Covalent modification of subtilisin Bacillus lentus Cysteine mutants with enantiomerically pure chiral auxiliaries causes remarkable changes in activity

Article abstract of DOI:10.1016/S0968-0896(00)00121-8

Methanethiosulfonate reagents may be used to introduce virtually unlimited structural modifications in enzymes via reaction with the thiol group of cysteine. The covalent coupling of enantiomerically pure (R) and (S) chiral auxiliary methanethiosulfonate ligands to cysteine mutants of subtilisin Bacillus lentus induces spectacular changes in catalytic activity between diastereomeric enzymes. Amidase and esterase kinetic assays using a low substrate approximation were used to establish k(cat)/K(M) values for the chemically modified mutants, and up to 3-fold differences in activity were found between diastereomeric enzymes. Changing the length of the carbon chain linking the phenyl or benzyl oxazolidinone ligand to the mutant N62C by a methylene unit reverses which diastereomeric enzyme is more active. Similarly, changing from a phenyl to benzyl oxazolidinone ligand at S166C reverses which diastereomeric enzyme is more active. Chiral modifications at S166C and L217C give CMMs having both high esterase k(cat)/K(M)'s and high esterase to amidase ratios with large differences between diastereomeric enzymes. Copyright (C) 2000 Elsevier Science Ltd.

Full text of DOI:10.1016/S0968-0896(00)00121-8

Bioorganic & Medicinal Chemistry 8 (2000) 1957±1968
Covalent Modi®cation of Subtilisin Bacillus lentus Cysteine
Mutants with Enantiomerically Pure Chiral Auxiliaries
Causes Remarkable Changes in Activity
Michael Dickmany and J. Bryan Jones*
Department of Chemistry, University of Toronto, 80 St. George St, Toronto, Ontario, Canada M5S 3H6
Received 3 March 2000; accepted 21 April 2000
AbstractÐMethanethiosulfonate reagents may be used to introduce virtually unlimited structural modi®cations in enzymes via
reaction with the thiol group of cysteine. The covalent coupling of enantiomerically pure (R) and (S) chiral auxiliary meth-
anethiosulfonate ligands to cysteine mutants of subtilisin Bacillus lentus induces spectacular changes in catalytic activity between
diastereomeric enzymes. Amidase and esterase kinetic assays using a low substrate approximation were used to establish kcat/KM
values for the chemically modi®ed mutants, and up to 3-fold di€erences in activity were found between diastereomeric enzymes.
Changing the length of the carbon chain linking the phenyl or benzyl oxazolidinone ligand to the mutant N62C by a methylene unit
reverses which diastereomeric enzyme is more active. Similarly, changing from a phenyl to benzyl oxazolidinone ligand at S166C
reverses which diastereomeric enzyme is more active. Chiral modi®cations at S166C and L217C give CMMs having both high
esterase kcat/KM's and high esterase to amidase ratios with large di€erences between diastereomeric enzymes. # 2000 Elsevier
Science Ltd. All rights reserved.
Introduction
In this paper, we endeavor to explore changes in enzyme
catalysis induced by the covalent attachment of enantio-
merically pure MTS ligands derived from chiral auxiliaries
to cysteine mutants of SBL (Scheme 1). We selected
mandelic acid and several oxazolidinones constructed
from glycine, valine, phenylglycine, phenylalanine and
cis-1-amino-indanol. We covalently linked the homo-
chiral MTS ligands to cysteine mutants of SBL to create
sets of diastereomeric chemically modi®ed mutants
(CMMs) allowing the observation of enzyme activity
changes due solely to di€erences in the chiral environment
at one site. This methodology acts as a very ®ne and
precise probe of enzymatic catalysis, since any di€er-
ences between diastereomeric enzymes are solely attri-
butable to the changed absolute con®guration of the
ligand.
Many methods for improving the activity and enantio-
selectivity of hydrolases have been investigated. They
include extreme temperatures,1 solvent engineering,2
structural variation of the substrate,3 imprinting,4 lyo-
protectants,5 chemical modi®cation,6 site-directed7 and
random8 mutagenesis. However, the chemical modi®cation
of mutant enzymes has been underused as a method for
generating new hydrolases with novel properties.9 We
have applied this strategy of site-directed mutagenesis
combined with chemical modi®cation to enhance the
activity and alter the speci®city of subtilisin Bacillus len-
tus (SBL).10 Using this technique, cysteine is introduced
at well de®ned locations near important pockets around
the active site, and the thiol is reacted speci®cally with a
methanethiosulfonate (MTS) reagent. This methodology
o€ers almost unlimited possibilities for structural varia-
tion. In addition, wild-type (WT) SBL contains no natural
cysteine, so the structural modi®cation of the cysteine
mutant occurs solely at the targeted site.
Enantiomerically pure MTS ligands, 1a±i, were synthe-
sized and used to chemically modify the N62C, S156C,
S166C and L217C mutants of SBL. These residues were
targeted on the basis of SBL's X-ray crystal structure.11
N62C is in the S2 pocket near His-64.12 S156C and S166C
are at the bottom of the S1 pocket. However, S156C is
surface exposed and S166C is buried pointing into the
*Corresponding author. Tel.: +1-416-978-3859; fax: +1-416-978-
1553; e-mail: jbjones@chem.utoronto.ca
yPresent address: College Universitaire de Saint-Boniface, 200 ave. de
la Cathedrale, Winnipeg, Manitoba, Canada, R2H 0H7.
0
pocket. L217C is found in S1 which is where the leaving
group is bound. A kinetic assay of amidase and esterase
activity was conducted on these new diastereomeric
0968-0896/00/$ - see front matter # 2000 Elsevier Science Ltd. All rights reserved.
PII: S0968-0896(00)00121-8

1958
M. Dickman, J. B. Jones / Bioorg. Med. Chem. 8 (2000) 1957±1968
CMMs in order to investigate their properties and to
probe any changes in selectivity.
give (R)-4 which was protected as its methoxymethyloxy
ether, (R)-5, in excellent yield (90% for two steps). The
ester, (R)-5, was reduced with LiBH4 to the alcohol, (R)-7
(98%), which was converted to the mesylate, (R)-9, and
then to the bromide, (R)-11 (80% for two steps), using
the same conditions as for the methyl ether analogue. This
bromide was reacted with NaSSO2CH3 in DMF for 2
days to give (R)-12 in 61% yield. The alcohol was
deprotected by the action of TFA/H2O to give the MTS
reagent, (R)-1b, in 82% yield. The methanethiosulfonate
(S)-1b was made in an analogous fashion from (S)-
mandelic acid.
Results
Synthesis of MTS reagents 1a±i
For the synthesis of the mandelate based MTS ligands, (R)-
mandelic acid, (R)-2, was O-methylated with Me2SO413 in
NaOH/H2O to give (R)-3 in 37% yield (Scheme 2). The
acid, (R)-3, was reduced in 72% yield with borane in THF
to alcohol, (R)-6, which was converted quantitatively to
mesylate, (R)-8, in CH2Cl2. The mesylate was converted to
bromide, (R)-10 (73%), by the action of LiBr in re¯uxing
acetone, and methanethiosulfonate, (R)-1a, was formed
in 84% yield from bromide, (R)-10, using NaSSO2CH3
in DMF. The methanethiosulfonate (S)-1a was made in
an analogous fashion from (S)-mandelic acid.
The synthesis of oxazolidinone-based methanethiosulfo-
nate ligands is shown in Scheme 3. Oxazolidinones have
been widely used as chiral auxiliaries in asymmetric
synthesis, and the degree of asymmetric induction can be
excellent in chemical transformations ranging from alky-
lations to aldol reactions to Diels±Alder additions.14 The
commercially available oxazolidinones, 13-(R)-16, were
alkylated with 1,3-dibromopropane or 1,2-dibromoethane
in DMSO/KOH15 to give the bromides, 17-(R)-22, which
A similar approach allowed the synthesis of (R)-1b
(Scheme 2). (R)-Mandelic acid, (R)-2, was esteri®ed to
Scheme 1. The corresponding author (S) MTS ligands follow the same code scheme [i.e. (S)-a, (S)-b, (S)-d, (S)-e, (S)-f, (S)-g, (S)-h, (S)-i].
Scheme 2. Reagents. (i) Me2SO4, NaOH, H2O, 37%; (ii) MeOH, H+; (iii) MOM-Cl, CH2Cl2, Et3N (90%, 2 steps); (iv) For (R)-3: BH3, THF, 82%;
For (R)-5: LiBH4, THF, 97%; (v) MeSO2Cl2, Et3N; Dor (R)-8: 100%; (vi) LiBr, acetone; For (R)-10: 84%; For (R)-11: 78% 2 steps; (vii) NaS-
SO2CH3, DMF; For (R)-12: 61%; (vii) TFA, H2O, 82%.

