Probing the specificity of the S1', leaving group, site of subtilisin Bacillus lentus using an enzyme-catalyzed transesterification reaction

Article abstract of DOI:10.1016/S0957-4166(98)00022-6

Subtilisin Bacillus lentus catalyzes transesterifications between N- acetyl-L-phenylalanine vinyl ester and a wide range of alcohols. Reaction yields are high when primary alcohols are used, and quantitative with methanol. With chiral alcohols, the reaction is enantioselective, and the stereoselectivity is reversed on going from open chain secondary alcohols to β-branched primary alcohols. A model is proposed to account for this change in absolute configuration preference.

Full text of DOI:10.1016/S0957-4166(98)00022-6

Pergamon
Tetrahedron: Asymmetry 9 (1998) 551–561
0
Probing the specificity of the S1 , leaving group, site of subtilisin
Bacillus lentus using an enzyme-catalyzed transesterification
reaction
Richard C. Lloyd, Michael Dickman and J. Bryan Jones ∗
Department of Chemistry, University of Toronto, 80 St. George St., Toronto, Ontario, M5S 3H6, Canada
Received 21 October 1997; accepted 7 December 1997
Abstract
Subtilisin Bacillus lentus catalyzes transesterifications between N-acetyl-L-phenylalanine vinyl ester and a wide
range of alcohols. Reaction yields are high when primary alcohols are used, and quantitative with methanol. With
chiral alcohols, the reaction is enantioselective, and the stereoselectivity is reversed on going from open chain
secondary alcohols to β-branched primary alcohols. A model is proposed to account for this change in absolute
configuration preference. © 1998 Elsevier Science Ltd. All rights reserved.
Hydrolase-catalyzed transesterifications of alcohols using vinyl or isopropenyl acetate as the acyl
donor are widely used to resolve racemic alcohols and to effect stereoselective acylation of prochiral
and meso diols.1 While lipases have enjoyed wide success in this regard, serine proteases have had
more limited applications, with the greatest successes to date being reported for the regioselective
acylation of carbohydrates,2 nucleosides,3 alkaloids4 and steroids.5 Subtilisins are amongst the most
stable, inexpensive and ubiquitous of serine proteases. However, in view of their ease of availability
and low cost, they appear underused as preparative catalysts. Accordingly, we have initiated a program
to extend the synthetic potential of subtilisins. In this paper, we evaluate the spectrum of applicability
06
of subtilisin Bacillus lentus (SBL) in transesterification reactions, and probe the specificity of the S1
site of SBL in such reactions. The prospects in this regard were encouraging, since kinetic studies7 on
subtilisin-catalyzed transesterifications between vinyl acetate and various alcohols, and related effects of
solvent on enzyme specificity, have been reported.8 Also, recent studies have documented some of the
potential of subtilisins in organic synthesis.9
Initially, we examined catalysis by SBL of the reactions between vinyl and isopropenyl acetate and a
range of alcohols. However, these reactions were too slow to be of preparative value. We presumed that
this was the result of the low affinity of SBL for small acyl groups and that better results might be achieved
with larger acyl groups that would interact more favorably with S1. The recent report of the preparation
∗
Corresponding author. E-mail: jbjones@alchemy.chem.utoronto.ca
0957-4166/98/$19.00 © 1998 Elsevier Science Ltd. All rights reserved.
PII: S0957-4166(98)00022-6

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R. C. Lloyd et al. / Tetrahedron: Asymmetry 9 (1998) 551–561
of N-acetyl-L-phenylalanine vinyl ester10 (1) pointed to a solution to the small acyl group problem, since
the benzyl side chain of 1 would target the S1 pocket of SBL, thereby improving acyl enzyme formation.
0
The specificity of 1 for S1 would also eliminate competition between the S1 and S1 binding sites for
0
the incoming nucleophile, thereby permitting an unequivocal study of the spatial restrictions of the S1
pocket. The vinyl ester 1 was prepared in reasonable (51%) yield and excellent (>99%) enantiomeric
excess via the palladium acetate-catalyzed coupling11 of vinyl acetate with N-acetyl-L-phenylalanine.
