PREPARATION OF TERTIARY ALCOHOLS, RESOLUTION OF TERTIARY ALCOHOLS AND STEREOSELECTIVE DEUTERATION OR TRITIATION BY RETROALDOLASES

Abstract

The present invention is directed to methods for catalyzing a chemical reaction by retroaldolases, corresponding uses of retroaldolases and to novel retroaldolases. The methods and retroaldolases have utility in (i) preparing tertiary alcohols, in (ii) chiral resolution of tertiary alcohols by retroaldol cleavage, and in (iii) deuteration or tritiation of carbonyl compounds.

Claims

1-15. (canceled)

16. A method for catalyzing a chemical reaction selected from the group consisting of: (i) preparing tertiary alcohols, optionally chiral tertiary alcohols, by an aldol reaction; (ii) chiral resolution of tertiary alcohols by retroaldol cleavage; and (iii) deuteration or tritiation of carbonyl compounds, comprising the steps of: (a) providing a retroaldolase selected from the group consisting of: (aa) a retroaldolase comprising an amino acid sequence selected from the group consisting of SEQ ID NOs: 3 to 28; (ab) a retroaldolase comprising an amino acid sequence having an amino acid sequence identity of at least 70% or 80%with an amino acid sequence selected from the group consisting of SEQ ID NOs: 3 to 28; (ac) a retroaldolase comprising a functional derivative and/or functional fragment of (aa) and/or (ab); and (ad) a retroaldolase according to any of (aa) to (ac), wherein in SEQ ID NOs: 3 to 28, position 50 is tyrosine, position 82 is lysine and position 109 is asparagine and/or position 179 is tyrosine or phenylalanine, for catalyzing the chemical reaction (i), (ii) or (iii), (b) providing at least one substrate for the chemical reaction (i), (ii) or (iii) selected from the group consisting of (ba) (baa) an aldehyde-comprising substrate and a ketone-comprising substrate, or (bab) two ketone-comprising substrates, which substrates react in the aldol reaction (i) to form a tertiary alcohol; (bb) tertiary alcohols for chiral resolution by retroaldol cleavage (ii); and (bc) carbonyl compounds, for deuteration or tritiation (iii); (c) contacting the retroaldolase of (a) with the substrate of (b) under conditions that allow enzymatic activity of the retroaldolase and the chemical reaction to proceed, and (d) optionally purifying the product of the chemical reaction.

17. The method according to claim 16, further comprising the step (e) of modifying the retroaldolase of (a), wherein step (e) is performed after step (a) and before step (c).

18. A method for modifying a retroaldolase for catalyzing a chemical reaction selected from the group consisting of: (i) preparing tertiary alcohols, optionally chiral tertiary alcohols, by an aldol reaction; (ii) chiral resolution of tertiary alcohols by retroaldol cleavage; and (iii) deuteration or tritiation of carbonyl compounds, comprising the steps of: (a) providing a retroaldolase selected from the group consisting of a. a retroaldolase comprising an amino acid sequence selected from the group consisting of SEQ ID NOs: 3 to 28; b. a retroaldolase comprising an amino acid sequence having an amino acid sequence identity of at least 70% or 80% with an amino acid sequence selected from the group consisting of SEQ ID NOs: 3 to 28; c. a retroaldolase comprising a functional derivative and/or functional fragment of a. and/or b.; and d. a retroaldolase according to any of a. to c., wherein in SEQ ID NOs: 3 to 28, position 50 is tyrosine, position 82 is lysine and position 109 is asparagine and/or position 179 is tyrosine or phenylalanine; (b) modifying at least one amino acid position in any one of the above retroaldolases a. to d.; (c) providing at least one substrate of interest for at least one of the above reactions (i) to (iii), and (d) contacting the at least one substrate of interest of (c) with at least one of the modified retroaldolases a. to d. under conditions that allow enzymatic activity of the retroaldolase and the reaction to proceed, and (e) identifying at least one modified retroaldolase that catalyzes at least one of the reactions (i) to (iii).

19. The method according to claim 18, wherein in step (b) of claim 18, the retroaldolase is modified in one or more of the following positions of SEQ ID NO: 3 to 28: in position 11 by glycine, phenylalanine or alanine; in position 111 by isoleucine, leucine or valine; in position 132 by phenylalanine; in position 183 by valine or tyrosine; and/or in position 209 by isoleucine or alanine.

20. The method according to claim 16, wherein the aldol reaction and/or the deuteration or tritiation reaction is a stereospecific reaction.