M. Dickman, J. B. Jones / Bioorg. Med. Chem. 8 (2000) 1957±1968
1959
were converted to the methanethiosulfonates, 1c-(R)-1h,
in 38±61% yield over two steps. The MTS reagents (S)-
1d-(S)-1h were made in an identical manner from the (S)
oxazolidinones.
titration18 of their active sites, Ellman's titration19 of
residual thiol (ꢀ2% in all cases), ES-MS after FPLC
puri®cation (mol. wt.Æ6 mass units in all cases), and by
nondenaturing gradient gels, which all showed one band.
The (1R,2S) oxazolidinone, (R)-24, of cis-1R-amino-2S-
indanol, (R)-23, was prepared in quantitative yield by the
reaction of (R)-23, triphosgene and Et3N in CH2Cl2
(Scheme 4).16 (R)-24 was then alkylated with 1,3-dibromo-
propane to make bromide, (R)-25, which was reacted
with NaSSO2CH3 to give (R)-1i (49% yield for two
steps). MTS reagent (S)-1i was synthesized from cis-1S-
amino-2R-indanol in the same manner.
Amidase and esterase kinetic assays were conducted on
these new diastereomeric CMMs. Both assays were run
using a low substrate concentration in order to obtain a
speci®city constant (kcat/KM) that gave us an idea of the
performance of the CMMs and allowed us to compare
diastereomeric enzymes. (At low substrate concentration,
(kcat/KM)=ninitial/[Enzyme][Substrate]). The results are
presented in Table 1.
Enzyme kinetic assay
Discussion
Subtilisin mutants were obtained from Genencor Inc.17
and modi®ed with the homochiral MTS reagents.
Characterization of the new CMMs was done by PMSF
Chiral auxiliaries are employed in asymmetric organic
synthesis to block one diastereotopic face of a molecule
Table 1. Kinetic assaya of SBL CMMs
Amidase assay kcat
1
/
Esterase assay kcat/
1
KM (mM 1 s
)
KM (mM 1 s
)
Enzyme
(R)
(S)
(R) (S)
WTb
209Æ15
92Æ7
3560Æ540
4380Æ655
N62Cb
N62C-a
N62C-b
N62C-c
N62C-d
N62C-e
N62C-f
N62C-g
N62C-h
N62C-i
218Æ9
226Æ11
220Æ9
5156Æ131
3571Æ73
5483Æ106
3054Æ171
187Æ10
181Æ6
9185Æ407
333Æ13
458Æ13
245Æ3
185Æ4
262Æ5
165Æ3
284Æ5
308Æ7
150Æ1
244Æ7
335Æ7
228Æ6
5440Æ78
13870Æ920
4995Æ87
4098 Æ151
6564Æ157
3261Æ163
4120Æ159
7591Æ209
3279Æ135
3635Æ58
6149Æ202
4675Æ143
S166Cb
84Æ4
75Æ1
350Æ41
S166C-a
S166C-b
S166C-c
S166C-d
S166C-e
S166C-f
S166C-g
S166C-h
S166C-i
72Æ2
48Æ2
26Æ1
15Æ1
1677Æ16
1061Æ18
1246Æ48
929Æ27
4898Æ196
75Æ1
101Æ3
22Æ1
76Æ1
64Æ2
52Æ1
37Æ1
80Æ2
47Æ1
4215Æ157
4076Æ111
1495Æ134
4281Æ96
2150Æ107
1488Æ54
4475Æ196
3964Æ90
3277Æ134
4069Æ165
5446Æ211
4556Æ170
Scheme 3. Reagents: (i) KOH, DMSO, Br(CH2)nBr; (ii) NASSo2CH3,
DMF.
104Æ2
35Æ1
20Æ1
L217Cb
L217C-a
L217C-b
L217C-c
L217C-d
L217C-e
L217C-f
L217C-i
51Æ4
85Æ1
5540Æ798
204Æ5
175Æ3
144Æ4
227Æ6
10140Æ230
9147Æ167
8075Æ144
8714Æ324
5917Æ200
105Æ3
120Æ4
73Æ2
104Æ2
184Æ3
79Æ2
8315Æ171
8015Æ413
6435Æ169
7914Æ272
9296Æ665
6696Æ255
5128Æ163
7321Æ330
118Æ4
171Æ7
S156Cb
S156C-a
S156C-b
S156C-e
147Æ8
Ðc
102Æ2
85Æ3
88Æ2
98Æ1
90Æ2
92Æ4
2468Æ45
2284Æ81
1796Æ63
1928Æ59
2528Æ68
2179Æ38
aThe amidase assay was done at 0.05 and 0.1 mM N-Suc-AAPF-pNA
in 0.1 M Tris at pH 8.6, and the esterase assay was conducted at 0.015
and 0.03 mM N-Suc-AAPF-SBn in 0.1 M Tris at pH 8.6. Assay errors
are the mean standard errors from sets of six replicates.
bkcat/KM obtained by full kinetic run of 8 substrate concentrations and
calculation of independent kcat and KM values. Errors were obtained
from the curve-®tting errors in kcat and KM
.
cDetermination of esterase kcat/KM for S156C was impossible due to
rapid reaction between the mutant and Ellman's reagent.
Scheme 4. Reagents: (i) Triphosgene, CH2Cl2, Et3N, 100%; (ii) KOH,
DMSO, Br(CH2)3Br; (ii) NaSSO2CH3, DMF.

1960
M. Dickman, J. B. Jones / Bioorg. Med. Chem. 8 (2000) 1957±1968
thus forcing the reaction to the other face which results
in the formation of solely one diastereomer. Combining
this negative, blocking methodology with the positive,
accelerating e€ect of enzymes seemed a natural mix. In
fact, the covalent coupling of enantiomerically pure (R)
and (S) chiral auxiliary MTS ligands to SBL cysteine
mutants has caused remarkable changes in enzyme activ-
ity. We can attribute these changes uniquely to the di€er-
ence in spatial orientation at the ligand stereocentre when
comparing diastereomeric enzymes. The extraordinary
di€erences in catalytic activity between diastereomeric
enzymes can be compared in Figures 1a±c.
N62C
Of the N62C CMMs, the N62C-e set of diastereomeric
CMMs is remarkable in displaying both high catalytic
activity and a large di€erence between diastereomers.
N62C-(R)-e is both an excellent amidase (2.2-fold better
than WT) and an excellent esterase (3.9-fold better than
WT). In addition, the (S)-diastereomer is a good amidase
(308 mM 1 s 1) and esterase (6564 mM 1 s 1), but not
as good as the (R)-diastereomer. Thus, there is a large
di€erence between the two diastereomeric CMMs with
respect to esterase performance ((R) is 2.1-fold better than
(S)) and a moderate di€erence in amidase activity. At the
same time, the achirally modi®ed mutant (N62C-c) is only
as good an amidase (181 mM 1 s 1) as WT and a poorer
esterase (9185mM 1 s 1) than N62C-(R)-e. These obser-
vations indicate that not only does the addition of a
phenyl group at the 4 position of the oxazolidinone ring
increase enzyme activity, but that the addition must be
(R)-phenyl. Thus, the (R)-e modi®cation at N62C is
a€ecting the enzyme in a unique manner. Individual kcat
and KM values were determined for the three enzymes,
N62C-c and the N62C-e set, and these results are presented
in Table 2 along with WT values for comparison. It is
obvious that the kinetic assay using the low substrate
approximation slightly underestimates the kcat/KM values,
but the ratios of catalytic activity between diastereomeric
enzymes remain approximately the same.
Modi®cation of N62C with (R)-1e, (S)-1e and 1c decreases
KM indicating better binding of the substrate, and in the
case of amidase activity, it is this KM e€ect that is the
source of the increased kcat/KM, since these N62C
CMMs have similar kcat values to the WT. However, the
changes in esterase activity for these enzymes are more
complex. N62C-(R)-e and N62C-c show signi®cantly
higher kcat and lower KM values than WT, giving overall
5.4-fold and 3.7-fold better esterase activity than WT,
respectively. The N62C-(S)-e CMM does not display
these characteristics. While it does bind the substrate
very well and achieve half its maximum turnover rate at
low substrate concentration (KM=0.15 mM), its kcat
(1106 s 1) is much lower than WT. Therefore, it appears
that a 4R-phenyl substitution on the oxazolidinone
improves overall catalytic performance by increasing
kcat and lowering KM.
In an attempt to improve on these results, the ethyl linked
phenyl and benzyl oxazolidinone N62C CMMs were pre-
pared (N62C-g and N62C-h). Surprisingly, there was a
reversal of which modi®cation made the best enzyme. In
the case of the propyl linked CMMs (N62C-e and
N62C-f), the (R) modi®cation was the best amidase and
esterase for both phenyl and benzyl groups. However, the
(S) modi®cation was the best when these same groups
were ethyl linked. This brings to mind the ¯ipping of
substrate preference for transesteri®cation reactions
catalyzed by WT from (S) to (R) and back to (S) for
secondary alcohols, b-branched primary alcohols and g-
branched primary alcohols respectively.20 However, in the
present situation, the substrate does not change. Rather,
the ability of the enzyme to convert substrate to product is
altered depending upon the stereocentre of the covalently
Figure 1. Comparison of speci®city constants for N62C, S166C and
L217C CMMs.

