Sulfone-stabilized carbanions for the reversible covalent capture of a posttranslationally-generated cysteine oxoform found in protein tyrosine phosphatase 1B (PTP1B)

Article abstract of DOI:10.1016/j.bmc.2016.03.054

Redox regulation of protein tyrosine phosphatase 1B (PTP1B) involves oxidative conversion of the active site cysteine thiolate into an electrophilic sulfenyl amide residue. Reduction of the sulfenyl amide by biological thiols regenerates the native cystei

Full text of DOI:10.1016/j.bmc.2016.03.054

Accepted Manuscript
Sulfone-stabilized carbanions for the reversible covalent capture of a posttrans-
lationally-generated cysteine oxoform found in protein tyrosine phosphatase 1B
(
PTP1B)
Zachary D. Parsons, Kasi Viswanatharaju Ruddraraju, Nicholas Santo, Kent S.
Gates
PII:
S0968-0896(16)30212-7
DOI:
http://dx.doi.org/10.1016/j.bmc.2016.03.054
BMC 12903
Reference:
To appear in:
Bioorganic & Medicinal Chemistry
Received Date:
Revised Date:
Accepted Date:
8 February 2016
16 March 2016
27 March 2016
Please cite this article as: Parsons, Z.D., Ruddraraju, K.V., Santo, N., Gates, K.S., Sulfone-stabilized carbanions for
the reversible covalent capture of a posttranslationally-generated cysteine oxoform found in protein tyrosine
phosphatase 1B (PTP1B), Bioorganic & Medicinal Chemistry (2016), doi: http://dx.doi.org/10.1016/j.bmc.
2
016.03.054
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Sulfone-stabilized carbanions for the reversible covalent
capture of a posttranslationally-generated cysteine oxoform
found in protein tyrosine phosphatase 1B (PTP1B)
‡
‡
‡
Zachary D. Parsons , Kasi Viswanatharaju Ruddraraju , Nicholas Santo , and Kent S.
‡
,§,
Gates *
‡
University of Missouri
Department of Chemistry
125 Chemistry Building
Columbia, MO 65211
§
University of Missouri
Department of Biochemistry
Columbia, MO 65211
*
To whom correspondence should be addressed: email: gatesk@missouri.edu; phone:
(573) 882-6763; FAX: (573) 882-2754
1

Abstract: Redox regulation of protein tyrosine phosphatase 1B (PTP1B) involves
oxidative conversion of the active site cysteine thiolate into an electrophilic sulfenyl
amide residue. Reduction of the sulfenyl amide by biological thiols regenerates the
native cysteine residue. Here we explored fundamental chemical reactions that may
enable covalent capture of the sulfenyl amide residue in oxidized PTP1B. Various
sulfone-containing carbon acids were found to react readily with a model peptide sulfenyl
amide via attack of the sulfonyl carbanion on the electrophilic sulfur center in the
sulfenyl amide. Both the products and the rates of these reactions were characterized.
The results suggest that capture of a peptide sulfenyl amide residue by sulfone-stabilized
carbanions can slow, but not completely prevent, thiol-mediated generation of the
corresponding cysteine-containing peptide. Sulfone-containing carbon acids may be
useful components in the construction of agents that knock down PTP1B activity in cells
via transient covalent capture of the sulfenyl amide oxoform generated during insulin
signaling processes.
TOC Graphic
2

1
. Introduction
Type 2 diabetes is an immense and growing worldwide health problem and
1-5
6,7
there is a need for new therapeutic agents in this area. The enzyme protein tyrosine
phosphatase 1B (PTP1B) is an important negative regulator of insulin signaling in
8,9
mammalian cells and studies with PTP1B knockout mice established this enzyme as a
10-13
therapeutic target for the treatment of type 2 diabetes.
However, it has proven
challenging to develop traditional reversible inhibitors that are both bioavailable and
selective for PTP1B over other members of the highly homologous protein tyrosine
14
phosphatase enzyme family. More than ten years of intense effort has failed to produce
14-19
successful clinical candidates.
As a result, some have classified PTP1B as an
20,21
“undruggable target”.
Here we describe chemical studies designed to explore an
alternate approach that has been suggested for the chemical knockdown of PTP1B
activity in cells involving covalent capture of a post-translationally modified, oxidized
22
form of the enzyme that is generated transiently as part of the insulin signaling cascade.
Binding of the peptide hormone insulin to the insulin receptor stimulates
production of H O , which causes transient inactivation PTP1B via oxidation of the active
2
2
23-29
site cysteine residue.
Multiple crystallographic analyses have shown that the catalytic
cysteine in oxidized PTP1B exists as a cyclic, acyl sulfenamide (commonly referred to as
2
4,30,31
a cysteine sulfenyl amide residue, Scheme 1).
biological thiols regenerate the catalytically-active enzyme
cellular responses to insulin via dephosphorylation of the insulin receptor and insulin
Reactions of oxidized PTP1B with
25,32,33
that can terminate
8,15-19,34,35
receptor substrates.
Importantly, the sulfenyl amide residue in oxidized PTP1B presents a unique
22,36,37
electrophilic sulfur center that may be exploited in drug and probe design.
Covalent
3