21. The method according to claim 16, wherein the retroaldolase catalyzes the reaction ##STR00011## wherein at least one of R.sup.1 or R.sup.2 is an electron withdrawing residue, and R.sup.1 and R.sup.2 are independently selected from the group consisting of (i) linear or branched, substituted or non-substituted (C.sub.2-20)alkyl ether, (C.sub.3-20)alkenyl ether, (C.sub.3-20)alkynyl ether and (C.sub.4-20)carbocyclic ether, wherein the ether is bonded to formula (I) via its carbon atom; (ii) linear or branched, substituted or non-substituted (C.sub.1-20)alkyl, (C.sub.2-20)alkenyl, (C.sub.2-20)alkynyl; (iii) substituted or non-substituted carbocycle selected from the group consisting of (C.sub.3-10)carbocycle a non-substituted phenyl and a para-substituted phenyl that is substituted by a substituent selected from the group consisting of Cl, F, Br, substituted or non-substituted methyl, (CF.sub.3), ethyl, propyl or cyclopropyl; (iv) substituted or non-substituted (C.sub.3-6)heterocycle and (C.sub.7-C10)carbo- or hetero-bicycle having 1 to 3 heteroatoms each independently selected from N, O and S,; and an electron withdrawing group;wherein R.sup.1 and/or R.sup.2 are bonded directly to formula (I), via O, or via a (H.sub.a).sub.b-linker, wherein a is an integer from 0 to 2 and b is an integer from 1 to 10; R.sup.3 and R.sup.4 are independently selected from the group consisting of (i) hydrogen, F, Cl, Br, R.sup.8, N(R.sup.8).sub.e, OR.sup.8, S(R.sup.8), P(R.sup.8).sub.f and C(R.sup.8).sub.d, wherein e is 1 or 2, f is an integer from 1 to 4, d is an integer from 1 to 3, and R.sup.8 is independently selected from the group consisting of (aa) hydrogen, F, Cl, Br, NO.sub.2, and oxo; (bb) linear or branched, substituted or non-substituted (C.sub.2-20)alkyl ether, (C.sub.3-20)alkenyl ether, (C.sub.3-20)alkynyl ether and (C.sub.4-20)carbocyclic ether; (cc) linear or branched, substituted or non-substituted (C.sub.1-20)alkyl, (C.sub.2-20)alkenyl, (C.sub.2-20)alkynyl; (dd) substituted or non-substituted carbocycle selected from the group consisting of (C.sub.3-10)carbocycle; and (ee) substituted or non-substituted (C.sub.3-6)heterocycle and (C.sub.7-10)carbo- or hetero-bicycle having 1 to 3 heteroatoms each independently selected from N, O and S,; and (ii) an electron withdrawing group wherein the electron withdrawing group is bonded directly to formula (II), via O, or via a (CH.sub.a).sub.b-linker, wherein a is an integer from 0 to 2 and b is an integer from 1 to 10;; R.sup.5 is hydrogen or C(R.sup.8).sub.d, wherein d is an integer from 1 to 3, and R.sup.8 is as defined above.

22. A retroaldolase selected from the group consisting of (a) a retroaldolase comprising an amino acid sequence having an amino acid sequence identity of at least 70% or 80 with SEQ ID NO: 3, wherein the retroaldolase is modified in position 111 of SEQ ID NO:3 by isoleucine, leucine or valine and in position 132 of SEQ ID NO: 3 by phenylalanine; with the proviso that the retroaldolase does not comprise one of sequences SEQ ID NO:1 and SEQ ID NO: 2; (b) a retroaldolase comprising functional fragments and/or functional derivatives of (a); and (c) a retroaldolase according to (a) or (b), wherein in SEQ ID NO: 3 or 4, position 50 is tyrosine, position 82 is lysine and position 109 is asparagine and/or position 179 is tyrosine or phenylalanine, wherein the retroaldolase of (a), (b) and (c) catalyzes the preparation of tertiary alcohols, optionally chiral tertiary alcohols, by an aldol reaction.

23. The retroaldolase according to claim 22, wherein the retroaldolase also catalyzes a reaction selected from the group consisting of (i) chiral resolution of tertiary alcohols by retroaldol cleavage; and (ii) deuteration or tritiation of carbonyl compounds,.

24. The retroaldolase according to claim 22, wherein the retroaldolase is modified in one or more of positions 11, 183 and 209 of SEQ ID NO: 3.

25. The retroaldolase according to claim 22, wherein the retroaldolase is selected from the group consisting of (a) a retroaldolase comprising an amino acid sequence according to SEQ ID NOs: 5 to 28; (b) a retroaldolase comprising an amino acid sequence having an amino acid sequence identity of at least 70% or 80% with SEQ ID NOs: 5 to 28, with the proviso that the retroaldolase does not comprise one of sequences SEQ ID NO:1 and SEQ ID NO: 2; and (c) a retroaldolase comprising functional fragments and/or functional derivatives of any of (a) and/or (b).

26. The retroaldolase according to claim 22, wherein the retroaldolase catalyzes the production of the tertiary alcohols or the deuterated or tritiated carbonyl compounds stereospecifically.