M. Dickman, J. B. Jones / Bioorg. Med. Chem. 8 (2000) 1957±1968
Table 2. Kinetic parameters of WT and selected SBL CMMsa
1961
Amidase
Esterase
kcat/KM
(mM 1 s
Enzyme
kcat
1
KM
(mM)
kcat/KM
(mM 1 s
kcat/KM
(mM 1 s 1) assay
kcat
1
KM
(mM)
kcat/KM
1
1
1
(s
)
)
(s
)
)
(mM
s
1) assay
WT
153Æ4
163Æ2
164Æ2
193Æ3
0.73Æ0.05
0.26Æ0.01
0.41Æ0.02
0.63Æ0.03
209Æ15
627Æ26
400Æ20
307Æ16
Ð
1940Æ180
2894Æ117
1106Æ45
3447Æ66
0.54Æ0.07
0.15Æ0.02
0.15Æ0.02
0.26Æ0.01
3560Æ540
Ð
N62C-(R)-e
N62C-(S)-e
N62C-c
458Æ13
308Æ7
181Æ6
19293Æ2895
7373Æ1098
13258Æ710
13868Æ920
6564Æ157
9185Æ407
aNotation as in Table 1.
linked ligand as well as the number of bonds present in the
link between the enzyme backbone and the stereocentre.
oxazolidinone modi®cations ((R)-e and (R)-g) produce
S166C CMMs that are better than the (S) analogues,
but the (S)-benzyl oxazolidinones ((S)-f and (S)-h) give
signi®cantly better S166C CMMs than the (R).
S166C
Modi®cations at S166C produced many sets of diaster-
eomeric CMMs with largedi€erences in activity. Primarily,
the 1a, 1b, 1f, 1g, 1h and 1i modi®cations produced
CMMs with greater than 2-fold variations in amidase
between diastereomeric CMMs. The largest di€erence
of any set of CMMs was achieved with S166C-b which
has a [kcat/KM (R)]/[kcat/KM (S)] ratio of 3.2. Notably,
the modi®cations with the phenyl and benzyl oxazolidi-
nones at S166C reverse which diastereomeric CMM has
greater catalytic activity in a way similar to the same
modi®cations at N62C. However, at S166C the reversal is
caused by the addition of a methylene unit directly to the
stereocentre of the oxazolidinone ligand. The (R)-phenyl
Though none of these CMMs showed signi®cantly greater
than WT activity, S166C-(S)-g and S166C-(S)-i are good
1
esterases (4069 mM 1 s
and 4556mM 1 s 1, respec-
tively) and have high esterase/amidase ratios of 110 and
97 making them good candidates to be superior peptide
ligation catalysts (Fig. 2a). S166C-(S)-a and S166C-(S)-b
have relatively high esterase/amidase ratios (48 and 62)
compared to S166C (4) and WT (23), but these two
CMMs are very poor esterases. Interestingly, for chiral
modi®cations at S166C, the (S)-ligand consistently gives
a CMM with a higher esterase to amidase ratio than the
(R)-ligand, except in the case of the 1f where the two
diastereomeric enzymes have similar ratios.
Table 3. Electro-spray mass spectral Data for CMMsa
Enzyme
Calculated mass
Found mass
(R)
(S)
(R)
(S)
N62C-a
N62C-b
N62C-c
N62C-d
N62C-e
N62C-f
N62C-g
N62C-h
N62C-i
S166C-a
S166C-b
S166C-c
S166C-d
S166C-e
S166C-f
S166C-g
S166C-h
S166C-i
L217C-a
L217C-b
L217C-c
L217C-d
L217C-e
L217C-f
L217C-i
S156C-a
S156C-b
S156C-e
26853
26839
26846
26888
26922
26936
26908
26922
26934
26880
26866
26873
26915
26949
26963
26935
26949
26961
26854
26840
26847
26889
26923
26937
26935
26880
26866
26949
26853
26839
26855
26841
26850
26889
26921
26939
26910
26924
26937
26881
26862
26877
26915
26950
26964
26937
26951
26964
26850
26842
26847
26892
26922
26938
26937
26883
26866
26949
26854
26838
26888
26922
26936
26908
26922
26934
26880
26866
26889
26921
26939
26907
26924
26936
26886
26872
26915
26949
26963
26935
26949
26961
26854
26840
26916
26951
26963
26934
26949
26964
26850
26840
26889
26923
26937
26935
26880
26866
26949
26892
26923
26940
26937
26883
26868
26949
Figure 2. Changes in esterase to amidase activity ratios for S166C and
L217C CMMs.
aMol. wt.Æ6 mass units in all cases.

1962
M. Dickman, J. B. Jones / Bioorg. Med. Chem. 8 (2000) 1957±1968
L217C
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. Compounds were iden-
While no large di€erences were observed between
diastereomeric CMMs, the chiral modi®cations at
L217C produced many CMMs that could be used as
peptide ligation catalysts due to their high esterase/
amidase ratio (Fig. 2b). L217C-(S)-d has a very high
esterase kcat/KM (9296 mM 1 s 1) and a low amidase
value (104 mM 1 s 1) giving it a relatively high esterase/
amidase ratio of 89. L217C-(R)-f has a similar ratio of
88 and a good esterase kcat/KM (6435 mM 1 s 1). While it
is true that the unmodi®ed L217C mutant has the highest
ratio in the group (109), this is mitigated by its lower
esterase kcat/KM (5540 mM 1 s 1). Encouragingly, all of
these CMMs should catalyze the formation of peptide
bonds from an ester acyl donor and amine nucleophile
very eciently.
1
ti®ed by their H (200 MHz) and 13C (50.3 MHz) NMR
spectra, run using a Varian Gemini NMR spectrometer,
their IR spectra using a Perkin±Elmer FTIR Spectrum
1000 instrument, and HRMS data were acquired using a
Micromass 70-250S (double focussing) mass spectro-
meter for EI spectra and a Micromass ZAB-SE for FAB
spectra. Enantiomeric excesses of methanethiosulfonates
((R)-1a, (S)-1a, (R)-1b and (S)-1b) were determined by
HPLC on a Chiralcel OJ column using a hexane: iso-
propanol eluent system. Enantiomeric excesses (ee) of
bromides ((R)-18, (S)-18, (R)-19, (S)-19, (R)-20, (S)-20,
(R)-21, (S)-21, (R)-22, (S)-22, (R)-25 and (S)-25) were
determined by HPLC on a Chiralcel OD column using
the same eluent system. The standard substrate N-Succi-
nyl AlaAlaProPhe-paranitroanilide (N-Suc-SSPF-pNA)
and N-Succinyl AlaAlaProPhe-thiobenzyl ester (N-Suc-
SSPF-SBn) were purchased from BACHEM California.
S156C
Modi®cation of S156C by 1a, 1b and 1e revealed no
enzymes with either high activity or large di€erence
between diastereomers. This is not surprising, because
the S156C side chain is surface exposed, so it is probable
that the ligand modi®ers either point out of, or are not
closely associated with the S1 pocket. For this reason,
the kinds of subtle variations expected due to spatial
orientation were not found at S156C. As a result, no
further modi®cations were made of this mutant.
Preparation of methanethiosulfonate reagents
(R)-2-Methoxy-2-phenyl-ethylmethanethiosulfonate ((R)-
1a). (R)-Mandelic acid (4.678 g, 30.75 mmol) was dis-
solved in 6 M NaOH (50 mL, 300 mmol) and dimethyl
sulfate (14.6 mL, 154 mmol) was added over 1 h so that
the temperature stayed at 50 ꢁC. After another 1 h of
stirring, H2O (50 mL) was added, and the solution was
acidi®ed to pH 1 with 12 M HCl. The mixture was satu-
rated with NaCl, extracted with EtOAc (3Â100 mL), and
the extracts dried with Na2SO4. After ®ltration and eva-
poration under reduced pressure, the solid was pulverized,
re¯uxed in hexanes (100 mL) for 15min and hot ®ltered.
The insoluble (R)-mandelic acid (2.71 g, 58%) was recov-
ered, and the hexanes evaporated under reduced pressure
to give (R)-2-methoxy-mandelic acid, (R)-3 (1.91 g, 37%)
which was used directly in the next step.
Conclusion
It has been found that the modi®cation of cysteine
mutants of SBL with enantiomerically pure MTS ligands
e€ects considerable changes in enzyme activity. Amidase
and esterase kinetic assays using a low substrate approx-
imation, found up to 3-fold di€erences in activity between
diastereomeric enzymes. N62C-(R)-e was particularly
remarkable. Its amidase kcat/KM was 1.56-fold better than
its diastereomer, N62C-(S)-e, and 3-fold better than WT.
Also, the esterase kcat/KM of N62C-(R)-e was 2.6-fold
better than its diastereomer and 5.4-fold better than
WT. Changing the length of the carbon chain linking
the phenyl or benzyl oxazolidinone ligand to N62C by a
methylene unit reverses which diastereomeric enzyme is
more active. In a similar fashion, changing from a
phenyl to benzyl oxazolidinone ligand at S166C reverses
which diastereomeric enzyme is more active. Work is in
progress investigating the peptide ligation and trans-
esteri®cation capabilities of the CMMs discussed in this
paper. In addition, the attachment of enantiomerically
pure ligands containing charged groups to SBL mutants
is being pursued.
(R)-3 (1.91 g, 11.46 mmol), was placed under Ar and dry
THF (15 mꢁL) was added. The resulting solution was
.
cooled to 0 C and 1 M BH3 THF (17.2 mL, 17.2 mmol)
was added over 1 min. The ice bath was removed, and the
reaction was allowed to warm to 20 ꢁC. After stirring
overnight, the reaction mixture was poured into a stirred
mixture of EtOAc (200mL)/saturated aqueous NaHCO3
(100mL). The aqueous layer was saturated with NaCl
and extracted with EtOAc (3Â150 mL). The combined
EtOAc fractions were dried with MgSO4, ®ltered and
evaporated under reduced pressure. Flash chromato-
graphy was conducted using a step gradient (25% EtOAc/
75% hexanes to 33% EtOAc/67% hexanes) to give (R)-2-
methoxy-2-phenyl-1-ethanol, (R)-6 (1.26 g, 72%), as a col-
25
d
25
d
orless oil. ‰ꢀŠ
114.6ꢁ (c 1.27, EtOH) [lit.21 ‰ꢀŠ
117.3ꢁ
(c 1.006, EtOH)]; IR, 1H NMR and 13C NMR data were
identical to the literature.21
Experimental
The N62C, L217C, S166C, and S156C mutants of
subtilisin Bacillus lentus were prepared and puri®ed by
the general method.17 Spectrophotometric measure-
ments were made on a Perkin±Elmer Lamda 2 spectro-
photometer.
(R)-6 (1.25 g, 8.213 mmol) and Et3N (2.29 mL,
16.43 mmol) were dissolved in CH2 Cl2 (20 mL) under Ar
and cooled to 0 ꢁC. MsCl (0.95 mL, 12.27 mmol) was
added over 1 min, and stirred for 10 min. The ice bath
was removed, and the solution was stirred overnight.

