Synthesis and biological evaluation of novel irreversible serine protease inhibitors using amino acid based sulfonyl fluorides as an electrophilic trap

Article abstract of DOI:10.1016/j.bmc.2011.02.014

We have designed and synthesized novel irreversible serine protease inhibitors containing aliphatic sulfonyl fluorides as an electrophilic trap. These substituted taurine sulfonyl fluorides derived from taurine or protected amino acids were conveniently s

Full text of DOI:10.1016/j.bmc.2011.02.014

Bioorganic & Medicinal Chemistry 19 (2011) 2397–2406
Contents lists available at ScienceDirect
Bioorganic & Medicinal Chemistry
journal homepage: www.elsevier.com/locate/bmc
Synthesis and biological evaluation of novel irreversible serine protease
inhibitors using amino acid based sulfonyl fluorides as an electrophilic trap
⇑
Arwin J. Brouwer, Tarik Ceylan, Anika M. Jonker, Tima van der Linden, Rob M. J. Liskamp
Department of Medicinal Chemistry and Chemical Biology, Utrecht Institute for Pharmaceutical Sciences, Faculty of Science, Utrecht University, PO Box 80082, 3508 TB
Utrecht, The Netherlands
a r t i c l e i n f o
a b s t r a c t
Article history:
We have designed and synthesized novel irreversible serine protease inhibitors containing aliphatic sul-
fonyl fluorides as an electrophilic trap. These substituted taurine sulfonyl fluorides derived from taurine
or protected amino acids were conveniently synthesized from b-aminoethanesulfonyl chlorides using KF/
18-crown-6 or from b-aminoethanesulfonates using DAST. Their potency of irreversible inhibition of ser-
ine proteases is described in different enzyme assays using chymotrypsin leading to binding affinities up
Received 15 November 2010
Revised 2 February 2011
Accepted 9 February 2011
Available online 13 February 2011
to 22 lM.
Keywords:
Ó 2011 Elsevier Ltd. All rights reserved.
Serine protease
Irreversible inhibitor
Peptido sulfonyl fluoride
Electrophilic trap
Chymotrypsin
1. Introduction
sulfonamides,10 oligopeptidosulfonamides11 and recently in the
synthesis of sulfonyl azides.12 These sulfonyl chlorides were
relatively easy to prepare and turned out to be more stable than
anticipated. The versatile synthesis and good stability of the sulfo-
nyl chlorides, enticed us to attempt preparation of amino acid
derived sulfonyl fluorides as possible functionalized analogs of the
commonly used protease inhibitors PMSF and AEBSF (Fig. 1).
To our knowledge the synthesis of other functionalized sulfonyl
fluorides has not been described in the literature together with
applications as protease inhibitors or in other areas. This may be
due to a relative lack of synthetic procedures for the mild prepara-
tion of aliphatic sulfonyl fluorides.
Recently, we reported the first amino acid derived sulfonyl
fluorides and their potential as irreversible serine protease inhib-
itors was demonstrated by some preliminary investigations on
the reactivity.13 Herein, the ability of protease inhibition of the
amino acid based sulfonyl fluorides will be evaluated using dif-
ferent enzyme assays. In addition, their synthesis and character-
ization is described, as well as further functionalization on the
N-terminus.
Irreversible enzyme inhibitors usually bind covalently to an en-
zyme using an electrophilic moiety (‘warhead’). The Michael
acceptor is perhaps the most widespread electrophilic moiety pres-
ent in both many naturally occurring and man-made compounds.1
The plethora of applications of the Michael acceptor is at least
partly made possible by virtue of ‘tuning’ the reactivity of the
Michael acceptors, which ranges from very reactive Michael accep-
tors such as acrylonitrile, via those present in natural products for
example helenalin,2 to the vinylsulfone3 and vinylsulfonamide.4
Despite the availability of several other electrophiles for incorpora-
tion into molecules towards biologically active and relevant com-
pounds such as the chloromethylketone, aldehyde, beta-lactone,
epoxide, epoxyketone, acyloxymethyl ketone, epoxysuccinate and
borate moieties,5 this number is limited. Furthermore, the reacti-
vity of a potential electrophile is very important for selectivity
and discriminativity. It is hypothesized that in general for selective
biological applications one needs low-reactivity electrophiles,
which do not react immediately with all kinds of abundantly pres-
ent nucleophiles of for example proteins, as well as those present
in the aqueous environment. Previously, we have developed an
efficient synthesis of amino acid derived sulfonyl chlorides6 which
we have successfully employed for use in peptide-peptidomimetic
hybrids,7 synthetic receptors,8 ligands for catalysis,9 cyclic
⇑
Corresponding author. Tel.: +31 30 253 7396.
E-mail address: r.m.j.liskamp@uu.nl (R.M.J. Liskamp).
Figure 1. The structures of PMSF and AEBSF.
0968-0896/$ - see front matter Ó 2011 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmc.2011.02.014

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A. J. Brouwer et al. / Bioorg. Med. Chem. 19 (2011) 2397–2406
Table 1
2. Results and discussion
2.1. Chemistry
Yields (%) for the synthesis of sulfonyl fluorides 7b–f from Cbz- or Fmoc-protected
amino acids
Amino acid
3
4
6a
7c
7a,d
Natural Cbz- or Fmoc-protected amino acids with a variety of
side chains were chosen as starting materials for the sulfonyl fluo-
ride synthesis. First, Cbz- and Fmoc-protected amino acids 1b–f
(Ala, Val, Leu and Phe) were reduced to the corresponding alcohols
2b–f (Scheme 1) using sodium borohydride treatment of the in situ
prepared mixed anhydride.14
Mesylates 3b–f were obtained in reasonable to high yields
(Table 1) by reaction of alcohols 2b–f with methanesulfonyl
chloride in the presence of a base (Et3N).
Cbz-Ala-OH (b)
Cbz-Val-OH (c)
Cbz-Leu-OH (d)
Cbz-Phe-OH (e)
Fmoc-Val-OH (f)
82
50
91
61
51
44
87e
71
65
Nd
45
67
30b
65
40
76
62
Nd
65
58a
86e
86
80e
90
a
Yield over two steps.
Yield obtained using the TBAF procedure.
Yield obtained using the KF/18-crown-6 ether procedure.
Yield obtained using the DAST procedure.
Yields from earlier work.6
b
c
d
e
Next, thioacetates 4b–f were prepared in good to high yields
(65–91%) by reaction with in situ prepared cesium thioacetate.
Oxidation of the thioacetates using aqueous acetic acid and hydro-
gen peroxide and subsequent addition of sodium acetate afforded
sodium sulfonates 5b–f. These sodium sulfonates were treated
with a phosgene solution, to yield sulfonyl chlorides 6b–f. For
the synthesis of their corresponding sulfonyl fluorides we used
three different synthesis routes.13 The first method, a reaction with
TBAF,15 afforded sulfonyl fluoride 7f only in low yield (30%). Fur-
thermore, it was found that the yields were not reproducible
due to varying quantities of residual water in the TBAF-solution.
The second method, reaction with potassium fluoride using 18-
crown-6,16 was more successful, leading to reproducible and
higher yields (45–67%, 7b, 7d, and 7e). For the third method
diethylamino sulfur trifluoride (DAST)17 was used to convert the
sodium sulfonates (5b–e) directly into sulfonyl fluorides 7b–e in
good yields (40–76%, 2 steps). Besides omitting one reaction step,
another advantage of using DAST is that the acidic phosgene reac-
tion is avoided, and therefore acid labile protecting groups for
functional amino acids can be used.
was also evident from 19F NMR and from a 13C–F coupling in the
13C NMR spectra.
It is expected that selective protease inhibitors can be prepared
by further functionalization of the sulfonyl fluorides. To explore
the possibility for coupling reactions at the N-terminus, the Cbz-
group was cleaved from sulfonyl fluorides 7a and 7d using HBr
in acetic acid (Scheme 2).