The transesterification reaction investigated is shown in Scheme 1. SBL was immobilized in a potas-
sium chloride matrix (KCl–SBL).12 The KCl–SBL preparation was more active than SBL lyophilized
from phosphate buffer alone, and enzyme activity was higher when KCl–SBL was lyophilized from
phosphate buffer than when lyophilized from Tris.13 KCl–SBL was most active in tert-butanol and
acetonitrile. The activity of KCl–SBL was also surveyed in isopropyl ether, tert-butyl methyl ether,
cyclohexane and octane with 1 as the substrate, but in none of these solvents did the yields of the reaction
approach those attained in tert-butanol and acetonitrile.
Scheme 1.
A spectrum of primary, secondary, achiral and chiral alcohols (2) were surveyed. In general, reactions
were carried out at 50°C for 24 hours to allow comparisons of the yields obtained. All reactions were
verified as being enzyme-catalyzed by running blank reactions containing the same reactants in the
absence of SBL. In no case was a background reaction observed.14 The results of the alcohol screen
are recorded in Table 1.
0
It can be seen that the S1 site of SBL exhibits a broad structural tolerance for primary alcohol
nucleophiles, with the highest yields being obtained for those with the least steric bulk. In fact,
the reaction with the smallest alcohol, methanol (2a), was essentially quantitative. There appears
0
to be little restriction on the steric bulk accommodated by S1 as even the largest nucleophile of
the series, 4-biphenylmethanol (2h), gave excellent (75%) conversion to 3h. However, alkyl chain
branching is somewhat deleterious, with 2- (2k) and 3-methylpentanol (2l) being lower yielding (58
and 55% respectively) than n-pentanol (2b, 86%). Furthermore, for the β-branched achiral alcohol,
cyclohexylmethanol (2i), the yield (66%) is lower than with benzyl alcohol (2d, 78%), to which it is
structurally very similar, or with the other benzylic alcohols 2g (70%) and 2h (75%). From this it would
appear that the nature of the β-branch is also important. Branching at an sp3 hybridized center engenders
more steric hindrance than at an sp2 hybridized center. Reactions with the secondary alcohols 2m, 2n and
2o gave lower yields (37%, 51%, 20% respectively) even when the standard reactio0n time was increased
threefold, indicating that branching at an α-carbon is even less well tolerated by S1 .
0
The stereoselectivity of the S1 pocket towards racemic alcohols was also explored. In the reactions
with the racemic primary alcohols 2j, 2k and 2l, some stereoselectivity is manifest in the products 3j, 3k
and 3l respectively. However, when secondary alcohols are used, the stereoselectivity is much greater,
with products of very high d.e. obtained in the reactions with (ꢀ)-2-octanol (2o, >99% d.e.), and (ꢀ)-
1-phenylethanol (2m, 95%). Interestingly, the reaction with (ꢀ)-endo-norborneol (2q) proceeded to give
the ester 3q with good d.e. (82%) while the (ꢀ)-exo-isomer (2p) yielded 3p of only 25% d.e.
Due to the expense of 1 it was decided to investigate the activity of SBL with the less costly, and

R. C. Lloyd et al. / Tetrahedron: Asymmetry 9 (1998) 551–561
553
Table 1
Results of the alcohol screen in Scheme 1 reactions
readily synthesized11 achiral analog, 3-phenylpropionic acid vinyl ester (4). When 4 was used as the acyl
donor with a representative selection of racemic alcohols, the reaction yields and e.e.s were similar to
those obtained with 1, thus demonstrating that the activity of the enzyme was not significantly altered
by changing the acyl donor. In fact, an increase in e.e. (from 30% to 50%) is observed for the primary
alcohol used, 2-phenylpropanol (2j).
It is possible to increase the enantioselectivity of an enzyme-catalyzed reaction by lowering the
temperature at which it is carried out.15 Consequently, we investigated this approach as a means of
increasing the d.e. of the products of reactions between 1 and the β-branched primary alcohols 2j and
2k. The results are shown in Table 2. Excellent yields were obtained when using acetonitrile as the
solvent, although in the series of reactions with 2k this was accompanied by a loss in enantioselectivity
at 4°C. Cooling of this reaction to −20°C retained the high yield and restored the d.e. to the level of the
initial screen. Reactions carried out in tert-butanol showed a modest increase in the d.e. of the products,

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R. C. Lloyd et al. / Tetrahedron: Asymmetry 9 (1998) 551–561
Table 2
Temperature studies on Scheme 1 reactions using racemic primary alcohols
although this was at the expense of yield, probably due to reduced substrate concentration, resulting from
freezing of the medium.
