The controlled introduction of multiple negative charge at single amino acid sites in subtilisin Bacillus lentus

Article abstract of DOI:10.1016/S0968-0896(99)00167-4

The use of methanethiosulfonates as thiol-specific modifying reagents in the strategy of combined site-directed mutagenesis and chemical modification allows virtually unlimited opportunities for creating new protein surface environments. As a consequence

Full text of DOI:10.1016/S0968-0896(99)00167-4

Bioorganic & Medicinal Chemistry 7 (1999) 2293±2301
The Controlled Introduction of Multiple Negative Charge at
Single Amino Acid Sites in Subtilisin Bacillus lentus
Benjamin G. Davis, a Xiao Shang, a Grace DeSantis, a Richard R. Bott b and
J. Bryan Jones a,*
aDepartment of Chemistry, University of Toronto, 80 St. George Street, Toronto, ON, Canada, M5S 3H6
bGenencor International, Inc., 925 Page Mill Rd., Palo Alto, CA 94304-1013, USA
Received 25 February 1999; accepted 6 April 1999
AbstractÐThe use of methanethiosulfonates as thiol-speci®c modifying reagents in the strategy of combined site-directed muta-
genesis and chemical modi®cation allows virtually unlimited opportunities for creating new protein surface environments. As a
consequence of our interest in electrostatic manipulation as a means of tailoring enzyme activity and speci®city, we have adopted
this approach for the controlled incorporation of multiple negative charges at single sites in the representative serine protease,
subtilisin Bacillus lentus (SBL). A series of mono-, di- and triacidic acid methanethiosulfonates were synthesized and used to modify
0
cysteine mutants of SBL at positions 62 in the S2 site, 156 and 166 in the S1 site and 217 in the S1 site. Kinetic parameters for these
chemically modi®ed mutant (CMM) enzymes were determined at pH 8.6 under conditions which ensured complete ionization of the
0
unnatural amino acid side-chains introduced. The presence of up to three negative charges in the S1, S1 and S2 subsites of SBL
resulted in up to 11-fold lowered activity, possibly due to interference with oxyanion stabilization of the transition state of the
hydrolytic reactions catalyzed. Each unit increase in negative charge resulted in a raising of KM and a reduction of kcat. However,
no upper limit was observed for increases in KM, whereas decreases in kcat reached a limiting value. Comparison with sterically
similar but uncharged CMMs revealed that electrostatic e€ects of negative charges at positions 62, 156 and 217 are detrimental, but
are bene®cial at position 166. These results indicate that the ground-state binding of SBL to the standard substrate, Suc-AAPF-
pNA, to SBL is reduced, but without drastic attenuation of catalytic eciency, and show that SBL tolerates high levels of charge at
single sites. # 1999 Elsevier Science Ltd. All rights reserved.
Introduction
Introductions of charge by protein engineering have
typically been limited to naturally occurring amino
acids such as negatively charged aspartate4,6,9,11±16 or
positively charged arginine4,7,9 or lysine.4,5,9,12 Chemical
modi®cation allows a greater variety of charged groups
to be introduced, but the reactions used for their intro-
duction are often non-speci®c in nature.19 For example,
the reaction of cyclic anhydrides with trypsin caused
indiscriminate modi®cation of lysine residues, the e€ects
of which could only be interpreted in general terms.17
Furthermore, although ingenious molecular biological
techniques have been developed for the introduction of
non-natural charged amino acids into proteins,20±22 they
are not yet suitable for large-scale synthesis or routine
application. In addition, the use of charged amino acids
in such techniques can result in poor levels of incor-
poration.20,21
The introduction of charge into wild-type (WT)
enzymes, through random1±3 or site-directed4±16 muta-
genesis or chemical modi®cation,17,18 can be used to
broaden substrate speci®city or to enhance catalytic
activity. The successful tailoring of speci®city towards
charged substrates has con®rmed the validity of
exploiting the electrostatic attraction between com-
plementary ions as a viable strategy for improving
binding. For example, the introduction of negative
charge into the active sites of subtilisin BPN0,4 aspartate
aminotransferase6 and l-lactate dehydrogenase11 has
expanded their structural speci®cities towards substrates
with positively charged side chains.
The use of site-directed mutagenesis combined with
chemical modi®cation23±28 of single sites o€ers a poten-
tial solution to these problems. In this technique
cysteine is introduced at preselected positions and its
Key words: Mutagenesis; electrostatic e€ects; enzymes and enzyme
reactions; kinetics.
* Corresponding author. Tel.: +1-416-978-3589; fax: +1-416-978-
1553; e-mail: jbjones@alchemy.chem.utoronto.ca
0968-0896/99/$ - see front matter # 1999 Elsevier Science Ltd. All rights reserved.
PII: S0968-0896(99)00167-4

2294
B. G. Davis et al. / Bioorg. Med. Chem. 7 (1999) 2293±2301
Results and Discussion
Synthesis of carboxyalkyl methanethiosulfonates 1b±e
Previous work29,31 has demonstrated that, of the meth-
ods available,36±41 direct nucleophilic displacement of a
primary alkyl bromide by methanethiosulfonate ion
provides the most ecient method for the preparation
of alkyl methanethiosulfonates. This general method
was therefore adopted as the basis for the preparation
of all of 1b±e. The aliphatic monocarboxylate MTS 1b42
was prepared from 5-bromopentanoic acid and NaS-
SO2CH3 in 80% yield.
Scheme 1.
The aromatic dicarboxylate MTS 1c was prepared from
toluene-3,5-dicarboxylic acid (2) via
a precursor
thiol residue is then reacted with methanethiosulfonate
(MTS) reagents (Scheme 1). Methanethiosulfonate
reagents react speci®cally and quantitatively with
thiols.29,30 Recently, using the serine protease subtilisin
Bacillus lentus (SBL) as our model, we have used this
technique to improve enzyme activity31 and alter speci-
®city.32,33 SBL is a near-ideal enzyme for evaluating the
validity of this strategy since its does not contain a nat-
ural cysteine. In the current work we describe the use of
this method to introduce locally high charge density
into SBL through the incorporation of single residues
bearing multiple charges.
benzylic bromide 3 as shown in Scheme 2. Treatment of
3 with NaSSO2CH3 gave the protected aromatic MTS 4
in 60% yield. Hydrolysis of 4 with TFA gave 1c as a
white solid (91% yield, 44% overall yield from 2).