After treatment with HBr, an ion exchange resin was used to ob-
tain the non-hygroscopic hydrochloride salts 8a–c. Taurine derived
hydrochloride salt 8a was successfully coupled to an amino acid
(Boc-Phe-OH) and a b-aminoethanesulfonyl chloride (6e) leading
to sulfonyl fluorides 9a and 10 in high yields (80% and 73%, respec-
tively). As a proof of principle, two ‘bulky’ amino acids (Boc-
Phe-OH and Boc-Leu-OH) were coupled to the more sterically
demanding leucine derived sulfonyl fluoride 7d, after Cbz-deprotec-
tion. Dipeptidosulfonyl fluorides 9b and 11 were both isolated in
high yields (74% and 73%, respectively), showing that in principle
any sequence can be prepared. The ability of a sulfonyl fluoride to re-
sist these harsh deprotection conditions and amine nucleophiles in
basic conditions, points to its relative stability as a ‘reactive’ group.
All sulfonyl fluorides (7a–f) were characterized by 1H, 13C and
2D NMR spectroscopy, elemental analysis (CHNF) and mass spec-
troscopy. Besides elemental analysis, the presence of the fluorine
2.2. Biological evaluation
Preliminary data on the reactivity clearly indicated the potential
of the amino acid based sulfonyl fluorides as possible serine and
cysteine protease inhibitors.13 Thus, the next step was to examine
the sulfonyl fluorides for inhibition of a serine protease. Chymo-
trypsin was chosen as a target enzyme for evaluation of the sulfo-
nyl fluorides. Chymotrypsin is one of the most widely studied
Scheme 1. Synthesis of amino acid based sulfonyl fluorides 7b–f starting from N-
protected amino acids.
Scheme 2. Modification of the N-terminus of sulfonyl fluorides 7a and 7d.

A. J. Brouwer et al. / Bioorg. Med. Chem. 19 (2011) 2397–2406
2399
0.25
0.23
0.21
0.19
0.17
0.15
0.13
0.11
0.09
0.07
0.05
0
5
10
15
20
25
30
Minutes
Figure 2. Progress curve of a chymotrypsin enzyme assay for comparison of the inhibitory activity of sulfonyl fluorides 7a–f and PMSF (j, control; N, 7a; h, 7b; ꢀ, 7c; 4, 7d;
ꢀ, 7e; +, 7f; O, PMSF). [E] = 1.0 M, [S] = 0.25 mM, [I] = 12.5 M.
l
l
substrate. PMSF was used as a reference inhibitor. The optimal chy-
motrypsin and substrate concentrations were found to be 1.0 and
12.5 lM, respectively. Sulfonyl fluorides 7a–f and PMSF were first
pre-incubated for 1 h with chymotrypsin before addition of the
substrate. Subsequently, the liberation of p-nitroaniline from Bz-
L-Tyr-pNA was measured at 405 nm during 30 min to determine
the residual enzyme activity. We were very pleased to see that
our sulfonyl fluorides were indeed capable of decreasing the activ-
ity of chymotrypsin (Fig. 2).
Figure 3. Structure of Fmoc-protected phenylalanine derived sulfonyl chloride 6g.6
members of the serine protease class of enzymes. First, sulfonyl
fluorides 7a–f (Fig. 8) were evaluated by an activity based enzyme
assay. For this a colorimetric assay in 96-well plates was developed
using a phosphate buffer (0.05 M, pH 7.0), and Bz-L-Tyr-pNA as a
Cbz-phenylalanine derived sulfonyl fluoride 7e was clearly the
best inhibitor, which might have been expected since the S1
0.17
0.15
0.13
0.11
0.09
0.07
0.05
0
5
10
15
20
25
30
Minutes
Figure 4. Progress curve of a chymotrypsin enzyme assay for comparison of the inhibitory activity of phenylalanine derived sulfonyl chlorides 6e and 6g, and sulfonyl
fluorides 7e and 8c (j, control; N, 6e; h, 6g; ꢀ, 8c; 4, 7e). [E] = 1.0 M, [S] = 0.25 mM, [I] = 12.5 M.
l
l

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A. J. Brouwer et al. / Bioorg. Med. Chem. 19 (2011) 2397–2406
subsite of chymotrypsin has a preference for aromatic side chains.
The inhibitory effect of 7e was even comparable to PMSF, which is
a known potent inhibitor. The inhibition results of the other sulfo-
nyl fluorides also showed the influence of the sulfonyl fluoride side
chain on the inhibition. Aliphatic side chain containing sulfonyl
fluorides 7b–d and 7f (Fig. 8) showed the weakest inhibition, most
likely due to low acceptance of the aliphatic group in the S1 sub-
site. Taurine derivative (7a), without side chain, was clearly inhib-
iting stronger. These results also showed that the aromatic ring
from the Cbz-group from 7e is not responsible for the high affinity
since the weakly binding sulfonyl fluorides 7b–d also contain a
Cbz-group. The influence of the protecting group on the binding
also seemed to be small since both Fmoc- and Cbz-valine derived
sulfonyl fluorides gave similar inhibition. In order to further inves-
tigate the inhibition of phenylalanine derived sulfonyl fluoride 7e,
other derivatives were also evaluated. Thus, 7e was compared with
Cbz-deprotected sulfonyl fluoride 8c, Cbz-protected sulfonyl chlo-
ride 6e and Fmoc-protected sulfonyl chloride 6g6 (Figs. 3 and 4).
As expected both sulfonyl chlorides (6e and 6f) showed only very
weak inhibition, most likely due to fast hydrolysis of the much more
reactive sulfonyl chloride moiety compared to the much more stable
sulfonyl fluorides. The decrease in inhibitory activity of Cbz-
deprotected sulfonyl fluoride 8c compared to 7e was remarkable.
Apparently the Cbz-group in 7e does participate in binding, or the
positively charged nitrogen from 8c reduces the binding affinity,
possibly due to repulsion in the chymotrypsin binding pocket. Since
taurine sulfonyl fluoride 7a was the second best inhibitor, it was
compared in the enzyme assay with Boc-Phe-Tau-F (9a), and
Cbz-protected sulfonamide containing sulfonyl fluoride 10, both
with an aromatic side chain on the P2-position (Fig. 5).
0.17
0.15
0.13
0.11
0.09
0.07
0.05
0
5
10
15
20
25
30
Minutes
Figure 5. Progress curve of a chymotrypsin enzyme assay for comparison of the inhibitory activity of taurine derived sulfonyl fluorides 7a, 9a and 10. (j, control; N, 7a; h,
9a; ꢀ, 10). [E] = 1.0
l
M, [S] = 0.25 mM, [I] = 12.5 lM.
0.25
0.23
0.21
0.19
0.17
0.15
0.13
0.11
0.09
0.07
0.05
0
10
20
30
40
50
60
Minutes
Figure 6. Progress curve of a papain enzyme assay for comparison of the inhibitory activity of sulfonyl fluorides 7a–e (j, control; N, 7a; h, 7b; ꢀ, 7c; 4, 7d; ꢀ, 7e).
[E] = 8.0 M, [S] = 1.0 mM, [I] = 2.5 M.
l
l

A. J. Brouwer et al. / Bioorg. Med. Chem. 19 (2011) 2397–2406
2401
200
150
100
50
0
-35
-25
-15
-5
5
15
25
35
[I] (uM)
Figure 7. A Dixon plot of inhibition of chymotrypsin by sulfonyl fluoride 7e ([S] (mM): j, 1.5; N, 1.7; ꢀ, 1.9; h, 2.3; 4, 2.5).
was expected for sulfonyl fluorides 7a–e. We were very pleased
Table 2
Inhibitor constants and kinactivation values of sulfonyl fluorides 7a–e (R-W[CH2SO2]-F)
to see clear inhibition for all sulfonyl fluorides, already at a rela-
tively low concentration of inhibitor (2.5 lM) (Fig. 6).
and PMSFa
kinact (minꢁ1
)
As expected, aliphatic side chain containing sulfonyl fluorides
7b, 7c and 7d were the weakest inhibitors. Phenylalanine derived
sulfonyl fluoride 7e was slightly better, but the best inhibitor
was again the taurine derived one (7a). The lack of an aliphatic
or aromatic side chain makes 7a the best inhibitor. It is expected
that lysine and arginine derived sulfonyl fluorides will inhibit
much stronger.