Absolute configurations of the products of the enzyme-catalyzed reactions were determined on
diastereoisomerically pure samples of 3m, 3n and 3o that were chemically prepared from N-acetyl-L-
phenylalanine and enantiomerically pure 2m, 2n and 2o. Absolute configurations of the enzyme product
esters 3m, 3n and 3o were then determined by HPLC coinjection or by NMR analysis of samples spiked
with the reference compounds. When enantiomerically pure alcohols were not available for the synthesis
of reference samples, absolute configurations were obtained by hydrolysis of the product ester and
comparison of the optical rotation of the alcohol obtained with the literature reference standards.16 In
the case of 3-methyl-1-pentanol (2l) the alcohol was oxidized to the known acid17 in order to assign
0
the absolute configuration. From the results, a distinct enantioselectivity pattern for the S1 pocket was
discovered. For open chain secondary alcohols, a preference for the S-enantiomer was evident, while a
reversal of selectivity, favoring the R-enantiomer, was apparent for β-branched primary alcohols. In the
case of the single γ-branched primary alcohol used (2l), the selectivity reversed once more, with the
product ester 3l incorporating an alcohol moiety with the S-configuration.
The way lipases control enantioselectivity of transesterifications with chiral alcohols18 has been
extensively studied and, based on the size and orientation of the substituents at the stereocenter, empirical
rules have been proposed to predict which enantiomer of the alcohol will react preferentially. A model
to explain why subtilisin Carlsberg is specific for the S-enantiomer of secondary alcohols has also been
formulated7a and X-ray studies have shown the active sites of subtilisin Carlsberg and lipases to be
approximate mirror images of one another19 in accord with the opposite enantioselectivity of these
enzymes for secondary alcohols.
Building on these Kaz0lauskas18g and Klibanov7a model proposals, we find that the reversals in
stereospecificity of the S1 pocket encountered in our SBL-catalyzed transesterification reactions with
open chain alcohols can be rationalized by the models depicted in Fig. 1. Figure 1(a) is the Klibanov
subtilisin model and also represents the orientation of substituents in the SBL active site when the
nucleophile is a secondary alcohol. Figure 1(b) represents the SBL picture for β-branched primary
alcohols and is similar to the Pseudomonas cepacia lipase model proposed by Kazlauskas. Figure 1(c)
shows that SBL will be specific for the S-enantiomer of 3-methyl-1-pentanol (2l), as observed, if the
orientation of the alkyl groups remain the same as in Fig. 1(b). It is also interesting to note that although

R. C. Lloyd et al. / Tetrahedron: Asymmetry 9 (1998) 551–561
555
Fig. 1. Model to predict and explain the specificity of SBL for (a) secondary alcohols (b) β-branched primary alcohols (c)
γ-branched primary alcohols. The size of ‘R’ denotes the relative size of the substituents
SBL is highly specific for the S-enantiomer of open chain secondary alcohols, it has a preference for the
R-enantiomers of the cyclic norborneols (2p and 2q).
In conclusion, our experiments show that, with a specific acyl donor, SBL can be a very useful
transesterification catalyst in organic solvents. While we cannot quantify the degree of improvement
conferred by using 1 in place of vinyl acetate, we estimate that the rate increase with N-acetyl-L-
phenylalanine vinyl ester as the acyl donor is >100 fold. There was no detectable yield after 24 hours
in attempted transesterifications using vinyl acetate under the Table 1 conditions. SBL exhibits broad
0
specificity in the S1 pocket and can be used to catalyze transesterifications of vinyl esters such as 1 and
0
4 with a wide range of alcohols in good to excellent yields. The enantioselectivity of the S1 pocket is
excellent for secondary alcohols, but diminishes the further the stereocenter is removed from the OH-
group of a chiral acyclic alcohol nucleophile. Furthermore, the enantiomeric preferences of these SBL-
catalyzed reactions are predictable using simple models of the type formulated for other hydrolases.