27. The retroaldolase according to claim 22, wherein the retroaldolase catalyzes the reaction ##STR00012## wherein at least one of R.sup.1 or R.sup.2 is an electron withdrawing residue, and R.sup.1 and R.sup.2 are independently selected from the group consisting of (i) linear or branched, substituted or non-substituted (C.sub.2-20)alkyl ether, (C.sub.3-20)alkenyl ether, (C.sub.3-20)alkynyl ether and (C.sub.4-20)carbocyclic ether, wherein the ether is bonded to formula (I) via its carbon atom; (ii) linear or branched, substituted or non-substituted (C.sub.1-20)alkyl, (C.sub.2-20)alkenyl, (C.sub.2-20)alkynyl,; (iii) substituted or non-substituted carbocycle selected from the group consisting of (C.sub.3-10)carbocycle,; (iv) substituted or non-substituted (C.sub.3-6)heterocycle and (C.sub.7-C10)carbo- or hetero-bicycle having 1 to 3 heteroatoms each independently selected from N, O and S; an electron withdrawing group, wherein R.sup.1 and/or R.sup.2 are bonded directly to formula (I), via O, or via a (CH.sub.a).sub.b-linker, wherein a is an integer from 0 to 2 and b is an integer from 1 to 10;; R.sup.3 and R.sup.4 are independently selected from the group consisting of (i) hydrogen, F, Cl, Br, R.sup.8, N(R.sup.8).sub.e, OR.sup.8, S(R.sup.8), P(R.sup.8).sub.f and C(R.sup.8).sub.d, wherein e is 1 or 2, f is an integer from 1 to 4, d is an integer from 1 to 3, and R.sup.8 is independently selected from the group consisting of (aa) hydrogen, F, Cl, Br, NO.sub.2, and oxo; (bb) linear or branched, substituted or non-substituted (C.sub.2-20)alkyl ether, (C.sub.3-20)alkenyl ether, (C.sub.3-20)alkynyl ether and (C.sub.4-10)carbocyclic ether; (cc) linear or branched, substituted or non-substituted (C.sub.1-20)alkyl, (C.sub.2-20)alkenyl, (C.sub.2-20)alkynyl; (dd) substituted or non-substituted carbocycle selected from the group consisting of (C.sub.3-10)carbocycle; and (ee) substituted or non-substituted (C.sub.3-6)heterocycle and (C.sub.7-C10)carbo- or hetero-bicycle having 1 to 3 heteroatoms each independently selected from N, O and S; and (ii) an electron withdrawing group; wherein the electron withdrawing group is bonded directly to formula (II), via O, or via a (CH.sub.a).sub.b-linker, wherein a is an integer from 0 to 2 and b is an integer from 1 to 10; R.sup.5 is hydrogen or C(R.sup.8).sub.d, wherein d is an integer from 1 to 3, and R.sup.8 is as defined above.

28. The retroaldolase according to claim 22, wherein the retroaldolase catalyzes the preparation of the tertiary alcohols without the step of decarboxylation and/or the preparation of cyanides.

29. The method according to claim 16, wherein the reaction of deuteration or tritiation of carbonyl compounds is at the a-position of the carbonyl group of the carbonyl compounds.

30. The method according to claim 18, wherein the reaction of deuteration or tritiation of carbonyl compounds is at the a-position of the carbonyl group of the carbonyl compounds.

31. The method according to claim 16, wherein the reaction of deuteration or tritiation of carbonyl compounds comprises the regio- and/or stereoselective deuteration or tritiation of carbonyl compounds.

32. The method according to claim 18, wherein the reaction of deuteration or tritiation of carbonyl compounds comprises the regio- and/or stereoselective deuteration or tritiation of carbonyl compounds,

33. The method according to claim 16, with the proviso that the carbonyl compounds in the deuteration or tritiation reaction are not acetone.

34. The method according to claim 18, with the proviso that the carbonyl compounds in the deuteration or tritiation reaction are not acetone.

35. The method according to claim 16, wherein the retroaldolase is selected from the group of SEQ ID NO: 30, SEQ ID NO: 34, a functional derivative or functional fragment thereof.

36. The method according to claim 16, wherein in (baa) the aldehyde-comprising substrate is a nucleophilic aldehyde-comprising substrate, and the ketone-comprising substrate is an electrophilic ketone-comprising substrate.

37. The method according to claim 18, wherein the retroaldolase is modified in at least one of positions 11, 111, 132, 183 and 209 of SEQ ID NO: 3 to 28.

38. The method according to claim 17, wherein the retroaldolase is modified in at least one of positions 11, 111, 132, 183 and 209 of SEQ ID NO: 3 to 28.

39. The method according to claim 38, wherein the retroaldolase is modified in one or more of the following positions of SEQ ID NO: 3 to 28: in position 11 by glycine, phenylalanine or alanine; in position 111 by isoleucine, leucine or valine; in position 132 by phenylalanine; in position 183 by valine or tyrosine; and/or in position 209 by isoleucine or alanine.