Sulfonyl fluoride
R
Ki (lM)
7b
7f
7d
7c
7a
Cbz-Ala
Fmoc-Val
Cbz-Leu
Cbz-Val
Cbz-Gly
Cbz-Phe
No inhibition
No inhibition
341 35.6
255 6.8
nd
nd
0.053 0.0017
0.043 0.006
0.13 0.006
0.33 0.03
104 10.7
22 1.6
7e
PMSF
13 0.8b
0.32 0.04b
Next, the chymotrypsin binding affinities were examined for
sulfonyl fluorides 7a–f (Fig. 8), and PMSF as a reference inhibitor.
These inhibitors were subjected to an assay with different inhibitor
and substrate concentrations. The remaining activities were mea-
sured and used in Dixon plots (Fig. 7), from which the Ki values
were determined.18 The Ki values found (Table 2) for the sulfonyl
fluorides were in agreement with the results from the comparison
assay (Fig. 2).
a
Ki values were determined using Dixon plots.18
b
Literature Ki value: 28 l .
M; kinact value: 0.32 minꢁ1 19
As expected, the best Ki values were found for phenylalanine
derived sulfonyl fluoride 7e (22
lM) and PMSF (13 lM, literature:
28
l
M),19 showing that the binding affinity of 7e is similar to
PMSF. We think that these low micromolar Ki values are very
encouraging and that the affinity for the active site can be im-
proved by modification of the N-terminus, for example, by intro-
duction of (an) amino acid residue(s). Lineweaver–Burk plots
from sulfonyl fluorides 7a and 7e showed that the mode of inhibi-
tion was competitive for the substrate.
Figure 8. Structures of sulfonyl fluorides 7a–f.
kinactivation values were determined by plotting residual enzyme
activity as a function of preincubation time. The lowest values
were found for aliphatic sulfonyl fluorides 7c and 7d (0.043 and
0.053 minꢁ1, respectively). Glycine derived sulfonyl fluoride 7a
was clearly better (0.13 minꢁ1), but the highest kinactivation value
was obtaꢁin1ed for phenylalanine derived sulfonyl fluoride 7e
(0.33 min ). The obtained kinactivation value was equal to PMSF
(0.32 minꢁ1, literature value19: 0.32 minꢁ1).
Both derivatives were inhibiting less good than 7a, of which sul-
fonamide 10 was clearly the better one.
To determine whether the sulfonyl fluorides were also capable
of inhibiting cysteine proteases, an enzyme assay was performed
with papain, which is the most widely studied member of the cys-
teine protease class of enzymes. For this a colorimetric assay in 96-
well plates was used. In this assay a phosphate buffer (0.10 M, pH
6.5) containing EDTA (1.5 mM) was used, and Bz-L-Arg-pNA
(1.0 mM) as a substrate. DTT, normally used in papain enzyme as-
says to activate the enzyme, was not used because DTT can cleave
the covalent papain-inhibitor linkage. A relatively high concentra-
3. Conclusions
tion of papain (8.0 lM) was used because only 10% of the papain
was active without activation. Since papain has a preference for ba-
sic residues in the S1 subsite, low inhibition and low selectivity
In conclusion, we have developed a successful synthesis of
substituted b-aminoethanesulfonyl fluorides, which can be pre-
pared starting from, in principle, any Cbz- or Fmoc-protected

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A. J. Brouwer et al. / Bioorg. Med. Chem. 19 (2011) 2397–2406
amino acid. Different procedures were used for fluorination of
which DAST and KF/18-crown-6 were the best. By using DAST for
introduction of the fluorine atom, strong acidic conditions were
avoided, which allows the use of acid labile protecting groups pres-
ent in functional amino acid derivatives. The described sulfonyl flu-
orides were capable of irreversible competitive inhibition of the
serine protease chymotrypsin. In addition, the affinities were
found to be dependent on the side chain of the inhibitor, which
points at the possibilities for tuning the selectivity and affinity.
66.8 (CH2 (Cbz)), 71.6 (SOCH2), 128.1, 128.2, 128.5, 136.2 (Ar-C
(Cbz)), 155.5 (C@O (Cbz)). Anal. Calcd for C12H17NO5S: C, 50.16;
H, 5.96; N, 4.87. Found: C, 49.99; H, 6.05; N, 4.81; ESI-MS: m/
z = 309.95 [M+Na]+.
4.1.2. Cbz-Val-w[CH2O]-Ms (3c)
The reaction was performed on a 25 mmol scale and purified
using column chromatography (EtOAc/hexanes, 1:6) to yield a
white solid (7.77 g, 50%). Depending on the purity of the crude
mesylate, it can be purified either by crystallization or by column
chromatography. Rf = 0.54 (EtOAc/hexanes, 5:95); mp = 64 °C; 1H
NMR (300 MHz, CDCl3): d = 1.00 (2d, J = 7.7 Hz and J = 8.5 Hz, 6H,
CH(CH3)2), 1.89 (m, 1H, CH(CH3)2), 2.93 (s, 3H, SO2CH3), 3.71 (m,
1H, NCH), 4.27 (d, J = 4.1 Hz, 2H, SOCH2) 4.74 (br s, 1H, NH), 5.13
(s, 2H, CH2 (Cbz)), 7.36 (m, 5H, C6H5 (Cbz)); 13C NMR (75 MHz,
CDCl3): d = 18.3, 19.2 (CH(CH3)2), 28.9 (CH(CH3)2), 37.2 (SO2CH3),
55.5 (NCH), 66.8 (CH2 (Cbz)), 69.3 (SOCH2), 128.0, 128.1, 128.5,
136.3 (Ar-C), 165.1 (C@O (Cbz)). Anal. Calcd for C14H21NO5S: C,
53.32; H, 6.71; N, 4.44. Found: C, 53.19; H, 6.74; N, 4.28; ESI-MS:
m/z = 353.95 [M+K]+.
The binding affinities were good, up to 22
lM, of which the best
one was comparable to PMSF (28
l
M),19 as well as the rate
constants. Hardly any chymotrypsin inhibition was observed with
substituted b-aminoethanesulfonyl chlorides, most likely due to
fast hydrolysis. This shows the importance of the moderate
reactivity needed for a suitable electrophilic trap for use in serine
protease inhibitors. The sulfonyl fluorides were also capable of
inhibition of the cysteine protease papain. We believe that these
sulfonyl fluorides will become a new important class of irreversible
serine and/or cysteine protease inhibitors of which the affinity can
be further improved and/or tuned by using different amino acid
side chains and/or by extending the N-terminus. Currently, we
are preparing and evaluating small libraries of N-terminal
functionalized amino acid based sulfonyl fluorides for finding
inhibitors with higher affinities for biologically more relevant
serine proteases.