While much more needs to be done before SBL can achieve the breadth of applicability enjoyed by
lipases in transesterifications, the current results represent substantial progress. Further improvements in
preparative SBL applications are currently being sought, including increasing the enzyme’s activity and
tailoring its specificity by chemical modification of SBL mutants.20
1. Experimental section
Subtilisin Bacillus lentus (SBL) was obtained as a crude solution from Genencor International Inc.
and was purified according to a previously published procedure.20c Vinyl acetate was distilled before
use. All alcohols used as substrates were used as received from Aldrich or Lancaster. t-BuOH was
dried by distillation from Mg turnings and I2. Melting points were determined using an Electrothermal
IA9000 series digital melting point apparatus, and are uncorrected. Optical rotation data were obtained
using a Perkin–Elmer 243B polarimeter. Products of enzyme-catalyzed reactions were identified by
1
their H NMR spectra, run in CDCl3 using a Varian Gemini 200 MHz NMR spectrometer, and
HRMS data, acquired using a Micromass 70-250S (double focussing) mass spectrometer for EI spectra,
and a Micromass ZAB-SE for FAB spectra (where indicated). 13C NMR spectra were obtained at
50.3 MHz using the Varian Gemini NMR spectrometer. Diastereoisomeric and enantiomeric excesses
were determined by HPLC, using a Chiralcel OD column and a hexane–isopropanol eluent system
1
(60:1–12:1 range, flow rate 1mL/min), or by H NMR spectroscopy using a Varian Unity 500 MHz
NMR spectrometer. Values quoted are judged to be accurate to ꢀ2%.

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R. C. Lloyd et al. / Tetrahedron: Asymmetry 9 (1998) 551–561
1.1. N-Acetyl-L-phenylalanine vinyl ester (1)
Using the method of Lobell and Schneider,11 a mixture of N-acetyl-L-phenylalanine (1.012 g, 4.883
mmol), Pd(OAc)2 (219 mg, 0.2 eq), KOH (27 mg, 0.1 eq) and 45 mL vinyl acetate (100 eq) was stirred
for 24 h at 20°C under N2. The reaction mixture was then added to Et2O (300 mL), stirred for 10 min
and filtered. After evaporation of the filtrate, the crude product was purified by flash chromatography
using a step gradient (25:75 EtOAc:hexanes followed by 50:50 EtOAc:hexanes) to give N-acetyl-L-
phenylalanine vinyl ester (1) (581 mg, 51%, >99% e.e.), which was recrystallized from EtOAc:hexanes.
M.p. 90.0–91.0°C [lit.10 56.0–58.0°C]; [α]D25=+32.7 (c 1.05, CHCl3); νmax (KBr) 3318, 1770, 1753
cm−1; 1H NMR (CDCl3) δ 7.34–7.07 (6H, m), 6.11 (1H, br d, J 7.7 Hz), 5.00–4.91 (2H, m), 4.66 (1H,
dd, J 6.2, 1.9 Hz), 3.25–3.08 (2H, m), 1.98 (3H, s); 13C NMR (CDCl3) δ 169.8, 168.8, 140.6, 135.4,
129.0, 128.4, 127.0, 99.0, 52.9, 37.4, 22.8; HRMS calcd for C13H16NO3 (M+1+) 234.113019. Found:
234.112903.
1.2. 3-Phenylpropionic acid vinyl ester (4)21
Following the method described for 1 above, but using 3-phenylpropionic acid (209 mg, 1.39 mmol),
Pd(OAc)2 (62 mg, 0.2 eq), KOH (8 mg, 0.1 eq) and vinyl acetate (13 mL, 100 eq). The crude product
was purified by flash chromatography using a step gradient (hexanes followed by 5:95 EtOAc:hexanes)
to yield 3-phenylpropionic acid vinyl ester (4) in 70% yield. νmax (neat) 1756 cm−1; 1H NMR (CDCl3)
δ 7.37–7.20 (6H, m), 4.90 (1H, dd, J 14.1, 1.6 Hz), 4.59 (1H, dd, J 6.4, 1.6 Hz), 3.02 (2H, t, J 7.8 Hz),
2.74 (2H, t, J 7.8 Hz); 13C NMR (CDCl3) δ 170.3, 141.6, 140.6, 129.0, 128.8, 126.9, 98.2, 36.1, 31.2;
HRMS calcd for C11H12O2 (M+) 176.083730. Found: 176.083816.
1.3. Preparation of SBL in a KCl matrix12
SBL (52.5 mg) was dissolved in 50 mM phosphate buffer pH 8.4 (30 mL). KCl (10.78 g) was added
to the solution and dissolved with swirling. The resulting solution was flash frozen in liquid N2 and
lyophilized for 48 h. The dry mixture was stored in a dessicator at −20°C prior to use.
