Stereocontrolled transformations of organophosphorus acid esters

A variety of phosphoric acid triesters and their derivatives are used as pesticides. Although there are no natural phosphorotriesters, those artificial ones undergo decomposition in the soil, implying that some microorganisms exist which are capable of hydrolysing them. The first report on a stereoselective enzymatic phos-photriester hydrolysis was published in 1973, when Dudman and Zerner succeeded

Table 9

Enzymatic acetylation of hydroxymethylphosphine P-boranes o er r

Solvent

Enzyme

Recovered alcohol 85

Acetate 86

Ref.

Table 9

Enzymatic acetylation of hydroxymethylphosphine P-boranes

Solvent

Enzyme

Recovered alcohol 85

Acetate 86

(%)

[«]o

(%)

(%)

[«]o

(%)

Abs. conf.

1

Et

C-C6H12

CAL

B1

—2B.B

92

R

S8

n.r.

B4

S

6

98

2

n-Bu

C-C6H12

CAL

2B

— 1S.B

91

R

62

n.r.

SS

S

10

98

B

•-Pr

C-C6H12

CAL

B2

—10.1

l9

R

61

n.r.

6l

S

B

98

4

í-Bu

C-C6H12

CAL

44

+2.9

SB

R

42

n.r.

n.d.

S

6

98

S

EtO

i-P^O

CAL-B

B8

-18.9

20

n.d.

B8

+1l

n.d.

n.d.

n.r.

99

6

• -PrO

•-Pr2O

AK

S1

—9.1

14

S

21

+12

Bl

R

n.r.

99

l

• -PrO

•-Pr2O

CAL-B

4B

—l.9

12

S

B9

+l

18

R

n.r.

99

8

• -PrO

C-C6H12

CAL-B

B1

—24.1

Bl

S

49

+ 11

29

R

n.r.

in the enzymatic preparation of optically active n-butyl methyl p-nitrophenyl phosphate starting from the racemic substrate. The enzyme involved in this reaction was an ingredient of the horse or beef serum.101 Later on, phosphotriesterase, an enzyme found in certain native soil bacteria (Pseudomonas diminuta and Flavobacterium sp.), was shown to degrade organophosphates.102 Raushel et al. used this enzyme for hydrolysis of the phosphonothionate 92 and found out that it hydrolysed exclusively the (S)-enantiomer of the substrate. The reaction proceeded with inversion of configuration at phosphorus and gave the (S)-enantiomer of the thioacid 93 (Equation 44).103

Phosphotriesterase from P. diminuta (PTE) was found to exhibit high hydrolytic activity towards various types of tetracoordinated phosphorus acid esters. Apart from the phosphonothionate 92, phosphoric acid triesters 94 (Equation 45),104 benzenephosphonic acid diester 95 (Equation 46)105 and methyl-phenylphosphinic acid ester 96 (Equation 47)106 were also stereoselectively hydrolysed under kinetic resolution conditions. Of course, in the case of the latter three kinds of substrates, half of the reacting ester was irreversibly lost due to the formation of achiral phosphorus acids.

In the case of the phosphotriesters 94, the use of engineered mutants of phos-photriesterase allowed not only to enhance but even to reverse the stereoselectivity of the native enzyme. The ees of the recovered esters exceeded 95%.104

In turn, for the phosphonic acid diester 95, stereoselectivity of the native phosphodiesterase was enhanced by over three orders of magnitude by alteration of the pKa values of the leaving group phenol. For example, for X = CO2Me, Y = H the stereoselectivity was 5000 higher than for X = NO2, Y = 2-F.105

Both types of manipulation were applied to achieve the required enantiomer and the highest enantioselectivity in the hydrolysis of the phosphinic acid diester 96.106

Finally, non-racemic phosphorothioic and phosphonothioic acids 98 were obtained via a PTE-catalysed stereoselective hydrolysis of the prochiral substrates 97 (Equation 48).107 The absolute configurations of the thioacids 98 depended on whether native PTE or its mutants were used.

In addition to phosphotriesterase from P. diminuta (PTE) discussed above, two other types of enzymes were found to exhibit phosphotriesterase activity. Interestingly, both are peptidases - the enzymes which in nature hydrolyse a peptide bond. The first one - organophosphorus acid anhydrolase (OPAA) from Alteromonas sp. JD6.5 - is a proline dipeptidase; its original activity is to cleave a dipeptide bond with a prolyl residue at the carboxy terminus.108 109 The second one - aminopeptidase P (AMPP) from Escherichia coli - is a proline-specific peptidase that catalyses hydrolysis of N-terminal peptide bonds containing a proline residue.110'111

OPAA was found to exhibit stereoselectivity towards phosphorotriester substrates 94, with a preference for the (S)-enantiomer. Surprisingly, the selectivity was most apparent for the substrates in which the non-hydrolysing substituents did not differ too much. For example, for 94 (R1 = Me, R2 = Et) the chiral preference was 112-fold, while for 94 (R1 = Me, R2 = i-Pr) it was 100-fold.108 Similarly, p-nitrophenyl analogues of sarin 99 and soman 100 (fluorine replaced by the p-nitrophenoxy group) were stereoselectively hydrolysed by OPAA with a 2- to 4-fold preference for the (R)-enantiomer. In the case of the soman analogue 100, the enzyme also exhibited an additional preference for the configuration of the stereogenic carbon atom, which depended on the configuration at phosphorus.109

In turn, AMPP was found to stereoselectively hydrolyse the phosphonoth-ionate 101, exhibiting preference towards the (S)-enantiomer. The hydrolysis

proceeded with inversion of configuration at phosphorus, similar to that shown in Equation 44.111

All the enzymes discussed above belong to the class of dimetalloenzymes.112 In this context, it should be mentioned that serine-type hydrolases are irreversibly inhibited by organophosphorus esters, among them highly toxic chemical warfare agents. However, in some cases, for example of human butyrylcholi-noesterase, the inhibited enzyme could be reactivated by proper mutations.113 Moreover, such mutations were found to confer phosphotriesterase activity in this enzyme!114

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