40. The method according to claim 18, wherein the retroaldolase of step 3(a)(b) has at least 90% sequence identity to an amino acid sequence selected from SEQ ID NO. 3 to 28.

41. The method according to claim 16, wherein the retroaldolase of step 1(ab) has at least 90% sequence identity to an amino acid sequence selected from SEQ ID NO. 3 to 28.

42. The method accordingly to claim 21, wherein R.sup.1, R.sup.2, R.sup.3, and R.sup.4 are independently selected from the group consisting of substituted or non-substituted methyl, ethyl, propyl, (C.sub.3)carbocycle or (C.sub.5-6)carbocycle.

43. The method according to claim 42, wherein R.sup.1, R.sup.2, R.sup.3, and R.sup.4 are independently selected from the group consisting of a non-substituted or a para-substituted phenyl that is substituted by a substituent selected from the group consisting of Cl, F, Br, substituted or non-substituted methyl, (CF.sub.3), ethyl, propyl and cyclopropyl.

44. The method accordingly to claim 21, wherein R.sup.1, R.sup.2 and the electron withdrawing group are independently selected from the group consisting of COOH, COOMe, COOEt, CF.sub.3, CHF.sub.2 or CCl.sub.3.

45. The method according to claim 21, wherein the electron withdrawing group is selected from the group consisting of COOR.sup.6, CR.sup.7.sub.d, S(O).sub.2OH, CONR.sup.1R.sup.2, wherein (aa) R.sup.6 is selected from the group consisting of hydrogen, R.sup.1, substituted or non-substituted methyl, ethyl and propyl; (bb) R.sup.7 is selected from the group consisting of hydrogen and halogens, wherein at least one of R.sup.7 is a halogen and the remaining residues are hydrogen, and wherein d is an integer from 1 to 3;

46. The method according to claim 45, wherein R.sup.7 is selected from F, Cl and Br.

47. The method according to claim 21, wherein the tertiary alcohol (III) is a chiral tertiary alcohol and the stereogenic carbon atoms (2) and (3) of tertiary alcohol (III) are (R,R)-, (S,R)-, (R,S)- or (S,S)-configured.

48. The retroaldolase according to claim 22, wherein the retroaldolase has at least 90% sequence identity with SEQ ID NO. 3.

49. The retroaldolase according to claim 22, wherein the reaction of deuteration or tritiation of carbonyl compounds is at the -position of the carbonyl group of the carbonyl compounds.

50. The retroaldolase according to claim 22, wherein the reaction of deuteration or tritiation of carbonyl compounds comprises the regio- and/or stereoselective deuteration or tritiation of carbonyl compounds.

51. The retroaldolase according to claim 22, with the proviso that the carbonyl compounds in the deuteration or tritiation reaction are not acetone.

52. The retroaldolase according to claim 24, wherein the retroaldolase is modified in position 11 by glycine, phenylalanine or alanine; in position 183 by valine or tyrosine; in position 209 by isoleucine or alanine; and combinations thereof.

53. The retroaldolase according to claim 25, wherein the retroaldolase comprises an amino acid sequence having an amino acid sequence identity of at least 90% with SEQ ID NOs: 5 to 28.

54. The retroaldolase according to claim 27, wherein R.sup.1 , R.sup.2, R.sup.3, and R.sup.4 are independently selected from the group consisting of substituted or non-substituted methyl, ethyl, propyl, (C.sub.3)carbocycle or (C.sub.5-6)carbocycle.

55. The retroaldolase according to claim 27, wherein R.sup.1, R.sup.2, R.sup.3, and R.sup.4 are independently selected from the group consisting of a non-substituted or a para-substituted phenyl that is substituted by a substituent selected from the group consisting of Cl, F, Br, substituted or non-substituted methyl, (CF.sub.3), ethyl, propyl and cyclopropyl.

56. The retroaldolase according to claim 27, wherein R.sup.1, R.sup.2 and the electron withdrawing group are independently selected from the group consisting of COOH, COOMe, COOEt, CF.sub.3, CHF.sub.2 or CCl.sub.3.

57. The retroaldolase according to claim 27, wherein the electron withdrawing group is selected from the group consisting of COOR.sup.6, CR.sup.7.sub.d, S(O).sub.2OH, CONR.sup.1R.sup.2, wherein (aa) R.sup.6 is selected from the group consisting of hydrogen, R.sup.1, substituted or non-substituted methyl, ethyl and propyl; (bb) R.sup.7 is selected from the group consisting of hydrogen and halogens, wherein at least one of R.sup.7 is a halogen and the remaining residues are hydrogen, and wherein d is an integer from 1 to 3;

58. The retroaldolase according to claim 57, wherein R.sup.7 is selected from F, Cl and Br.

59. The retroaldolase according to claim 27, wherein the tertiary alcohol (III) is a chiral tertiary alcohol and the stereogenic carbon atoms (2) and (3) of tertiary alcohol (III) are (R,R)-, (S,R)-, (R,S)- or (S,S)-configured.