4.1.3. Cbz-Leu-w[CH2O]-Ms (3d)
The reaction was performed on a 39.5 mmol scale to yield a yel-
low oil. The crude mesylate was directly used in the synthesis of
thioacetate 4d. A small sample was purified using column chroma-
tography (EtOAc/hexanes, 1:6) for characterization. Rf = 0.78
(EtOAc/hexanes, 1:9); mp = 57 °C; 1H NMR (300 MHz, CDCl3):
d = 0.93 (d, J = 6.3 Hz, 6H, CH2CH(CH3)2), 1.43 (m, 2H,
CH2CH(CH3)2), 1.70 (m, 1H, CH2CH(CH3)2), 2.92 (s, 3H, SO2CH3),
3.94 (m, 1H, NCH), 4.14 (dd, Jgem = 9.9 Hz, Jvic = 4.0 Hz, 1H, SOCHa),
4.25 (dd, Jgem = 9.9 Hz, Jvic = 3.0 Hz, 1H, SOCHb), 4.95 (br s, 1H,
CONH), 5.10 (s, 2H, CH2 (Cbz)), 7.34 (m, 5H, C6H5 (Cbz)). Anal. Calcd
for C15H23NO5S: C, 54.69; H, 7.04; N, 4.25. Found: C, 54.66; H, 7.11;
N, 4.21; ESI-MS: m/z = 367.95 [M+K]+.
4. Experimental
Peptide grade solvents for synthesis were purchased from Bio-
solve (The Netherlands) and were stored on molecular sieves
(4 Å). Reactions were carried out at ambient temperature unless
stated otherwise. TLC analysis was performed on Merck pre-coated
Silica Gel 60 F-254 (0.25 mm) plates. Spots were visualized with
UV light, ninhydrin or Cl2-TDM.20 Solvents were evaporated under
reduced pressure at 40 °C. Column chromatography was per-
4.2. General procedure for the synthesis of thioacetates 4b–g
formed on ICN Silica Gel 60 (32–63 lm). Melting points were mea-
sured on a Büchi Schmelzpunktbestimmungsapparat (according to
dr. Tottoli) and are uncorrected. Electrospray mass spectra were re-
corded on a Shimadzu LC/MS-QP-8000 spectrometer, or a Finnigan
LCQ Deca XP MAX spectrometer. Elemental analyses were carried
out at Kolbe Mikroanalytisches Laboratorium (Mülheim an der
Ruhr, Germany). 1H NMR (300 MHz), 13C NMR (75 MHz) and COSY
spectra were recorded on a Varian G-300 spectrometer. Chemical
shifts are reported in ppm relative to TMS (0 ppm) or DMSO
(2.50 ppm) for the 1H NMR and to CDCl3 (77 ppm) or DMSO
(39.5 ppm) for the 13C NMR spectra as internal standards. 13C
NMR spectra were recorded using the attached proton test (APT)
pulse sequence.
Thioacetates 4b–g were prepared following our earlier pub-
lished protocol.6 All products were purified by recrystallization
(from EtOAc/hexanes) and/or by column chromatography (10%
hexanes/EtOAc). Compounds 4e–g were synthesized and charac-
terized as described before.6
4.2.1. Cbz-Ala-w[CH2S]-Ac (4b)
The reaction was performed on a 31 mmol scale to yield off-
white crystals (6.20 g, 91%). Rf = 0.30 (CH2Cl2); mp = 57 °C; 1H
NMR (300 MHz, CDCl3): d = 1.18 (d, J = 6.6 Hz, 3H, CHCH3), 2.32
(s, 3H, COCH3), 3.04 (d, J = 5.2 Hz, 2H, SCH2), 3.93 (m, 1H, NCH),
4.90 (br s, 1H, NH), 5.08 (s, 2 H, CH2 (Cbz)), 7.34 (m, 5H, C6H5
(Cbz)); 13C NMR (75 MHz, CDCl3): d = 20.0 (CHCH3), 30.4 (COCH3),
34.8 (SCH2), 47.0 (NCH), 66.5 (CH2 (Cbz)), 128.0, 128.4, 136.4 (Ar-
C), 155.5 (C@O (Cbz)), 195.6 (COCH3). Anal. Calcd for C13H17NO3S:
C, 58.40; H, 6.41; N, 5.24. Found: C, 58.25; H, 6.29; N, 5.14; ESI-MS:
m/z = 289.95 [M+Na]+.
4.1. General procedure for the synthesis of mesylates 3b–g
Mesylates 3b–g were prepared following our earlier published
protocol.6 All products were recrystallized from CH2Cl2/hexanes,
except 3d, which was used directly in the next reaction without
purification. Compounds 3e–g were synthesized and characterized
by as described.6
4.2.2. Cbz-Val-w[CH2S]-Ac (4c)
The reaction was performed on a 36 mmol scale and purified by
column chromatography followed by crystallization to yield white
crystals (6.93 g, 65%). Rf = 0.35 (CH2Cl2); mp = 69 °C; 1H NMR
(300 MHz, CDCl3): d = 0.94 (2d, J = 6.9 Hz (both), 6H, CH(CH3)2),
1.82 (m, 1H, CH(CH3)2), 2.27 (s, 3H, COCH3), 2.97 (dd, Jgem = 9.9 Hz,
Jvic = 4.0 Hz, 1H, SOCHa), 3.06 (dd, Jgem = 9.9 Hz, Jvic = 3.0 Hz, 1H,
SOCHb), 3.64 (m, 1H, NCH), 4.83 (d, J = 8.3 Hz, 1H, NH), 5.12 (2d,
J = 12.1 Hz (both), 2H, CH2 (Cbz)), 7.33 (m, 5H, C6H5 (Cbz)); 13C
NMR (75 MHz, CDCl3): d = 17.8, 19.1 (CH(CH3)2), 30.4 (COCH3),
31.6 (SCH2), 32.0 (CH(CH3)2), 56.5 (NCH), 66.5 (CH2 (Cbz)), 127.8,
4.1.1. Cbz-Ala-w[CH2O]-Ms (3b)
The reaction was performed on a 55.2 mmol scale to yield off-
white crystals (11.9 g, 82%). Rf = 0.39 (EtOAc/CH2Cl2, 1:9);
mp = 86 °C; 1H NMR (300 MHz, CDCl3): d = 1.24 (d, 3H, CHCH3),
2.96 (s, 3H, SO2CH3), 4.02 (m, 1H, NCH), 4.15 (dd, Jgem = 10.2 Hz,
Jvic = 4.4 Hz, 1H, SOCHa), 4.24 (br dd, 1H, SOCHb), 4.92 (br s, 1H,
NH), 5.10 (s, 2H, CH2 (Cbz)), 7.35 (m, 5H, C6H5 (Cbz)); 13C NMR
(75 MHz, CDCl3): d = 17.1 (CHCH3), 37.2 (SO2CH3), 46.1 (NCH),

A. J. Brouwer et al. / Bioorg. Med. Chem. 19 (2011) 2397–2406
2403
127.9, 128.3, 136.6 (Ar-C (Cbz)), 156.3 (C@O (Cbz)),195.9 (COCH3).
Anal. Calcd for C15H21NO3S: C, 60.99; H, 7.17; N, 4.74. Found: C,
61.02; H, 7.20; N, 4.65; ESI-MS: m/z = 317.95 [M+Na]+, 333.95
[M+K]+.
C6H5 (Cbz)); 13C NMR (75 MHz, CDCl3): d = 21.5, 22.8
(CH2CH(CH3)2), 24.7 (CH2CH(CH3)2), 42.2 (CH2CH(CH3)2), 46.9
(NCH), 67.0 (CH2 (Cbz)), 69.0 (SCH2), 128.0, 128.2, 128.5, 136.0
(Ar-C (Cbz)), 155.5 (C@O (Cbz)); ESI-MS: m/z = 396.75
[M+CH3CN+Na]+.
4.2.3. Cbz-Leu-w[CH2S]-Ac (4d)
The reaction was performed on a 38.4 mmol scale and purified
by column chromatography to yield a white solid (6.80 g, 58%).
