Measuring stereoselectivity in lipase-catalyzed acidolysis reactions by ultra-high resolution 13C nuclear magnetic resonance

Article abstract of DOI:10.1007/s11746-001-0370-4

Elucidating the stereoselectivity of lipases in synthetic reactions of triacylglycerols has hitherto been carried out using traditional analytical techniques to determine the composition of the reaction products. These methods are laborious and are not always appropriate for analysis of certain triacylglycerol types. A direct method, utilizing a stereospecific deuterium-labeled triacylglycerol substrate, has been developed where the stereoisomeric composition of the reaction product is determined by ultra-high resolution ¹³C nuclear magnetic resonance (NMR) spectroscopy. Through lipase-catalyzed transesterification of deuterium-labeled trilauroylglycerol with oleic acid, chemical shifts were induced in the ¹³C NMR spectrum by the deuterium atom and olefinic double bonds, enabling unambiguous stereospecific assignment of triacylglycerol species. By this method of analysis, we found an effect of the degree of reaction conversion on the extent of stereoisomerism in the triacylglycerol product. Stereoselectivity was greatest (for sn-1) with lipase from Rhizomucor miehei. Lipases from Rhizopus niveus, Candida rugosa, Carica papaya, and the cutinase from Fusarium sp. were also found to exhibit stereoselectivity, with preference for either sn-1 or sn-3 acyl exchange.

Full text of DOI:10.1007/s11746-001-0370-4

Measuring Stereoselectivity in Lipase-Catalyzed
Acidolysis Reactions by Ultra-High Resolution
13C Nuclear Magnetic Resonance
Ian C. Chandlera,*, Oliver W. Howarthb, and David H.G. Croutb
aUnilever Research, Colworth Laboratory, Sharnbrook, Bedford, MK44 1LQ, United Kingdom,
and bDepartment of Chemistry, University of Warwick, Coventry, CV4 7AL, United Kingdom
ABSTRACT: Elucidating the stereoselectivity of lipases in syn- form diastereomers, which are then separated chromatograph-
thetic reactions of triacylglycerols has hitherto been carried out
using traditional analytical techniques to determine the compo-
sition of the reaction products. These methods are laborious and
are not always appropriate for analysis of certain triacylglycerol
types. A direct method, utilizing a stereospecific deuterium-
labeled triacylglycerol substrate, has been developed where the
stereoisomeric composition of the reaction product is deter-
mined by ultra-high resolution 13C nuclear magnetic resonance
(NMR) spectroscopy. Through lipase-catalyzed transesterifi-
cation of deuterium-labeled trilauroylglycerol with oleic acid,
chemical shifts were induced in the 13C NMR spectrum by the
deuterium atom and olefinic double bonds, enabling unambigu-
ous stereospecific assignment of triacylglycerol species. By this
method of analysis, we found an effect of the degree of reaction
conversion on the extent of stereoisomerism in the triacylglyc-
erol product. Stereoselectivity was greatest (for sn-1) with lipase
from Rhizomucor miehei. Lipases from Rhizopus niveus, Can-
dida rugosa, Carica papaya, and the cutinase from Fusarium sp.
were also found to exhibit stereoselectivity, with preference for
either sn-1 or sn-3 acyl exchange.
ically. These analytical methods cannot be applied in all situ-
ations or only with difficulty, particularly when short-chain
acyl groups are present. Here we describe a new method for
analyzing stereochemistry by ultra-high resolution 13C nu-
clear magnetic resonance (NMR) spectroscopy, using a re-
mote carbon–carbon double bond as a reporter group modify-
ing the chemical shift of a carbonyl group. The principle is
illustrated by application to the stereochemical analysis of
lipase-catalyzed triacylglycerol interesterifications.