M. Dickman, J. B. Jones / Bioorg. Med. Chem. 8 (2000) 1957±1968
1963
The reaction was then poured into EtOAc (200 mL)/
saturated aqueous NaHCO3 (100 mL), and then stirred
and saturated with NaCl. The aqueous layer was extrac-
ted with EtOAc (3Â150 mL), and the combined organic
fractions were dried with MgSO4. After ®ltration and
evaporation under reduced pressure, the crude product
was puri®ed by ¯ash chromatography using 50%
EtOAc/50% hexanes to give (R)-2-methoxy-2-phenyl-1-
(S)-1a was prepared in the same manner as (R)-1a. From
(S)-10 (1.00 g, 4.649 mmol) was obtained (S)-1a (0.961 g,
1
84%, eeꢂ98%). Its H NMR and 13C NMR data were
identical to (R)-1a. ‰ꢀŠ25 +93.8ꢁ (c 1.002); HRMS (FAB+)
d
m/z: calcd for C10H14O3S2+H, 247.0463; found, 247.0474.
(R)-2-Hydroxy-2-phenyl-ethylmethanethiosulfonate ((R)-
1b). (R)-Mandelic acid (2.568 g, 16.87 mmol) and 2,2-
dimethoxypropane (5.1 mL, 41.48 mmol) were dissolved
in MeOH (100mL) and 12 M HCl (100mL) was added.
The resulting solution was stirred for 20h under a CaCl2
tube and evaporated to dryness under reduced pressure.
EtOAc (100mL) and saturated aqueous NaHCO3
(100mL) were added, and the aqueous phase was extrac-
ted with EtOAc (3Â100 mL). The organic fractions were
dried with MgSO4, and evaporated under reduced pres-
sure to give (R)-methyl mandelate, (R)-4, quantitatively
(2.78 g) as a white solid which was of sucient purity
for the next step.
ethylmethanesulfonate, (R)-8, quantitatively (1.88 g)
97.4ꢁ (c 1.36, CHCl3); 1H NMR
25
d
as a colorless oil. ‰ꢀŠ
(CDCl3) d 7.30±7.40 (5H, m), 4.47±4.52 (1H, m),
4.20±4.36 (2H, m), 3.30 (3H, s), 2.99 (3H, s); 13C
NMR (CDCl3) d 136.6, 128.8, 126.9, 81.5, 72.7, 57.0,
37.6.
(R)-8 (1.88 g, 8.160 mmol) and LiBr (3.54 g, 40.76 mmol)
were re¯uxed in freshly distilled acetone (20 mL) for 20 h
under a CaCl2 drying tube. After cooling and evaporation
to dryness under reduced pressure, hexanes (30 mL) were
added and the mixture ®ltered. The ®ltrate was evaporated
under reduced pressure, and ¯ash chromatography of the
crude product was done using a step gradient (hexanes to
5% EtOAc/95% hexanes) to give (R)-2-methoxy-2-
(R)-4 (1.695 g, 10.20 mmol) and Huenig's base (6.22 mL,
35.70 mmol) were dissolved in dry CH2Cl2 (25 mL) at
0 ꢁC under Ar. MOM-Cl (2.32 mL, 30.55 mmol) was
dripped into the solution over 1 min, and the reaction
was stirred at 20 ꢁC for 16 h. The solution was poured
into a mixture of EtOAc (200 mL)/ice/3 M HCl (100 mL)
and stirred for 5 min. The aqueous layer was extracted
with EtOAc (3Â150 mL), and the combined organic frac-
tions were dried with MgSO4. Flash chromatography was
performed using a step gradient (10% EtOAc/90% hex-
anes to 25% EtOAc/75% hexanes) to give (R)-2-methy-
phenyl-1-ethyl bromide, (R)-10, (1.284 g, 73%), as a
71.6 ꢁ(c 1.26, MeOH) [lit.22 for the
25
d
colorless oil. ‰ꢀŠ
25
d
(S) enantiomer ‰ꢀŠ +73 (MeOH)]; 1H NMR (CDCl3)
d 7.31±7.40 (5H, m), 4.36±4.42 (1H, m), 3.45±3.60 (2H,
m), 3.32 (3H, s); 13C NMR (CDCl3) d 139.0, 128.6,
128.5, 126.7, 83.4, 57.2, 36.2; HRMS (EI) m/z: calcd for
C9H11OBr, 213.9993; found, 213.9988.
(R)-10 (1.28 g, 5.951 mmol) and sodium methanethio-
sulfonate (1.04 g, 7.752 mmol) were dissolved in dry DMF
(10 mL) under Ar and heated to 70ꢁC. After stirring for
24h, the DMF was evaporated under reduced pressure.
The crude product was dissolved in EtOAc, ®ltered, and
the ®ltrate was evaporated under reduced pressure. ¯ash
chromatography using a step gradient (5% EtOAc/95%
hexanes to 33% EtOAc/67% hexanes) gave the title
loxymethoxy methyl mandelate, (R)-5 (1.935 g, 90%), as a
133.5ꢁ (c 1.41, CHCl3); [lit.23 for the
25
d
colorless oil. ‰ꢀŠ
(S) enantiomer ‰ꢀŠ25 +5.9ꢁ (c 1.11, CHCl3); IR, 1H NMR
d
and 13C NMR data were identical to the literature.23
(R)-5 (1.924 g, 9.152mmol) was dissolved in dry THF
(50 mL) at 0 ꢁC under Ar, and LiBH4 (0.498g, 22.87 mmol)
was added. The reaction was stirred for 16 h at 20 ꢁC,
and then poured into a stirred mixture of EtOAc
(200mL)/saturated aqueous NaHCO3 (150mL). After the
reaction had subsided, the aqueous layer was extracted
with EtOAc (3Â200 mL), and the combined organic frac-
tions were dried with MgSO4. The crude product was
puri®ed by ¯ash chromatography using a step gradient
(25% EtOAc/75% hexanes to 33% EtOAc/67% hex-
compound, (R)-1a (1.235g, 84%, eeꢂ98%), as a colorless
25
d
oil. ‰ꢀŠ
90.4ꢁ (c 0.94, CHCl3); 1H NMR (CDCl3) d 7.31±
7.39 (5H, m), 4.42±4.48 (1H, m), 3.41±3.46 (2H, m), 3.27
(3H, s), 3.24 (3H, s); 13C NMR (CDCl3) d 139.0, 128.7,
128.5, 126.6, 82.3, 56.9, 50.3, 43.4; HRMS (FAB+) m/z:
calcd for C10H14O3S2+H, 247.0463; found, 247.0470.
(S)-2-Methoxy-2-phenyl-ethylmethanethiosulfonate ((S)-
1a). (S)-3 was prepared in the same manner as the (R)-3.
From (S)-mandelic acid (4.00 g, 26.29 mmol) was obtained
(S)-1 (1.301 g, 30%). (S)-6 was prepared in the same manner
as the (R)-6. From (S)-3 (1.20 g, 7.221 mmol) was obtained
(S)-6 (0.903 g, 82%). Its IR, 1H NMR and 13C NMR data
anes) to give (R)-2-methyloxymethoxy-2-phenyl-1-etha-
189.9ꢁ
25
d
nol, (R)-7 (1.63 g, 98%), as a colorless oil. ‰ꢀŠ
20
d
(c 1.72, CHCl3); [lit.24 for the (S) enantiomer ‰ꢀŠ
+196ꢁ (c 2.67, CHCl3)]; IR, H NMR and 13C NMR
1
data were identical to the literature.
were identical to (R)-6. ‰ꢀŠ25 +115.0ꢁ (c 1.26, EtOH).
(R)-2-Methyloxymethoxy-2-phenyl-1-ethylmethanesulfo-
nate, (R)-9, was prepared in the same manner as (R)-8.
(R)-7 (1.530g, 8.396 mmol) was converted quantitatively
d
(S)-8 was prepared in the same manner as the (R)-8. From
(S)-6 (0.883g, 5.802 mmol) was obtained (S)-8 (1.33 g,
25
d
to (R)-9 (2.175 g). ‰ꢀŠ
141.6ꢁ (c 1.10, CHCl3); 1H NMR
100%). Its 1H NMR and 13C NMR data were identical to
(CDCl3) d 7.35 (5H, s), 4.89±4.95 (1H, m), 4.56±4.65 (2H,
AB q), 4.25±4.40 (2H, m), 3.36 (3H, s), 2.95 (3H, s); 13C
NMR (CDCl3) d 136.6, 128.7, 127.1, 94.4, 75.5, 72.3, 55.6,
37.4.
25
d
(R)-8. ‰ꢀŠ +95.0ꢁ (c 1.70, CHCl3).
(S)-10 was prepared in the same manner as (R)-10. From
(S)-8 (1.33 g, 5.773 mmol) was obtained (S)-10 (1.02 g,
81%). Its 1H NMR and 13C NMR data were identical to
(R)-2-Methyloxymethoxy-2-phenyl-1-ethyl bromide, (R)-
11, was prepared in the same manner as (R)-10. (R)-9
25
d
(R)-10. ‰ꢀŠ +72.4ꢁ (c 1.15, MeOH).