The aliphatic di- and tricarboxylates 1d,e were prepared
from Meldrum's acid (5a) using 1,2-dibromoethane to
introduce a brominated linker group as shown in
Scheme 3. The low pKa of 5a43,44 allowed the use of
mildly basic conditions compatible with this choice of
linker. For the sake of simplicity, we chose methyl
Meldrum's acid (5b) as a starting material for 1d in
which one alkylation site is blocked as a methyl group.45
The synthesis of 1e utilized this position to introduce a
third carboxylate moiety.
Using the X-ray structure of SBL34 (Fig. 1) as our guide,
four sites were chosen for mutation because of their
seminal positions in the active site. Two of these, N62
(subtilisin BPN0 numbering) and L217, occupy positions
that are equidistant from S221 of the catalytic triad, in
Alkylation of 5b with 1,2-dibromoethane a€orded bro-
mide 6b in 71% yield. Treatment of 6b with NaS-
SO2CH3 in DMF at 50ꢀC led to the displacement of the
remaining bromide and resulted in the formation of
protected dicarboxylate MTS 7b. Hydrolysis of 7b using
acidic ion exchange resin allowed the successful forma-
tion of the aliphatic diacidic MTS 1d (79% yield, 37%
overall yield from 5b).
the S2 and S10 pockets, respectively. The other two
35
sites, S156 and S166, are located at the base of the S1
pocket and their side chains are directed towards SBL's
surface and catalytic triad, respectively. The MTS
reagents 1a±e were selected to modify these positions.
The synthesis of the triacidic reagent 1e required the
construction of a protected tricarboxylate 5c before
Figure 1. The active site of SBL showing the residues of the catalytic
triad, Ser221 (orange), His64 (blue) and Asp32 (red). The irreversible
phenylmethylsulfonyl inhibitor (pink) forms a bond to the Og atom of
Ser221 and its phenyl ring occupies the S1 binding site. The residues
chosen for mutation, Asn62 in the S2 site, Ser156 and Ser166 in the S1
Scheme 2. (a) (i) Im2CO, DMF, 40ꢀC then DBU, t-BuOH, 84%, (ii)
NBS, azobis(cyclohexanecarbonitrile), CCl4, Á, 96%; (b) NaS-
SO2CH3, DMF, 50ꢀC, 60%; (c) CF3COOH, CH2Cl2, 91%.
0
site and Leu217 in the S1 ' site are highlighted in yellow.34

B. G. Davis et al. / Bioorg. Med. Chem. 7 (1999) 2293±2301
2295
steps. Complete deprotection of 7c using CF3COOD in
1
D2O was followed by H NMR, and resulted in the for-
mation of target 1e (70% yield, 23% overall yield from 5a).
Preparation of chemically modi®ed mutants (CMMs)
MTS reagents 1a±e were used to modify the chosen SBL
cysteine mutants, N62C, S156C, S166C and L217C
under conditions described previously.31±33 These reac-
tions proceeded rapidly and quantitatively, as judged by
the monitoring of changes in speci®c activity and by
titration of residual free thiols with Ellman's reagent,46
respectively. The structure of the charged CMMs was
con®rmed by ES±MS. Non-reducing native PAGE was
used to determine the purity of all the enzymes, which
appeared as single bands. Consistent with the introduc-
tion of negative charge, each of the CMMs showed
retarded mobility in the direction of the cathode relative
to WT. The active enzyme concentration of the resulting
CMM solutions was determined by active site titration
with a-toluenesulfonyl ¯uoride (PMSF) using a ¯uoride
ion-sensitive electrode.47
Scheme 3. (a) K2CO3, DMF then BrCH2COOBut, 59%; (b) K2CO3,
DMF then Br(CH2)2Br, 71% for 6b, 66% for 6c; (c) NaSSO2CH3,
DMF, 50ꢀC, 83% for 7b, 86% for 7c; (d) Dowex 50W(H+), p-diox-
ane, H2O, 79%; (e) CF3COOD, D2O, 50ꢀC, 70%.
Kinetic e€ects of site speci®c modi®cation
The e€ects of modi®cation upon SBL were assessed by
the determination of kcat and KM for the hydrolysis of
succinyl-AAPF-p-nitroanilide (Suc-AAPF-pNA). Our
usual assay pH of 8.6 ensured complete ionization of
the unnatural amino acid side-chains introduced. The
kinetic parameters of the 20 CMMs generated are com-
pared with those of WT and unmodi®ed mutants in
Table 1 and Figure 2.
elaboration. Alkylation of Meldrum's acid (5a) with
tert-butyl bromoacetate allowed the formation of 5c
with moderate selectivity in 59% yield. Elaboration of
tricarboxylate 5c was carried out using 1,2-dibro-
moethane and then NaSSO2CH3 in an analogous man-
ner to that used for the synthesis of 1d and gave
protected tricarboxylate MTS 7c in 57% yield over 2
Table 1. Kinetic parametersa for modi®ed enzymes
Entry
Enzyme
Pocket
R
Level of charge
kcat (s±1
)
KM (mM)
kcat/KM (s±1 mM±1
)
1
2
3
4
5
6
7
8
SBL-WT
N62C
Ð
S2
Ð
H
a
b
c
d
e
H
a
b
c
d
e
H
a
b
c
d
e
Ð
0
1
1
2
2
3
0
1
1
2
2
3
0
1
1
2
2
3
0
1
1
2
2
3
153‹4
174‹9
119‹4
106‹2
113‹7
90‹4
129‹3
41‹1
89‹6
54‹1
69‹2
63‹2
55‹2
125‹4
87‹2
76‹1
61‹1
53‹1
74‹2
42‹1
25‹1
0.73‹0.05
1.90‹0.20
0.93‹0.07
1.01‹0.05
1.00‹0.10
1.47‹0.14
1.46‹0.06
0.80‹0.04
1.80‹0.20
1.03‹0.06
0.81‹0.06
1.65‹0.11
1.48‹0.08
0.85‹0.06
1.20‹0.07
1.08‹0.04
1.39‹0.10
1.67‹0.06
1.87‹0.08
0.50‹0.05
1.34‹0.08
1.52‹0.06
1.60‹0.20
2.26‹0.10
2.46‹0.11
209‹15
92‹11
128‹11
105‹6
113‹13
61‹6
88‹4
51‹3
50‹6
52‹3
85‹7
38‹3
37‹3
147‹11
73‹5
70‹3
44‹3
32‹1
39‹2
84‹9
19‹1
31‹1b
29‹4
30‹2
31‹2
0
L217C
S156C
S166C
S1
9
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
S1
H
a
b
c
d
48‹1b
47‹3
67‹2
76‹2
e
Michaelis±Menten constants were measured at 25ꢀC according to the initial rates method in 0.1 M Tris±HCl bu€er at pH 8.6, 0.005% Tween 80,
1% DMSO, Suc-AAPF-pNA as the substrate.