MATERIALS AND METHODS
Materials. All chemicals, substrates, and solvents were pur-
chased in the highest available purity from the Sigma-Aldrich
group (Poole, United Kingdom). Lipase (EC 3.1.1.3) from
Rhizopus niveus [Lipase N (4,500 Lipase Units/g)] was pur-
chased from Amano Pharmaceutical Co. (Nagoya, Japan). Li-
®
pase from R. miehei, supported on Duolite resin [Lipozyme
IM (100,000 LU/g)] was a gift from Novo Nordisk A/S
(Bagsværd, Denmark). Lipase from Candida rugosa [Lipase
OF360 (360,000 LU/g)] was purchased from Meito Sangyo
Paper no. J9811 in JAOCS 78, 953–958 (September 2001).
KEY WORDS: Acidolysis, chemical shifts, chiral, cutinase,
deuterium, lipase, NMR, stereoselectivity, triacylglycerol, (Tokyo, Japan). Lipase from Carica papaya was extracted
triglyceride.
from papain obtained from Sigma-Aldrich Ltd. Cutinase from
Fusarium solani pisi was donated by the Biotechnology unit
of Unilever Research (Vlaardingen, The Netherlands).
Asymmetric acylglycerol molecules (containing different Macroporous polypropylene enzyme support (Accurel
moieties in positions sn-1 and sn-3) exist as R and S stereo- EP100, particle size distribution 200–400 µm) was purchased
isomers. In lipase-catalyzed hydrolysis of di- and triacyl- from Akzo Nobel GmbH (Obernburg, Germany). All other
glycerols, it has been observed that certain lipases exhibit a solvents and chemicals were of analytical reagent (AR) qual-
preference for one stereoisomer over the other (1–4). Stereo- ity and purchased from Fisher Scientific Ltd. (Loughborough,
selectivity has also been reported in lipase-catalyzed acidoly- United Kingdom) or Sigma-Aldrich Ltd.
sis reactions under certain conditions of low water activity,
Immobilization of lipases onto Accurel EP100. All lipases
particularly with the lipase from Rhizomucor miehei, making were used in immobilized form. Unless supplied already im-
possible the synthesis of chiral triacylglycerols (5). A number mobilized, the lipases were adsorbed onto Accurel EP100,
of methods for the stereochemical analysis of triacylglycerols following the method described by Bosley and Peilow (11).
have been described (6–10). Lipase stereoselectivity in mi-
Preparation of C. papaya latex. The method described by
croaqueous reactions is currently determined by indirect Giordani et al. (12) was followed. The latex was used directly
chemical analysis. For example, the triacylglycerol products as an acidolysis catalyst and had a hydrolysis activity of 5500
of a reaction can be quantified after partial “hydrolysis” by LU/g (on tributyrin).
Grignard reagents and derivatization with chiral reagents to
Synthesis of stereospecifically labeled triacylglycerol sub-
strates. A known method for the production of enantiomeri-
cally pure triacylglycerols was adapted so that an atom of
*To whom correspondence should be addressed.
E-mail: Ian.Chandler@Unilever.com
Copyright © 2001 by AOCS Press
953
JAOCS, Vol. 78, no. 9 (2001)

954
I.C. CHANDLER ET AL.
dc = 100 × % oleic acid in total triacylglycerol/% oleic acid
in total triacylglycerol at equilibrium
[1]
13C NMR. 13C NMR spectra at high resolution were ob-
tained using a Bruker ACP400 spectrometer (Karlsruhe, Ger-
many). Spectra were obtained at the highest available resolu-
tion (0.015 Hz/data point), aided by a sample longer than usual
(4 cm), temperature control (298 K), minimal decoupling
power, and Lorentzian to Gaussian resolution enhancement.
