This chapter covered the characterization of the solvent-free aza-Michael addition of benzylamine (1) and trans-ethyl crotonate (2) towards rac-3 as the first step in the chemo-enzymatic synthesis of ethyl-(S)-β-amino butyrate. The following observations have been made:
• The reaction pathway was proven to proceed solely via aza-Michael addition in a first step and an aminolytic side reaction as a second, subsequent step.
• The reactions can be considered irreversible within limits of detection.
• The aza-Michael addition follows simple second-order kinetics. Kinetic parameters for both aza-Michael addition and aminolysis were determined in the temperature range from 40-70 ◦C.
• Activation energies ofEA,1 = 40.4 kJ mol−1 andEA,2 = 54.3 kJ mol−1 were deter-mined for both steps and allow the simulation of reactions carried out at temper-atures not investigated experimentally.
• At a starting molar ratio of 2:1 of substrates1and2, a maximum space-time yield of 1.06 kg L−1 d−1 was achieved at the highest temperature tested in batch mode of 70◦C. Lower temperatures only slightly improve selectivity while the necessary reaction time to obtain maximum yields is considerably elongated.
4 Biocatalytic aminolysis 1
Candida antarctica lipase B (CALB) immobilized on acrylic resin, which is commercially available as Novozym 435 from Novozymes, Denmark, catalyzes the kinetic resolution of rac-3with benzylamine (1) according to Figure 4.1. The reaction can be carried out under solvent-free conditions at elevated temperatures.
NH
Figure 4.1: Novozym 435-catalyzed aminolysis for the kinetic resolution ofrac-3.
The enantioselectivity of an enzyme-catalyzed reaction is mainly determined by the structure of the active site and the interactions of particular active site amino acids with the substrate. Improvement of the enantioselectivity of a biocatalyst can effectively be achieved using random mutagenesis strategies, rational design as well as combinations thereof [9]. In addition, several physicochemical parameters commonly influence the enantioselectivity of enzyme-catalyzed kinetic resolutions, in particular solvent, temper-ature and pressure [18]. These parameters are addressed in this chapter and in chapter 7. In literature, a large number of enzyme-catalyzed kinetic resolutions are described with a majority being related to the hydrolase-catalyzed resolution of prochiral alcohols [33]. The separation of racemic carboxylic acids or esters can also be readily achieved via esterification or transesterification using the same class of enzymes. Kinetic resolu-tions of prochiral amines or esters with amines as nucleophiles have been realized, even though less frequently. The mechanism generally proceeds via the well-known catalytic triad mechanism of lipases, which is depicted in Figure 4.2 on the next page.2
The nucleophile attacking the acyl-intermediate in step I can be either an amine or an alcohol. Aminolysis reactions usually proceed more slowly compared to interesterifica-tions with alcohols, while at the same time enantioselectivities are generally improved.
The lower reaction rates are probably due to the higher pka value of amines causing a decreased hydrogen transfer rate from the neutral amine to the histidine residue in step I of Figure 4.2. The better selectivity can be explained by an increased stability of the produced amide [26].
1Parts of this chapter have been published in [123]
2Abbreviated form, the acyl-intermediate of step I is itself formed via an activated intermediate.
4 Biocatalytic aminolysis
Figure 4.2: Mechanism of ester cleavage via acyl-enzyme intermediate. The nucleophile can either be an amine or an alcohol. Figure according to [26].
With regard to the economic feasibility of a process, factors such as the selectivity of the biocatalytic reaction and enzyme kinetics play a crucial role. Both these criteria and enzyme stability largely depend on the reaction system applied. In solvent-free reactions as investigated here, high space-time yields can generally be achieved and thus carry an inherent advantage compared to solvent-systems. This chapter is devoted to the characterization of such a solvent-free system for the production of an industrially interestingβ-amino acid precursor via kinetic resolution of a racemicβ-amino acid ester.
