Synthesis of Optically Active - American Chemical Society

Chapter 24

Downloaded by EAST CAROLINA UNIV on September 18, 2015 | http://pubs.acs.org Publication Date: June 14, 2009 | doi: 10.1021/bk-2009-1009.ch024

Synthesis of Optically Active α-Methyl Amino Acids Using Biotransformation as a Key Step Masanobu Yatagai, Takayuki Hamada, Hiroyuki Nozaki, Shinji Kuroda, Kenzo Yokozeki, and Kunisuke Izawa AminoScience Laboratories, Ajinomoto Company, Inc., 1-1 Suzuki-cho, Kawasaki-ku, Kawasaki-shi 210-8681, Japan

A new method for the synthesis α-methyl-L-cysteine was realized by the enzymatic resolution of racemic 2,4-dimethyl4-methoxycarbonylthiazoline, which can be derived from chloroacetone in 4 steps. α-Methyl-L-cysteine hydrochloride with high optical purity was successfully obtained by hydrolyzing (4R)-2,4-dimethyl-4-methoxycarbonylthiazoline. In an alternative approach, a new enzymatic synthesis of α -methyl-L-serine was discovered in the stereoselective hydroxymethylation of L-alanine. The chemical conversion of α-methyl-L-serine to α-methyl-L-cysteine is also demonstrated.

394

© 2009 American Chemical Society

In Asymmetric Synthesis and Application of -Amino Acids; Soloshonok, V., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 2009.

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α-Methyl-a-amino acids are a class of nonproteinogenic α,α-disubstituted α-amino acids, and some compounds, such as (S)-a-methyl tyrosine, (5)isovaline, and (S)-a-methyl aspartic acid, are found in nature (1). These amino acids are well known as important building blocks, especially in bioorganic chemistry.

(S)-A-ME-TYR

(S)-ISOVALINE

(S)-A-ME-ASP

(S)-A-ME-DOPA

Figure 1. Examples of a-methyl a-amino acids

The peptides in a-methyl-a-amino acids show limited rotation around N Cct (φ) and Ca-carbonyl (ψ) bonds, and this stabilizes the conformation of the peptide backbone (2). These effects make them remarkably resistant to enzymatic degradation.

Ο

Η

Ο

I

II

R

C H

3

H

Figure 2. Peptide bond with α-methyl-a-amino acid

The antihypertensive drug (S)-ot-methyl DOPA, which inhibits an aromatic amino acid decarboxylase, is a typical drug application (3).

α-Methyl Cysteine α-Methyl cysteine plays an important role in peptide chemistry by forming constrained cyclic structure with disulfide bridging. Natural products with thiazoline rings exhibit antitumor and anti-HIV activities. Thiazoline com-


396 pounds derived from α-methyl cysteine have also been reported as drug candidates. (4)


DEFERITRIN

DESFERRITHIOCIN

Figure 3. Drug candidates derivedfrom α-methyl cysteine

Synthetic Methods for α-Methyl Cysteine Many methods for the synthesis of α-methyl cysteine have been reported so far. Schôllkopf s alkylation of chiral bislactim ether (5) and Pattenden's selfreproduction of L-cysteine's chirality (4a) are well-known techniques for the asymmetric synthesis of α-methyl cysteine. These methods offer a convenient approach to the chiral form, but require relatively expensive reagents such as lithium base. The enzymatic hydrolysis of α,α-disubstituted malonate and subsequent Curtius rearrangement has been demonstrated for the effective synthesis of protected chiral α-methyl cysteines (6). Optical resolution of racemic intermediates has also been extensively studied (7). In these approaches, sulfur atom is introduced by reaction with sodium hydrogensulfide, tertiarybutylthiol, benzylthiol, or thioacetic acid. However, these sulfur reagents often cause odor issues and require special care to contain them (8).

New Synthetic Method for a-Methyl-L-Cysteine The goal of our study was to satisfy the following concepts and develop a new method for the synthesis of α-methyl-L-cysteine 1 with a potential industrial application. This method does not require expensive alkylating reagents. The sulfur atom is imported from an odorless material. Chirality is realized by enzymatic resolution or synthesis. Our preliminary routes to a-methyl-Lcysteine 1 are outlined in Scheme 1. Odorless thiourea 3 was selected as a sulfurizing reagent and converted to 2amino-4-methyl-4-methoxycarbonylthiazoline 4. Optical resolution of this racemic intermediate 4 was studied. An alternative synthetic route was also studied, in which chiral α-methyl-L-cysteine 1 could be derived from a-methylL-serine 6. Soil samples were screened to find a new enzyme to catalyze the enantioselective hydroxymethylation of alanine 5.