Description

[0118] The following Figures and Examples serve to illustrate the invention and are not intended to limit the scope of the invention as described in the appended claims.

[0119] FIG. 1: shows a HPLC chromatogram for the analysis of the product of Example 1 (ethyl (S)-2-hydroxy-4-oxo-2-phenethylpentanoate) compared to the racemic aldol reaction product under the following conditions: Chiralcel OD-H, n-hexane/i-PrOH=9:1, flow rate 0.6 mL/ min, =210 nm.

[0120] FIG. 2: shows a HPLC chromatogram for the analysis of the resolution product of Example 3 under the following conditions: Chiralcel OD-H, n-hexane/i-PrOH=9:1, flow rate 0.6 mL/min, =230 nm.

[0121] FIG. 3: shows the 1H-NMR comparison of the reaction products of Example 4.

[0122] FIG. 4A-B: is a schematic representation of the active site of RA95.5-8F (SEQ ID NO: 1). Lysine 83 is covalently bonded to the substrate analog 1-(6-methoxynaphthalen-2-yl)butane-1,3-dione, shown in dark grey. FIG. 4A highlights residues important for catalysis, and FIG. 4B shows residues important for stereoselectivity and substrate recognition (the figure was created using the program Pymol (Schrdinger, Cambridge, Mass., USA) and the PDB access code 5AN7).

Example 1: Synthesis of (S)-2-hydroxy-4-oxo-2-phenethylpentanoate

[0123] ##STR00003##

[0124] In a 100 mL Erlenmeyer flask, an acetone solution (2 M final concentration) containing ethyl 2-oxo-4-phenylbutanoate (0.12 mmol, 24.75 mg) was mixed with buffer solution (25 mM HEPES, 100 mM NaCl, pH 7.5) containing RA95.5-8F (SEQ ID NO: 1, final concentration 1 M) and the reaction mixture was incubated at 29 C. 600 rpm in an orbital shaker. Reaction progress was monitored by HPLC, and conversion quantified using solutions of the purified aldol adduct as external standard. After 24 h reaction time, the reaction mixture was saturated with NaCl, extracted with EtOAc, and the organic phase dried over sodium sulfate. Solvent was removed under vacuum. The crude material was purified by flash chromatography (EtAcO/Hexane 1:2) to obtain ethyl (S)-2-hydroxy-4-oxo-2-phenethylpentanoate (21.87 mg, 70.1% isolated yield) as a colorless oil. The stereochemistry of the product (R/S=0.4:99.6) was established by chiral HPLC by comparison with published data (Tetrahedron Lett. 2010, 51, 1884-1886): Chiralcel OD-H, n-hexane/i-PrOH=9:1, flow rate 0.6 mL/min, =210 nm.

Example 2: Stereoselective Aldol Reactions

[0125] The following reactions were performed with the catalysts listed in Table 1 below.

##STR00004##

##STR00005##

Reaction 1

[0126] Reaction conditions: 2.0 M acetone, 5.0 mM ethyl 2-(4-nitrophenyl)-2-oxoacetate; analysis by chiral HPLC analysis: Chiralcel OD-H column, =210 nm, i-PrOH:Hexane=10:90, 0.6 mL/min

Reaction 2

[0127] Reaction conditions: 2.0 M acetone, 5.0 mM 2,2,2-trifluoro-1-phenylethan-1-one; analysis by chiral HPLC analysis: Chiralcel OD-H column, =210 nm, i-PrOH:Hexane=3:97, 1.0 mL/min

TABLE-US-00001 TABLE 1 Catalysts used in reactions 1 and 2 above Reaction 1 Reaction 2 Selectivity Selectivity Catalyst (R/S) (R/S) RA95.5-8F (SEQ ID NO: 1) 51.8:48.2 68.9:31.1 RA95.5-8F F112I (SEQ ID NO: 30) 85.4:4.6 63.2:36.8 RA95.5-8F F112L (SEQ ID NO: 31) 79.5:20.5 73.1:26.9 RA95.5-8F F112V (SEQ ID NO: 32) 85.3:14.7 67.0:31.0 RA95.5-8F F184V (SEQ ID NO: 33) 37.4:62.6 40.9:59.1 RA95.5-8F L210A (SEQ ID NO: 53) 60.9:39.1 75.9:24.1 RA95.5-8F L210I (SEQ ID NO: 52) 56.9:43.1 62.0:38.0 RA95.5-8F I133F (SEQ ID NO: 34) 72.4:27:6 68.8:31.2 RA95.5-8F V12G F112I 88.8:11.2 63.5:36:6 (SEQ ID NO: 35) RA95.5-8F V12F F112I 52.8:47.2 70.3:29.7 (SEQ ID NO: 36) RA95.5-8F V12I F112L 53.5:46.5 81.0:19.0 (SEQ ID NO: 37) RA95.5-8F V12A F112V 90.5:9.5 69.5:30.5 (SEQ ID NO: 38)