4.1 Selectivity of Novozym 435
The Novozym 435-catalyzed, solvent-free kinetic resolution ofrac-3with benzylamine as depicted in Figure 4.1 has been reported to proceed enantioselectively with an E-value of 27 at 60◦C by Weiß and Gr¨oger [138, 140]. TheE-value was calculated according to Equations 1.1 and 1.2 on page 3 from X/eep and ees/eep-values at a single conversion point, respectively.
The enantioselectivity of the same Novozym 435-catalyzed reaction is shown in Figure 4.3 as a conversion vs. ee-plot. The reaction was carried out with an initial molar fraction of χrac−3 = 0.5 at 60 ◦C. Approximately 60 % conversion are needed to obtain an attractive ee(S)−3 of > 99% in the solvent-free reaction. Thus, (S)-3 is also slowly converted to the corresponding amide (S)-4. Equation 4.1 can be used to determine the value ofE by nonlinear regression according to the procedure suggested by Rakels et al.
[102].
eep = ees [(1+ee(1−ees)E
s) ]1/(E−1)−1 (4.1)
An E-value of 32 was determined using this method. Among other phenomena, diffu-sional limitations and side reactions can interfere in the accurate determination of E
4.1 Selectivity of Novozym 435
Figure 4.3: Selectivity of the Novozym 435-catalyzed solvent-free kinetic resolution of rac-3 via aminolysis at 60 ◦C (χ0,rac−3 = 0.5, χ0,1 = 0.5, 0.152 gN435 g−1).
(): ee of substrate 3; (N): ee of product 4.
[120]. A possible diffusion limitation using an immobilized enzyme on a porous carrier was excluded by observing equal reaction rates using pestled lipase Novozym 435 instead of the intact carrier (see section 4.3 on page 33). The intrinsic enantioselectivity E of the enzyme may also be altered by spontaneous side reactions. Such a side reaction is to be expected here, since a thermal aminolysis reaction was observed in the Michael addition in section 3.1 on page 17, even though it proceeded very slowly. Therefore, an intrinsic enantioselectivity E of the enzyme and an apparent enantioselectivity E’ is defined. The E-value as an intrinsic parameter depends on the values of kcat/Km for each enantiomer and is constant by definition. In the case of a solvent-free reaction, the composition of the medium changes with conversion and, thus, can have an impact on the enantioselectivity. However, the effect is considered negligible here, since fitting of the experimental data using the equations introduced previously for the calculation of E is possible.
4.1.1 Temperature effect on selectivity
The effect of temperature on enantioselectivity was investigated in the range from 40-80◦C in Figure 4.4. The respective apparent enantioselectivityE’ was again determined by nonlinear regression using Equation 4.1 as described above for each temperature. The determined E-values could be used to fit eeS and eeP as a function of conversion.
Both E and E’ are depicted as a function of temperature in Figure 4.5. E’ was calculated from the rate constant of the overall reaction obtained from initial rate mea-surements, the rate constant for the spontaneous aminolysis (see Appendix, Table B.1 on page 111) and the apparentE’-value using the software Selectivity-KRESH.3A dramatic linear decrease of bothEandE’ with increasing temperature is observed in the analyzed temperature range. Thus, mainly the loss of intrinsic enzyme selectivity accounts for the observed effect while the thermal side reaction does not contribute significantly to
31995 by K. Faber, M. Mischitz, and A. Kleewein. http://www-orgc.tu-graz.ac.at/.
4 Biocatalytic aminolysis
(a)eeof substrate3
0.6
(b) eeof substrate3(zoom)
0.0 0.2 0.4 0.6 0.8 1.0
Figure 4.4: Temperature dependence of enantioselectivity of Novozym 435 in solvent-free kinetic resolution of rac-3 via aminolysis. χ0,rac−3 = 0.5, χ0,1 = 0.5, 0.152 gN435 g−1.