397 NH,

B R ^ X O Î M E "

2

^

jS

Br H

2

C

N ^ /

*•

0

H N—^ Τ*»

M

e

enzyme

2

2

2

4

N ^ C 0

2

H

E

N

Z

*

M

^

H

2

N

V ^

C

0

-
U l

N h 2

16

N

•

MeOH 60°C,24h

HCI ^N^COaH

^ C 0 M e onc.HCI 2

—(/ jS \ ^ 20 87%

C

• 100°C,16h

H

S

^

24 87%

Scheme 6. Acidic hydrolysis of2 4"dimethyU4-methoxycarbonylthiazoline f

Enantioselective Hydrolysis of 2,4-DimethyI-4-Methoxycarbonylthiazoline Commercially available enzymes were screened for the enantioselective hydrolysis of 2,4-dimethyl-4-methoxycarbonyllthiazoline 20 (Scheme 6). The substrate 20 was added to 10% wt/wt enzyme and reacted in phosphate buffer at 30°C for 24 hours. The optical purity of the products was then monitored. In the screening, three proteases stereoselectively hydrolyzed an ester and afforded 22 in higher than 90% ee (Table J). The following issues were noted for scale-up. Ester hydrolysis proceeded stereoselectively in a diluted condition (1% w/v), but predominant hydrolysis of the thiazoline ring was observed under a higher concentration (15% w/v). After the hydrolysis of the enantiomer was completed, 22 also tended to be hydrolyzed.


400 enzyme .C0 Me (lo%wt/wt)

N

2

1

j

aq.KH P0 pH7,30°C 1-24h 2

20

C

N ^ ° 2 7 s

M

e

-X 7

N^> +

2

H

—? Τ*Χ

4

C 0

δ

^

22


Scheme 7. Enantioselective hydrolysis of2,4-dimethyl-4methoxycarbonylthiazoline

Table 1. Optical Yields of 22 in Screening Enzymes Reaction Time

Enzyme lh

4h

24h

Protease Ρ

39% ee (40%)

80% ee (51%)

91% ee (68%)

Prolether FG-F

23% ee (27%)

61%ee(42%)

99% ee (58%)

Orientase 22BF

-

96% ee (52%)

99% ee (75%)

Conversion of 2,4-dimethyl-4-methoxycarbony lthiazoline is given in the parentheses,

Synthesis of a-Methyl-L-Cysteine Racemic oc-methyl-P-chloroalanine methyl ester 16 was condensed with thioacetamide 18 in 87% yield, and (4/?)-2,4-dimethyl-4-methoxycarbonylthiazoline 22 was obtained in 97% ee via enzymatic resolution. Although the purification of the hydrochloride of α-methyl-L-cysteine 1 was difficult in the final step, crystallization of 25 was identified as an effective purification process. The isolated carboxylic acid 25 was hydrolyzed under acidic conditions, and the hydrochloride of α-methyl-L-cysteine 1 was obtained in good yield and higher than 99%ee(14).

ot-Methyl-L-Serine A new enzymatic synthesis of a-methyl-L-serine 6 and chemical conversion to α-methyl-L-cysteine 1 were also studied (Scheme 9). Wilson and Snell reported the hydroxymethylation of alanine as a reversible reaction (15). An enzyme, a-methyl-L-serine hydroxytransferase, which was isolated from soil bacterium, catalyzed the reversible cleavage of a-methyl-Lserine to formaldehyde and D-alanine (Scheme 10). In this reaction, tetrahydrofolate is added as a cofactor, which takes in the formaldehyde to form


401 • H

2

N ^ C 0

— M

I

C0 ME

N

j r C R

N H J

M E

2

E

0

2

—

2

~

-

H

N

1Q0°C, 24H

ο-^

1

32%

^ —99%EE

Scheme 8. Synthesis of a-methyl-L-cysteine

H

2

N

V

X 0

H

2

HCHO

E

'Β

N

Z

Y

M

H

E

H

2

N ^ C 0

°

2

H

H

6

2

«

N ^ C 0

2

2

M E

6

HCI

>

H N. 2

HS"