Rf = 0.40 (CH2Cl2); mp = 52 °C; 1H NMR (300 MHz, CDCl3):
d = 0.90 (d, J = 6.6 Hz, 6H, CH(CH3)2), 1.28, 1.65 (2 m, 3H,
CHCH2CH(CH3)2), 2.29 (s, 3H, COCH3), 2.97 (dd, Jgem = 14.0 Hz,
Jvic = 7.2 Hz, 1H, SOCHa), 3.11 (dd, Jgem = 14.0 Hz, Jvic = 4.7 Hz, 1H,
SOCHb), 3.90 (m, 1H, NCH), 4.75 (br d, 1H, NH), 5.14 (2d,
J = 12.4 Hz (both), 2H, CH2 (Cbz)), 7.33 (m, 5H, C6H5 (Cbz)); 13C
NMR (75 MHz, CDCl3): d = 22.1, 22.9 (CH(CH3)2), 24.8 (CH(CH3)2),
30.4 (COCH3), 34.2 (SCH2), 43.5 (CH2CH(CH3)2), 49.2 (NCH), 66.5
(CH2 (Cbz)), 127.9, 128.0, 128.4, 136.5 (Ar-C), 155.9 (C@O (Cbz)),
195.9 (COCH3). Anal. Calcd for C16H23NO3S: C, 62.11; H, 7.49; N,
4.53. Found: C, 62.08; H, 7.45; N, 4.42; ESI-MS: m/z = 332.00
[M+Na]+, 347.95 [M+K]+.
4.4. General procedures for the synthesis of sulfonyl fluorides
7a–f
Procedure 1 (using KF): To a solution of the sulfonyl chloride
(6a–b and 6d–f; 4,0 mmol) in acetonitrile (20 mL) was added
potassium fluoride (465 mg, 8,0 mmol) and 18-Crown-6 (53 mg,
0,20 mmol). After stirring overnight at rt, the mixture was concen-
trated in vacuo and purified by column chromatography.
Procedure 2 (using DAST): Thioacetates 4b–f were oxidized to so-
dium sulfonates 5b–f following an earlier described protocol.6 To a
suspension of sulfonate salt (2.69 mmol) in DCM (14 mL) was
added 640 lL DAST (4.84 mmol). After stirring overnight at rt,
the mixture was concentrated in vacuo and purified by column
chromatography.
Procedure 3 (using TBAF): To a solution of the sulfonyl chloride
(6a or 6f) (10.0 mmol, 3.91 g) in THF (200 mL) was added a tetra-
butylammonium fluoride solution in THF (15 mL, 1.0 M). After stir-
ring for 2 h at rt, HCl (200 mL, 1.0 M) was added and the THF was
evaporated in vacuo. After addition of dichloromethane (400 mL),
the layers were separated and the water layer was extracted with
dichloromethane (400 mL). After drying (Na2SO4) and concentra-
tion in vacuo of the combined organic layer, the sulfonyl fluoride
was purified by column chromatography.
4.3. General procedure for the synthesis of sulfonyl chlorides
6b–g
Sulfonyl chlorides 6b–g were prepared following our earlier
published protocol.6 After concentration of the reaction mixture,
the residue was taken up in dichloromethane (50 mL) and washed
once with water (30 mL) for removal of the salts. After separation,
the organic layer was dried (Na2SO4) and concentrated in vacuo. All
products were purified by a fast silica plug. Compounds 6a and 6e–
g were synthesized and characterized by us before.6
4.4.1. Cbz-Tau-w[CH2SO2]-F (7a)
Eluent for column chromatography: CH2Cl2. Yield procedure 1:
70% (720 mg, white solid), yield procedure 2: 42% (110 mg, white
solid), yield procedure 2 via the crude sulfonyl chloride: 60%
(15.7 g, over 2 steps); yield procedure 3: 31% (328 mg, white solid).
Rf = 0.30 (CH2Cl2); mp = 124 °C; 1H NMR (300 MHz, CDCl3): d = 3.62
(m, 2H, SO2CH2), 3.72 (m, 2H, NCH2), 5.12 (s, 2H, CH2 (Cbz)), 5.38
(br s, 1H, NH), 7.35 (m, 5H, C6H5); 13C NMR (CDCl3) d = 36.6
(NCH2), 50.6, 50.8 (SO2CH2), 67.3 (CH2 (Cbz)), 128.1 128.4 128.6
135.8 (Ar-C), 156.1 (C@O (Cbz)); 19F NMR (282 MHz, CDCl3):
d = ꢁ120.9 (s). Anal. Calcd for C10H12FNO4S: C, 45.97; H, 4.63; N,
5.36; F, 7.27. Found: C, 46.20; H, 4.57; N, 5.25; F, 7.21.
4.3.1. Cbz-Ala-w[CH2SO2]-Cl (6b)
The reaction was performed on a 6.0 mmol scale to yield a
white solid (1.07 g, 61%). Eluent silica plug: hexanes/EtOAc, 1:9,
the purified product hydrolyzed clearly faster than sulfonyl chlo-
rides with different side chains. Rf = 0.34 (hexanes/CH2Cl2, 1:9);
mp = 85 °C; 1H NMR (300 MHz, CDCl3): d = 1.44 (br d, 3H, CHCH3),
3.22 (br s, 1H, CHCH3), 3.82, 4.08 (2 br d, 2H, CH2SO2Cl), 4.34 (m,
1H, NCH), 5.12 (s, 2H, CH2 (Cbz)), 5.28 (m, 1H, NH), 7.35 (m, 5H,
C6H5 (Cbz)); 13C NMR (75 MHz, CDCl3): d = 19.6 (CHCH3), 44.4
(NCH), 67.0 (CH2 (Cbz)), 69.5 (CH2SO2Cl), 128.1, 128.2, 128.3,
128.5, 128.6, 128.8, 128.9, 129.3, 135.2, 135.9 (Ar-C (Cbz)), 155.3
(C@O (Cbz)); ESI-MS: m/z = 313.15 [M+Na]+.
4.4.2. Cbz-Ala-w[CH2SO2]-F (7b)
Eluent for column chromatography: hexanes/CH2Cl2, 5:95. Yield
procedure 1: 65% (900 mg, white solid), yield procedure 2: 65%
(180 mg, white solid). Rf = 0.30 (CH2Cl2); mp = 85 °C; 1H NMR
(300 MHz, CDCl3): d = 1.42 (d, J = 6.9 Hz, 3H, CHCH3), 3.51, 3.56
4.3.2. Cbz-Val-w[CH2SO2]-Cl (6c)
The reaction was performed on a 3.0 mmol scale to yield a
white solid (490 mg, 51%). Eluent silica plug: hexanes/EtOAc,
3:10, the purified product contained some N-formylated side prod-
uct which could be more easily separated after conversion to the
sulfonyl fluoride. Rf = 0.45 (CH2Cl2); mp = 74 °C; 1H NMR
(300 MHz, CDCl3): d = 0.99 (2d, J = 6.7 Hz (both), 6H, CH(CH3)2),
2.10 (m, 1H, CH(CH3)2), 3.87–4.08 (m, 2H, CH2SO2Cl), 4.12 (m,
1H, NCH), 5.12 (s, 2H, CH2 (Cbz)), 5.21 (br s, 1H, NH), 7.34 (m, 5
H, C6H5 (Cbz)); 13C NMR (75 MHz, CDCl3): d = 17.9, 18.8 (CH(CH3)2),
31.4 (CH(CH3)2), 53.6 (NCH), 67.1 (CH2SO2Cl, CH2 (Cbz)), 127.8,
128.0, 128.1, 128.2, 128.5, 136.0 (Ar-C (Cbz)), 155.7 (C@O (Cbz)).