capture of oxidatively-inactivated PTP1B by an appropriate nucleophile has the potential
to block regeneration of the catalytically active enzyme, thus knocking down cellular
22
activity of this enzyme. This strategy may yield selectivity arising from the insulin
signaling process itself, whereby the nucleophilic agent reacts with only the small subset
38
of PTP enzymes that are involved in, and oxidatively regulated by, insulin signaling.
Scheme 1. Oxidative inactivation of PTP1B, thiol-mediated recovery of enzyme activity,
and capture of the oxidized enzyme by a sulfone-stabilized carbanion.
The choice of nucleophile for trapping oxidized PTP1B may be crucial. For
example, many common nucleophiles such as thiols, amines, and phosphines may not be
ideal for capturing the sulfenyl amide because the S-S, S-N, and S-P bonds resulting from
their reactions with the electrophilic sulfur center would be unstable in the presence of
3
3,39
biological thiols or water.
Consequently, these nucleophiles may not effectively block
regeneration of active enzyme under physiological conditions. On the other hand,
4

reactions of carbon nucleophiles with the sulfenyl amide would yield carbon-sulfur
bonds, which are expected to be relatively stable under physiological conditions (Scheme
1
). Along these lines, carbon nucleophiles derived from 1,3-diketones such as dimedone
5,5-dimethyl-1,3-cyclohexanedione) have long been used as tools for the covalent
capture of the electrophilic sulfur center in peptide and protein sulfenic acids (Scheme
(
37,40-46
2
).
Additionally, carbon nucleophiles derived from 1,3-diketones have been shown
to capture of the electrophilic sulfur center in a dipeptide model of the protein sulfenyl
36,47
amide.
Scheme 2. Reaction of dimedone with a sulfenic acid residue.
Sulfone-stabilized carbon anions are excellent nucleophiles whose properties are
48-50
distinctly different from those of 1,3-diketone-derived enolates.
The sulfonyl group is
a powerful electron-withdrawing group that stabilizes a-carbanions by an inductive
4
8,51-53
effect.
more reactive in protic media than aprotic media.
properties prompted us to examine the reactions of sulfone-stabilized carbanions with the
Sulfone-stabilized carbanions are polarizable species that are, in some cases,
51,54,55
These interesting chemical
56
synthetic dipeptide 1 containing a sulfenyl amide functional group. We found that β-
ketosulfones, β-disulfones, and β-cyanosulfones react readily with the electrophilic sulfur
center in the sulfenyl amide to generate adducts containing the expected carbon-sulfur
bonds. Our results reveal that the a-sulfonyl sulfide adducts 2 derived from the reaction
5

of 1 with sulfone-based carbanions slowed, but did not completely prevent, the ability of
thiols to generate the reduced cysteine dipeptide 11. Overall, these results suggest that
covalent capture of the sulfenyl amide residue in PTP1B by sulfone-stabilized carbanions
has the potential to transiently inhibit thiol-mediated regeneration of catalytic activity
from oxidized PTP1B.
2
2
1
. Results and Discussion
.1 Reactions of sulfone-based carbon nucleophiles with the peptide sulfenyl amide
.
The dipeptide sulfenyl amide 1 was prepared by the method of Morin and
36,56,57
Gordon.
Reactions of 1 with the sulfone-containing carbon acids A-I were carried
out in a solvent system composed of 3:1 methanol and HEPES buffer (50 mM, pH 7)
containing NaCl (100 mM), and EDTA (1 mM). In this predominantly organic solvent
mixture, the sulfenyl amide-containing dipeptide 1 is reasonably stable and, in the
absence of added nucleophile, persists for several hours as judged by thin layer
chromatographic analysis (TLC).
O
O
BocHN
N
H
COOMe
COOMe
BocHN
H
H
3:1 CH3OH/buffer
24 ûC, 1-5 min
N
H
COOMe
O
O
S
BocHN
+
S
R
EWG
S
R
EWG
+
N
O
O
H
S
S
HN
COOMe
S
O
S
O
O
EWG
R
O
R = Ph or CH3
EWG = SO2R or COR or CN
NHBoc
1
2, Single Addition
3, Double Addition
Scheme 3. Reactions of sulfone-derived carbanions with the sulfenyl amide residue in
the dipeptide 1.
The reactions of the sulfones A-I (22 mM) with the dipeptide 1 (20 mM) were
rapid, with all starting sulfenyl amide consumed within 10 min as judged by TLC. In
6