%

*C0 H 2

A

Scheme 9. Synthesis of a-methyl-L-cysteine via a-methyl-L-serine

H N. *C0 H N £ 2 2

%

, , A-METHYLSERINE

2 2

H

2

HYDROXY M E THY ITRANSFERASE

J HO

N

X 0

V

2

2

H

2

|

•Τ

HN^

> ^

„

Ν

TETRAHYDROFOLATE 1

Λ

5,10-METHYLENETETRAHYDROFOLATE

Scheme 10. Reversible hydroxymethylation of D-alanine (12)


402 aminal, 5.10-methylenetetrahydrofolate. This aminal is also reversibly hydro lyzed to formaldehyde and tetrahydrofolate. We expected to find a new enzyme to catalyze the enantioselective hydroxymethylation of L-alanine from the screening of soil samples.


Finding New Enzymes One hundred stocked soil samples were screened for new enzymes with the ability to catalyze hydroxymethylation of alanine. In this screening, micro organisms were obtained from soil enrichment cultures, and the cell-free extracts were assayed for the ability of enzymes to release formaldehyde from a-methyl-DLserine 27 (Scheme 11). Six kinds of enzymes were shown to release formaldehyde. Three enzymes were characterized to be independent with regard to tetrahydro folate co-factor, and thus three quite new enzymes were obtained. Three were identified to be Bosea sp., Ralstonia sp., and Variovoraxparadoxus, respectively.

H N ^ ^ C 0 H microorganism 2

H

°

H N. Χ 0 Η

H

> — • τ -.A 2

27

2

2

S

Scheme 11. Screening soil samples for the metabolism of a-methyl-DL-serine

The enzymes were isolated from the cell-free extracts, using three chromatographies, such as ion-exchange, hydrophobic interaction, and hydroxyapatite chromatography, and were finally confirmed to be electrophoretically pure. The enzymes were then analyzed to determine their amino acid sequences, and the encoding genes were cloned. Eventually, the cloned genes were respectively transferred to E.coli. and overexpressed to make recombinant cells. The three kinds of recombinant cells were used for the synthesis of a-methyl-L-serine 6.

Enzymatic Synthesis of a-Methyl-L-Serine The recombinant cells were respectively mixed with L-alanine 28 and formaldehyde, and reacted in phosphate buffer at 30°C (Scheme 12). The results are summarized in Table 2. As described in Table 2, optically pure a-methyl-L-serine 6 was obtained in all cases with excellent reaction yields (16). A small amount of D-alanine was


403 HN

C0 H

2

2

Recombinant cell

V

2

+

Ύ'

0

=

H N ^C0 H 2

N

2

• H

100mM KPB (pH7.4) 0.1 mM PLP, 30°C

28

/ 6


Scheme 12. Synthesis of a-methyl-L-serine using recombinant cells

observed in the reaction mixture, but these enzymes did not catalyze the hydroxymethylation of D-alanine in separate experiments. An enzyme derived from Bosea sp. was used for kg-scale synthesis and a-methyl-L-serine 6 was successfully obtained.

Table 2. Synthesis of α-Methyl-L-Serine Using Recombinant Cells

Varivorax

L-Ala (mmol) 15

HCHO (mmol) 14

a -Me-L-Ser yield (%) 98

Bosea

15

15

95

Ralstonia

60

60

89

Origin

Synthesis of α-Methyl-L-Cysteine from a-Methyl-L-Serine The chlorination conditions for α-methyl serine 6 were screened. In the first trials using thionyl chloride or phosphorous pentachloride, the desired βchlorinated product was not obtained. These results suggest that the bulkiness around the quaternary carbon could interfere with the substitution on the β-carbon. As depicted in Scheme 13, a methansulfonate 29 was prepared from amethyl-L-serine 6 in several steps, and reacted with thioacetamide 18. Under the same conditions as in the reaction of a-methyl-P-chloroalanine methylester 16, no desired product was observed in the reaction mixture. NH

H N_C0 Me 2

2

ΜβθΛ

2 99

H

2

S «

"

e 0 H

60°C, 16h

N^

Synthesis of Optically Active - American Chemical Society

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