(2t, Jgem = 14.7 Hz, Jvic = 5.3 Hz, JH F = 5.3 Hz, 1H, SOCHa), 3.70
(dd, Jgem = 14.7 Hz, Jvic = 3.5 Hz, 1H, SOCHb), 4.25 (m, 1H, NCH),
5.10 (s, 2H, CH2 (Cbz), 5.28 (s, 1H, NH), 7.34 (m, 5H, C6H5 (Cbz));
13C NMR (75 MHz, CDCl3): d = 19.2 (CH3), 43.3 (NCH), 55.1, 55.3
(CH2SO2), 67.0 (CH2 (Cbz)), 128.1, 128.3, 128.5, 135.9 (Ar-C
(Cbz)), 155.2 (C@O (Cbz)); 19F NMR (282 MHz, CDCl3): d = ꢁ115.6
(s). Anal. Calcd for C11H14FNO4S: C, 47.99; H, 5.13; N, 5.09; F,
6.90. Found: C, 48.11; H, 5.08; N, 5.15; F, 6.82; ESI-MS: m/
z = 313.95 [M+K]+.
3
a
4.3.3. Cbz-Leu-
w
[CH2SO2]-Cl (6d)
4.4.3. Cbz-Val-w[CH2SO2]-F (7c)
The reaction was performed on a 3.0 mmol scale to yield a
white solid (440 mg, 44%). Eluent silica plug: hexanes/EtOAc, 3:7;
Rf = 0.40 (hexanes/CH2Cl2, 2:8); mp = 45 °C; 1H NMR (300 MHz,
CDCl3): d = 0.95 (d, J = 5.8 Hz, 6H, CH2CH(CH3)2), 1.68, 1.71 (2 m,
3H, CH2CH(CH3)2), 3.86 (dd, Jgem = 14.0 Hz, Jvic = 3.3 Hz, 1H, SOCHa),
4.08 (dd, Jgem = 14.0 Hz, Jvic = 6.1 Hz, 1H, SOCHb), 4.25 (m, 1H, NCH),
5.10 (s, 2H, (CH2 (Cbz)), 5.21 (d, J = 8.0 Hz, 1H, NH), 7.34 (m, 5H,
Eluent for column chromatography: hexanes/CH2Cl2, 3:7. Yield
procedure 2: 40% (120 mg, white solid). Rf = 0.45 (CH2Cl2);
mp = 123 °C; 1H NMR (300 MHz, CDCl3): d = 0.98 (2d, J = 6.7 Hz
(both), 6H, CH(CH3)2), 2.07 (m, 1H, CH(CH3)2), 3.57, 3.62 (2t,
3
a
Jgem = 14.8 Hz, Jvic = 4.3 Hz, JH F= 4.3 Hz, 1H, SOCHa), 3.71 (dd,
Jgem = 14.8 Hz, Jvic = 7.4 Hz, 1H, SOCHb), 4.00 (m, 1H, NCH), 5.05,
5,08 (d, J = 8.8 Hz, 1H, NH), 5.12 (s, 2H, CH2 (Cbz)), 7.35 (m, 5 H,

2404
A. J. Brouwer et al. / Bioorg. Med. Chem. 19 (2011) 2397–2406
C6H5 (Cbz)); 13C NMR (75 MHz, CDCl3): d = 18.0, 19.2 (CH(CH3)2),
31.1 (CH(CH3)2), 52.5, 52.6 (CH2SO2Cl), 52.7 (NCH), 67.1 (CH2
(Cbz)), 128.0, 128.2, 128.5, 136.0 (Ar-C (Cbz)), 155.7 (C@O (Cbz));
19F NMR (282 MHz, CDCl3): d = ꢁ116.3 (s). Anal. Calcd for
2 ꢂ 8 (600 mg, Cl-form) was added. Stirring for 5 min at rt, fol-
lowed by filtration and concentration in vacuo, afforded the HCl-
salt. The HCl-salts (8a–c) were obtained as white solids in very
high yields (90–100%).
C
13H18FNO4S: C, 51.47; H, 5.98; N, 4.62; F, 6.26. Found: C, 52.01;
H, 6.10; N, 5.26; F, 5.64; ESI-MS: m/z = 325.95 [M+Na]+, 342.30
4.6. General procedure for coupling of an amino acid to Cbz-
deprotected sulfonyl fluorides 8a and 8b
[M+K]+.
4.4.4. Cbz-Leu-
w
[CH2SO2]-F (7d)
To a solution of HCl-salt (1.0 mmol, 8a, 8b) in dichloromethane
Eluent for column chromatography: hexanes/CH2Cl2, 3:7. Yield
procedure 1: 45% (530 mg, white solid), yield procedure 2: 76%
(334 mg, white solid). Rf = 0.40 (hexanes/CH2Cl2, 1:9); mp = 62 °C;
1H NMR (300 MHz, CDCl3): d = 0.93 (3 lines, 6H, CH2CH(CH3)2),
1.49, 1.70 (2 m, 3H, CH2CH(CH3)2), 3.58, 3.64 (2t, Jgem = 14.8 Hz,
(40 mL) was added a Boc-protected amino acid (1.0 mmol), BOP
(464 mg, 1.05 mmol), and DiPEA (385 lL, 2.33 mmol). The mixture
was stirred overnight at rt, and if necessary additional DiPEA was
added to keep the mixture basic. After concentration, EtOAc
(35 mL) was added the organic layer was washed with 1.0 M
KHSO4 (3 ꢂ 20 mL), 5% NaHCO3 (3 ꢂ 20 mL, and brine. Drying
(Na2SO4), followed by column chromatography afforded sulfonyl
fluorides 9a, 9b and 11 as white solids.
Jvic = 5.1 Hz, 3JH F = 5.1 Hz, 1H, SOCHa), 3.70, 3.74 (2t, Jgem = 14.8 Hz,
a
Jvic = 5.6 Hz, 3JH F = 1.4 Hz, 1H, SOCHb), 4.20 (m, 1H, NCH), 5.11 (s,
b
2H, (CH2 (Cbz)), 5.15 (s, 1H, NH) 7.35 (m, 5H, C6H5 (Cbz)); 13C
NMR (75 MHz, CDCl3): d = 21.5, 22.8 (CH2CH(CH3)2), 24.7
(CH2CH(CH3)2), 41.9 (CH2CH(CH3)2), 45.8 (NCH), 54.6, 54.7
(SCH2), 67.1 (CH2 (Cbz)), 128.0, 128.3, 128.6, 135.9 (Ar-C (Cbz)),
155.5 (C@O (Cbz)); 19F NMR (282 MHz, CDCl3): d = ꢁ115.0 (s). Anal.
Calcd for C14H20FNO4S: C, 52.98; H, 6.35; F, 5.99; N, 4.41. Found: C,
53.06; H, 6.41; F, 6.03; N, 4.29; ESI-MS: m/z = 339.95 [M+Na]+,
355.95 [M+K]+.
4.6.1. Boc-Phe-Tau-F (9a)
Eluent for column chromatography: hexanes/ethyl acetate, 5:1.
Yield: 80% (213 mg, white solid). Rf = 0.27 (acetone/CH2Cl2; 5:95);
mp = 128 °C; 1H NMR (300 MHz, CDCl3/CD3CN): d = 1.41 (s, 9H,
C(CH3)3), 2.88 (m, 1H, PhCHa), 3.00 (dd, Jgem = 13.8 Hz, Jvic = 6.3 Hz,
1H, PhCHb), 3.42–3.67 (m, 4H, SO2CH2CH2), 4.28 (m, 1H, NCH), 5.21
(d, 1H, NHBoc), 7.00 (s, 1H, NHCH2CH2SO2), 7.16 (m, 5H, Ar-CH);
13C NMR (75.5 MHz, CDCl3/CD3CN): d = 27.8, (C(CH3)3), 33.5
(NHCH2), 38.0 (CH2Ph), 49.5, 49.7 (CH2SO2), 55.3 (NCH) 79.8
(C(CH3)3) 126.6, 128.3, 128.9, 136.3 (Ar-C), 155.1 (C@O (Boc)),
172.1 (CHC(O)NH); 19F NMR (282 MHz, CDCl3): d = ꢁ121.6 (s).