Stereoselectivity factor (SF). Using Microsoft (Redmond,
WA) Excel Solver, integration data from 13C NMR spectra
were quantitatively correlated with triacylglycerol composi-
tion data from gas chromatography analyses. A measure of
the extent of stereoselectivity and the configuration of the
products was obtained from the equation:
SCHEME 1
SF = OLaLa*/(OLaLa* + LaLaO*)
[2]
deuterium (2H1) was incorporated nonstereospecifically onto
the sn-3 glyceryl carbon (13) (Scheme 1). 1,2:5,6-Di-O-
where O is oleate, La is laurate, and * represents the deuterated
glyceryl position (sn-3). An SF value of 1.00 represents total
selectivity for sn-1 (S), 0.00 represents total selectivity sn-3 (R),
and 0.50 represents a racemic triacylglycerol composition.
isopropylidene- -mannitol [1, 5 g] was dissolved in cold ethyl
D
acetate (100 mL) and oxidized by stepwise addition of lead (iv)
tetra-acetate (8.7 g) over 20 min. The product, 1,2-O-isopropyl-
idene-
D
-glyceraldehyde [2, 3.6 g] was recovered by distilla-
RESULTS AND DISCUSSION
tion, dissolved in ethanol (50 mL), and reduced with sodium
[2H4] borohydride (0.35 g, 98% atom 2H) to 1,2-O-isopropyli- Method development. In seeking a simpler and more direct
dene- -glycerol (3, 1.1 g). The protecting group was removed method for determining the stereochemistry of lipase-catalyzed
D
by acid hydrolysis to yield sn-[3-2H1]-glycerol (4, 0.6 g). The interesterification in triacylglycerols, the use of stereospecifi-
deuterium atom was assigned to the sn-3 carbon by application cally labeled glycerol was envisaged. A triacylglycerol such as
of IUPAC rules on the stereospecific numbering of tri- trilaurin 5 (Scheme 2), prepared from such a labeled glycerol,
acylglycerols (14). Trilauroyl-sn-[3-2H1]-glycerol was pro- might undergo lipase-catalyzed fatty acid exchange against a
duced by complete acylation of 4 with lauroyl chloride in the different acid, oleic acid 6 for example, to give enantiomeric
presence of pyridine. Mass spectrometry data: m/z 658 (molec- products arising from exchange of the sn-1 and sn-3 fatty acyl
+
ular ion + NH4 ); compares with m/z 657 (molecular ion + groups, respectively. Elucidation of the stereochemistry of this
+
NH4 ) for unlabeled trilaurin, indicating monodeuteration.
process could then be based on determining the ratio of fatty
Enzyme-catalyzed reactions of triacylglycerol and free acid exchange at the deuterated and nondeuterated positions
fatty acid. All reactants and solvents were equilibrated to a (Scheme 2).
water activity of 0.11, at ambient temperature (20°C), over
One option considered for determining this ratio was by the
saturated lithium chloride in a sealed vessel for at least 7 d, as use of MS, relying on C–C bond fragmentation between the 1,2-
described by Valivety et al. (15). It is assumed that, provided and 2,3-positions in the glycerol component. This fragmenta-
sufficient time is allowed for equilibration, the water activity tion would give ions that would consist of nondeuterated and
of the sample equals that of the salt solution. The acidolysis monodeuterated species in a ratio directly related to the stere-
reactions of trilauroyl-sn-[3-2H1]-glycerol (73 mg; 114 µmol) ospecificity of fatty acid exchange. However, the required frag-
and oleic acid (97 mg; 343 µmol) were performed and ana- mentation was difficult to achieve and produced the required
lyzed according to the method described in Chandler et al. ions only in very low abundance and with low reproducibility.