a
b
Based on total protein concentration.48

2296
B. G. Davis et al. / Bioorg. Med. Chem. 7 (1999) 2293±2301
Position 62 in the S2 pocket is the most tolerant of
mutation and modi®cation (Fig. 2(a)) and mutation to
cysteine reduces kcat/KM by only a factor of 2 (Table 1,
Entry 2). Upon modi®cation, activity is partially
restored and values of kcat/KM for N62C-a,b,c are raised
approximately 1.5-fold relative to N62C (Table 1,
entries 3±5). Modi®cations with aliphatic di- and tri-
carboxylate MTS reagents 1d,e elicit further drops in
kcat/KM, with N62C-d being 3.5-fold lower than WT.
However, despite the increased charge, this kcat/KM
drop is less marked for N62C-e.
The deleterious e€ect of negative charges in the S2
pocket upon kcat is apparent in the 1.3-fold decrease
observed for N62C-a (Table 1, entry 3) relative to WT.
However, as the level of negative charge increases, kcat
values do not decrease further to any signi®cant extent.
In fact, of all the CMMs, the kcat level of N62C-e (129
s
1, Table 1, Entry 7) is the highest. In contrast, KM
values increase continually with each additional charge,
reaching values for N62C-d (Table 1, Entry 6) and
N62C-e (Table 1, Entry 7) that are 2-fold higher than
WT.
0
The e€ects of mutation at position 217 in the S1 pocket
(Fig. 2(b)) are intrinsically more dramatic than at all
three other sites since the value of kcat/KM for L217C is
4-fold lower than WT. The introduction of a single
negative charge only a€ects kcat/KM a little and leads to
near-identical kcat/KM values for L217C-a,b (Table 1,
Entries 9,10). As negative charge increases further, two
opposite trends are observed, with the kcat/KM value for
aromatic L217C-c being raised 1.6-fold relative to
L217C-a,b, while those for aliphatic L217C-d,e are
reduced by 1.4-fold.
These slight kcat/KM changes seen at position 217 are
the result of larger, but counteracting changes in each
of kcat and KM. For example, while L217C-a has the
highest value of kcat, it also has the highest KM value
(both 2.2-fold higher than L217C). As at position
62, when the level of negative charge increases,
from L217C-a to L217C-e, kcat values decrease only
slightly and remain 1.3- to 1.7-fold higher than L217C
(Table 1, entries 10±13). KM values increase unevenly
to 2-fold higher than WT. Interestingly, the underlying
cause of the out-of-line kcat/KM of L217C-c is an unu-
sually low KM (0.81 mM, entry 11), which may be a
consequence of complementary aromatic interactions
between the substrate and the phenyl ring of side
chain c.
The e€ects of mutation and modi®cation at positions
156 and 166 in the S1 pocket are shown in Figure 2(c)
and (d). Mutation at position 156 to cysteine causes a
1.4-fold drop in kcat/KM (S156C, Table 1, entry 14).
From S156C-a to S156C-d kcat/KM decreases mono-
tonically to a value that is 6-fold lower than WT.
The additional negative charge present in S156C-e par-
tially restores this value, to only 5.4-fold lower than
WT.
Figure 2. Altered speci®city patterns relative to WT as the level of
negative charge increases in N62C, L271C, S156C and S166C mutants
and CMMs with suc-AAPF-pNA as the substrate: (a) The kcat/KM
for N62C CMMs alternate at moderately reduced levels, 1.5- to 3.5-
fold lower than WT, which are established by the initial mutation to
s
Mutation and modi®cation at position 166 leads to the
least active negatively charged CMMs, with kcat/KMs 6
to 11-fold lower than WT. This partly re¯ects the
intrinsically lower kcat/KM value of the unmodi®ed
mutant S166C, which is already 2.5-fold lower than
WT. However, the presence of the sulfonatoethyl side
chain in S166C-a causes a dramatic drop to a value that
is 11-fold lower than WT. kcat/KM is increased 1.5-fold
for S166C-b and remains steady as the level of negative
charge increases from S166C-c to S166C-e.
N62C (RˆH). (b) L217C CMMs show steady but lower levels of kcat
/
KM, 4- to 5.5-fold lower than WT, which are again established by the
initial mutation to cysteine. The exception is L217C-c which is only
2.5-fold lower than WT, possibly due to favourable binding of sub-
strate to the phenyl ring of the aromatic side chain introduced by
modi®cation. (c) From the small reduction caused by mutation to
S156C (RˆH), kcat/KMs decrease monotonically to 6-fold lower than
WT for S156C-d. The kcat/KM of S156C-e is partially restored. (d) kcat
/
KM decreases only 2.5-fold upon mutation to S166C (RˆH) but
decreases dramatically to 11-fold lower than WT when the negatively
charged sulfonatoethyl side chain a is introduced. In parallel to N62C
and L217C CMMs, kcat/KM for S166C CMMs does not decrease fur-
ther to any signi®cant extent as the level of negative charge increases.
The kcat values for S156C and S166C CMMs are simi-
lar to those found for L217C CMMs, typically 2 to

B. G. Davis et al. / Bioorg. Med. Chem. 7 (1999) 2293±2301
2297
2.5-fold lower than WT. As at positions 62 and 217, the
detrimental e€ect of a single negative charge on kcat is
not ampli®ed by the introduction of additional negative
charges. In fact, kcat values for S166C CMMs increase
steadily from 6-fold lower than WT for S166C-a (Table
1, entry 21) to 2-fold lower than WT for S166C-e (Table
1, entry 25).