1.4. A general method for SBL-catalyzed transesterifications
KCl–SBL powder (438.2 mg), N-acetyl-L-phenylalanine vinyl ester (1) (9.8 mg, 42 µmol) and benzyl
alcohol (2d) (45 µL, 434 µmol) were mixed in a glass vial under N2. Dry t-BuOH (2 mL) was added and
the contents stirred at 50°C for 24 h under nitrogen. The reaction was terminated by filtration through
Celite, evaporated, and the residue purified by flash column chromatography (EtOAc:hexanes 25:75
followed by 50:50 EtOAc:hexanes) to yield N-acetyl-L-phenylalanine benzyl ester (3d) (10.0 mg, 78%,
1
>99% e.e.) as white crystals, m.p. 76.0–78.0°C. [α]D27=+17.8 (c 0.82, CHCl3); H NMR δ 7.41–7.19
(8H, m), 6.98 (2H, m), 5.91 (2H, br d, J 7.4 Hz) 5.15, (2H, m), 4.92 (1H, m), 3.12 (2H, d, J 6.0 Hz), 1.98
(3H, s); HRMS calcd for C18H19NO3 (M+) 297.136494. Found: 297.136298.The following compounds
were prepared using the above procedure, except where stated.
1.5. N-Acetyl-L-phenylalanine methyl ester (3a)22,23
From 1 (10.3 mg, 44.2 µmol) and 2a (20 µL, 494 µmol). Reaction performed in MeCN at
room temperature. White crystals (9.6 mg, 98%, >99% e.e.), m.p. 84.5–86.0°C [lit.23 86.0–87.0°C].

R. C. Lloyd et al. / Tetrahedron: Asymmetry 9 (1998) 551–561
557
[α]D25=+105.6 (c 0.46, CHCl3) [lit.23 [α]D25=+101.5 (c 1.00, CHCl3)]; 1H NMR δ 7.31–7.23 (3H, m),
7.12–7.05 (2H, m), 5.90 (1H, br d, J 5.9 Hz), 4.88 (1H, app q, J 5.9 Hz), 3.73 (3H, s), 3.12 (2H, m), 1.98
(3H, s); HRMS calcd for C12H15NO3 (M+) 221.105194. Found: 221.104113.
1.6. N-Acetyl-L-phenylalanine pentyl ester (3b)
From 1, (10.7 mg, 45.9 µmol) and 2b (50 µL, 460 µmol). Colorless oil (11.2 mg, 86%, >99% e.e.).
[α]D26=+59.7 (c 0.71, CHCl3); H NMR δ 7.30–7.22 (3H, m), 7.15–7.07 (2H, m), 5.90 (1H, d, J 6.0
1
Hz), 4.88 (1H, app q, J 6.0 Hz), 4.10 (2H, dt, J 3.0, 6.0 Hz), 3.13 (2H, d, J 6.0 Hz), 1.99 (3H, s), 1.60
(2H, m), 1.31 (4H, m), 0.91 (3H, t, J 7.0 Hz); HRMS calcd for C16H23NO3 (M+) 277.167794. Found:
277.166817.
1.7. N-Acetyl-L-phenylalanine nonyl ester (3c)
From 1, (9.9 mg, 42.4 µmol) and 2c (75 µL, 430 µmol). Colorless oil (9.0 mg, 64%, >99% e.e.).
[α]D29=+38.2 (c 0.67, CHCl3); 1H NMR δ 7.32–7.22 (3H, m), 7.12–7.07 (2H, m), 5.92 (1H, br d, J 7.2
Hz), 4.87 (1H, m), 4.09 (2H, dt, J 2.5, 6.6 Hz), 3.12 (2H, d, J 6.0 Hz), 1.99 (3H, s), 1.59 (2H, m), 1.27
(10H, m) 0.88 (3H, t, J 6.4 Hz); HRMS calcd for C20H32NO3 (M+1+) 334.238219. Found: 334.237586.
1.8. N-Acetyl-L-phenylalanine cinnamyl ester (3e)
From 1, (10.5 mg, 45.0 µmol) and 2e (60 mg, 447 µmol). White crystals (12.3 mg, 85%, >99% e.e.),
m.p. 91.0–93.0°C. [α]D25=+4.9 (c 0.88, CHCl3); 1H NMR δ 7.41–7.20 (8H, m), 7.13–7.08 (2H, m), 6.64
(1H, d, J 16.0 Hz), 6.21 (1H, dt, J 16.0, 6.8 Hz), 5.93 (1H, br d, J 8.0 Hz), 4.92 (1H, m), 4.77 (2H, br d,
J 6.6 Hz), 3.14 (2H, d, J 5.8 Hz) 2.00 (3H, s); HRMS calcd for C20H21NO3 (M+) 323.152118. Found:
323.152144.