Example 3: Resolution of Tertiary Chiral Alcohols

[0128] ##STR00006##

[0129] In a 2.0 mL centrifuge tube, an acetonitrile solution (27 L) containing rac-ethyl 2-hydroxy-2-(6-methoxynaphthalen-2-yl)-4-oxopentanoate (95 g, final concentration 300 M) was mixed with buffer solution (973 L; 25 mM HEPES, 100 mM NaCl, pH 7.5) containing F112I/L210A RA95.5-8F (SEQ ID NO: 50, final concentration 3 M) and the reaction mixture was incubated at 29 C. Aliquots taken at 0 and 48 h reaction time were analyzed by chiral HPLC: Chiralcel OD-H, n-hexane/i-PrOH=9:1, flow rate 0.6 mL/min, =230 nm. Selective cleavage of one of the two enantiomers was observed, with a final enantiomeric ratio of 2:98.

Example 4: Deuteration of Cyclohexanone

[0130] Buffered solutions (989.6 L; HEPES 25 mM, NaCl 100 mM, pH=7.5) containing no catalyst (A), RA95.5-8F (SEQ ID NO: 1, 2 M; B), and F112I RA95.5-8F (SEQ ID NO: 30, 2 M; C) were dried by lyophilization in 2.0 mL centrifuge tubes and the resulting solid was subsequently resuspendded in D.sub.2O (989.6 L; 99.90% purity). Cyclohexanone (10.4 L, 100 mM final conc.) was added and the reactions were incubated at 29 C. After 2 h reaction time, the reactions were extracted with deuterated chloroform (1.0 mL). The organic phases were dried with Na.sub.2SO.sub.4, centrifuged, and analyzed by .sup.1H-NMR. Significant deuteration is not detected in absence of catalyst or using RA95.5-8F. In contrast, in presence of F112I RA95.5-8F (SEQ ID NO: 30), single deuteration of carbon-2 and carbon-6 of cyclohexanone (i.e. the positions in alpha of the carbonyl function) is observed, thus yielding cyclohexan-1-one-2,6-d.sub.2.

Example 5: Protein Expression

[0131] RA95.5-8 (SEQ ID NO: 2), RA95.5-8F (SEQ ID NO: 1) and its variants (SEQ ID NOs: 30 - 53) were subcloned into the commercial pET-29b(+) vector (Novagen). Individual variants were produced as C-terminally His-tagged proteins in E. coli BL21-Gold(DE3) and purified by affinity chromatography. To ensure monoclonality, single-colony streakouts of the most active clones were prepared from the library master plates. Single colonies were used to prepare precultures, of which 0.5 mL were inoculated in 500 mL of LB medium containing 30 g mL.sup.1 kanamycin sulfate. The bacterial cultures were incubated at 37 C. and 220 rpm until a D.sub.600 nm of 0.4 was reached. Following induction of enzyme production with 0.1 mM IPTG, the cells were incubated for an additional 5 h at 37 C., at which point they were harvested. Cell pellets were stored at 20 C. before lysis. Upon thawing, the pellets were resuspended in 25 mM HEPES, 300 mM NaCl, pH 7.5, containing 1 mg mL.sup.1 egg white lysozyme and incubated for 1 h at 4 C. The cells were then lysed by sonication, and cell debris was removed by centrifugation. Ni-NTA beads (Qiagen, Venlo, Netherlands) were equilibrated with 25 mM HEPES, 300 mM NaCl, pH 7.5, and the soluble protein fraction was loaded onto the column. The samples were washed once with the same buffer without imidazole and subsequently with buffer containing 20 mM and 40 mM imidazole. The protein was finally eluted using 200 mM imidazole. The sample was dialyzed at 4 C. against 25 mM HEPES, 100 mM NaCl, pH 7.5. Protein concentration was determined from the absorbance at 280 nm using calculated extinction coefficients (Gasteiger E., et al., The Proteomics Protocols Handbook, Humana Press (2005), pp. 571-607).