Anal. Calcd for C16H23FN2O5S: C, 51.32; H, 6.19; N, 7.48; F, 5.07.
Found: C, 51.40; H, 6.21; N, 7.37; F, 5.01; ESI-MS: m/z = 275.0
[MꢁBoc+Na]+.
4.4.5. Cbz-Phe-w[CH2SO2]-F (7e)
Eluent for column chromatography: hexanes/CH2Cl2, 2:8. Yield
procedure 1: 67% (640 mg, white solid), yield procedure 2: 62%
(520 mg, white solid). Rf = 0.39 (CH2Cl2); mp = 124 °C; 1H NMR
(300 MHz, CDCl3): d = 3.05 (m, 2H, CHCH2C6H5), 3.55, 3.60 (2t,
3
a
Jgem = 14.6 Hz, Jvic = 4.5 Hz, JH F = 4.5 Hz, 1H, SOCHa), 3.75 (dd,
Jgem = 14.6 Hz, Jvic = 5.9 Hz, 1H, SOCHb), 4.32 (m, 1H, NCH), 5.09
(s, 2H, CH2 (Cbz)), 5.21 (d, J = 7.4 Hz, 1H, NH), 7.15–7.36 (m, 10H,
2xC6H5); 13C NMR (75 MHz, CDCl3): d = 38.9 (CHCH2CH6H5), 48.9
(NCH), 52.8, 53.0 (CH2SO2F), 127.4, 128.0, 128.3, 128.5, 129.0,
129.2, 135.7, 135.9 (2 ꢂ C6H5), 155.4 (C@O (Cbz)); 19F NMR
(282 MHz, CDCl3): d = ꢁ115.7 (s). Anal. Calcd for C17H18FNO4S: C,
58.11; H, 5.16; N, 3.99; F, 5.41. Found: C, 58.20; H, 5.22; N, 3.86;
F, 5.26; ESI-MS: m/z = 389.90 [M+K]+.
4.6.2. Boc-Phe-Leu-w[CH2SO2]-F (9b)
The reaction was performed on 0.43 mmol scale to yield a white
solid (138 mg, 74%). Eluent column chromatography: acetone/
CH2Cl2, 1:99. Rf = 0.59 (acetone/CH2Cl2, 4:96); mp = 172 °C; 1H
NMR (300 MHz, CDCl3): d = 0.88–0.94 (m, 6H, CH(CH3)2), 1.20–
1.69 (m, 3H, CH2CH(CH3)2), 1.42 (s, 9H, (CH3)3), 3.06 (d,
J = 7.2 Hz, 2H, CH2C6H5), 3.34–3.39 (m, 1H, CHaSO2F), 3.58–3.65
(ddd, Jgem = 14.9 Hz, Jvic = 5.3 Hz, 3JH F = 1.0 Hz, 1H, CHbSO2F),
b
4.4.6. Fmoc-Val-
w
[CH2SO2]-F (7f)
Sulfonyl fluoride 7f was purified by column chromatography
(acetone/CH2Cl2, 1:9) followed by crystallization (CH2Cl2/hexanes).
Yield procedure 3: 30% (211 mg, white solid). Rf = 0.46 (CH2Cl2);
mp = 183 °C; 1H NMR (300 MHz, CDCl3/acetone-d6): d = 0.98 (d,
J = 7.3 Hz, 6H, CH(CH3)2), 2.05 (m, 1H, CH(CH3)2), 3.68, 3.81 (2 m,
2H, SO2CH2), 4.10 (m, 1H, NCH), 4.22 (m, 1H, CH (Fmoc)), 4.41
(m, 2H, CH2 (Fmoc)), 5.00 (d, J = 8.3 Hz, 1H, NH), 7.29–7.77 (m,
8H, Ar-CH (Fmoc)); 13C NMR (75 MHz, CDCl3/acetone-d6):
d = 17.4, 18.6 (CH(CH3)2), 31.5 (CH(CH3)2), 46.9 (CH (Fmoc)), 52.1
(NCH), 52.2, 52.3 (SO2CH2), 66.2 (CH2 (Fmoc)), 119.5, 124.6,
124.7, 126.5, 126.5, 127.3, 140.9, 143.4, 143.6 (Ar-C (Fmoc)),
155.6 (C@O (Fmoc)); 19F NMR (282 MHz, CDCl3): d = ꢁ116.2 (s);
In the 1H NMR spectrum, also broad, low-intensity signals were
observed, presumably due to the presence of the minor Fmoc-
rotamer. Anal. Calcd for C20H22FNO4S: C, 61.36; H, 5.66; N, 3.58;
F, 4.85. Found: C, 61.27; H, 5.57; N, 3.49; F, 4.78; ESI-MS: m/
z = 413.9 [M+Na]+.
4.25–4.42 (m, 2H, NCH (Phe), NCH (Leu)), 4.89 (br s, 1H, NHBoc),
6.30 (d, J = 8.5 Hz, 1H, NH (Leu)), 7.20–7.34 (m, 5H, Ar-CH); 13C
NMR (75 MHz, CDCl3): d = 21.4, 22.9 (CH(CH3)2), 24.6 (CH(CH3)2),
28.2 ((CH3)3), 37.7 (CH2CH(CH3)2), 41.4 (CH2C6H5), 43.8 (NCH
(Leu)), 54.4, 54.6 (CH2SO2F), 56.0 (NCH (Phe)), 80.6 (C(CH3)3),
127.1, 128.7, 129.2, 136.4 (Ar-C), 155.5 (C@O (Boc)), 171.4 (C@O
(Phe)); 19F NMR (282 MHz, CDCl3): d = ꢁ114.8 (s). Anal. Calcd for
C20H31N2O5SF: C, 55.79; H, 7.26; N, 6.51. Found: C, 55.28; H,
7.23; N, 6.37; ESI-MS: m/z = 331.13 [MꢁBoc+H]+.
4.6.3. Cbz-Phe-w[CH2SO2]-Tau-F (10)
Cbz-Tau-F (7a, 653 mg, 2.5 mmol) was Cbz-deprotected using
the general procedure. To the HBr-salt, obtained after evaporation
of HBr, was added Cbz-Phe
dichloromethane (25 mL) and N-methylmorpholine (825
w
[CH2SO2]-Cl (6e, 920 mg, 2.5 mmol),
L,
l
7.5 mmol). The mixture was stirred for 40 min at rt, during which
the mixture turned into a milky suspension. After evaporation of
the dichloromethane, ethyl acetate (50 mL) and methanol (5 mL)
were added. Washing with KHSO4 (30 mL), water and brine was
followed by drying (Na2SO4) and concentration in vacuo. To the
crude product was added dichloromethane (40 mL), and the flask
was rotated for 1 h at rt. Filtration followed by drying of the resi-
due afforded sulfonyl fluoride 10 as a white solid (73%, 837 mg).