In contrast, the introduction of negative charge at posi-
tion 166 partially restores some of the activity lost
through the introduction of near-isosteric uncharged
side chains. Therefore, the drastically lowered kcat/KMs
of CMMs S166C-a±e relative to WT are, in fact, a result
of steric or hydrophobic e€ects. Mutation analysis of
subtilisin BPN0 has shown that kcat/KM decreases dra-
matically when the optimal binding volume of the S1-
pocket is exceeded.51 The e€ect of introducing even
small groups at position 166 of SBL52 is to ®ll the S1-
pocket and this dramatically decreases kcat/KM. For
example, uncharged CMM S166C-S-ethyl has a kcat/KM
13.5-fold lower than WT.49 Molecular mechanics ana-
lysis of S166C CMMs has shown that charged side
chains introduced at position 166 may orientate them-
selves towards external solvent.33 This serves to reduce
the volume of the S1 pocket that is occupied by the side
chain. The existence of such an orientation for S166C-
a±e, which is lacking in uncharged CMM counterparts,
might, in part, explain the bene®cial e€ects of introdu-
cing charge. As a result, charged CMM S166C-S-
EtSO3 , side chain a, has a kcat/KM only 11-fold lower
than WT.
The KM values for both S156C and S166C CMMs
increase steadily with increasing negative charge and are
largest for S166C-e (KM 2.46 mM, 3.5-fold higher than
WT, Table 1, entry 25). Consistent with its surface-
exposed nature these e€ects are less pronounced at
position 156, with KM increasing to only 2.5-fold higher
than WT for S156C-e (Table 1, entry 19).
Kinetic e€ects of negative charge
To separate the contribution of electrostatics from steric
e€ects, a comparison of these charged CMMs with
those containing sterically similar uncharged side
chains49 was made. For example, N62C-a was com-
pared with N62C-S-ethyl, N62C-b,d,e were compared
with N62C-S-n-pentyl and N62C-c was compared with
N62C-S-benzyl. Figure 3 illustrates the results of intro-
ducing charge to these near-isosteric systems. This pro-
vides an estimate of the e€ect of negative charge upon
the kinetics of SBL when corrected for underlying steric
and hydrophobic e€ects.
Conclusions
In summary, we have devised short and ecient syn-
thetic routes to three novel multiply charged methane-
thiosulfonates, 1c, d and e. Such compounds, as well
as being of interest in our approach to the controlled
tailoring of enzyme activity, may prove useful in the
study of ion channels. The use of MTS reagents in
techniques such as the substituted-cysteine accessibility
method (SCAM)53±55 has allowed aspects of membrane
ion channel topology and conformation to be deter-
mined. In particular the use of charged MTS reagents
has given an invaluable insight into ion speci®city56 and
mechanism of action.57±61
Two di€ering trends emerge from Figure 3. At positions
62, 217 and 156, the electrostatic contribution of each of
side chains a±e is detrimental to kcat/KM. The reductions
caused are similar for each side chain, vary little from
site to site and increase with the level of negative charge
introduced. These reduced kcat/KM values resulting from
the introduction of negative charge are consistent with
earlier ®ndings.12,31±33 Such e€ects may be attributed, in
part, to destabilization of the tetrahedral oxyanion
intermediate that is formed in the rate limiting step of
catalysis.50
Using our established methodology, we selectively
modi®ed the cysteine thiols of SBL mutants, N62C,
S156C, S166C, and L217C, with these reagents. With-
out exception, mutation and modi®cation at all four
sites led to reduced catalytic eciency in the hydrolysis
of Suc-AAPF-pNA. However these reductions do not
exceed 11-fold relative to WT and the lowest kcat values
determined were only 6-fold reduced. This reduced e-
ciency is manifested largely through decreased binding
interactions, i.e. decreased KM values, that increase with
the level of charge introduced. In contrast, kcat values
corresponding to the introduction of multiple charge are
similar to, if not higher than, those for single charge.
Comparison with near-isosteric uncharged CMMs
revealed that electrostatic e€ects are important at posi-
tions 62, 217 and 156. However at position 166 steric
e€ects dominate and the introduction of negative charge
is, in fact, bene®cial.
Figure 3. The e€ects of introducing negative charge to CMMs: ln
(kcat/KM), with suc-AAPF-pNA as the substrate, of the negatively
charged N62C, L217C and S156C CMMs decreases relative to that of
near-isosteric uncharged CMMs as the level of negative charge
increases (from side chain a to e). In contrast, this value for the cor-
responding S166C CMMs increases with increasing negative charge.
The hydrolysis of di€erent substrates, including those
containing basic residues, and pH-activity pro®les, are
being investigated. The pKa e€ects and speci®city con-
sequences will be presented in due course.

2298
B. G. Davis et al. / Bioorg. Med. Chem. 7 (1999) 2293±2301
Experimental
were combined, washed with water and 10% K2CO3
(aq), dried (MgSO4), ®ltered and the solvent removed.
The residue was puri®ed by ¯ash chromatography
(EtOAc:hexane, 1:50) to a€ord a colorless oil which
solidi®ed upon standing to give di-tert-butyl toluene-
Mutants of subtilisin Bacillus lentus (SBL) were gene-
rated, and WT and mutant enzymes puri®ed as described
previously.32,33 NaSSO2CH3 (mp 269±269.5ꢀC (dec.)
(lit.,29 mp 272±273.5ꢀC)) and toluene-3,5-dicarboxylic
acid (2)62 (mp 294.5±296 ꢀC (water) (lit.,62 mp 287±
288ꢀC)) were prepared according to literature methods.
DMF was distilled under N2 from CaH2 and stored
over molecular sieves under N2 before use. CCl4 was
fractionally distilled before use. Sulfonatoethyl methane-
thiosulfonate (1a) was purchased from Toronto
Research Chemicals (2 Brisbane Rd., Toronto, ON,
Canada). All other chemicals were used as received
from Sigma-Aldrich or Baker. All ¯ash chromato-
graphy was performed using silica gel (Whatman, 60 A,
230±400 mesh). Melting points were determined using
an Electrothermal IA9000 series digital melting point
apparatus and are uncorrected. IR spectra were recor-
ded on Bomem MB or Perkin±Elmer FTIR Spectrum
1000 spectrophotometers. 1H NMR and 13C NMR
spectra were recorded on a Varian Gemini 200 NMR
spectrometer at 200 and 50.3 MHz, respectively. ES±
MS data were acquired using a PE SCIEX API III Bio-
molecular mass spectrometer. All other MS and HRMS
data, were acquired using Micromass 70±250S or
Micromass ZAB-SE mass spectrometers according to
the ionization methods indicated. Microanalyses were
performed by Canadian Microanalytical Service Ltd.