each case, two major products were isolated by column chromatography on silica gel.
Spectroscopic analyses indicated that these products corresponded to the single and
double addition products 2 and 3 (Scheme 3). The single-addition products 2 were
obtained as mixtures of diastereomers. For example, product 2a resulting from reaction
of 1 with 1.1 equiv methanesulfonylacetone A was a 2:1 mixture of the two possible
diastereomers (Figure 1). The double-addition products 3b-i were characterized by a
doubling of the integrals for peptide-associated resonances and the absence of a
1
resonance for the a-proton in the H-NMR (resonance “k” in Figure 1). Mass
spectrometric analyses were also consistent with the proposed structures.
Presumably, these reactions proceed via an initial ionization of the parent carbon
acids to afford the sulfone-stabilized carbanions. Then, nucleophilic attack of an
equivalent of sulfonyl carbanion on the electrophilic sulfur center in 1 affords the
observed single-addition products. Subsequently, double-addition products may form
after loss of the remaining acidic a-proton present in the single-addition products 2a-i,
followed by reaction with a second equivalent of electrophile 1. The mass balances in
these reactions were generally good, with the isolated products accounting for 69-93% of
the input dipeptide 1. In general, reactions leading to the double-addition products 3b-i
seemed to be faster than the initial reactions leading to the single-addition products 2a-i.
This speculation is based on the observation that double-addition products tended to
predominate even though a slight excess of sulfone over 1 was employed in the reactions.
A similar propensity for the generation of double addition products was reported
58
previously in the alkylation of sulfone-stabilized anions.
7

Scheme 4. No evidence was observed for generation of significant concentrations of
sulfenic acid 4 under the reaction conditions employed in these studies.
(g)
a
b
h
O
H
d
Me
O
N
e
N
H
c
CO
2
g
O
f
i
S
(d)
j
l
k*
S
O
O
O
(j)
(l)
(
a)
TMS
(
k)
(
i)
(f)
(
e,c)
H O
(h)
(b)
2
(
f)
1
Figure 1. H-NMR spectrum of 2a.
It was recently proposed that the sulfenyl amide 1 exists in equilibrium with the
ring-opened sulfenic acid 4 (Scheme 4), when dissolved in a 2:1 (v/v) aqueous-organic
solvent mixture containing acetonitrile and phosphate buffered saline (phosphate, 10 mM,
46
pH 7; NaCl, 137 mM; KCl, 2.7 mM). This suggestion was based on the observed rapid
decomposition (< 15 min) of 1 in this solvent system affording products reasonably
expected to arise from dimerization of the putative sulfenic acid 4. With this in mind, it
was interesting for us to consider whether the sulfenic acid 4 might be a significant
8

contributor to the reactivity of 1 in our solvent mixture containing a relatively small
fraction of aqueous buffer (3:1 methanol and HEPES buffer).
We made several observations suggesting that, in our solvent mixture, significant
concentrations of the sulfenic acid 4 are not generated. First, as noted above, we did not
observe significant decomposition of 1 over the course of several hours when carbon
nucleophiles were omitted from the reaction mixture. Second, we observed no reaction
when 1 (12 mM) was stirred with methyl iodide (2 M) at 24 ˚C for 8 h. This result is
significant because we and others have shown that methyl iodide, in this concentration
range, effectively traps sulfenic acid intermediates to generate the corresponding methyl
46,59
sulfoxide.
Finally, we showed that 1 reacted rapidly with the carbon nucleophiles D
and F in dry organic solvent (acetone), where generation of significant concentrations of
the sulfenic acid were not possible. The reactions in dry solvent were complete in less
than 5 min and gave the same products observed in our standard 3:1 organic-aqueous
solvent mixture. Specifically, the reaction of 1 with D (1.1 eq.) in dry acetone containing
K CO (0.5 eq.) provided 2d and 3d in 83% and 8% yields respectively (5 min at 24 ˚C).
2
3
Similarly, reaction of 1 with F (1.1 eq.) in dry acetone yielded 2f and 3f in 16% and 78%
yields at 24 ˚C in 5 min. These reactions in dry solvent confirm that the sulfenyl amide
group is a competent electrophile, capable of reacting directly with sulfone-stabilized
carbanions. While it is not possible to rule out some involvement of the sulfenic acid 4
(or an analogous sulfenyl methyl ester) in our 3:1 methanol-buffer system, the evidence
suggests: (1) that substantial amounts of the sulfenic acid are not present in our reaction
mixtures and, (2) that the sulfenic acid is not an obligate intermediate in the reaction of
sulfenyl amide 1 with sulfonyl-stabilized carbanions. Our 3:1 (v/v) organic-aqueous
solvent conditions may be complementary to the 1:2 (v/v) organic-aqueous system used
9