Example 6: Expression of Aldolase Libraries

[0132] Plasmids were transformed in BL21-Gold (DE3) cells, plated on Petri plates (LB media, 30 mg.Math.L.sup.1 kanamycin) and incubated overnight at 37 C. Ninety six-well micro titer plates, containing 150 L of LB media with 30 mg.Math.L.sup.1 kanamycin per well, were inoculated with single colonies using sterile tooth picks. Two wells were inoculated with clean toothpicks as blank controls, and four wells were inoculated with a single colony of RA95.5-8F (SEQ ID NO: 1) as a reference of 100% of activity. The plates were covered with air permeable membranes (Breathe Easy, Diversified Biotech) and incubated overnight at 30 C. and 900 rpm Pre-warmed (37 C.) 96 deep-well plates (Deepwell plate 96/2000 L, Eppendorf) containing 1.5 mL LB-medium (30 mg.Math.L.sup.1 kanamycin) per well were inoculated with the pre-culture (24l per well), covered with an air permeable membrane (Breathe Easy, Diversified Biotech), and incubated at 37 C. and 600 rpm After 135 min, protein expression was induced with an IPTG solution (30 m, final concentration 0.1 mM), and the plates were incubated for additional 5 h at 37 C. and 600 rpm The cells were harvested by centrifugation (4000 rpm, 4 C., 15 min) and the supernatant was completely discarded. The pellets were suspended in 400 L of assay buffer (25 mM HEPES 100 mM NaCl, pH 7.5) supplemented with 1 mg.Math.mL.sup.1 lysozyme from chicken egg. The plate was incubated (600 rpm, room temperature) for 1 h and stored overnight at 20 C. The plates were thawed and incubated (600 rpm, room temperature) for 1 h and the suspensions were cleared by centrifugation (4000 rpm, 20 C., 20 min).

Example 7: Spectroscopic Analysis of Aldolase Libraries

[0133] For the assay, 20 L of the cleared lysates were transferred to a 96-well microtiter plate containing in each well 174 L of assay buffer (25 mM HEPES 100 mM NaCl, pH 7.5) and 6 L of an acetonitrile solution of enantiopure or racemic ethyl 2-hydroxy-2-(6-methoxynaphthalen-2-yl)-4-oxopentanoate (0.03 mol, 9.5 g, final concentration 150 M), or an alternative aldol adduct resulting of the addition of a nucleophile ketone of interest to ethyl 2-(6-methoxynaph-thalen-2-yl)-2-oxoacetate. Enzymatic activity was measured in a UV plate reader (Thermofisher Scientific Varioscan) monitoring the absorbance decrease at 350 nm.

Example 8: Chromatographic Analysis of Aldolase Libraries

[0134] For the assay, 20-100 L of the cleared lysates were transferred to a 96-well polypropylene multi-well plate (Deepwell plate 96/20004, Eppendorf) containing in each well 180-100 L of a buffered solution (25 mM HEPES 100 mM NaCl, pH 7.5) with the nucleophilic ketone (5-3000 mM final concentration) and the electrophilic ketone (1-50 mM). The plate was incubated for 48 h at 20-29 C. Conversion was determined by HPLC analysis of reaction samples (20 L) diluted in acetonitrile (180 L). The crude reactions of active variants (i.e. yielding >30% conversion) were extracted by addition of 300 L methyl tert-butyl ether and vigorous shaking for 2 min. After centrifugation (3 min, 12 C., 2500 r.c.f.) a fraction of the organic phase (200 L) was transferred to a fresh multi-well plate (MicroWell, Nunc). The solvent was evaporated under a flow of air subsequently under reduced pressure (1-2 mbar) for 10 min. The crude products were resuspended in 150 L of heptane/isopropanol mixture of variable composition. The solutions were transferred to a 96-well filter plate (0.2 m pore-size PTFE membranes, AcroPrep, Pall Corporation) and centrifuged (2 min, 12 C., 2500 r.c.f.) into 96-well polypropylene plates (MicroWell, Nunc). The filtered solutions were transferred into glass vials and 30 L samples were injected and analyzed by chiral HPLC.

Example 9: Analysis of Purified Variants of RA95.5-8F (SEQ ID NO: 1)

[0135] Spectroscopic analysis: Reactions were carried out at 29 C. in aqueous buffer (25 mM HEPES, 100 mM NaCl, pH 7.5) in 1 mL sealed quartz cuvettes using RA95.5-8F (SEQ ID NO: 1) or its variants (SEQ ID NOs: 30-53) as catalysts. Acetonitrile at a final concentration of 2.7% was included as co-solvent to facilitate substrate solubility. The retro-aldol cleavage of rac-ethyl 2-hydroxy-2-(6-methoxynaphthalen-2-yl)-4-oxopentanoate to give ethyl 2-(6-methoxynaphthalen-2-yl)-2-oxoacetate and acetone was monitored spectroscopically at 350 nm (=8641 M.sup.1 cm.sup.1) using a Perkin Elmer Lambda 35 UV-vis spectrometer equipped with a Peltier system for temperature control. The data were corrected for the buffer-catalyzed background reaction measured under the same conditions. Steady-state kinetic parameters were derived by fitting the experimental data to the Michaelis-Menten equation: v.sub.0/[E]=k.sub.cat.Math.[S]/(K.sub.M+[S]), where v.sub.0 is the initial rate, [E] is the enzyme concentration, K.sub.M is the Michaelis constant, and [S] is the substrate concentration.