1964
M. Dickman, J. B. Jones / Bioorg. Med. Chem. 8 (2000) 1957±1968
(2.035 g, 7.817 mmol) was converted to (R)-11 (1.536 g,
56%). Its 1H NMR and 13C NMR data were identical to
25
25
d
80%). ‰ꢀŠ
130.9ꢁ (c 1.29, MeOH); 1H NMR (CDCl3) d
(R)-12. ‰ꢀŠ +153.4ꢁ (c 2.43, CHCl3).
d
7.35 (5H, s), 4.82±4.88 (1H, m), 4.57±4.66 (2H, AB q),
3.49±3.65 (2H, m), 3.43 (3H, s); 13C NMR (CDCl3) d 139.0,
128.6, 128.5, 126.9, 94.5, 77.7, 55.8, 36.2; HRMS (EI) m/z:
calcd for C10H13O2Br, 244.0099; found, 244.0091.
The title compound, (S)-1b, was prepared in the same
manner as (R)-1b. From (S)-12 (0.526 g, 1.903 mmol) was
obtained (S)-1b (0.419 g, 95%, eeꢂ98%), as white crys-
tals, which were recrystallized from ether/hexanes. Its 1H
(R)-2-Methyloxymethoxy-2-phenyl-1-ethylmethanethio-
sulfonate, (R)-12, was prepared in the same manner as
(R)-1a. (R)-10 (1.458 g, 5.948 mmol) was converted to
NMR and 13C NMR data were identical to (R)-1b. Mp
25
47.0±48.0 ꢁC; ‰ꢀŠ +63.3ꢁ (c 1.676, CHCl3); HRMS
d
(FAB+) m/z: calcd for C9H12O3S2+H, 233.0307; found,
233.0311.
25
d
1
(R)-12 (1.005g, 61%). ‰ꢀŠ
149.6ꢁ (c 2.23, CHCl3); H
NMR (CDCl3) d 7.36 (5H, s), 4.88±4.94 (1H, m), 4.56 (2H,
s), 3.48±3.51 (2H, m), 3.40 (3H, s), 3.23 (3H, s); 13C NMR
(CDCl3) d 139.0, 128.7, 128.6, 126.9, 94.3, 76.3, 55.9, 50.5,
43.4; HRMS (FAB+) m/z: calcd for C11H16O4S2+H,
277.0569; found, 277.0600.
N-(30-Methanethiosulfonatopropyl)-2-oxazolidinone (1c).
To a cooled solution (15±20 ꢁC) of 1,3-dibromopropane
(6.4 mL, 63.05 mmol) in dry DMSO (5 mL) was added
powdered KOH (0.920 g, 16.40 mmol). 2-Oxazolidinone
(1,100 g, 12.63 mmol) was added in small amounts over
5 min, and the reaction was stirred for 4 h at 20 ꢁC. The
mixture was diluted with ether (100 mL) and H2O
(20 mL), and the aqueous phase was extracted with
ether (3Â50 mL). After drying with MgSO4, the crude
product was puri®ed by ¯ash chromatography using a
step gradient (25% EtOAc/75% hexanes to 50%
(R)-12 (0.864g, 3.126 mmol) was suspended in H2O
(10 mL) and tri¯uoroacetic acid (10 mL) was added at
0 ꢁC. The solution was stirred at 20ꢁC for 40 h, and the
volatiles were evaporated under reduced pressure to near
dryness. H2O (20mL) was added, and the suspension was
evaporated to dryness. Finally, toluene (50mL) was added,
and the solution was evaporated to dryness. The crude
product was puri®ed by ¯ash chromatography using a step
gradient (25% EtOAc/75% hexanes to 33% EtOAc/67%
hexanes) to give the title compound, (R)-1b (0.689 g, 95%,
EtOAc/50% hexanes) to give N-(30-bromopropyl)-2-
1
oxazolidinone, 17 (1.48 g, 56%). IR (neat) 1747 cm
;
1H NMR (CDCl3) d 4.30 (2H, t, J=7.2 Hz), 3.57 (2H, t,
J=8.2 Hz), 3.33±3.43 (4H, q), 2.03±2.17 (2H, m); 13C
NMR (CDCl3) d 158.4, 61.7, 45.0, 43.0, 30.4, 29.9;
HRMS (FAB+) m/z: calcd for C6H10NO2Br, 207.9972;
found, 207.9957.
eeꢂ98%), as white crystals. An analytical sample was
25
d
recrystallized from ether/hexanes. Mp 48.5±49.5 ꢁC; ‰ꢀŠ
63.1ꢁ (c 0.89, CHCl3); IR (neat) 3470cm 1; H NMR
1
(CDCl3) d 7.38 (5H, s), 5.00±5.06 (1H, m), 3.44±3.49 (2H,
m), 3.26 (3H, s), 2.60 (1H, br s); 13C NMR (CDCl3) d 141.5,
128.7, 128.5, 125.9, 73.0, 50.5, 44.8; HRMS (FAB+) m/z:
calcd for C9H12O3S2+H, 233.0307; found, 233.0326.
The title compound, 1c, was prepared in the same manner
as (R)-1a. 17 (1.316g, 6.325 mmol) was converted to 1c
(1.013g, 67%). It was recrystallized from EtOAc/ether.
Mp 36±37.5 ꢁC; IR (neat) 1748cm 1; 1H NMR (CDCl3) d
4.32 (2H, t, J=7.4 Hz), 3.56 (2H, t, J=8.4 Hz), 3.35 (2H,
t, J=6.7 Hz), 3.31 (3H, s), 3.14 (2H, t, J=7.0 Hz), 1.96±
2.10 (2H, m); 13C NMR (CDCl3) d 158.5, 61.7, 50.4, 44.6,
42.9, 33.2, 27.6; HRMS (FAB+) m/z: calcd for C7H13
NO4S2+H, 240.0364; found, 240.0365.
(S)-2-Hydroxy-2-phenyl-ethylmethanethiosulfonate ((S)-
1b). (S)-4 was prepared in the same manner as (R)-4.
From (S)-mandelic acid (3.176 g, 20.87 mmol) was
obtained crude (S)-4 (3.45 g, quantitative) which was
used directly in the next step.
(S)-5 was prepared in the same manner as (R)-5. From
(S)-4 (3.45 g, 20.76 mmol) was obtained (S)-5 (3.014 g,
N-(30-Methanethiosulfonatopropyl)-(R)-4-isopropyl-2-ox-
azolidinone ((R)-1d). N-(30-Bromopropyl)-(R)-4-isopro-
pyl-2-oxazolidinone, (R)-18, was prepared in the same
manner as 17. From (R)-4-isopropyl-2-oxazolidinone
1
69%). Its H NMR and 13C NMR data were identical
25
d
to (R)-5. ‰ꢀŠ +131.6ꢁ (c 1.74, CHCl3).
(0.518 g, 4.011 mmol) was obtained (R)-18 (0.626 g, 62%,
25
d
(S)-7, was prepared in the same manner as (R)-7. From
(S)-5 (2.995 g, 14.25 mmol) was obtained (S)-7 (2.565 g,
eeꢂ98%), as a colorless oil. ‰ꢀŠ
2.7ꢁ (c 1.87, CHCl3);
IR (neat) 1748 cm 1; H NMR (CDCl3) d 4.20 (1H, t,
J=8.8 Hz), 4.04 (1H, dd, J=9.0, 5.3), 3.69±3.77 (1H, m),
3.47±3.58 (1H, m), 3.40 (2H, t, J=6.5 Hz), 3.06±3.20
(1H, m), 2.25±1.99 (3H, m), 0.86 (6H, t, J=7.4 Hz); 13C
NMR (CDCl3) d 158.3, 62.7, 59.6, 40.6, 30.2, 27.7, 17.5,
14.2; HRMS (FAB+) m/z: calcd for C9H16NO2Br,
250.0441; found, 250.0419.
1
99%) Its 1H NMR and 13C NMR data were identical to
25
d
(R)-7. ‰ꢀŠ +193.2ꢁ (c 1.30, CHCl3).
(S)-9 was prepared in the same manner as (R)-9. From
(S)-7 (2.467g, 13.54 mmol) was obtained (S)-9 (3.486g,
99%). Its 1H NMR and 13C NMR data were identical to
(R)-9. ‰ꢀŠ25 +135.5ꢁ (c 1.40, CHCl3).
d
The title compound, (R)-1d was prepared in the same
manner as (R)-1a. (R)-18 (0.530g, 2.119 mmol) was con-
(S)-11, was prepared in the same manner as (R)-11. From
(S)-9 (3.486g, 13.39 mmol) was obtained (S)-11 (2.822g,
25
d
verted to (R)-1d (0.492g, 83%). ‰ꢀŠ
22.3ꢁ (c 1.37,
86%). Its 1H NMR and 13C NMR data were identical to
CHCl3); IR (neat) 1744cm 1; H NMR (CDCl3) d 4.25
(1H, t, J=9.0Hz), 4.07 (1H, dd, J=9.0, 5.4 Hz), 3.73±
3.81 (1H, m), 3.50±3.65 (1H, m), 3.33 (3H, s), 3.07±3.21
(3H, m), 1.98±2.13 (3H, m), 0.90 (3H, d, J=7.0 Hz),
0.86 (3H, d, J=6.8 Hz); 13C NMR (CDCl3) d 158.6,
1
25
d
(R)-11. ‰ꢀŠ +125.8ꢁ (c 1.21, MeOH).
(S)-12 was prepared in the same manner as (R)-12. From
(S)-11 (0.863g, 3.521 mmol) was obtained (S)-12 (0.541g,