46
by Gupta and Carroll. Dissolution of 1 in the 1:2 organic-aqueous system of Gupta and
Carroll may enable access to significant concentrations of the sulfenic acid 4, while the
3
:1 organic-aqueous solvent condition used here may allow 1 to remain largely intact,
thus serving as a faithful chemical model for the active site of oxidized PTP1B where, at
least under the conditions used for X-ray crystallographic analyses, the sulfenyl amide
24,30,31
form predominates over the sulfenic acid (Scheme 1).
The equilibrium between
cysteine sulfenyl amide and sulfenic acid in peptides and proteins under various aqueous
solvent conditions and protein environments deserves further study.
10

Table 1. Products obtained from the reaction of the sulfenyl amide-containing dipeptide
with various sulfone-stabilized carbon nucleophiles. (The dipeptide is truncated in
1
these structures. Complete structures of the side chains in the single addition and
double addition products are shown in Scheme 3).
11

2
.2 Reaction of sulfone-based carbon nucleophiles with other electrophilic sulfur
compounds. For the purposes of comparison, we examined the reactions of the sulfone-
based carbon nucleophiles B, C, D, and F with the electrophilic sulfur centers in the
cysteine disulfide 5. Additionally, we examined the reactivity of C, E, and F with the
60
synthetically-used sulfenylating agent N-(phenylthio)succinimide 6. Stirring the
disulfide 5 with 1.1 equiv of the carbon acids B, C, D, or F in our standard 3:1 methanol
and HEPES buffer gave no reaction over the course of 8 h, as judged by TLC. On the
other hand, the acyl sulfenamide 6 displayed reactivity that was quite similar to the
sulfenyl amide residue in 1. For example, stirring 6 with 1.1 equiv E for 30 min
produced a mixture of the single addition and double addition products 8 and 9 in 34%
and 51% yields, respectively (Table 2). Sulfones E and F gave exclusively the double-
addition products 7 and 10. The results suggest that, for some applications, 6 might
provide a reasonable model for the reactivity of the electrophilic sulfur center in the
sulfenyl amide residue.
12

Table 2. Products resulting from the reaction of sulfone-stabilized carbanions with 6.
2
.3 Kinetics of reactions between sulfone-based carbon nucleophiles and 1. We
36
employed a spectrophotometric assay to measure the rates at which sulfone-based
carbon nucleophiles reacted with 1. This assay exploits the rapid reaction of 1 with the
highly colored thiol, 5-thio-2-nitrobenzoic acid (TNB, the reduced form of Ellman’s
reagent) and enabled us to monitor the amount of 1 remaining as a function of time,
following addition of a large molar excess of the carbon nucleophile (10-fold or greater)
in a solvent mixture composed of 1:1 (v/v) methanol and buffer (Tris, 50 mM, Bis-Tris,
50 mM, sodium acetate, 100 mM, and diethylenetriaminepentaacetic acid DTPA, 10 mM,
pH 7.0). Under these conditions, we found that 1 was stable over the time-course of the
kinetic assays (10 min, Fig. S1 panel A). We determined the reaction rates of 1 with
three representative sulfones for which reaction products had been spectroscopically
characterized: b-ketosulfone A, b-sulfonylsulfone D, and b-cyanosulfone F (Fig. S1). To
13

further explore structure-activity relationships, we also examined the reaction of 1 with
the b-ketosulfones J, K, L, and M (Fig. S1).
We first confirmed the molecularity of these reactions using pseudo-first order
kinetic analysis. To that end, we employed a large molar excess of •-ketosulfone L over
sulfenyl amide 1 and determined the pseudo first-order rate constants associated with
capture of 1 by L at various molar excesses of L (Fig. 2A). A replot of these reaction
2
rate constants versus molar concentration of L revealed a straight line (r = 0.9998),
consistent with an overall second-order process. The rate constant for this process was
-1
-1
1
5.8 ± 0.5 M s . It is worth noting that in these experiments, because a minimum 10-
fold molar excess of sulfone over 1 was employed, these kinetic data likely reflect the
rate of monoadduct formation – that is, the rate of initial nucleophilic attack of carbanion
on sulfenyl amide. We then determined approximate second-order rate constants for
capture of 1 by the remaining sulfones A, D, F, J, K, and M (Fig. S1). In those
experiments, we employed a single concentration of carbon nucleophile under pseudo
first-order conditions and calculated apparent second-order rate constants from the data.
All kinetic data fit well to first-order processes, and the apparent second-order rate
-1
-1
constants measured for these sulfones ranged from 9 to 64 M s (Table 3).
To probe the origins of the observed kinetic trends, we spectrophotometrically
determined the pK values of sulfones A, D, F, J, K, L and M (Fig. S3). Interestingly
a
there was no clear correlation between the acidity of the sulfone-derived carbon acids and
the rates at which they reacted with 1 (Table 3). For example, although disulfone D and
the b-cyanosulfone F have nearly identical aqueous pK values (10.9 and 11.0,
a
respectively), the b-cyanosulfone F was found to react with 1 approximately 5-fold faster
-1
-1
than disulfone D (approximate second-order rate constants 12 and 64 M s ,
14

respectively). In general, previous work has shown that the nucleophilicities of
carbanions and enolates do not correlate strongly with the pK of the corresponding
a
51
carbon acids. This observation was recapitulated in our recent work describing covalent
36
capture of 1 by 1,3-diketone-derived carbon acids as well as recent work from Carroll’s
46
group.
Figure 2. Kinetics of capture of sulfenyl amide 1 by sulfone L.
15