4.1 Selectivity of Novozym 435 the overall decrease of selectivity in the analyzed temperature range. Ottosson et al. an-alyzed temperature effects on enantioselectivity in a lipase catan-alyzed transesterification of secondary alcohols and found that both enthalpic and entropic changes contribute to the temperature dependence of E [96, 97]. Therefore, depending on the order of magnitude of each term, negative, neutral or positive effects ofE with temperature may occur. Indeed, several examples of an at first glance somewhat unexpected increasing E value with temperature have been described in literature [8, 94]. Generally though, E decreases with increasing temperature which corresponds to the observation made here.
30 40 50 60 70 80 90 a function of temperature in the Novozym 435-catalyzed solvent-free kinetic resolution of rac-3 via aminolysis. (): E’; (♦): E.
χ0,rac−3 = 0.5, χ0,1 = 0.5, 0.152 gN435 g−1.
4.1.2 Selectivity in solvent system
The influence of different solvents was investigated by carrying out reactions in five dif-ferent solvents of difdif-ferent polarity. The enantioselectivity E was calculated from the average of eeS and eeP-pairs. The respective conversion vs. ee-plots can be found in the appendix ( B.1 on page 111). Figure 4.6 shows the obtained E-values as a function of solvent polarity expressed as logP. Significant differences were observed depending on the solvent used with E-values ranging from 12 in n-hexane to 34 in tetrahydrofuran.
A trend of decreasing enantioselectivity with increasing solvent hydrophobicity is seem-ingly observed. A correlation of solvent hydrophobicity and enantioselectivity has been reported previously, amongst others by Sakurai et al. and Terradas et al. [110, 127].
Hirose et al. even observed a complete change of enantiopreference in a lipase catalyzed kinetic resolution from 99 % (R)-preference in diisopropylether to 91 % (S)-preference in cyclohexane [44]. However, solvent hydrophobicity alone is not a generally valid in-dicator [30]. A correlation of the molecular size of the solvent with enantioselectivity was observed by Ottosson et al. [96]. Similar trends but with significant scattering of the data were found here when plotting E-value vs. other polarity scales (π∗, ET(30);
data not shown).4 Therefore, a larger number of data from further solvents is required
4See section 4.4.1 on page 35 for details on polarity scales
4 Biocatalytic aminolysis
in order to show if the observed correlation with logP can be verified or if it is merely coincidental.
Figure 4.6: Dependence of en-antioselectivity on solvent polarity expressed as logP. ACN: acetoni-trile; THF: tetrahydrofuran; Tol:
toluene; DIPE: diisopropylether;
nHex: n-hexane. 0.2 M of 1 and rac-3, 45 mgN435 ml−1, 60 ◦C.
0 1 2 3 4 5
0 10 20 30 40
ACN THF
DIPE Tol E [-] nHex
logP
4.1.3 Selectivity in solvent-free system
In a solvent-free reaction system polarity of the medium depends on the initial mole fraction χof the two substrates rac-3 and 1 and was found to decrease withχrac-3 (see section 4.4.1 on page 35). However, only minor differences were observed in enantio-selectivity using different substrate mole fractions in the range from χrac−3 = 0.5−0.8.
Atχrac−3 = 0.33 a significantly lowerE’-value is noted (Table 4.1).
Table 4.1: Apparent enantioselectivityE’ at different initial substrate mole fractions.
χrac−3 0.33 0.5 0.6 0.67 0.8
E’ 17 31 32 31 30
Simulations using the kinetic model introduced in section 4.4.5 on page 42 revealed, however, that the apparent loss of selectivity is caused entirely by the chemical side reaction. Figure 4.7 shows the experimental and simulated conversion vs. ee-plot of a reaction with an initial molar fraction of χrac−3 = 0.33. The term for the uncatalyzed side reaction was erased from the model and a new conversion vs. ee-plot simulated.
The analogous procedure was applied for the reaction with an initial mole fraction of χrac−3 = 0.67. Simulated data of the latter reaction did not reveal noticeable differences with and without consideration of the side reaction due to the high reaction rate of
4.2 Stability of Novozym 435