(Delta, B.C., V4G 1G7, Canada). Solvents were
removed in vacuo.
3,5-dicarboxylate (4.58 g, 84%) as a white solid; mp
1
86.5±87.5ꢀC (hexane); IR (®lm) 1712 cm
(CˆO),
1606, 1476 cm 1 (Ar CˆC); 1H NMR (CDCl3) d 1.60 (s,
18H, C(CH3)3), 2.43 (s, 3H, CH3), 7.95 (br s, 2H, H-2,
H-6), 8.38 (br s, 1H, H-4); 13C NMR (CDCl3) d 21.4
(CH3), 28.2 (C(CH3)3), 81.4 (C(CH3)3), 127.7, 132.1,
133.7, 138.1 (Ar), 165.2 (COO).
NBS (0.521 g, 2.93 mmol) and 1,10-azobis(cyclohex-
anecarbonitrile) (30 mg, 0.12 mmol) were added to
solution of this diester (0.712 g, 2.44 mmol) in CCl4 (10
mL) and heated under re¯ux under N2. After 3 h a sec-
ond portion of initiator (30 mg, 0.12 mmol) was added.
After a further 3 h the reaction solution was cooled and
®ltered. The ®ltrate was washed with sat. NaHCO3 (aq),
dried (MgSO4), ®ltered and the solvent removed. The
residue was partially puri®ed by ¯ash chromatography
(EtOAc:hexane, 1:50) to a€ord crude 3,5-di(tert-butoxy-
carbo)benzylbromide (3) (0.872 g, 96%). A solution of 3
(0.872 g, 2.35 mmol) and NaSSO2CH3 (0.327 g, 2.44
mmol) in DMF (1 mL) was heated at 50ꢀC under N2.
After 1 h the reaction solution was cooled, diluted with
water (5 mL) and extracted with ether (15 mL Â 3). The
combined extracts were washed with brine, dried
(MgSO4) and the solvent removed. The residue was
puri®ed by ¯ash chromatography (EtOAc:hexane, 1:8)
to give 3,5-di(tert-butoxycarbo)benzyl methanethio-
sulfonate (4) (0.570 g, 60%) as a colorless oil; IR (®lm)
4-Carboxybutyl methanethiosulfonate (1b).42 A solution
of 5-bromopentanoic acid (1.238 g, 6.84 mmol) and
NaSSO2CH3 (0.916 g, 6.84 mmol) in DMF (6 mL) was
heated at 70ꢀC under N2. After 2 h the solution was
cooled, water (15 mL) added and the resulting mixture
extracted with ether (30 mL Â 3). The organic fractions
were combined, washed with brine, dried (MgSO4), ®l-
tered and the solvent removed. The residue was puri®ed
by ¯ash chromatography (ether:CH2Cl2:AcOH, 40:
120:1) to give 1b (1.167 g, 80%) as a white solid; mp
1
1
1717 cm (CˆO), 1604, 1477, 1456 cm (Ar CˆC),
1
1
1328, 1135 cm (S±SO2); H NMR (CDCl3) d 1.59 (s,
18H, C(CH3)3), 3.07 (s, 3H, SO2CH3), 4.43 (s, 2H,
CH2), 8.13 (s, 2H, H-2, H-6), 8.48 (s, 1H, H-4); 13C
NMR (CDCl3) d 28.2 (C(CH3)3), 40.0 (CH2), 51.3
(SO2CH3), 82.1 (C(CH3)3), 130.2, 133.2, 133.5, 135.7
(Ar), 164.3 (COO).
A solution of 4 (0.941 g, 2.30 mmol) in CF3COOH:
CH2Cl2 (1:1 v/v, 10 mL) was stirred at room tem-
perature for 3 h, during which time a white pre-
cipitate was formed. The solvents were removed and the
residue triturated with CH2Cl2 (5 mL). The resulting
mixture was ®ltered, and the residue washed with
CH2Cl2 and dried under vacuum to give 1c (0.611 g,
91% from 4) as a white solid; mp 199.5±200 (ꢀC (dec.);
IR (KBr) 1716, 1693 cm 1 (CˆO), 1605, 1461 cm 1 (Ar
CˆC) , 1319, 1128 cm 1 (S-SO2); 1H NMR (acetone-d6)
d 3.29 (s, 3H, SO2CH3), 4.69 (s, 2H, CH2), 8.36 (d,
J=1.4 Hz, 2H, H-2, H-6), 8.61 (t, J=1.7 Hz, 1H, H-4);
13C NMR (acetone-d6) d 40.0 (CH2), 51.2 (SO2CH3),
130.8, 132.5, 135.2, 138.5 (Ar), 166.3 (COOH); MS m/z
(EI+): 290 (M+, 2), 273 (M+-OH, 4), 210 (M+-
CH3SO2H, 100), 179 (M+-SSO2CH3, 5); HRMS m/z
(FAB+): Found 290.9987 (M+H+), C10H11O6S2
requires 290.9998.
61±62.5ꢀC [lit.,42 mp 69±71ꢀC]; IR (KBr) 1703 cm
1
1
1
(CˆO), 1311, 1125 cm (S±SO2); H NMR (CDCl3) d
1.70±2.00 (m, 4H, H±2, H±3), 2.43 (t, J=6.9 Hz, 2H, H-
4), 3.20 (t, J=6.8 Hz, 2H, H-1), 3.34 (s, 3H, SSO2CH3),
8.82 (br s, 1H, COOH); 13C NMR (CDCl3) d 23.4, 28.9,
33.1, 35.9 ((CH2)4) , 50.7 (SSO2CH3), 178.7 (COOH);
MS m/z (EI+): 213 (M+H+, 2), 195 (M+H+-H2O,
11), 133 (50), 115 (M+-CH3SO2±H2O, 100%); HRMS
m/z (EI+): Found 213.0251 (M+H+); C6H13O4S2
requires 213.0255.