Table 3. Reaction rates of sulfone-stabilized carbanions with 1.
ktrap (M-1 s-1)a
pKab
Compound
J
Structure
O
O
O
O
S
O
31c
9.2
O
O
S
18c
K
L
9.3
9.3
O
O
O
S
15.8 ± 0.5
O
O
O
S
20c
M
A
D
9.7
10.0
10.9
O
O
S
O
O
9c
O
O
O
S
S
12c
64c
O
O
S
F
11.0
N
a
Second-order rate constants for trapping the model sulfenyl amide
o
in 1:1 CH OH:Buffer B (pH 7.0), 23 ± 2 C.
b
3
Aqueous pK values were determined as reported in the text.
a
c
Approximate second-order rate constants for trapping the model
o
sulfenyl amide in 1:1 CH OH:Buffer B (pH 7.0), 23 ± 2 C.
3
2
.4. Reaction of products 2a, 2d, and 2f with 1,4-dithio-D-threitol (DTT). The
stability of the series of adducts 2 resulting from covalent capture of 1 by the sulfone-
stabilized nucleophiles is an important consideration (in the Conclusions, we provide
16

some discussion regarding why the monoadducts 2 may be more relevant to the enzyme).
In the context of the enzyme, conversion of the oxidized PTP1B sulfenyl amide to a
stable sulfone adduct would prevent regeneration of the catalytically active form of the
enzyme. Of special concern is whether reactions of the adducts 2 with thiols can
regenerate the native cysteine residue, analogous to catalytically active PTP1B.
Therefore, we exposed the sulfone-dipeptide adducts 2a, 2d, and 2f to the thiol-
containing reducing agent, 1,4-dithio-D-threitol (DTT, 20 mM) in our standard solvent
mixture, composed of 3:1 methanol and buffer (HEPES 50 mM, NaCl 100 mM, and
EDTA 1 mM, pH 7) at 37 ˚C (Scheme 5). Under these conditions, the b-cyanosulfone
adduct 2f released the cysteine-containing peptide 11 in a modest 24% yield after 10 min.
The b-ketosulfone adduct 2a and b-disulfone adduct 2d gave only traces of 11 after a
reaction time of 10 min. On the other hand, the unmodified sulfenyl amide-containing
dipeptide 1 afforded a 92% yield of 11 after 10 min under these conditions. However,
following extended incubation with DTT (5 h), adducts 2f, 2d and 2a gave 94%, 77% and
58% yields of 11, respectively. Overall, these results show that covalent capture of 1 by
sulfone-derived carbon nucleophiles may impede, but not completely prevent, thiol-
dependent generation of the cysteine dipeptide 11. From the data, it is possible to
estimate a half-life of approximately 25 min for 2f at 37 ˚C in the presence of 25 mM
DTT. These results stand in contrast to our earlier results with diketone adducts of 1,
where enolization may have stabilized the diketone-peptide adducts against thiol-
36
mediated reversion to the cysteine dipeptide.
17

Scheme 5. Reaction of adducts 2 with DTT.
3
. Conclusions
The sulfone-stabilized carbanions examined here reacted readily with the sulfenyl
amide residue in the dipeptide 1. The products were consistent with a mechanism
involving ionization of the carbon acid, followed by nucleophilic attack of the resulting
carbanion on the electrophilic sulfur center in the sulfenyl amide residue of 1. These
monoadduct products 2 may then react with a second equivalent of 1 to give the double-
addition products 3. The double-addition reaction is probably not relevant to the enzyme
PTP1B, as steric constraints seem unlikely to permit two molecules of the enzyme to be
30
linked via their occluded active site cysteine residues through a single carbon atom.
-1
-1
Rate constants for the reaction of the sulfones with 1 ranged from 9–64 M s .
These rate constants are larger than those reported recently for the reaction of 1,3-
57
diketone-derived nucleophiles with 1 and are comparable to the rates ascribed to the
46
reaction of cyclic sulfone-stabilized nucleophiles with the sulfenic acid 4. By way of
further comparison, rates reported for the reaction of dimedone with sulfenic acid
61
-1 -1 46
residues are in the range of 0.03 to 12 M s . Importantly, rate constants in the range
-1
-1
of 9–64 M
s
may allow micromolar concentrations of these agents to compete
18