(5). From the analysis of data, a degree of reaction conver-
sion (dc) was calculated, where:
As an alternative, the use of NMR was explored in the
anticipation that a combination of deuteration shifts and
SCHEME 2
JAOCS, Vol. 78, no. 9 (2001)

LIPASE STEREOSELECTIVITY MEASURED BY 13C NMR
955
substituent shifts might reveal the positional specificity of fatty rect was obtained by determining the spectrum of a mixture
acid exchange. This hope was realized when the carbonyl re- of deuterated and nondeuterated trilaurin in unequal propor-
gion of the 13C NMR spectra of products derived from deuter- tions. From the relative intensities of the signals attributable
ated trilaurin by interesterification against oleic acid was ana- to deuterated and nondeuterated material, it was possible to
lyzed at ultra-high resolution. Stereochemical analysis was assign them unambiguously. The resonances in region b arise
made possible by the operation of two effects: chemical shift from lauryl carbonyl groups attached to the sn-1 position, and
changes arising from long-range effects of the olefinic double magnetically equivalent lauryl groups attached at the sn-3 po-
bond in oleyl components, and deuteration shifts affecting acyl sition in undeuterated glycerol remaining in the deuterated
substituents at the labeled sn-3 position. The laurate-oleate sample used for preparation of the trilaurin. In region c are
shift differences arise from the effects of the dipoles at the seen resonances attributable to oleyl groups attached to the
ends of the oleyl double bond, and it should be noted that the sn-3 position. The chemical shift is the net result of a positive
influence of the reporter olefinic bond is transmitted over eight deuteration shift superimposed on a negative shift arising
carbon–carbon single bonds. However, this method of stereo- from the long-range effect of the unsaturation in the oleyl side
chemical characterization relies on the presence of detectable chain (17–19). Region d encompasses the resonances attrib-
shift differences between the reaction substrates and products, utable to oleyl groups attached at the sn-1 position and oleyl
and therefore would not be suitable for the study of reactions groups attached to the sn-3 position in material derived from
involving the exchange of long-chain saturated acyl groups.
residual unlabeled glycerol. Resonances attributable to acyla-
13C NMR spectra at high resolution were obtained. The tion at the 2-position are seen in region e near δ 172.74. Only
chemical shift of a given carbonyl resonance was determined a single resonance is seen as the lipase from R. miehei does
by the environment (acyl group attached to a primary or sec- not catalyze exchange at the sn-2 position. It should be noted
ondary center) and by the two structural factors noted above, that the range of chemical shifts of signals attributable to acyl
namely, the nature of the side chain and the position of the groups attached at the sn-1 and sn-3 positions is only 50 ppb.
carbonyl group relative to the deuterated position.
The percent deuteration, the percent conversion, and
In Figure 1 is shown the carbonyl region of the 13C NMR the stereoselectivity of the interesterification are readily de-
spectrum of the product of interesterification of trilaurin with rived from the integrals of the signals in the regions a to e
oleic acid catalyzed by the lipase from R. miehei. In the re- (Fig. 1). Thus, the stereoselectivity is given by the ratio
gion δ 172.7–173.18 ppm, the carbonyl carbon atom reso- OLaLa*/(OLaLa* + LaLaO*) and the asterisk indicates that the
nances fall into five groups. At the lowest field, the signal a is corresponding residue is attached to the deuterated position. The
attributable to the lauryl groups attached to the deuterated other species potentially present are LaLaLa*, OLaO*, LaLaLa,
sn-3 position. The deuteration shift is +7 ppb and is unexpect- OLaLa, and OLaO. The proportions of these were adjusted,
edly positive. This can be attributed to a small reduction in γ- using Microsoft Excel Solver, to give the best fit to the five re-
gauche shielding at the carbonyl carbon atom, which arises solved signals. For the example shown in Figure 1, the calcu-
from the shorter C–D bond distance compared with the C–H lated stereoselectivity is 0.38, the extent of deuteration is 81%,
bond distance (16). Confirmation that this assignment is cor- and there have been 56% single replacements of lauryl groups
b
e
a
c
d
FIG. 1. 13C Nuclear magnetic resonance (NMR) spectrum, in the range δ 172.7–173.25 ppm,
of the product of interesterification of trilauroyl-sn-[3-2H1]-glycerol 5 against oleic acid 6 cat-
alyzed by the lipase from Rhizomucor miehei. Signals a–e have been attributed to chemical
shifts according to the vicinity of the carbonyl group to unsaturated bonds or deuterium atoms.
These are discussed fully within the text.
JAOCS, Vol. 78, no. 9 (2001)

956
I.C. CHANDLER ET AL.