1.9. N-Acetyl-L-phenylalanine 5-phenylpentyl ester (3f)
From 1, (10.6 mg, 45.5 µmol) and 2f (77 µL, 457 µmol). Colorless oil (12.7 mg, 77%, >99% e.e.).
[α]D26=+47.1 (c 0.81, CHCl3); 1H NMR δ 7.43–7.05 (10H, m), 5.91 (1H, br d, J 7.2 Hz), 4.87 (1H, m),
4.08 (2H, dt, J 6.9, 2.6 Hz), 3.10 (2H, d, J 6.9 Hz), 2.61 (2H, t, J 7.2 Hz), 1.97 (3H, s), 1.63 (4H, m),
1.33 (2H, m); HRMS calcd for C22H27NO3 (M+) 353.199094. Found: 353.199583.
1.10. N-Acetyl-L-phenylalanine 2-naphthalenemethyl ester (3g)
From 1, (10.6 mg, 45.5 µmol) and 2g (73.4 mg, 464 µmol). White crystals (11.0 mg, 70%, >99% e.e.),
m.p. 122.0–123.5°C. [α]D25=+5.8 (c 0.53, CHCl3); H NMR δ 7.87–7.78 (4H, m), 7.52 (2H, m), 7.41
1
(1H, dd, J 8.6, 1.6 Hz), 7.17 (3H, m), 6.98 (2H, m), 5.92 (1H, br d, J 7.6 Hz), 5.35 (1H, d, J 12.2 Hz),
5.26 (1H, d, J 12.2 Hz), 4.95 (1H, m), 3.12 (2H, d, J 5.8 Hz), 1.99 (3H, s); HRMS calcd for C22H21NO3
(M+) 347.152144. Found: 347.151309.
1.11. N-Acetyl-L-phenylalanine 4-biphenylmethyl ester (3h)
From 1, (10.3 mg, 44.2 µmol) and 2h (80.6 mg, 437 µmol). White crystals (12.3 mg, 75%, >99% e.e.),
m.p. 123.5–125.0°C. [α]D26=−3.4 (c 0.44, CHCl3); 1H NMR δ 7.62–7.57 (4H, m), 7.51–7.34 (4H, m),

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R. C. Lloyd et al. / Tetrahedron: Asymmetry 9 (1998) 551–561
7.26–7.20 (4H, m), 7.02–6.95 (2H, m), 5.90 (1H, br d, J 8.0 Hz), 4.94 (1H, m), 3.14 (2H, d, J 6.0 Hz),
1.99 (3H, s); HRMS calcd for C24H23NO3 (M+) 373.167794. Found: 373.166087.
1.12. N-Acetyl-L-phenylalanine cyclohexylmethyl ester (3i)
From 1, (11.6 mg, 49.8 µmol) and 2i (61 µL, 498 µmol). White crystals (10.1 mg, 66%, >99% e.e.),
m.p. 80.0–82.0°C. [α]D26=+51.4 (c 0.84, CHCl3); H NMR δ 7.35–7.22 (3H, m), 7.16–7.07 (2H, m),
1
5.91 (1H, br d, J 7.2 Hz), 4.90 (1H, m), 3.91 (2H, m), 3.12 (2H, d, J 5.8 Hz), 2.00 (3H, s), 1.80–1.55 (6H,
br m), 1.35–1.13 (3H, br m), 1.07–0.82 (2H, br m); HRMS calcd for C18H26NO3 (M+H+) 304.191269.
Found: 304.191834.
1.13. N-Acetyl-L-phenylalanine 2-phenyl-1-propyl ester (3j)
From 1, (10.1 mg, 43.3 µmol) and (ꢀ)-2j (60 µL, 429 µmol). Colorless oil (7.5 mg, 53%, 30% d.e.).
1H NMR δ 7.37–7.18 (8H, m), 7.01–6.83 (2H, m), 5.87 (1H, br d, J 8.0 Hz), 4.84 (1H, m), 4.18 (2H,
m), 3.08–2.94 (3H, m), 1.95 (3H, s), 1.28 (3H, d, J 7.2 Hz); HRMS calcd for C20H23NO3 (M+1+)
326.175619. Found: 326.177031.
1.14. N-Acetyl-L-phenylalanine 2-methyl-1-pentyl ester (3k)
From 1, (10.3 mg, 44.2 µmol) and (ꢀ)-2k (52 µL, 419 µmol). Colorless oil (7.5 mg, 58%, 26% d.e.).
