×10-2

Effect of S?
ONO2O
EtO P
EtO O

<NO2

4.0×10-1

k (min-1mole-1) hydrolysis in 50% acetone at 25 ºC
Ketelaar, J.A.A. and Gersmann, H.R. Recl. Trav. Chim. Pays-Bas, 1958,, 77, 973

SPS

SC6F5O

SPS

OR

Effect of leaving group?
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The reactions in both of the loading and coupling steps finishedin 30 min at r.t..
→ The leaving group should not be critical for high reactivity.
Knouse, K. W.; Baran, P.; et al. Science, 2018, 361, 1234

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35

The answer lies in ring moiety

Effect of S?
OSPS

SC6F5OSP

Which is faster for hydrolysis?

SC6F5O

Effect of ring?

OSOPOMe

MeOvs.

OP

MeO

OMe

Westheimer, F. H. Acc. Chem. Res. 1968, 1, 70

Effect of leaving group?
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The rate of hydrolysis: Five-membered ring vs. acyclic phosphate
AOO

B MeO P O

OMe

MeOPOOOO POMe
OH2HOHOpseudorotation

OPOOMeOO P OMe-O OH
2O
7:3kA/kB > 106

OMeOPMeOOMeOH2OHOOH

MeOOPPOOMeOMe

The rate of hydrolysis of methyl ethylene phosphate A (kA) exceedsthat of trimethyl phosphate B (kB) by 106.
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Westheimer, F. H. Acc. Chem. Res. 1968, 1, 70

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TBP intermediate of five-membered ring phosphate in hydrolysis

OO
P99 ºOOMe

The ring angle at phosphorus (O-P-O) is 99 º determined by
X-ray crystallography.
The five-membered ring is highly strained.
↓90 ºOO POOMe
OH2

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The formation of the TBP intermediatewith a 90 º angle at phosphorus(ring in the equatrial–apical conformation)
largely decrease the strain energy.

Westheimer, F. H. Acc. Chem. Res. 1968, 1, 70

M2 Fujiyoshi

38Driving force: ring strain
OMe
O
MeO P
OMe
OH

O-O

O

O P O
OMe
OH

PGO

MeO

OMe

𝛥GA – 𝛥GB
= 5~6 kcal/mol

OP

MeO

𝛥G‡A
= 𝛥H‡A – T𝛥S‡A

𝛥GA

HOHO

𝛥G‡A – 𝛥G‡B
= – 8.5 kcal/mol

OMe𝛥G‡B
𝛥GB= 𝛥H‡B – T𝛥S‡BO

HOOMe

MeOOPPO𝛥H‡A – 𝛥H‡B= – 7.6 kcal/mol

OMe

The difference of 𝛥H‡ is comparable to that of 𝛥G‡.
The release of ring strain accounts for the rapid hydrolysis of five-membered ring phosphate A
compared with acyclic one B.
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Westheimer, F. H. Acc. Chem. Res. 1968, 1, 70
Chang, N.; Lim, C. J. Am. Chem. Soc. 1998, 120, 2156

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39Answer: The best reactivity of 𝜓 reagent

Ring strain

HMeDecrease
the enthalpy of activationin rate limitting step
DBUMeMeS S R OHO PXHS SHOO PRX
HH
MeTS1Ring strain freeMe-H
MeS SOPORXH

The driving force of 𝜓 reagent should be the ring strain.
It should decrease the activation energy compared with acyclic phosphate.
This is why 𝜓 reagent shows the high reactivity.
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40SummaryMe

Me-X = SC6F5H
MeDBU

MeMeS S R OHO P
X
HS S
HOO P
RXH

H
MeH

MeMeS SRO P OH
SC6F5HMe2)The cleavage of P–Sbond accompanied bypseudorotation
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MeMeS SO PHMe1)The direction ofnucleophilic addition

MeCoupling reagent-S SOO PRXH

X = OR'

Stereoselectivity

HS SO PORH

H

Chemoselectivity
1)𝛥G‡ in TS1
Cys << Ser< Thr << Tyr << Lys
2)TS2 𝛥G‡ (Cys)> TS1 𝛥G‡ (Ser)