TABLE 1
Stereoselectivity of Reactions of Trilauroylglycerol and Oleic Acid Catalyzed
by Rhizomucor mieheia Lipase at Varying Degrees of Conversion (dc)
Triacylglycerol composition
(normalized to 100%)
dcb
(%)
LaLaO/
OLaLa
% eed
LaLaLa
OLaO
OOO
SF
(configuration)e
14
25
34
44
69
80
64
53
42
21
18
31
40
48
52
2
4
7
10
22
0
1
1
1
4
1.00
1.00
0.86
0.73
0.67
100 (S)
100 (S)
72 (S)
46 (S)
34 (S)
aThis lipase was prepared using recombinant DNA technology: The genetic coding for the lipase was
transferred from the source organism to a host, Aspergillus niger.
bPercent conversion defined as 100 × [oleate in triacylglycerol/oleate in the triacylglycerol at equi-
librium].
cStereoselectivity factor = OLaLa*/(OLaLa* + LaLaO*).
d% ee of OLaLa [note: % ee = 100 × (2SF − 1) where SF = stereoselectivity factor and ee = enan-
tiomeric excess].
e(S) stereoisomer arises from sn-1 exchange.
by oleyl groups. An indication of the consistency of the method cation product was lowered. Because at equilibrium the con-
is that the deuterium content as deduced from the 13C NMR data centrations of enantiomers must be equal (e.g., OLaLa =
agrees well with the ratio determined by integration of the peaks LaLaO), the stereoselectivity is time dependent, as observed.
attributable to the glycerol protons in the 1H NMR spectrum of
the same sample (data not shown).
Lipase from R. niveus. At very low conversion, a high de-
gree of selectivity for sn-1 was observed for lipase from R.
In Tables 1 and 2 are shown the results of interesterifica- niveus. At a degree of conversion of >60% the product
tions between trilaurin and oleic acid catalyzed by the lipases stereoisomerism is lost; this also agrees with previous experi-
from R. miehei, R. niveus, C. rugosa, and C. papaya, and by ments (5). In comparing stereoselectivities between different
the cutinase from F. solani pisi.
enzymes, the extent of conversion must be taken into account.
Lipase from R. miehei. Under the conditions applied, the However, when stereoselectivities are compared at the same
lipase from R. miehei exhibited high stereoselectivity (for po- percent conversions (interpolating where necessary), it can
sition sn-1). This agreed with previous findings (5). However, be seen that the lipase from R. miehei does indeed exhibit a
in using the earlier analytical methods it was not possible to higher stereoselectivity than the lipase from R. niveus. It
determine the effect of the degree of reaction conversion. At should be noted, however, that the reaction conditions had
low conversions, stereoselectivity was essentially complete. been optimized for catalysis by R. miehei lipase only.
However, as the interesterification reaction proceeded toward
Lipases from C. rugosa and C. papaya, and Fusarium cuti-
equilibrium, the stereochemical integrity of the interesterifi- nase. These enzymes all have similar, but low, stereoselectivi-
TABLE 2
Stereoselectivity of Reactions of Trilauroylglycerol and Oleic Acid Catalyzed by Lipases
(or cutinase) from Various Sources
Triacylglycerol composition
(normalized to 100%)
Source
dcb
(%)
LaLaO/
OLaLa
% eed
organisma
LaLaLa
OLaO
OOO
SF
(configuration)e
R. niveus
R. niveus
C. rugosa
C. papaya
F. solani pisi
F. solani pisi
7
63
55
49
23
52
88
27
37
45
68
36
10
47
42
34
29
47
2
27
18
18
5
0
0
3
3
1
2
0.97
0.53
0.44
0.47
0.36
0.42
94 (S)
6 (S)
12 (R)
6 (R)
28 (R)
16 (R)
15
aLipases were expressed by the native organism. Organisms include Rhizopus niveus, Candida ru-
gosa, Carica papaya, and Fusarium solani pisi.
bPercent conversion defined as 100 × [oleate in triacylglycerol/oleate in the triacylglycerol at equi-
librium].
c Stereoselectivity factor = OLaLa*/(OLaLa* + LaLaO*).
d% ee of OLaLa [note: % ee = 100 × (2SF − 1)].
e (S), (R) stereoisomers arise from sn-1 and sn-3 exchange, respectively. See Table 1 for other abbre-
viations.
