Screening For The Regio And Stereospecific Hydroxylation Of Pyrrolidine

We were interested in the discovery and development of biocatalysts for the regio- and stereoselective hydroxylation of pyrrolidine to 3-hydroxypyrrolidine, since both (S)- and (R)-3-hydroxypyrrolidines are useful intermediates for the preparation of several pharmaceuticals (Figure 15.1), and they are difficult to prepare by chemical syntheses. To avoid random screening of a large number of microorganisms, we concentrated on microorganisms with selected biodegradation abilities. Microorganisms that degrade n-alkanes often contain alkane hydroxy-lases that catalyze the hydroxylation of alkanes at a terminal position as the first degradation step. These alkane hydroxylases might also catalyze the desired hydroxylation of pyrrolidines. Moreover, the alcohol dehydrogenases in alkane-degrading microorganisms that catalyze the oxidation of terminal alcohols to the corresponding aldehydes during biodegradation may not be able to oxidize the desired product 3-hydroxypyrrolidine, which is a cyclic secondary alcohol. Therefore, we selected 70 n-alkane-degrading strains as the source of catalyst for n02 O

H3COOC

n02 O

H3COOC

h3c n ch3

hci h3c n ch3

Barnidipine, calcium antagonist

DX-9065a, anticoagulant h2n/,,

DX-9065a, anticoagulant h2n/,, hci h2o

Darifenacin, flor irritable bowel syndrome

RS-533, carbapenem antibiotics hci h2o

ABT-719, antibacterial drug

Figure 15.1 (S)- and (R)-3-hydroxypyrrolidines as pharmaceutical intermediates.

screening. By use of a miniaturized system, which allows for parallel inoculation, growth, and bioconversion on a microtiter plate,24 coupled with a fast and sensitive Liquid Chromatography-Mass Spectroscopy (LC-MS) detection, we found that 25 of the 70 strains catalyze the 3-hydroxylation of N-benzylpyrrolidine.25 Further investigation with 12 more active strains demonstrated complementary enantioselectivities (Table 15.1). Hydroxylation of N-benzylpyrrolidine with Pseudomonas oleovorans GPol, the prototype alkane hydroxylating strain that contains a well-known membrane-bound alkane hydroxylase (alkB) afforded 62% of (R)-N-benzyl-3-hydroxypyrrolidine in 52% enantiomeric excess (ee), whereas

TABLE 15.1 Enantioselectivity and Activity of the Hydroxylation of N-Benzylpyrrolidine to N-Benzyl-3-hydroxypyrrolidine with Several Alkane-Degrading Strains

Entry

Microorganisms

Product ee (%)

Relative Activity

1

HXN-11CC

VC (R)

4

2

HXN-4CC

65 (R)

0.5

3

P.putida P1

62 (R)

1

4

P. oleovorans GPo1

52 (R)

1

5

BC2C

4C (R)

1

6

HXN-15CC

25 (R)

3

V

HXN-5CC

10 (R)

11

S

HXN-2CC

53 (S)

6

9

HXN-1CC

10 (S)

3

1C

HXN-19CC

<10 (S)

10

11

HXN-1000

<10 (S)

1

12

HXN-6CC

C

3

"Relative activity was based on the activity with P. oleovorans GPol.

"Relative activity was based on the activity with P. oleovorans GPol.

Sphingomonas sp.

Sphingomonas sp.

Activity (U/g cdw)

ee ee ee after crystalization (%)

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