Primary Alcohols

Lipases usually show lower enantioselectivity toward primary alcohols than toward secondary alcohols. Only porcine pancreatic lipase (PPL) and PCL show high enantioselectivity toward a wide range of primary alcohols. An empirical rule can predict some of the enantiopreference of PCL toward pri-

Fig. 5. Three-dimensional active-site models for PCL proposed after overlaying energy-minimized structures of both good and bad substrates. (a) Lemke et al.'s model contains a spherical hydrophobic pocket and a tubelike hydrophobic pocket. (b) Grabuleda et al.'s model is slightly smaller. The reacting hydroxyl group goes in the HH site (hydrophilic), whereas the substituents go into the two hydrophobic sites, HS and HL. Both models predict the same absolute configuration. The drawing shows Grabuleda et al.'s model rotated by 180° about the vertical axis relative to Lemke et al.'s model.

Fig. 5. Three-dimensional active-site models for PCL proposed after overlaying energy-minimized structures of both good and bad substrates. (a) Lemke et al.'s model contains a spherical hydrophobic pocket and a tubelike hydrophobic pocket. (b) Grabuleda et al.'s model is slightly smaller. The reacting hydroxyl group goes in the HH site (hydrophilic), whereas the substituents go into the two hydrophobic sites, HS and HL. Both models predict the same absolute configuration. The drawing shows Grabuleda et al.'s model rotated by 180° about the vertical axis relative to Lemke et al.'s model.

Fig. 6. Proposed substrate binding site in three synthetically useful lipases. The catalytic Ser lies at the bottom of a crevice with the catalytic His on the left. Although the details of this crevice differ for each lipase, each crevice contains a large hydrophobic pocket (light gray) and smaller pocket (medium gray), labeled stereoselectivity pocket. This crevice is the alcohol binding site and the two pockets resemble the empirical rule in Fig. 1. The structure for CRL also shows the mouth of a tunnel that binds the acyl chain of an ester. Pictures were drawn with RasMac v2.6 (30) starting from the X-ray crystal structures for each lipase: CAL-B (31), CRL (32), and PCL (33).

Fig. 6. Proposed substrate binding site in three synthetically useful lipases. The catalytic Ser lies at the bottom of a crevice with the catalytic His on the left. Although the details of this crevice differ for each lipase, each crevice contains a large hydrophobic pocket (light gray) and smaller pocket (medium gray), labeled stereoselectivity pocket. This crevice is the alcohol binding site and the two pockets resemble the empirical rule in Fig. 1. The structure for CRL also shows the mouth of a tunnel that binds the acyl chain of an ester. Pictures were drawn with RasMac v2.6 (30) starting from the X-ray crystal structures for each lipase: CAL-B (31), CRL (32), and PCL (33).

mary alcohols (Fig. 8) (36). Like the foregoing secondary alcohol rule, the primary alcohol rule is based on the size of the substituents, but, surprisingly, the sense of enantiopreference is opposite; that is, the —OH of secondary alco-

Fig. 7. Substrate binding site in subtilisin Carlsberg. The labels and coloring show the amino acid residues of the catalytic triad and the residues forming the S1 binding site. This binding site is a shallow groove lined with nonpolar amino acid residues. The large hydrophobic substituent of secondary alcohols probably binds in this pocket. Note that the histidine of the catalytic triad lies to the right of the catalytic serine, whereas in lipases (Fig. 6), it lies to the left of the serine. This opposite chirality of the catalytic triad in part accounts for the opposite enantiopreference of lipases and subtilisins. Coordinates are from Brookhaven protein data bank file 1sbc (34) and the figure was created using RasMac v 2.6 (30).

Fig. 7. Substrate binding site in subtilisin Carlsberg. The labels and coloring show the amino acid residues of the catalytic triad and the residues forming the S1 binding site. This binding site is a shallow groove lined with nonpolar amino acid residues. The large hydrophobic substituent of secondary alcohols probably binds in this pocket. Note that the histidine of the catalytic triad lies to the right of the catalytic serine, whereas in lipases (Fig. 6), it lies to the left of the serine. This opposite chirality of the catalytic triad in part accounts for the opposite enantiopreference of lipases and subtilisins. Coordinates are from Brookhaven protein data bank file 1sbc (34) and the figure was created using RasMac v 2.6 (30).

Fig. 8. Empirical rule predicts the enantiopreference of PCL toward primary alcohols. (A) The empirical rule is based on the relative sizes of the substituents at the stereocenter. This rule is reliable only when there is no oxygen directly bonded at the stereocenter. Computer modeling (and the drawing) suggests that the large substituent, L, binds to different regions of the active site. Comparing this rule to the one in Fig. 1 shows that PCL favors opposite enantiomers in the case of primary and secondary alcohols. (B) PCL shows high enantioselectivity toward the primary alcohols shown. Reaction (hydrolysis of the diacetate in the first case, acylation of the diol in the second case) occurs at the arrow.