3,5-Dicarboxybenzyl methanethiosulfonate (1c). 1,10-
Carbonyldiimidazole (6.67 g, 0.0411 mol) was added to
a solution of toluene-3,5-dicarboxylic acid (2) (3.364 g,
0.0187 mol) in DMF (30 mL) and the resulting mixture
stirred at 40ꢀC under N2. After 1.5 h DBU (6.15 mL,
0.041 mol) and t-BuOH (7.7 mL, 0.0822 mol) were
added. After 24 h the solution was cooled, ether (150
mL) added and the mixture acidi®ed (HCl (aq), 1.5 M).
The ethereal layer was separated and the aqueous layer
further extracted (ether, 150 mL). The organic fractions
3,3-Dicarboxybutyl methanethiosulfonate (1d). Anhy-
drous K2CO3 (1.67 g, 12.0 mmol) was added to a solu-
tion of methyl Meldrum's acid (5b) (1 g, 6.33 mmol) in

B. G. Davis et al. / Bioorg. Med. Chem. 7 (1999) 2293±2301
2299
DMF (33 mL) under N2 and stirred vigorously. After 1
h the supernatant liquid was added dropwise to a solu-
tion of 1,2-dibromoethane (1.9 mL, 22.2 mmol) in DMF
(11 mL) under N2. After 89 h TLC (EtOAc:hexane, 1:3)
indicated conversion of starting material (Rf 0.3) to a
major product (Rf 0.5). The reaction mixture was added
to water (100 mL) and extracted with ether (100 mL Â
3). The organic fractions were combined, dried
(MgSO4), ®ltered and the solvent removed. The residue
was puri®ed by ¯ash chromatography (EtOAc:hexane,
1:4) to give 5-(20-bromoethyl)-2,2,5-trimethyl-1,3-dioxo-
cyclohexa-4,6-dione (6b) (1.183 g, 71%) as a white solid;
mL, 7.63 mmol) in DMF (5 mL) under N2. After a fur-
ther 52 h TLC (acetone:toluene, 1:9) indicated the con-
version of starting material (Rf 0.45) to major (Rf 0.5)
and minor (Rf 0.8) products. The reaction mixture was
added to water (100 mL) and extracted with ether (100
mL Â 3). The organic fractions were combined, dried
(MgSO4), ®ltered and the solvent removed. The residue
was puri®ed by ¯ash chromatography (EtOAc:hexane,
3:17 to 1:3) to give 5,5-di(tert-butoxycarbo)methyl-2,2-
dimethyl-1,3-dioxocyclohexa-4,6-dione (412 mg, 16%);
mp 103±105ꢀC (ether:hexane); H NMR CDCl3 d 1.41
1
(s, 18H, C(CH3)3Â2), 1.92 (s, 6H, C(CH3)2), 2.97 (s, 4H,
CH2COOButÂ2); 13C NMR CDCl3 d 28.5 (C(CH3)3),
29.2, 44.1 (CH2COOBut, C(CH3)2) 47.2 (C-5), 83.1
(C(CH3)3), 108.5 (C(CH3)2), 168.0, 168.9 (C-4, C-6,
COOBut); and a mixture of 5a and 5-(tert-butoxy-
carbo)methyl-2,2-dimethyl-1,3-dioxocyclohexa-4,6-dione
(5c). This mixture was puri®ed by repeated crystal-
lization from ether:hexane to give 5c (1.05 g, 59%) as a
white solid; mp 124±126ꢀC (ether:hexane); IR (KBr)
1772, 1755, 1712 cm 1 (CˆO); 1H NMR (CDCl3) d 1.43
(s, 9H, C(CH3)3), 1.80 (s, 6H, C(CH3)2), 3.09 (d, J 4Hz,
2H, CH2COOBut), 3.70 (t, J 4Hz, 1H, H-5); 13C NMR
CDCl3 d 18.7, 28.8, 33.0, 43.4 (CH2COOBut, C(CH3)2,
C-5), 28.5 (C(CH3)3), 82.8 (C(CH3)3), 105.6 (C(CH3)2),
165.6, 169.6 (C-4, C-6, COOBut); MS m/z (CI-): 257
([M-H] , 100), 200 (8), 159 (25) 143 (32%).
mp 84±85ꢀC (ether:hexane); IR (KBr) 1738, 1784 cm
1
1
(CˆO); H NMR (CDCl3) d 1.66 (s, 3H, CH3), 1.76,
1.78 (s  2, 3H  2, C(CH3)2), 2.61 (t, J=8 Hz, 2H, H-
10), 3.32 (t, J=8 Hz, 2H, H-20); 13C NMR (CDCl3) d
25.2, 26.6, 29.1, 30.1, 42.4 (CH3, C(CH3)2, C-10, C-20),
49.4 (C-5), 106.0 (C(CH3)2), 169.5 (C-4, C-6); MS m/z
(EI+): 249, 251 (M+-CH3, 5), 206, 208 (M+-OC(CH3)2,
14), 162, 164 (M+-C(O)OCO(CH3)2, 42), 69 (M+-
C(O)OCO(CH3)2-CH2Br, 100%).
NaSSO2CH3 (776 mg, 5.80 mmol) was added to a solu-
tion of 6b (1.18 g, 4.46 mmol) in DMF (40 mL) under
N2 and the resulting solution warmed to 50ꢀC. After
29 h the reaction solution was cooled and the solvent
removed. The residue was puri®ed by ¯ash chromato-
graphy (EtOAc:hexane, 3:7 to give 2-(20,20,50-trimethyl-
1,3-dioxocyclohexa-4,6-dionyl)ethyl methanethiosulfonate
(7b) (1.10 g, 83%) as a cloudy oil; IR (®lm) 1737, 1771
Anhydrous K2CO3 (300 mg, 2.17 mmol) was added to a
solution of 5c (400 mg, 1.55 mmol) in DMF (10 mL)
under N2 and stirred vigorously. After 1 h the super-
natant liquid was added dropwise to a solution of 1,2-
dibromoethane (0.7 mL, 8.06 mmol) in DMF (3 mL)
under N2 at 50ꢀC. After 70 h, TLC (EtOAc:hexane, 1:9)
indicated the conversion of starting material (Rf 0.1) to
a major product (Rf 0.3). The reaction mixture was
cooled, added to distilled water (50 mL) and extracted
with ether (50 mL Â 3). The organic fractions were
combined, dried (MgSO4), ®ltered and the solvent
removed. The residue was puri®ed by ¯ash chromato-
graphy (EtOAc:hexane, 1:9) to give 5-(20-bromoethyl)-
5-(tert-butoxycarbo)methyl-2,2-dimethyl-1,3-dioxocy-
clohexa-4,6-dione (6c) (372 mg, 66%) as a white solid;
mp 120±123ꢀC (ether:hexane); IR (KBr) 1773, 1731
cm 1 (CˆO); 1H NMR (CDCl3) d 1.40 (s, 9H, C(CH3)3),
1.80, 1.93 (s x 2, 3H Â 2, C(CH3)2), 2.41 (t, J=8 Hz,
2H, H-10), 3.11 (s, 2H, CHCOOBut), 3.32 (t, J=8 Hz,
2H, H-20); 13C NMR (CDCl3) d 25.2, 29.3, 29.9,
41.0, 41.4 (CH2COOBut, C(CH3)2, C-10, C-20), 28.5
(C(CH3)3), 51.2 (C-5), 83.2 (C(CH3)3), 107.8 (C(CH3)2),
167.8, 170.2 (C-4, C-6, COOBut); MS m/z (CI-): 287 (2),
257 (M-(CH2)2Br, 100), 142 (15) 79, 81 (Br, 91%).