M. Dickman, J. B. Jones / Bioorg. Med. Chem. 8 (2000) 1957±1968
1965
62.9, 59.2, 50.5, 40.5, 33.5, 27.9, 27.6, 17.6, 14.2; HRMS
(FAB+) m/z: calcd for C10H19NO4S2+H, 282. 0834;
found, 282.0842.
2.04±2.27 (2H, m); 13C NMR (CDCl3) d 158.0, 135.2,
128.9, 128.8, 127.1, 66.7, 56.6, 40.8, 38.5, 30.5, 30.2;
HRMS (FAB+) m/z: calcd for C13H16NO2Br, 298.0441;
found, 298.0416.
N-(30-Methanethiosulfonatopropyl)-(S)-4-isopropyl-2-ox-
azolidinone ((S)-1d). (S)-18 was prepared in the same
manner as (R)-18. From (S)-4-isopropyl-2-oxazolidinone
(0.504 g, 3.902 mmol) was obtained (S)-18 (0.558 g,
The title compound, (R)-1f, was prepared in the same
manner as (R)-1a. (R)-20 (0.364 g, 1.221 mmol) was con-
25
d
verted to (R)-1f (0.362g, 90%). ‰ꢀŠ
31.7ꢁ (c 1.33, CH
1
57%, eeꢂ98%)). Its H NMR and 13C NMR data were
Cl3); IR (neat) 1745 cm 1; 1H NMR (CDCl3) d 7.14±7.34
(5H, m), 3.98±4.21 (3H, m), 3.48±3.61 (1H, m), 3.32 (3H, s),
3.04±3.30 (4H, m), 2.61±2.73 (1H, m), 1.98±2.11 (2H, m);
13C NMR (CDCl3) d 158.2, 135.2, 128.9, 128.8, 127.1, 66.7,
56.1, 50.4, 40.7, 38.4, 33.3, 27.8; HRMS (FAB+) m/z:
calcd for C14H19NO4S2+H, 330.0834; found, 330.0834.
25
identical to (R)-18. ‰ꢀŠ +3.4ꢁ (c 3.42, CHCl3).
d
The title compound, (S)-1d, was prepared in the same
manner as (R)-1d. From (S)-18 (0.493g, 1.971 mmol) was
1
obtained (S)-1d (0.435g, 78%). Its H NMR and 13C
25
NMR data were identical to (R)-1d. ‰ꢀŠ +23.2ꢁ (2.27,
d
CHCl3); HRMS (EI) m/z: calcd for C10H19NO4S2+H,
282. 0834; found, 282.0833.
N-(30-Methanethiosulfonatopropyl)-(S)-4-benzyl-2-oxazo-
lidinone ((S)-1f). (S)-20 was prepared in the same manner
as (R)-20. From (S)-4-benzyl-2-oxazolidinone (0.504g,
2.844 mmol) was obtained (S)-20 (0.558g, 66%, eeꢂ
98%)). Its 1H NMR and 13C NMR data were identical to
N-(30-Methanethiosulfonatopropyl)-(R)-4-phenyl-2-oxa-
zolidinone ((R)-1e). N-(30-Bromopropyl)-(R)-4-phenyl-2-
oxazolidinone, (R)-19, was prepared in the same manner
as 17. From (R)-4-phenyl-2-oxazolidinone (0.322 g, 1.970
(R)-20. ‰ꢀŠ25 +14.1ꢁ (c 2.50, CHCl3). The title compound,
d
(S)-1f, was prepared in the same manner as (R)-1f. From
mmol) was obtained (R)-19 (0.370 g, 66%, eeꢂ98%), as
(S)-20 (0.449 g, 1.506 mmol) was obtained (S)-1f (0.458g,
92%). Its 1H NMR and 13C NMR data were identical to
25
d
a colorless oil. ‰ꢀŠ
35.8ꢁ (c 3.10, CHCl3); IR (neat)
25
(R)-1f. ‰ꢀŠ +29.9ꢁ (c 1.19, CHCl3); HRMS (EI) m/z:
1
1748cm 1; H NMR (CDCl3) d 7.26±7.45 (5H, m), 4.79
(1H, dd, J=8.8, 6.3 Hz), 4.63 (1H, dd, J=8.6, 8.6 Hz), 4.15
(1H, dd, J=8.6, 6.4 Hz), 3.30±3.54 (3H, m), 2.89±3.03 (1H,
m), 1.90±2.12 (2H, m); 13C NMR (CDCl3) d 158.2, 137.7,
129.3, 129.2, 126.9, 69.8, 60.3, 41.1, 30.2, 29.9; HRMS (EI)
m/z: calcd for C12H14NO2Br, 283.0208; found, 283.0197.
d
calcd for C14H19NO4S2+H, 330.0834; found, 330.0844.
N-(20-Methanethiosulfonatoethyl)-(R)-4-phenyl-2-oxazo-
lidinone ((R)-1g). N-(30-Bromoethyl)-(R)-4-phenyl-2-oxa-
zolidinone, (R)-21, was prepared in the same manner as
17, except 10 equiv of 1,2-dibromoethane and 3 equiv of
KOH were used. From (R)-4-phenyl-2-oxazolidinone
The title compound, (R)-1e, was prepared in the same
manner as (R)-1a. (R)-19 (0.346 g, 1.218 mmol) was con-
(0.261 g, 1.599 mmol) was obtained (R)-21 (0.387 g, 90%,
54.1ꢁ (c 1.80, CHCl3);
25
d
25
d
verted to (R)-1e (0.344 g, 89%). ‰ꢀŠ
70.5ꢁ (c 0.84,
eeꢂ98%), as a colorless oil. ‰ꢀŠ
CHCl3); IR (neat) 1746 cm 1; 1H NMR (CDCl3) d 7.26±
7.43 (5H, m), 4.81 (1H, dd, J=8.8, 6.6 Hz), 4.65 (1H, dd,
J=8.6, 8.6 Hz), 4.16 (1H, dd, J=8.6, 6.6 Hz), 3.40±3.55
(1H, m), 3.29 (3H, s), 2.90±3.15 (3H, m), 1.82±1.97 (2H,
m); 13C NMR (CDCl3) d 158.4, 137.5, 129.4, 129.3, 127.1,
69.9, 60.0, 50.6, 41.0, 33.4, 27.5; HRMS (FAB+) m/z:
calcd for C13H17NO4S2+H, 316.0678; found, 316.0678.
IR (neat) 1749 cm ;
1H NMR (CDCl3) d 7.26±7.46
1
(5H, m), 4.98 (1H, dd, J=8.8, 6.6 Hz), 4.67 (1H, dd,
J=8.8, 8.8 Hz), 4.16 (1H, dd, J=8.8, 6.6 Hz), 3.75±3.87
(1H, m), 3.42±3.53 (1H, m), 3.12±3.36 (2H, m); 13C
NMR (CDCl3) d 158.0, 137.4, 129.4, 129.3, 127.0, 70.0,
60.4, 43.8, 28.6; HRMS (EI) m/z: calcd for
C11H12NO2Br, 269.0051; found, 269.0055.
N-(30 -Methanethiosulfonatopropyl)-(S)-4-phenyl-2-oxa-
zolidinone ((S) - 1e). (S)-19 was prepared in the same
manner as (R)-19. From (S)-4-phenyl-2-oxazolidinone
(0.964 g, 5.911 mmol) was obtained (S)-19 (0.955 g,
57%, eeꢂ98%)). Its 1H NMR and 13C NMR data were
The title compound, (R)-1g, was prepared in the same
manner as (R)-1a. (R)-21 (0.392g, 1.462 mmol) was con-
25
verted to (R)-1g (0.320g, 73%). ‰ꢀŠ
28.8ꢁ (c 1.32,
d
CHCl3); IR (neat) 1749cm 1; 1H NMR (CDCl3) d 7.29±
7.43 (5H, m), 4.88 (1H, dd, J=8.9, 6.6 Hz), 4.67 (1H, dd,
J=8.8, 8.8 Hz), 4.18 (1H, dd, J=8.8, 6.5 Hz), 3.59±3.76
(1H, m), 3.28 (3H, s), 3.10±3.26 (3H, m); 13C NMR
(CDCl3) d 158.1, 137.3, 129.4, 129.3, 127.1, 69.9, 60.3, 50.7,
41.8, 33.6; HRMS (EI) m/z: calcd for C12H15NO4S2+H,
302.0521; found, 302.0529.
identical to (R)-19. ‰ꢀŠ25 +33.3ꢁ (c 2.50, CHCl3). The title
d
compound, (S)-1e, was prepared in the same manner as
(R)-1e. From (S)-19 (0.870 g, 3.062 mmol) was obtained
1
(S)-1e (0.814 g, 84%). Its H NMR and 13C NMR data
25
were identical to (R)-1e. ‰ꢀŠ +68.8ꢁ (c 1.21, CHCl3);
d
HRMS (EI) m/z: calcd for C13H17NO4S2+H, 316.0678;
found, 316.0683.
N-(20-Methanethiosulfonatoethyl)-(S)-4-phenyl-2-oxazoli-
dinone ((S)-1g). (S)-21 was prepared in the same manner
as (R)-21. From (S)-4-phenyl-2-oxazolidinone (0.381g,
2.335 mmol) was obtained (S)-21 (0.564g, 89%,
N-(30-Methanethiosulfonatopropyl)-(R)-4-benzyl-2-oxa-
zolidinone ((R)-1f). N-(30-Bromopropyl)-(R)-4-benzyl-2-
oxazolidinone, (R)-20, was prepared in the same manner as
17. From (R)-4-benzyl-2-oxazolidinone (0.499 g, 2.816
eeꢂ98%). Its 1H NMR and 13C NMR data were identical
25
to (R)-21. ‰ꢀŠ +54.6ꢁ (c 1.85, CHCl3). The title com-
d
pound, (S)-1g, was prepared in the same manner as (R)-
mmol) was obtained (R)-20 (0.454 g, 54%, eeꢂ98%), as
25
d
a colorless oil. ‰ꢀŠ
14.3ꢁ (c 2.06, CHCl3); IR (neat)
1g. From (S)-21 (0.532g, 1.969 mmol) was obtained (S)-
1g (0.450g, 76%). Its 1H NMR and 13C NMR data were
1
1751 cm
;
1H NMR (CDCl3) d 7.14±7.36 (5H, m),
3.96±4.21 (3H, m), 3.10±3.65 (5H, m), 2.61±2.72 (1H, m),
25
identical to (R)-1g. ‰ꢀŠ +27.8ꢁ (1.30, CHCl3); HRMS
d