[0136] Chromatographic analysis: Reactions were conducted in 1.5 mL centrifuge tubes incubated in a water bath thermostated at 29 C. The electrophilic ketone (1-50 mM final concentration) and the nucleophilic ketone (5-3000 mM final concentration) were mixed, and the enzyme solution (0.05-1.0 nmol, 0.1-10 M final concentration) in buffer (sufficient amount for 500 uL total volume, 25 mM HEPES 100 mM NaCl, pH=7.5) was added. Conversions were determined at 3 h and 24 h reaction time. Reaction monitoring was as follows: aliquots (20 L) were withdrawn, diluted with acetonitrile (120 L) and analyzed by HPLC. The reaction crudes were subsequently extracted by addition of 600 L methyl tert-butyl ether and vigorous shaking for 2 min. After centrifugation (3 min, 12 C., 2500 r.c.f.) a fraction of the organic phase (400 L) was transferred to a fresh multi-well plate (MicroWell, Nunc). The solvent was evaporated under a flow of air subsequently under reduced pressure (1-2 mbar) for 10 min. The crude products were resuspended in 150 L of heptane/isopropanol mixture of variable composition. The solutions were transferred to a 96-well filter plate (0.2 m pore-size PTFE membranes, AcroPrep, Pall Corporation) and centrifuged (2 min, 12 C., 2500 r.c.f.) into 96-well polypropylene plates (Micro-Well, Nunc). The filtered solutions were transferred into glass vials and 30 L samples were injected and analyzed by chiral HPLC.

Activities

[0137] Activities (k.sub.cat/K.sub.M) of RA95.5-8F and variants were determined as described above (standard errors <10%).

TABLE-US-00002 RA95.5-8F: 60 M.sup.1s.sup.1 (100%); RA95.5-8F F112L: 210 M.sup.1s.sup.1 (350%); RA95.5-8F F112I: 310 M.sup.1s.sup.1 (520%) RA95.5-8F F112V: 170 M.sup.1s.sup.1 (290%).

Example 10: Synthesis of Ethyl 2-hydroxy-2-(2-Oxopropyl)Hexanoate

[0138] ##STR00007##

In a 100 mL Erlenmeyer flask, an acetone solution (2 M final concentration) containing ethyl 2-oxohexanoate (0.30 mmol, 47.46 mg) was mixed with buffer solution (25 mM HEPES, 100 mM NaCl, pH 7.5) containing RA95.5-8F (SEQ ID NO: 1, final concentration 2 M) and the reaction mixture was incubated at 29 C., 600 rpm in an orbital shaker. Reaction progress was monitored by HPLC, and conversion quantified using solutions of the purified aldol adduct as external standard. After 24 h reaction time, the reaction mixture was saturated with NaCl, extracted with EtOAc, and the organic phase dried over sodium sulfate. Solvent was removed under vacuum. The crude material was purified by flash chromatography (EtAcO/Hexane 1:3) to obtain ethyl 2-hydroxy-2-(2-oxopropyl) hexanoate (23.49 mg, 36.2% isolated yield) as a colorless oil.

Example 11: Substrate Diversity

[0139] The following reactions were performed with the catalysts listed in Table 2 below.

##STR00008##

##STR00009##

##STR00010##

Product formation was determined by LC-MS analysis [integration of peak corresponding to (M+Na).sup.+].

Reaction 1

[0140] Reaction conditions: 100 mM 2-pentanone, 5.0 mM ethyl 2-(4-nitrophenyl)-2-oxoacetate, 20% DMSO, analysis by LC-MS (Acquity UPLC System, Waters). Product formation determined by LC-MS analysis [integration of peak corresponding to (M+Na).sup.+].

Reaction 2

[0141] Reaction conditions: 2.0 M acetone, 5.0 mM 2,2-difluoro-1-phenylethan-1-one, analysis by LC-MS (Acquity UPLC System, Waters). Product formation determined by LC-MS analysis [integration of peak corresponding to (M+Na).sup.+].

Reaction 3

[0142] Reaction conditions: 100 mM 2-pentanone, 5.0 mM ethyl 2-oxo-4-phenylbutanoate, 20% DMSO, analysis by LC-MS (Acquity UPLC System, Waters). Product formation determined by LC-MS analysis [integration of peak corresponding to (M+Na).sup.+] and spectroscopic analysis.

TABLE-US-00003 TABLE 2 Reaction 1 Reaction 2 Reaction 3 Relative Relative Relative reaction reaction reaction Catalyst rate (%) rate (%) rate (%) RA95.5-8F (SEQ ID NO: 1) 47 51 9 RA95.5-8F I133F 75 26 100 (SEQ ID NO: 34) RA95.5-8F F112I 100 100 14 (SEQ ID NO: 30) RA95.5-8F F112L 72 46 18 (SEQ ID NO: 31) RA95.5-8F F112V 79 77 11 (SEQ ID NO: 32)