1
1
cm
(CˆO), 1300, 1133 cm
(S±SO2); 1H NMR
(CDCl3) d 1.68 (s, 3H, CH3), 1.78, 1.79 (sÂ2, 3HÂ2,
C(CH3)2), 2.47±2.55 (m, 2H, H-2), 3.08±3.16 (m, 2H, H-
1), 3.34 (s, 3H, SSO2CH3); 13C NMR CDCl3 d 24.9,
29.3, 30.0, 31.9, 49.2 (CH3, C(CH3)2, C-1, C-2), 49.2
(C-50), 51.2 (SSO2CH3), 106.1 (C(CH3)2), 169.5 (C-40,
C-60); MS m/z (EI+): 281 (M+-CH3, 1), 269 (2), 239
(M+-C3H5O, 3), 159 (100), 141 (56), 113 (96), 103
(23), 87 (78), 69 (M+-C(O)OCO(CH3)2-CH2SSO2CH3,
79%).
Dowex 50W(H+) resin (2.53 g) was added to a suspen-
sion of 7b (1.08 g, 3.65 mmol) in p-dioxan (3.5 mL) and
distilled water (35 mL) and stirred at room temperature.
After 68 h the reaction mixture was ®ltered and the sol-
vent removed. The resulting solid was recrystallized
from water:acetone:ethyl acetate to give 1d (738 mg,
79%) as a white solid; mp 109±111ꢀC; IR (KBr) 1706
cm 1 (CˆO), 1317, 1133 cm 1 (S-SO2); 1H NMR (D2O)
d 1.43 (s, 3H, H-4), 2.25±2.33 (m, 2H, H-2), 3.16±3.24
(m, 2H, H-1), 3.45 (s, 3H, SSO2CH3); 13C NMR (D2O)
d 20.3 (C-4), 32.1, 36.4 (C-1, C-2), 50.5 (SSO2CH3), 54.3
(C-3), 176.0 (COOH); MS m/z (EI+): 256 (M+, 6), 132
(M+H+-CH2SSO2CH3, 40), 116 (59), 87 (100%);
HRMS m/z (CI-): Found 254.9996 ([M-H]-); C7H11O6S2
requires 254.9997.
NaSSO2CH3 (143 mg, 1.07 mmol) was added to a solu-
tion of 6c (301 mg, 0.82 mmol) in DMF (20 mL) under
N2 and the resulting solution warmed to 50ꢀC. After
29 h the reaction solution was cooled and the solvent
removed. The residue was puri®ed by ¯ash chromato-
graphy (EtOAc:hexane, 1:3) and crystallized from ether
to give 2-(50-(tert-butoxycarbo)methyl-20,20-dimethyl-
1,3-dioxocyclohexa-4,6-dionyl)ethyl methanethiosulfo-
nate (7c) (280 mg, 86%) as a colorless crystalline solid;
mp 103±105ꢀC (ether:hexane); IR (KBr) 1772, 1738,
3,3,4-Tricarboxybutyl methanethiosulfonate (1e). Anhy-
drous K2CO3 (1.2 g, 8.68 mmol) was added to a solu-
tion of Meldrum's acid (5a) (1 g, 6.94 mmol) in DMF
(20 mL) under N2 and stirred vigorously. After 2 h the
supernatant liquid was added dropwise over a period of
1 h 30 min to a solution of tert-butylbromoacetate (1.14

2300
B. G. Davis et al. / Bioorg. Med. Chem. 7 (1999) 2293±2301
1
1
1
1717 cm (CˆO) 1314, 1129 cm (S±SO2); H NMR
CDCl3 d 1.41 (s, 9H, C(CH3)3), 1.83, 1.93 (sÂ2, 3HÂ2,
C(CH3)2), 2.33±2.41 (m, 2H, H-2), 3.10±3.18 (m, 2H, H-
1), 3.13 (s, 2H, CH2COOBut), 3.32 (s, 3H, SSO2CH3);
13C NMR (CDCl3) d 28.0 (C(CH3)3), 28.9, 29.2, 30.7,
37.9, 40.2 (CH2COOBut, C(CH3)2, C-1, C-2), 50.0 (C-
50), 50.6 (SSO2CH3), 82.8 (C(CH3)3), 107.3 (C(CH3)2),
167.2, 169.7 (C-40, C-60, COOBut); MS m/z (CI-): 395
([M-H] , 1), 381 (M -CH3, 2), 281 (M -H-CH2COO-
But, 5), 257 (M -(CH2)2 SSO2CH3, 100), 215 (45), 158
(37%).
(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. MS m/z
(ES±MS): N62C-a calculated 26,826, found 26,828;
S156C-a33 calculated 26,853, found 26,859; S166C-a33
calculated 26,853, found 26,851; L217C-a31 calculated
26,827, found 26,828; N62C-b calculated 26,819, found
26,820; S156C-b calculated 26,846, found 26,846; S166C-
b calculated 26,846, found 26,846; L217C-b calculated
26,820, found 26,820; N62C-c calculated 26,897, found
26,896; S156C-c calculated 26,924, found 26,928; S166C-
c calculated 26,924, found 26,928; L217C-c calculated
26,898, found 26,904; N62C-d calculated 26,863, found
26,870; S156C-d calculated 26,890, found 26,892; S166C-
d calculated 26,890, found 26,894; L217C-d calculated
26,864, found 26,866; N62C-e calculated 26,907, found
26,909; S156C-e calculated 26,934, found 26,939; S166C-
e calculated 26,934, found 26,939; L217C-e calculated
26,908, found 26911.