1966
M. Dickman, J. B. Jones / Bioorg. Med. Chem. 8 (2000) 1957±1968
(EI) m/z: calcd for C12H15NO4S2+H, 302.0521; found,
302.0534.
128.3, 126.2, 125.8, 80.8, 61.7, 39.3; HRMS (FAB+) m/z:
calcd for C10H9NO2+H, 176.0771; found, 176.0681.
N-(20-Methanethiosulfonatoethyl)-(R)-4-benzyl-2-oxazoli-
dinone ((R)-1h). N-(30-Bromoethyl)-(R)-4-benzyl-2-oxa-
zolidinone, (R)-22, was prepared in the same manner as
17, except 10 equiv of 1,2-dibromoethane and 3 equiv of
KOH were used. From (R)-4-benzyl-2-oxazolidinone
N-(30-Bromopropyl)-(3aR-cis)-3,3a,8,8a-tetrahydro-2H-
indeno[1,2-d]-oxazol-2-one, (R)-25, was prepared in the
same manner as 17. From (R)-24 (1.007 g, 5.748 mmol)
was obtained (R)-25 (1.11 g, 65%, eeꢂ98%), as a color-
25
d
less oil. ‰ꢀŠ
+31.3ꢁ (c 1.61, CHCl3); IR (neat)
(0.386 g, 2.178 mmol) was obtained (R)-22 (0.372 g,
1748 cm 1; 1H NMR (CDCl3) d 7.24±7.45 (4H, m), 5.31
(1H, dt, J=7.4, 3.1 Hz), 5.14 (1H, d, J=7.7 Hz), 3.23±
3.70 (6H, m), 2.12±2.34 (2H, m); 13C NMR (CDCl3) d
157.1, 140.5, 138.0, 129.8, 127.4, 125.8, 125.1, 77.1, 64.1,
41.0, 39.3, 30.4, 30.1; HRMS (FAB+) m/z: calcd for
C13H14NO2Br, 296.0285; found, 296.0254.
25
d
60%, eeꢂ98%), as a colorless oil. ‰ꢀŠ
16.7ꢁ (c 1.35,
CHCl3); IR (neat) 1748 cm 1; 1H NMR (CDCl3) d 7.12±
7.40 (5H, m), 3.81±4.30 (4H, m), 3.38±3.63 (3H, m),
3.11±3.20 (1H, m), 2.66±2.76 (1H, m); 13C NMR
(CDCl3) d 157.8, 135.2, 129.0, 127.3, 67.1, 56.9, 44.1,
38.7, 29.1; HRMS (EI) m/z: calcd for C12H14NO2Br,
284.0286; found, 284.0281.
The title compound, (R)-1i, was prepared in the same
manner as (R)-1a. (R)-25 (0.925 g, 3.123 mmol) was
converted to (R)-1i (0.882 g, 86%). It was recrystallized
The title compound, (R)-1h, was prepared in the same
manner as (R)-1a. (R)-22 (0.334g, 1.175mmol) was con-
25
from EtOAc/hexanes. Mp 94.0±95.0 ꢁC; ‰ꢀŠ +17.7ꢁ (c
d
verted to (R)-1h (0.363g, 98%). ‰ꢀŠ25 +4.5 (c 1.10, CHCl3);
1.28, CHCl3); IR (KBr) 1729 cm 1; 1H NMR (CDCl3) d
7.26±7.38 (4H, m), 5.32 (1H, dt, J=7.4, 3.0 Hz), 5.14
(1H, d, J=7.6 Hz), 3.36±3.69 (4H, m), 3.32 (3H, s), 3.14±
3.22 (2H, m), 2.10±2.23 (2H, m); 13C NMR (CDCl3) d
157.2, 140.6, 137.9, 129.7, 127.4, 125.8, 125.0, 77.2, 63.7,
50.4, 40.9, 39.2, 33.4, 27.5; HRMS (FAB+) m/z: calcd
for C14H17NO4S2+H, 328.0677; found, 328.0683.
d
1
IR (neat) 1748 cm
;
1H NMR (CDCl3) d 7.15±7.39
(5H, m), 4.02±4.29 (3H, m), 3.72±3.89 (1H, m), 3.14±
3.58 (4H, m), 3.38 (3H, s), 2.65±2.75 (1H, m); 13C NMR
(CDCl3) d 158.1, 135.2, 129.0, 127.3, 67.2, 57.0, 50.7, 42.0,
38.7, 33.9; HRMS (EI) m/z: calcd for C13H17NO4S2+H,
316.0677; found, 316.0683.
N-(20-Methanethiosulfonatoethyl)-(S)-4-benzyl-2-oxazoli-
dinone ((S)-1h). (S)-22 was prepared in the same manner
as (R)-22. From (S)-4-benzyl-2-oxazolidinone (0.371g,
2.094 mmol) was obtained (S)-22 (0.375g, 63%, eeꢂ
98%). Its 1H NMR and 13C NMR data were identical to
N-(30 -Methanethiosulfonatopropyl)-(3aS-cis)-3,3a,8,8a-
tetrahydro-2H-indeno[1,2-d]-oxazol-2-one ((S)-1i). (S)-24
was prepared in the same manner as (R)-24. From (1S, 2R)-
cis-1-amino-2-indanol (1.09 g, 7.306 mmol) was obtained
1
(S)-24 (1.27 g, quantitative). Its H NMR and 13C NMR
25
d
(R)-22. ‰ꢀŠ25 +15.6ꢁ (c 1.55, CHCl3). The title compound,
data were identical to (R)-24. Mp 205.0±207.0 ꢁC; ‰ꢀŠ
d
(S)-1h, was prepared in the same manner as (R)-1h. From
109.7ꢁ (c 1.30, CHCl3).
(S)-22 (0.328g, 1.154 mmol) was obtained (S)-1h (0.245g,
67%). Its 1H NMR and 13C NMR data were identical to
(R)-1h. ‰ꢀŠ
(S)-25 was prepared in the same manner as (R)-25. From
(S)-24 (1.023g, 5.839 mmol) was obtained (S)-25 (0.940g,
25
d
5.8ꢁ (c 1.20, CHCl3); HRMS (EI) m/z: calcd
for C13H17NO4S2+H, 316.0677; found, 316.0664.
1
54%, eeꢂ98%). Its H NMR and 13C NMR data were
25
d
identical to (R)-25. ‰ꢀŠ
30.5ꢁ (c 1.82, CHCl3).
N-(30 -Methanethiosulfonatopropyl)-(3aR-cis)-3,3a,8,8a-
tetrahydro-2H-indeno[1,2-d]-oxazol-2-one ((R)-1i). (1R,
2S)-cis-1-Amino-2-indanol (0.980 g, 6.569 mmol) was
placed in a round-bottomed ¯ask and a dry Ar atmosphere
was established. Dry CH2Cl2 (50 mL) and Et3N (1.9 mL,
13.63 mmol) were added, and the resulting solution was
cooled to 60 ꢁC. On addition of triphosgene (0.64 g,
2.157 mmol), the cooling bath was removed, and the
reaction was allowed to warm to 20ꢁC over 1 h. The reac-
tion was then poured into CH2Cl2 (100mL) and H2O
(50mL) and the aqueous phase was extracted with CH2Cl2
(3Â100 mL). After drying with MgSO4, the organic layer
was evaporated under reduced pressure to give (3aR-cis)-
3,3a,8,8a-tetrahydro-2H-indeno[1,2-d]-oxazol-2-one, (R)-24
(1.15 g, quantitative) as white crystals, which were of suf-
®cient purity for the next step in the reaction sequence.
An analytical sample was recrystallized from CH2Cl2/
The title compound, (S)-1i, was prepared in the same
manner as (R)-1i. From (S)-25 (0.840 g, 2.836 mmol) was
obtained (S)-1i (0.838 g, 90%). It was recrystallized from
1
EtOAc/hexanes. Its H NMR and 13C NMR data were
25
d
identical to (R)-1i. Mp 94.0±95.0 ꢁC; ‰ꢀŠ
18.7ꢁ (c 1.38,
CHCl3); HRMS (EI) m/z: calcd for C14H17NO4S2+H,
328.0677; found, 328.0694.
Site-speci®c chemical modi®cation
To 1.25 mL of a SBL mutant stored in MES bu€er
(10 mM MES, 1 mM CaCl2, pH 5.8) was added 0.75 mL
CHES bu€er (70 mM CHES, 5 mM MES, 2 mM CaCl2,
pH 9.5) at 20 ꢁC and one of the methanethiosulfonate
reagents (100 mL of a 0.5 M solution in CH3CN) in a
PEG (10,000) coated polypropylene test tube, and the
mixture agitated in an end-over-end rotator. After
30 min, all modi®cation reactions were negative to the
Ellman's test indicating the absence of free thiol. In order
to ensure complete reaction, a further 100 mL of meth-
anethiosulfonate solution was added and the reaction was
continued for another 30 min. The reaction solution was
puri®ed on a disposable desalting column (Pharmacia
hexanes. Mp 205.5±206.5 ꢁC; [lit.25 for enantiomer mp
25
d
205 ꢁC]; ‰ꢀŠ +107.7ꢁ (c 1.25, CHCl3); [lit.25 for enantio-
25
d
mer ‰ꢀŠ
79.4ꢁ (c 1.4, CHCl3)]. IR (KBr) 3255, 1752,
1
1707 cm 1; H NMR (acetone-d6) d 7.24±7.43(4H, m),
5.39 (1H, t, J=7.5 Hz), 5.21 (1H, d, J=7.0 Hz), 3.42 (1H,
dd, J=17.7, 6.2 Hz), 3.20 (1H, d, J=17.9 Hz), 2.90 (1H,
br s); 13C NMR (acetone-d6) d 159.1, 142.5, 141.0, 129.7,