1H NMR δ 7.29–7.22 (3H, m), 7.12–7.07 (2H, m), 5.91 (1H, br d, J 7.3 Hz), 4.89 (1H, m), 4.01–3.84
(2H, m), 3.11 (2H, d, J 5.8 Hz), 1.98 (3H, s), 1.74 (1H, m), 1.30 (4H, m), 0.89 (6H, m); HRMS calcd for
C17H25NO3 (M+) 291.183444. Found: 291.183350.
1.15. N-Acetyl-L-phenylalanine 3-methyl-1-pentyl ester (3l)
From 1, (10.3 mg, 44.2 µmol) and (ꢀ)-2l (52 µL, 429 µmol). Colorless oil (7.1 mg, 55%, 21% d.e.).
1H NMR δ 7.30–7.20 (3H, m), 7.14–7.05 (2H, m), 5.90 (1H, br d, J 8.1 Hz), 4.88 (1H, m), 4.13 (2H, m),
3.12 (2H, app d, J 6.1 Hz), 1.99 (3H, s), 1.70–1.10 (5H, br m), 0.87 (6H, m); HRMS calcd for C17H25NO3
(M+) 291.183444. Found: 291.184308.
1.16. N-Acetyl-L-phenylalanine 1-phenylethyl ester (3m)24
From 1, (9.9 mg, 42.4 µmol) and (ꢀ)-2m (52 µL, 431 µmol). Stirred at 50°C for 72 h. Colorless oil
(4.9 mg, 37%, 95% d.e.). [α]D22=+4.9 (c 0.49, CHCl3) [lit.24,25 [α]D25=−31.4 (c 0.5, CHCl3)]; 1H NMR
δ 7.38–7.25 (7H, m), 7.17–7.11 (2H, m), 6.80 (1H, m), 5.90 (2H, m), 3.10 (2H, m), 1.96 (3H, s), 1.57
(3H, d, J 6.6 Hz); HRMS calcd for C19H21NO3 (M+) 311.152144. Found: 311.153015.
1.17. N-Acetyl-L-phenylalanine 2-butyl ester (3n)24
From 1, (10.3 mg, 44.2 µmol) and (ꢀ)-2n (40 µL, 436 µmol). Stirred at 50°C for 72 h. Colorless oil
(5.9 mg, 51%, 46% d.e.). 1H NMR δ 7.32–7.22 (3H, m), 7.18–7.09 (2H, m), 5.90 (1H, br), 4.85 (2H, m),
3.11 (2H, m), 1.99 (s, minor diastereoisomer), 1.97 (s, major diastereoisomer), 1.56 (2H, m), 1.20 (3H,
m), 0.88 (3H, m); HRMS calcd for C15H21NO3 (M+) 263.152144. Found: 263.153343.

R. C. Lloyd et al. / Tetrahedron: Asymmetry 9 (1998) 551–561
559
1.18. N-Acetyl-L-phenylalanine 2-octyl ester (3o)24
From 1, (10.5 mg, 45.0 µmol) and (ꢀ)-2o (68 µL, 427 µmol). Stirred at 50°C for 72 h. Colorless
oil (2.9 mg, 20%, >99% d.e.). [α]D25=+47.1 (c 0.48, CHCl3) [lit.24 [α]D22=+53.3 (c 0.80, CHCl3)]; 1H
NMR δ 7.29–7.23 (3H, m), 7.15–7.10 (2H, m), 5.90 (1H, br d, J 7.2 Hz), 4.86 (2H, m), 3.11 (2H, app
d, J 5.8 Hz), 1.98 (3H, s), 1.49 (2H, m), 1.26 (8H, br s), 1.18 (3H, d, J 6.4 Hz), 0.89 (3H, t, J 6.3 Hz);
HRMS calcd for C19H29NO3 (M+) 319.214744. Found: 319.215998.
1.19. N-Acetyl-L-phenylalanine exo-norbornyl ester (3p)
From 1, (10.1 mg, 43.3 µmol) and (ꢀ)-2p (48.9 mg, 435 µmol). Stirred at 50°C for 72 h. Colorless oil
1
(6.6 mg, 50%, 25% d.e.). H NMR δ 7.29–7.22 (3H, m), 7.11–7.07 (2H, m), 6.21 (1H, br d, J 7.4 Hz),
4.79 (1H, m), 4.57 (1H, m), 3.06 (2H, m), 2.24 (2H, m), 1.94 (3H, s), 1.62 (1H, m), 1.42 (3H, m), 1.06
(2H, m); HRMS calcd for C18H23NO3 (M+) 301.167794. Found: 301.167151.