O
OR'R

H

SO S
P
H
ORMeR'O

To thiophosphoate
diesterHigh reactivity1)Strained five memberedring
2)Lower the entalpy of theactivation in TS1
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41Appendix

Appendix: Synthesis of 5’-O-DMT-nucleoside 3’-O-(2-thio-1,3,2-oxathiaphospholane)

PCl3OHHS

HNi-Pr2O

P N

P ClSSDMTOO

BaseO

OH
1. tetrazole
2. Sulfur

DMTOBaseOOO

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Separation bychromatography
SPS
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43Appendix: Phosphorylation of existing method

The introduction of phosphoryl
group to a secondary alocoholby existing methods was quitedifficult.
• Phosphoramidite• Sulfide was oxidized…
• Other reagents• Too harsh or invalid…

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44Appendix: Phosphorylation by ψ reagent

Ψ reagent provided thiophosphatetriester in high yield (90%).
SeO2 oxidation and hydrolysis ofphosphate thioester gave aphosphomonoester final productTagetitoxin.

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45Appendix: The rate of hydrolysis of some phosphorus acid esters
1J. A. A. Ketelaar and H. 9. Gersmann.

976

Table I
Results of hydrolysis
I measurements

No.

CompoundSolvent-log.
freq.
factor(min-1)

Activation
Energycal/mole-~

77 (1958) RECUEIL

Chemical studies on insecticides IV.

Activation
Entropy(Entr.
Units)--FreeEnergyof Activation
cal/molek(25" C)min-'rnole-'lk(37.50 c)
min-'mole-'liI

Inhibition
Cholinesterase'IGO

Ali-esterase 1
Is0I60Cholinesterase~

2.24 x 10-3> 10-1-

1.48 X lo-*10-3

3x 10-1

10-8201Hz07.94

15000

-32.2

24100

250% ethanol

8.76

15000

-28.5

23900

x 10-4
5.3 x 10-3

350% acetone

8.64

12400

-29.020500

4.0 X 10-1 9.5

450% ethanol

11.90

15800

-14.2

19500

1.86

5.5

8 x5SOYo acetone

11.96

14300

-13.9

17900

3.1 X 10

8.2 X 102 x 10-8

106S O Y O acetone

11.25

13400

-17.2

17900

3.4

7.8 X 104 x 10-85720% ethanol

10.01

18300

-22.824500

820% ethanol

8.97

8700

-27.5

16400

5 x 10-7

0.14975% ethanol

13.57

16100

- 6.617500> 10-2(0.151050% acetone

6.34

4100

-39.6

15400

x 10
3.5 x 10-4
3.7 x 1P
5.4 x 102.0 x 103

4 x 10-611150% ethanol

10.85

I6700

-19.0

21800

3.6 X lo-'

1250% ethanol

12.01

15200

-1 3.718700

6.8

1350% acetone

11.26

16700

-1 7.1

21200

8.5 X

x 10-1
1.9 x 10
3.4 x 10-1

H2010.76

15450

-19.6

20700

50% acetone

11.9619200

-13.9

22700

1.3 X lo-"

5.5Hz0

10.95

16600

-18.5

21600

1520% ethanol

13.79

17500

- 5.618600

9.1

2.7 X 101650% acetone

8.73

12100

-28.6

20100

9.7

1750% acetone

10.33

13900

-21.3

19700

1.35

I x 10-3

1850% acetone

5.215700

-44.7

18500

1.25 X 10

2 x 10-4

0.5

1925.% acetone

20.45

24500

-24.75165OO

2.6

5 x 10-6

1514-977

7.9x 10-1

x 1Oe

1.2

5 x 10-6x 10-3

6.8 X l @1.6 X

1.1x 10-2lo-'

1.4 X lo(

1.5 x 10-3

1.4 x 10-3

5 x 10-4

0.8( 1 ) 2.5 X

4 x 10-3

1.8(?)

3 x 10-3

1.50.25.) These values were taken from the work of Mendef and Myers9).

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46

Appendix: Ab initio calculations for MEP vs. TMP or EPP

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Appendix: Ab initio calculations for MEP vs. TMP or EPP

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48