M. Dickman, J. B. Jones / Bioorg. Med. Chem. 8 (2000) 1957±1968
1967
Biotech PD-10, Sephadex G-25 M) pre-equilibrated
Acknowledgements
with MES bu€er (5 mM MES, 2 mM CaCl2, pH 6.5).
The CMM was eluted with MES-bu€er (5.0 mL), dia-
lyzed (MWCO 12-14,000) against MES bu€er (10 mM
MES, 1 mM CaCl2, pH 5.8) then ¯ash frozen and stored
at 20 ꢁC. Modi®ed enzymes were analyzed by non-
denaturing gradient (8±25%) gels at pH 4.2, run towards
Generous funding for this project was provided by the
Natural Sciences and Engineering Council of Canada
(NSERC) and Genencor International Inc., who also
provided the WT, N62C, S156C, S166C and L217C
SBL enzymes. In addition, one of us (M.D.) thanks
NSERC for the award of a Postdoctoral Fellowship.
the cathode on the Pharmacia Phast-SystemTM 26
and
,
appeared as one single band. Each of the CMMs was
analyzed in parallel with its parent cysteine mutant and
the WT enzyme.
References and Notes
1. (a) Noritomi, H.; Almarsson, O.; Barletta, G. L.; Klibanov,
A. M. Biotechnol. Bioeng. 1996, 51, 95. (b) Sakai, T.; Kawa-
bata, I.; Kishimoto, T.; Ema, T.; Utaka, M. J. Org. Chem.
1997, 62, 4906. (c) Ullmann, D.; Bordusa, F.; Salchert, K.;
Jakubke, H.-D. Tetrahedron: Asymmetry 1996, 7, 2047. (d)
Holmberg, E.; Hult, K. Biotechnol. Lett. 1991, 13, 323. (e)
Phillips, R. S. Enzyme Microb. Technol. 1992, 14, 417. (vi)
Lam, L. K. P.; Hui, R. A. H. F.; Jones, J. B. J. Org. Chem.
1986, 51, 2047.
2. (a) Enzymatic Reactions in Organic Media; Koskinen, A.
M. P., Klibanov, A. M., Eds.; Blackie Academic and Profes-
sional: London, 1996. (b) Gutman, A. L.; Shapira, M. Adv.
Biochem. Eng./Biotechnol. 1995, 52, 87. (c) Griebenow, K.;
Klibanov, A. M. Biotechnol. Bioeng. 1997, 53, 351. (d) Bon-
neau, P. R.; Eyer, M.; Graycar, T. P.; Estell, D. A.; Jones, J.
B. Bioorg. Chem. 1993, 21, 431.
Enzyme characterization
Prior to ES-MS analysis, CMMs were puri®ed by FPLC
(BioRad, Biologic System) on a Source 15 RPC matrix
(17-0727-20 from Pharmacia) with 5% acetonitrile,
0.01% TFA as the running bu€er and eluted with 80%
acetonitrile, 0.01% TFA in a one step gradient. Elec-
trospray mass spectra were recorded on a PE SCIEX
API III Biomolecular Mass Analyzer (Table 3).
The free thiol content of N62C, L217C, S166C, S156C
and their CMMs, was determined spectrophotometrically
by titration with Ellman's reagent (E412=13,600
M
1 cm 1) in phosphate bu€er 0.25 M, pH 8.0.
3. (a) Gupta, A. K.; Kaslauskas, R. J. Tetrahedron: Asym-
metry 1993, 4, 879. (b) Sih, C. J.; Gu, Q.-M.; Holdgrun, X.;
Harris, K. Chirality 1992, 4, 91.
4. (a) Rich, J. O.; Dordick, J. S. J. Am. Chem. Soc. 1997, 119,
3245. (b) Russell, A. J.; Klibanov, A. M. J. Biol. Chem. 1988,
263, 11624.
5. (a) Dabulis, K.; Klibanov, A. M. Biotechnol. Bioeng. 1993,
41, 566. (b) Khmelnitsky, Y. L.; Welch, S. H.; Clark, D. S.;
Dordick, J. S. J. Am. Chem. Soc. 1994, 116, 2647.
The active enzyme concentration was determined as
previously described18 by monitoring ¯uoride release
upon enzyme reaction with a-toluenesulfonyl ¯uoride
(Aldrich Chemical Co. Inc.) as measured by a ¯uoride ion
sensitive electrode (Orion Research 96-09). The active
enzyme concentration determined in this way was used
to calculate kinetic parameters for each CMM.
6. (a) Scouten, W. H. Methods Enzymol. 1987, 135, 30. (b)
Polgar, L.; Bender, M. L. J. Am. Chem. Soc. 1966, 88, 3153.
(c) Wu, Z.-P.; Hilvert, D. J. Am. Chem. Soc. 1989, 111, 4513.
7. (a) Wong, C.-H.; Chen, S.-T.; Hennen, W. J.; Bibbs, J. A.;
Wang, Y.-F.; Liu, J. L.-C.; Pantoliano, M. W.; Whitlow, M.;
Bryan, P. N. J. Am. Chem. Soc. 1990, 112, 945. (b) Bonneau, P.
R.; Graycar, T. P.; Estell, D. A.; Jones, J. B. J. Am. Chem. Soc.
1991, 113, 1026. (c) Zhong, Z.; Liu, J. L.-C.; Dinterman, L. M.;
Finkelman, M. A. J.; Mueller, W. T.; Rollence, M. L.; Whitlow,
M.; Wong, C.-H. J. Am. Chem. Soc. 1991, 113, 683. (d) Estell, D.
A.; Graycar, T. P.; Wells, J. A. J. Biol. Chem. 1985, 260, 6518. (e)
Sears, P.; Wong, C.-H. Biotechnol. Prog. 1996, 12, 423.
8. (a) Reetz, M. T.; Zonta, A.; Schimossek, K.; Liebeton, K.;
Jaeger K.-E. Angew. Chem., Int. Ed. Engl. 1997, 36, 2830. (b)
Chen, K.; Arnold, F. H. Proc. Natl. Acad, Sci. USA 1993, 90,
5618. (c) Stemmer, W. P. C. Nature 1994, 370, 389.
9. Gron, H.; Bech, L. M.; Branner, S.; Breddam, K. Eur. J.
Biochem. 1990, 194, 897.
10. (a) DeSantis, G.; Berglund, P.; Stabile, M. R.; Gold, M.;
Jones, J. B. Biochemistry 1998, 37, 5968. (b) Berglund, P.;
DeSantis, G.; Stabile, M. R.; Shang, X.; Gold, M.; Bott, R.
R.; Graycar, T. P.; Lau, T. H.; Mitchinson, C.; Jones, J. B. J.
Am. Chem. Soc. 1997, 119, 5265.
11. X-ray structure solved by Rick Bott at Genencor Interna-
tional Inc. Brookhaven data base entry 1JEA of SBL.
12. Nomenclature according to Schechter, I.; Berger, A. Bio-
chem. Biophys. Res. Commun. 1967, 27, 157.
Kinetic measurements
Speci®city constants determined using the low substrate
approximation were measured at 0.05 and 0.1 mM N-Suc-
AAPF-pNA at 25ꢁC in 0.1 M Tris containing 0.005%
Tween 80 and 1% DMSO at pH 8.6 for amidase activity
(E410=8800 M 1 cm 1), and at 0.015 and 0.03mM N-Suc-
AAPF-SBn at 25ꢁC in 0.1 M Tris containing 0.005%
Tween 80 and 1% 37.5 mM DTNB in DMSO at pH 8.6
for esterase activity (E412=13,600 M 1 cm 1). A general
run consisted of equilibrating six plastic cuvettes con-
taining 980 mL of 0.1 M Tris, 0.005% Tween 80 at pH
8.6 to 25 ꢁC. The substrate (10 mL) in DMSO was
added and the cuvette was shaken twice before return-
ing it to the machine for zeroing. Immediately, the
enzyme (10 mL) in 20 mM MES, 1 mM CaCl2 at pH 5.8
was added and the cuvette was returned to the machine
with an eight second delay. The initial rate data was
recorded and used to calculate kcat/KM. Esterase data
was adjusted to account for background hydrolysis of
the substrate.
Michaelis±Menten constants were measured at 25 ꢁC by
curve ®tting (GraFit1 3.03) of the initial rate data
determined at eight concentrations (0.05±3.0 mM) of the
N-Suc-AAPF-pNA substrate for amidase activity and
eight concentrations (0.015±2.0 mM) of the N-Suc-
AAPF-SBn substrate for esterase activity.
13. Reeve, W.; Christo€el, I. J. Am. Chem. Soc. 1950, 72, 1480.
14. (a) Gage, J. R.; Evans, D. A. Org. Synth. 1990, 68, 77. (b)
Ager, D. J.; Prakassh, I.; Schaad, D. R. Aldrichimica Acta 1997,
30, 3.

1968
M. Dickman, J. B. Jones / Bioorg. Med. Chem. 8 (2000) 1957±1968
15. Isele, G. L.; Luttringhaus, A.. Synthesis, 266.
16. Sibi, M. P.; Deshpande, P. K.; LaLoggia, A. J.; Chris-
tensen, J. W. Tetrahedron Lett. 1995, 36, 8961.
17. Stabile, M. R.; Lai, G. W.; DeSantis, G.; Gold, M.; Jones,
J. B.; Mitchinson, C.; Bott, R. R.; Graycar, T. P.; Liu, C.-C.
Bioorg. Med. Chem. Lett. 1996, 6, 2501.
21. Aller, E.; Brown, D. S.; Cox, G. G.; Miller, D. J.; Moody,
C. J. J. Org. Chem. 1995, 60, 4449.
22. Casey, J. P.; Lewis, R. A.; Mislow, K. J. Am. Chem. Soc.
1969, 91, 2789.
23. Barrett, A. G. M.; Rys, D. J. J. Chem. Soc., Perkin Trans.
1 1995, 1009.
18. Hsia, C. Y.; Ganshaw, G.; Paech, C.; Murray, C. J. Anal.
Biochem. 1996, 242, 221.
19. Ellman, G. L.; Courtney, K. D.; Andres, V.; Featherstone,
R. M. Biochem. Pharmacol. 1961, 7, 88.
24. Ko, K.-Y.; Eliel, E. L. J. Org. Chem. 1986, 51, 5353.
25. Ghosh, A. K.; Duong, T. T.; McKee, S. P. J. Chem. Soc.
Chem. Commun. 1992, 1673.
26. Pharmacia Application File no. 300.
20. Lloyd, R.; Dickman, M.; Jones, J. B. Tetrahedron: Asym-
metry 1998, 9, 551.