effectively against millimolar concentrations of cellular thiols for reaction with oxidized
33
PTPs. We also examined the stabilities of the adducts 2 against thiols. Cells contain
62,63
approximately 1-5 mM glutathione (E
0
’ = –0.207 V).
We found that, under our
standard solvent conditions, the reactions of the adducts 2 with glutathione (5 mM) were
sluggish (data not shown). Therefore, in order to measure the relative reactivity of the
different adducts 2, we employed more stringent reaction conditions involving an
64
0
increased concentration (20 mM) of the strongly-reducing thiol DTT (E ’ = –0.327 V).
Our results showed that conversion of the sulfenyl amide 1 to the sulfone adducts 2
slowed, but did not completely prevent, the ability of thiols to generate the reduced
51,54,55
cysteine dipeptide 11. The polarizable nature of sulfone-stabilized carbanions
that
makes them excellent nucleophiles may also make them reasonably good leaving groups
in reactions of the adducts with thiols. From a pharmacological perspective, the thiol-
labile nature of the sulfone adducts 2 could be viewed as an advantage, given that some
65
degree of reversibility may be desirable in covalent drugs. At the same time, in the
context of PTP1B, the sulfone-cysteine adducts modeled by 2 may enjoy greater stability
if sequestered within the rather deep active site of the enzyme where the attachment to
66
the enzyme is less accessible to thiols.
The pharmacological pairing explored in this work, involving reaction of an
electrophilic protein target with a nucleophilic small molecule is quite unusual. Among
the few such examples in medicinal chemistry is the use of 2-pyridine aldoxime methyl
chloride (PAM) as an antidote to regenerate catalytic activity of acetylcholinesterases
67,68
inactivated by organophosphorus nerve agents.
This reaction involves nucleophilic
attack of PAM on the electrophilic adducts formed by nerve agents at the active site
serine residue of acetylcholinesterases. A second example involves the nucleophilic
19

attack of the arylamine group of sulfa drugs on 6-hydroxymethyl-7,8-dihydropterin
69
pyrophosphate bound at the active site of dihydropteroate synthase. The sulfur center in
the oxidized, sulfenyl amide form of PTP1B generated during insulin signaling may
represent a unique endogenous electrophilic protein target in the cell. Our results show
36
that carbon nucleophiles do not react with the electrophilic sulfur centers in disulfides.
As a result, the reaction of nucleophilic agents with oxidized PTP1B has the potential to
be an inherently selective process, with off-target attack on other redox-regulated proteins
37,47,57,70
being one obvious concern. Overall, our studies alongside other recent work
suggest that covalent capture of oxidized PTP1B by carbon nucleophiles may offer a
viable alternative to traditional inhibitors for the chemical knockdown of this enzyme
activity in cells.
4
4
. Experimental
.1. General
Unless otherwise noted, all reagents were purchased from commercial vendors
and used without further purification. Analytical thin layer chromatography was
performed on silica gel plates with UV indicator. Flash chromatography was carried out
1
using 230-400 mesh silica gel. H NMR spectra were recorded on either a Bruker DRX-
500 (500 MHz) or a DRX-600 (600 MHz) spectrometer with chemical shifts reported in d
13
ppm. C NMR spectra were obtained on the same instruments at 125 and 150 MHz,
respectively. Melting points were recorded on a Unitemp capillary melting point
apparatus and are uncorrected. Infrared spectra were recorded on a FT-IR spectrometer
20

using NaCl plates. High-resolution mass spectrometry (HRMS) was performed using
electrospray ionization (ESI) and time-of-flight (TOF) mass analysis.
4
.2. General procedure for synthesis of adducts 2a-2i and 3a-3i.
To a stirred solution of the sulfenyl amide peptide 1 (20 mg, 0.060 mmol) in 3 mL
of 3:1 methanol:HEPES buffer (HEPES, 50 mM, NaCl, 100 mM, EDTA, 1 M, pH 7.0)
was added the sulfone-containing nucleophile (1.1 equiv) and the mixture stirred at room
temperature (24 ˚C). When the reaction was judged complete by TLC analysis, methanol
was completely removed by blowing a stream of nitrogen gas on the solution, and the
resulting aqueous solution extracted with ethyl acetate or dichloromethane (2 × 2 mL).
2 4
The combined organic layers were dried over anhydrous Na SO and concentrated by
rotary evaporation. The products were isolated by column chromatography on silica gel
eluted with mixtures of either ethyl acetate-hexane or methanol-dichloromethane.
4
.2.1. (2S)-methyl 2-((2R)-2-((tert-butoxycarbonyl)amino)-3-((1-(methylsulfonyl)-2-
oxopropyl)thio)propanamido)-3-methylbutanoate (2a)
Yellow oil (19.5 mg, 69%) as an inseparable mixture of diastereomers (70:30). R
f
1
=
0.37 (40% ethyl acetate/hexanes). H NMR (CDCl
3
, 500 MHz) δ 7.01 (d, J = 8.5 Hz,
1
4
7
2
1
H), δ 6.85 (br, 0.5H), δ 5.52 (d, J = 7.0 Hz, 1H), δ 5.26 (s, 0.5H), δ 5.00 (s, 0.5H), δ
.46-4.50 (m, 2H), δ 3.73 (s, 2H), δ 3.71 (s, 1H), δ 3.38 (br, 0.5H), δ 3.26 (dd, J = 14.0,
.0 Hz, 0.5H), δ 3.17 (s, 2H ), δ 3.13 (s, 1H), δ 2.92 (dd, J = 14.2, 6.5 Hz, 1H), δ 2.50 (s,
H), δ 2.48 (s, 1H), δ 2.16-2.24 (m, 1H), δ 1.45 (s, 4H), δ 1.44 (s, 5H), δ 0.94 (dd, J =
1
3
3
6.5, 7.0 Hz, 6H); C NMR (CDCl , 125 MHz) δ 198.3, 198.1, 172.1, 171.9, 170.3,
21