A
solution of 7c (138 mg, 0.35 mmol) in
CF3COOD:D2O (7:3, 2 mL) was heated to 50ꢀC. After
32 h, H NMR spectroscopy showed the conversion of
starting material to a single product. The solution was
cooled and the solvent removed. The residue was pur-
i®ed by ¯ash chromatography (butan-1-ol:AcOH:water,
4:1:1) and ion exchange chromatography (Amberlyst
A21, 30% v/v CF3COOH (aq) as eluent) to give 1e (73
1
1
mg, 70%) as an amorphous solid; IR (KBr) 1706 cm
1
(CˆO) 1310, 1127 cm (S-SO2); 1H NMR (D2O) d
Active site titrations. The active enzyme concentration
was determined as previously described47 by monitoring
¯uoride ion release upon enzyme reaction with a-tolue-
nesulfonyl ¯uoride (PMSF) as measured by a ¯uoride
ion sensitive electrode (Orion Research 96-09). The
active enzyme concentration determined in this way was
used to calculate kcat values for each CMM except in the
case of S166C-c for which total protein concentration as
2.25±2.34 (m, 2H, H-2), 3.01 (s, 2H, H-4), 3.12±3.20 (m,
2H, H-1), 3.45 (s, 3H, SSO2CH3); 13C NMR (D2O) d
34.3, 37.7, 41.1 (C-1, C-2, C-4), 52.9 (SSO2CH3), 58.0
(C-3), 177.0, 177.1 (COOH, CH2COOH); MS m/z
(FAB-): 299 ([MH] , 42), 221 (21), 183 (40), 111 (49), 91
(100%). Anal. calcd for C8H12O8S2: C, 32.00; H, 4.03%;
found: C, 31.84; H 3.91%;
1
determined by absorbance at 280 nm (e280=23000 M
1 64
Site-speci®c chemical modi®cation. To approximately 25
mg of each of the SBL mutants in CHES bu€er (2.5 mL;
70 mM CHES, 5 mM MES, 2 mM CaCl2, pH 9.5) at
20ꢀC was added each of the methanethiosulfonate
reagents (100 mL of a 0.2 M solution: 1b in CH3CN:
H2O (1:9), 1a,c,d,e in water), in a PEG(MW 10,000)-
coated polypropylene test tube and mixed using an
end-over-end rotator. The progress of modi®cation was
followed using speci®c activity measurement, mon-
itored spectrophotometrically (10 mL aliquots in 0.1 M
Tris±HCl bu€er, pH 8.6, 0.005% Tween 80, and 1%
DMSO, with succinyl-AAPF-pNA (1 mg/mL) as
cm
)
was used.48
Kinetic measurements. Michaelis±Menten constants
were measured at 25(‹0.2)ꢀC by curve ®tting (GraFit1
3.03) of the initial rate data determined at eight or nine
concentrations (0.125±4.0 mM) of succinyl-AAPF-pNA
substrate in 0.1 M Tris±HCl bu€er containing 0.005%
1
Tween 80, 1% DMSO, pH 8.6 (e410=8800 M
cm 1).63
Acknowledgements
substrate at 25ꢀC, e410=8800 M 1 cm 1 63 on a Perkin±
)
Elmer Lambda 2 spectrophotometer. The reaction was
terminated when the addition of a further 100 mL of
methanethiosulfonate solution gave no further change
in speci®c activity, typically after 2 to 3 h. The reaction
solution was puri®ed on a disposable desalting column
(Pharmacia Biotech PD-10, Sephadex G-25 M) pre-
equilibrated with MES bu€er (5 mM MES, 2 mM
CaCl2, pH 6.5). The CMM was eluted with this bu€er
(3.5 mL), dialyzed against MES bu€er (10 mM MES, 1
mM CaCl2 pH 5.8, 1 L Â3) at 4ꢀC and subsequently
¯ash frozen and stored at 18ꢀC. The free thiol content
of all CMMs, was determined spectrophotometrically
Generous funding was provided by the Natural Sciences
and Engineering Research Council of Canada (NSERC)
and Genencor International Inc. who also provided
SBL-WT, -N62C, -S156C, -S166C and -L217C enzymes.
We also acknowledge the award of a scholarship from
NSERC (to G.D.). We thank Drs. Chris Murray,
Christian Paech, Thomas Graycar, and Colin Mitchinson
for helpful discussions.
References and Notes
1
1. Hermes, J. D.; Blacklow, S. C.; Knowles, J. R. Proc. Natl.
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lyzed by nondenaturing gradient (8±25%) gels at pH
4.2, run towards the cathode, on the Pharmacia Phast-
system and appeared as a single band. Each of the
CMMs showed reduced mobility relative to wild-type.
Prior to ES±MS analysis CMMs were puri®ed by FPLC

B. G. Davis et al. / Bioorg. Med. Chem. 7 (1999) 2293±2301
2301
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1
280 nm (e280=23,000 M cm 1). The KM value determined
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ethyl kcat 104 ‹2 s 1, KM 0.55 ‹ 0.04 mM, kcat/KM 189‹14
s
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mM, kcat/KM 379‹37 s 1mM 1; N62C-S-n-pentyl kcat 184‹5
1, KM 0.75‹0.05 mM, kcat/KM 245‹18 s 1mM 1; L217C-
S-n-pentyl kcat 87‹3 s 1; KM 0.52‹0.05 mM, kcat/KM
s
1
1
167‹17 s ; S156C-S-benzyl kcat 72‹2 s ; KM
1mM
0.59‹0.05 mM, kcat/KM 122‹11 s 1mM 1; S166C-S-ethyl
kcat 11.8‹0.5 s-1; KM 0.76‹0.08 mM, kcat/KM 15.5‹1.8
s
1mM 1; S166C-S-benzyl kcat 23.1‹0.5 s 1; KM 1.17‹0.06
mM, kcat/KM 19.7‹1.1 s 1mM
.
1
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