Rf = 0.29 (acetone/CH2Cl2; 5:95); mp = 172 °C; 1H NMR (300 MHz,
CD3CN): d = 2.42 (dd, Jgem = 13.8 Hz, Jvic = 9.2 Hz, 1H, CHaPh), 2.55
4.5. General procedure for the synthesis of Cbz-deprotected
sulfonyl fluorides 8a–c
To a solution of the sulfonyl fluoride (1.0 mmol) in dichloro-
methane (10 mL) was added a solution of HBr in acetic acid (33%,
6.0 mL). After stirring at rt for 30 min, the solvents were removed
in vacuo. The residue was dissolved in H2O (10 mL), and Dowex

A. J. Brouwer et al. / Bioorg. Med. Chem. 19 (2011) 2397–2406
2405
(dd, Jgem = 13.8 Hz, Jvic = 5.5 Hz, 2H, CHbPh), 2.83 (dd, Jgem = 14.6 Hz,
substrate solution (10
lL), enzyme solution (20
lL) and buffer
Jvic = 4.4 Hz, 1H, NCHCHaSO2), 2.90 (dd, Jgem = 14.6 Hz, Jvic = 8.3 Hz,
1H, NCHCHbSO2), 3.14 (m, 2H, NCH2), 3.37 (m, 2H, SCH2CH2),
3.83 (m, 1H, NCH), 4.56 (s, 2H, CH2 (Cbz)), 5.61 (m, 2H, 2 ꢂ NH),
6.83 (m, 10H, 2 ꢂ C5H5); 13C NMR (75 MHz, CDCl3): d = 38.4
(NCH2), 41.1 (NCHCH2pH), 50.4 (NCH), 52.3, 52.5 (CH2SO2F), 67.0
(CH2 (Cbz)), 127.8, 128.7, 129.0, 129.6, 130.5, 138.4, 138.9
(2 ꢂ C6H5) 157.4 (C@O (Cbz)); 19F NMR (282 MHz, CD3CN):
d = ꢁ121.4 (s). Anal. Calcd for C13H18FNO4S: C, 51.47; H, 5.98; F,
6.26; N, 4.62. Found: C, 52.01; H, 6.10; F, 5.64; N, 5.26; ESI-MS:
m/z = 325.95 [M+Na]+, 342.30 [M+K]+.
(160 lL). For the controls DMSO was added instead of inhibitor
solution. The liberation of p-nitroaniline was measured directly
at 405 nm for 15 min. Final concentrations in the wells were: en-
zyme: 0.5 lM; substrate: 0.25, 0.23, 0.21, 0.19, 0.17 or 0.15 mM;
inhibitor: 31.25, 15.63 or 7.81
lM. The assays were performed in
quadruplicate.
4.7.4. kinact determination
An enzyme solution (5.55
sin (1.25 mg, 0.05 mol) in buffer (9.0 mL). A stock solution of the
substrate (2.78 mM) was made by dissolving Bz-L-Tyr-pNA
(1.12 mg, 2.76 mol) in DMSO (994 L), which was diluted with
DMSO to a concentration of 278 M. For the inhibitor solution
(12.5 mM), the inhibitor (5.0 mol) was dissolved in DMSO
(400 L). In a typical assay, an enzyme–inhibitor mixture was first
made taking inhibitor solution (16 L), DMSO (84 L) and enzyme
solution (900 L). From this mixture 30 L was directly (t = 0 min)
added to a well containing 270 L substrate solution (278 M). At
t = 1 min, 30 L of the enzyme–inhibitor mixture was added to a
new well containing 270 L substrate solution. This procedure
lM) was made dissolving chymotryp-
l
4.6.4. Boc-Leu1-Leu2-
w
[CH2SO2]-F (11)
l
l
The reaction was performed on 0.71 mmol scale to yield a white
solid (206 mg, 73%). Eluent column chromatography: gradient
from acetone/CH2Cl2, 1:99, followed by acetone/CH2Cl2, 5:95.
Rf = 0.49 (acetone/CH2Cl2, 4:96); mp = 161 °C; 1H NMR (300 MHz,
CDCl3): d = 0.86–0.95 (m, 12H, (CH3)2 (Leu1,2)), 1.37–1.82 (m, 6H,
CH(CH3)2 (Leu1,2), NCHCH2 (Leu1,2)), 1.44 (s, 9H, (CH3)3), 3.56–
l
l
l
l
l
l
l
l
l
3
a
3.64 (2t, Jgem = 14.9 Hz, Jvic = 5.2 Hz, JH F = 5.2 Hz, 1H, CHaSO2F),
l
3.71–3.78 (ddd, Jgem = 14.9 Hz, Jvic = 5.8 Hz, 3JH F = 0.9 Hz, 1H,
l
b
CHbSO2F), 3.99–4.07 (m, 1H, NCH (Leu1)), 4.41 (m, 1H, NCH
(Leu2)), 4.74 (br d, 1H, NHBoc), 6.57 (d, J = 8.3 Hz, 1H, NH (Leu2));
13C NMR (75 MHz, CDCl3): d = 21.5, 22.9 ((CH3)2 (Leu1,2)), 24.7
(CH(CH3)2 (Leu1,2)), 28.2 ((CH3)3), 40.2, 41.7 (NCHCH2 (Leu1,2)),
44.0, 53.1 (NCH (Leu1,2), 54.4, 54.6 (CH2SO2F), 80.4 (C(CH3)3),
155.8 (C@O (Boc)), 172.6 (C@O (Leu1)); 19F NMR (282 MHz, CDCl3):
d = ꢁ115.0 (s); ESI-MS: m/z = 297.09 [MꢁBoc+H]+.
was repeated until t = 5 min. At t = 6, the liberation of p-nitroani-
line in all six wells was measured at 405 nm for 15 min. Final con-
centrations in the wells were: enzyme: 0.5
inhibitor: 20 M.
lM; substrate: 250 lM;
l
4.7.5. Papain assay
Papain was dissolved in buffersolution (80
lM) and after shaking
the solution for 5 min, it was centrifuged. The substrate (Bz-L-Arg-
pNA) was first dissolved in DMSO and then diluted twice
(50.0 mM) with buffer. The inhibitors were dissolved in DMSO and
4.7. Enzyme assays
diluted twice (125.0
itor solution (4.0 L), buffer solution (172.0
tion (20.0 L). For the controls a DMSO/buffer solution (1:1) was
used instead of the inhibitor solution. After 1 h pre-inhibition a
sample (98.0 L) was taken from each well and added to wells con-
taining substrate solution (2.0 L), and subsequently the liberation
p-nitroaniline was measured during 1 h. Final concentrations in
the wells were: enzyme: 8.0 M; substrate: 1.0 mM; inhibitor:
2.5 M.
l
M) with buffer. To each well was added inhib-
4.7.1. General remarks for kinetic experiments
l
lL) and enzyme solu-
Buffer solutions were prepared using distilled water. Buffer
solution for all chymotrypsin assays: sodium phosphate (0.05 M,
pH 7.0); for the papain assay: sodium phosphate (100 mM, pH
6.5) containing EDTA (1.5 mM). Papain stock solutions were centri-
fuged before use. Papain and chymotrypsin were purchased from
Sigma and were used without further purification. Bz-L-Arg-pNA
and Bz-L-Tyr-pNA were purchased from Bachem (Switzerland).
Kinetic enzyme assays were performed using 96-wells plates in a
l
l
l
l
l
l
Quant Biotek plate reader for 1 h at rt at 405 nm. All assays were
Acknowledgment
carried out in triplicate or quadruplicate.
We thank Hans Hilbers for measuring some of the mass spectra.
4.7.2. Progress curves
Chymotrypsin was dissolved in buffer (1.1 lM), the substrate
References and notes
(Bz-L-Tyr-pNA) was dissolved in DMSO (5.0 mM), and the inhibi-
tors were dissolved in DMSO (250 M). To each well was added
inhibitor solution (10.0 L) and enzyme solution (180.0 L). For
the controls DMSO was used instead of the inhibitor solution. After
1 h pre-inhibition a sample (95.0 L) was taken from each well and
added to wells containing substrate solution (5.0 L), and subse-
quently the liberation p-nitroaniline was monitored during
l
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4. Roush, W. R.; Cheng, J.; Knapp-Reed, B.; Alvarez-Hernandez, A.; McKerrow, J.
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An enzyme solution (5.0
trypsin (1.25 mg, 0.05 mol) in buffer (10 mL). A stock solution of
the substrate (5.0 mM) was made by dissolving Bz-L-Tyr-pNA
(2.03 mg, 5.0 mol) in DMSO (1.0 mL). Dilutions of this stock
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l
l
solution were made using DMSO, resulting in substrate concen-
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lmol) was dissolved
l
l
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