1
3
1
69.9, 155.8, 155.7, 80.9, 80.6, 73.8, 72.4, 57.7, 57.6, 54.2, 52.4, 52.3, 52.2, 37.3, 37.1,
-
1
6.6, 36.1, 31.0, 30.9, 30.8, 28.4, 19.3, 19.2, 17.7, 17.6; IR (cm ) 3416, 3054, 2979,
735, 1715, 1675, 1497, 1366, 1310, 1262, 1159, 1107, 734, 698; HRMS (ESI-TOF, [M
+
+
H] ) m/z calcd for C18
H
32
N
2
8
O S
2
469.1683, found 469.1678.
2-((2R)-2-((tert-butoxycarbonyl)amino)-3-((2-methoxy-1-
methylsulfonyl)-2-oxoethyl)thio)propanamido)-3-methylbutanoate (2b)
Colorless oil (17 mg, 58%) R = 0.32 (30% ethyl acetate/hexanes). H NMR
, 500 MHz) δ 7.16 (d, J = 8.5 Hz, 0.5H), δ 6.84 (d, J = 8.5 Hz, 0.5H), δ 5.49-5.54
m, 1H), δ 4.86 (s, 0.5H), δ 4.80 (s, 0.5H), δ 4.48-4.53 (m, 2H), δ 3.86 (s, 1H), δ 3.85 (s,
4
.2.2. (2S)-methyl
(
1
f
(CDCl
3
(
2
1
0
1
3
1
H), δ 3.74 (s, 1H), δ 3.73 (s, 2H), δ 3.41-3.45 (m, 0.5H), δ 3.28 (s, 1.5H ), δ 3.24 (s,
.5H), δ 3.07-3.18 (m, 1H), δ 2.17-2.24 (m, 1H), δ 1.46 (s, 5H), δ 1.45 (s, 4H), δ 0.90-
13
.96 (m, 6H); C NMR (CDCl
3
, 150 MHz) δ 171.9, 169.9, 169.8, 165.4, 164.9, 155.5,
55.1, 80.8, 80.5, 69.2, 57.4, 57.3, 54.3, 54.0, 53.9, 53.1, 52.3, 52.2, 37.2, 36.9, 36.5,
-1
6.3, 30.9, 29.7, 28.3, 28.2, 18.9, 17.5; IR (cm ) 3357, 3016, 2769, 2929, 1742, 1677,
+
487, 1377, 1315, 1217, 1162, 756, 667; HRMS (ESI-TOF, [M + H] ) m/z calcd for
C
18 33
H N
2
O
S
9 2
485.1627, found 485.1635.
4
.2.3. (3S,6R,12R,15S)-trimethyl
6,12-bis((tert-butoxycarbonyl)amino)-2,16-
dimethyl-9-(methylsulfonyl)-5,13-dioxo-8,10-dithia-4,14-diazaheptadecane-3,9,15-
tricarboxylate (3b)
1
Colorless oil (3 mg, 12%) R
f
= 0.24 (30% ethyl acetate/hexanes). H NMR
3
(CDCl , 500 MHz) δ 7.22 (br, 1H), δ 5.69 (d, J = 8.0 Hz, 1H), δ 5.55 (d, J = 8.0 Hz, 1H),
δ 4.51-4.55 (m, 4H), δ 3.92 (s, 3H), δ 3.75 (s, 6H), δ 3.36 (s, 3H), δ 3.23-3.32 (m, 4H), δ
1
3
2
.15-2.21 (m, 2H), δ 1.46 (s, 18H), δ 0.93 (dd, J = 13.0 Hz, 6.5 Hz, 12H); C NMR
22