JAOCS, Vol. 78, no. 9 (2001)

LIPASE STEREOSELECTIVITY MEASURED BY 13C NMR
957
TABLE 3
A Comparison of Data from Different Stereoselectivity Assays for Selected Lipases
Hydrolysis of trioleoylglycerol
(monolayer technique)b
Acidolysis of trilauroyl-
sn-[3-2H1]-glycerol
Lipase sourcea
dc
% ee
sn-1/3
dc
% ee
sn-1/3
R. miehei
6
6
—
6
82
55
—
14
72
1
1
—
1
14
—
7
55
28
100
—
94
12
16
1
—
1
3
3
R. arrhizusc
R. niveusc
C. rugosa
F. solani pisi
6
3
aOrganisms include Rhizomucor miehei, Rhizopus arrhizus, Rhizopus niveus, Candida rugosa, and
Fusarium solani pisi.
bRogalska, Cudrey, Ferrato, and Verger method (1).
cNo direct comparison is available. The results from tests with lipases from related Rhizopus species
are given.
ties at the degrees of conversion measured. With these cata- tween methods for the limited data available. Further valida-
lysts the stereopreference was found to be toward the sn-3 po- tion of the acidolysis technique with a wider range of lipases
sition, and strong sn-3 selectivity has been reported for both and reaction conditions is necessary.
the C. papaya latex lipase and the Fusarium cutinase (6,20).
Further splittings of the NMR signals. In some experi-
However, there is a notable discrepancy in the stereoprefer- ments, the NMR signals observed (as in Fig. 1) showed fur-
ence assignment for C. rugosa lipase, which has been shown ther fine splittings of the order of 3 ppb (Fig. 2), particularly
to exhibit sn-1 selectivity in the hydrolysis of triacylglycerols in cases, as here, corresponding to high conversions. These
(1,3). An insufficient number of experiments have been con- arise through the same through-bond dipolar processes that
ducted with C. rugosa lipase to be confident of our determi- bring about separation of the lauryl and oleyl carbonyl sig-
nation of sn-3 selectivity in the acidolysis reaction.
nals at a given glycerol site, except that in this case the per-
In Table 3 a comparison has been made (where data are turbation is caused by chain variations at the other sites trans-
available) between the hydrolytic method of stereoselectivity mitted through the glycerol backbone. Although the exact sig-
determination used by Rogalska et al. (1) and our acidolysis nal assignment has not been confirmed, the observed shifts
method. As already discussed, the degree of conversion can are approximately consistent with the attenuation factors de-
affect the enantiomeric excess obtained, which limits the cor- duced previously; therefore, all have the same sign (16,18).
relation of data, and the acidolysis exchange conditions were These extra shifts have not been reported previously and offer
only optimized for R. miehei lipase. Nevertheless, C. rugosa a potential method for assessing the chain distribution in an
data withstanding, there appears to be general agreement be- unknown di- or triacylglycerol mixture.
FIG. 2. 13C NMR spectrum, in the range δ 172.7–173.25 ppm, of the product of interesterifica-
tion of trilauroyl-sn-[3-2H1]-glycerol 5 against oleic acid 6 catalyzed by the lipase from Rhi-
zomucor miehei. The spectrum shows fine structure attributable to long-range effects on indi-
vidual carbonyl resonances of acyl groups attached at other positions, and these have been
enlarged to show the detail. The signals have the same chemical shifts as in Figure 1. See Fig-
ure 1 for abbreviation.
JAOCS, Vol. 78, no. 9 (2001)

958
I.C. CHANDLER ET AL.
10. Zhang, X.-M., and I.W. Wainer, Online Determination of Li-
ACKNOWLEDGMENTS
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range splittings of the type described, and we are grateful to Drs.
Paul Quinlan, Gerald McNeill, and John Bosley for valuable discus-
sions.
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[Received November 14, 2000; accepted May 29, 2001]
JAOCS, Vol. 78, no. 9 (2001)