1.20. N-Acetyl-L-phenylalanine endo-norbornyl ester (3q)
From 1, (10.1 mg, 43.3 µmol) and (ꢀ)-2q (51.1 mg, 455 µmol). Stirred at 50°C for 72 h. Colorless oil
(7.4 mg, 56%, 82% d.e.). 1H NMR δ 7.30–7.21 (3H, m), 7.13–7.08 (2H, m), 6.15 (1H, m), 4.88 (2H, m),
3.07 (2H, m), 2.40 (1H, m), 2.18 (1H, m), 1.97 (3H, s), 1.96 (1H, m), 1.56 (2H, m), 1.31(4H, m), 0.90
(1H, m); HRMS calcd for C18H23NO3 (M+) 301.167794. Found: 301.168322.
1.21. 3-Phenylpropionic acid methyl ester (5a)26
From 4, (10.2 mg, 57.9 µmol) and 2a (24 µL, 593 µmol). Reaction performed in MeCN at room
1
temperature. Colorless oil (8.9 mg, 94%). H NMR δ 7.32–7.15 (5H, m), 3.67 (3H, s), 2.96 (2H, t, J 7.4
Hz), 2.62 (2H, t, J 7.4 Hz); HRMS calcd for C10H12O2 (M+) 164.083730. Found: 164.084406.
1.22. 3-Phenylpropionic acid 2-phenylpropyl ester (5j)
From 4, (9.9 mg, 56.3 µmol) and (ꢀ)-2j (79 µL, 565 µmol). Colorless oil (7.9 mg, 52%, 50% e.e.).
1H NMR δ 7.35–7.14 (10H, m), 4.18 (2H, m), 3.06 (1H, m), 2.90 (2H, t, J 7.6 Hz), 2.59 (2H, t, J 7.4
Hz), 1.25 (3H, d, J 6.0 Hz); HRMS (+FAB) calcd for C18H21O2 (M+H+) 269.1542. Found: 269.1573.
1.23. 3-Phenylpropionic acid 1-phenylethyl ester (5m)24
From 4, (14.1 mg, 80.0 µmol) and (ꢀ)-2m (112 µL, 802 µmol). Stirred at 50°C for 72 h. Colorless
oil (8.6 mg, 45%, 90% e.e.). [α]D25=−39.0 (c 0.39, CHCl3) [lit.24 [α]D22=−47.9 (c 1.70, CHCl3)]; 1H
NMR δ 7.36–7.15 (10H, m), 4.89 (1H, q, J 7.5 Hz), 2.96 (2H, t, J 7.6 Hz), 2.59 (2H, t, J 7.6 Hz), 1.26
(3H, d, J 7.2 Hz); HRMS calcd for C17H18O2 (M+) 254.130680. Found: 254.131134.
1.24. 3-Phenylpropionic acid 2-octyl ester (5o)24,27
From 4, (10.5 mg, 59.6 µmol) and (ꢀ)-2o (95 µL, 597 µmol). Stirred at 50°C for 72 h. Colorless oil
(4.9 mg, 31%, >99% e.e.). [α]D25=+7.9 (c 0.47, CHCl3) [lit.24 [α]D22=+12.1 (c 0.80, CHCl3)]; 1H NMR
δ 7.33–7.19 (5H, m), 4.89 (1H, m), 2.95 (2H, t, J 7.4 Hz), 2.61 (2H, t, J 7.4 Hz), 1.48 (2H, m), 1.25

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R. C. Lloyd et al. / Tetrahedron: Asymmetry 9 (1998) 551–561
(8H, br s), 1.17 (3H, d, J 6.4 Hz), 0.88 (3H, t, J 6.4 Hz); HRMS (+FAB) calcd for C17H27O2 (M+H+)
263.2011. Found: 263.1980.
Acknowledgements
We thank the Natural Sciences and Engineering Research Council of Canada (NSERC) and Genencor
International Inc. for financial support, and NSERC for the award of a postdoctoral scholarship (to MD).
We also thank Dr. Per Berglund for helpful initial discussions.
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