Fig. 8. Empirical rule predicts the enantiopreference of PCL toward primary alcohols. (A) The empirical rule is based on the relative sizes of the substituents at the stereocenter. This rule is reliable only when there is no oxygen directly bonded at the stereocenter. Computer modeling (and the drawing) suggests that the large substituent, L, binds to different regions of the active site. Comparing this rule to the one in Fig. 1 shows that PCL favors opposite enantiomers in the case of primary and secondary alcohols. (B) PCL shows high enantioselectivity toward the primary alcohols shown. Reaction (hydrolysis of the diacetate in the first case, acylation of the diol in the second case) occurs at the arrow.

hols and the —CH2OH of primary alcohols point in opposite directions in the two models. Computer modeling suggests that the large substituent of primary alcohols does not bind in the same pocket as secondary alcohols (37).

Not all primary alcohols fit this rule. In particular, primary alcohols that have an oxygen at the stereocenter (e.g., glycerol derivatives) do not fit this rule. An example of how the empirical rule does not apply to primary alcohols with an oxygen at the stereocenter is the y-butyrolactones in Fig. 9 (38). For the trans isomer, PCL favors one enantiomer, but for the cis isomer, PCL favors the other. Computer modeling on other substrates containing an oxygen at the stereocenter suggested that Tyr29 might form a hydrogen bond to this oxygen (37). The formation of this hydrogen bond and its influence on enantioselectivity likely depends on differences in structure more subtle than just size of the substituents. For those primary alcohols without an oxygen at the stereocenter, the rule in Fig. 8 showed 89% reliability (correct for 54 of 61 examples). For primary alcohols that did contain an oxygen at the stereocenter, the rule showed only 37% reliability (10 of 27 examples).

For secondary alcohols, increasing the difference in the size of the substitu-ents often increased the enantioselectivity of PCL and other lipases as discussed in Subheading 3.1. However, for primary alcohols, this strategy was not reliable. Upon adding large substituents, the enantioselectivity sometimes increased (39), sometimes decreased, and sometimes remained unchanged (36).

Researchers have had difficulties finding a reliable rule for PPL-catalyzed reactions of primary alcohols (40-43). Two proposed rules, based on different sets of substrates, even predict opposite enantiomers. An example of the difficulty is shown in Fig. 10. Enantiopreference of PPL reversed upon changing from a trans to a cis configuration of the double bond in the 2-substituted 1,3-propandiol derivatives (44). PPL favored the (S)-enantiomer with high enantioselectivity for the trans isomer, the (S)-enantiomer with moderate enantioselectivity for the saturated analog, but the (K)-enantiomer with low to moderate enantioselectivity for the cis isomer. This reversal is difficult to explain using only the relative sizes of the substituents. Note that for secondary alcohols, the enantioselectivity of PPL also varied with the configuration of double bonds in the large substituent, but the enantiopreference remained the same (45). A similar division of primary alcohols into two groups, those with and without oxygens at the stereocenter, may resolve dilemma of enantiomeric rules for PPL. The rule in Fig. 8 was reliable for primary alcohols without an oxygen at the stereocenter (27 out of 31 substrates, 87% reliability), but not for those with an oxygen at the stereocenter (3 out of 9, 33% reliability).

Naemura et al. (23) used a box-type model to predict the fast reacting enantiomer of primary alcohols in PFL (lipase YS)-catalyzed reactions (Fig. 11). None of the primary alcohols contained oxygen at the stereocenter. They used the same model to predict the fast reacting enantiomer for secondary alcohols. This model, unlike the computer modeling mentioned earlier, predicts that the large substituent of both primary and secondary alcohols binds in the same region of the lipase.

Fig. 9. Examples of primary alcohols with oxygen directly bonded to the stereocenter. The primary alcohol rule does not apply to these types of alcohols. In the hydrolysis of the corresponding acetates, the absolute configuration of the favored enantiomer reverses in the cis and trans diastereomers. Thus, a rule based only on the size of the substituents cannot predict which enantiomer reacts faster.

Fig. 10. The presence and configuration of a double-bond change and even reverse the enantioselectivity in PPL-catalyzed hydrolyses. The ent indicates that the enanti-omer of the structure drawn reacts faster.

Fig. 10. The presence and configuration of a double-bond change and even reverse the enantioselectivity in PPL-catalyzed hydrolyses. The ent indicates that the enanti-omer of the structure drawn reacts faster.

Tanaka et al. (46) proposed a box-type model with a single binding site for lipase from Rhizopus delemar (not shown). They used this model to rationalize RDL-catalyzed hydrolyses of meso-bis(acetoxymethyl)cyclopentanes (meso diacetates of primary alcohols).

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