3
(CDCl , 125 MHz) δ 172.3, 172.2, 172.0, 170.0, 169.9, 165.2, 155.8, 155.5, 84.4, 80.7,
-
1
5
3
6
8
4
7.6, 57.5, 55.0, 53.6, 53.5, 52.4, 38.9, 34.9, 34.8, 31.4, 31.3, 28.5, 19.1, 17.9; IR (cm )
412, 3356, 3015, 2971, 2931, 2871, 1740, 1676, 1497, 1366, 1314, 1215, 1159, 754,
+
66; HRMS (ESI-TOF, [M + H] ) m/z calcd for C32
H
57
N
4
O
14
S 817.3033, found
3
17.3028.
.2.4. (2'S)-dimethyl
2,2'-(((2R,2'R)-3,3'-((1,1-dioxido-3-oxo-2,3-
dihydrobenzo[b]thiophene-2,2-diyl)bis(sulfanediyl))bis(2-((tert-
butoxycarbonyl)amino)propanoyl))bis(azanediyl))bis(3-methylbutanoate) (3c)
1
Colorless oil (20 mg, 78%) R
f
= 0.25 (40% ethyl acetate/hexanes). H NMR
(
CDCl
δ 5.60 (s, 1H) δ 5.55 (d, J = 7.5 Hz, 1H), δ 4.53-4.56 (m, 4H), δ 3.76 (s, 3H), δ 3.75 (s,
H), δ 3.16-3.31 (m, 4H), δ 2.15-2.22 (m, 2H), δ 1.45 (s, 9H), δ 1.43 (s, 9H), δ 0.92-0.95
3
, 500 MHz) δ 8.05 (d, J = 7.5 Hz, 1H), δ 7.92-7.97 (m, 2H), δ 7.83-7.86 (m, 1H),
3
1
3
(
m, 12H); C NMR (CDCl
3
, 150 MHz) δ 183.4, 172.1, 169.8, 169.7, 155.5, 155.4, 142.0,
1
3
1
37.2, 134.7, 130.1, 126.5, 122.4, 80.5, 80.4, 79.2, 57.6, 57.5, 53.3, 52.4, 52.3, 34.0, 33.8,
-1
1.2, 29.7, 28.3, 19.0, 17.9; IR (cm ) 3408, 3354, 3058, 2969, 2933, 2872, 1716, 1680,
+
504, 1363, 1262, 1212, 1158, 1021, 737, 705; HRMS (ESI-TOF, [M + H] ) m/z
calculated for C36
.2.5. (S)-methyl
butoxycarbonyl)amino)propanamido)-3-methylbutanoate (2d)
55
H N
4
O
13
S
3
:847.2928, found 847.2911.
4
2-((R)-3-((bis(phenylsulfonyl)methyl)thio)-2-((tert-
White solid (19.5 mg, 51%). Mp: 65-73 °C. R
f
= 0.42 (40% ethyl
1
acetate/hexanes). H NMR (CDCl
3
, 500 MHz) δ 8.16 (d, J = 7.5 Hz, 2H), δ 8.03 (d, J =
7
2
.5 Hz, 2H), δ 7.73 (t, J = 7.5 Hz, 1H), δ 7.67 (t, J = 7.0 Hz, 1H), δ 7.61 (t, J = 7.5 Hz,
H), δ 7.52 (t, J = 7.5 Hz, 2H), δ 7.20 (d, J = 8.5 Hz, 1H), δ 6.16 (s, 1H), δ 5.71 (d, J =
23

6
.5 Hz, 1H), δ 4.46 (dd, J = 8.0, 4.5 Hz, 1H), δ 4.37-4.41 (m, 1H), δ 3.74 (s, 3H), δ 2.65
13
(
br, 2H), δ 2.20-2.29 (m, 1H), δ 1.48 (s, 9H), δ 0.96 (dd, J = 12.0, 7.0 Hz, 6H); C NMR
(
CDCl , 125 MHz) δ 171.7, 169.7, 155.4, 137.7, 137.2, 135.0, 134.8, 130.4, 130.2, 129.1,
3
-
1
1
28.9, 85.6, 80.7, 58.0, 53.8, 52.3, 37.2, 30.7, 28.4, 19.2, 17.6; IR (cm ) 3413, 3369,
+
3
055, 2972, 2361, 2322, 1678, 1265, 1154, 747, 703; HRMS (ESI-TOF, [M + H] ) m/z
629.1661, found 629.1670.
.2.6. (2S,5R,11R,14S)-dimethyl 5,11-bis((tert-butoxycarbonyl)amino)-2,14-
diisopropyl-4,12-dioxo-8,8-bis(phenylsulfonyl)-7,9-dithia-3,13-diazapentadecane-
,15-dioate (3d)
White solid (11.5 mg, 40%). Mp: 108-113 °C. R
calcd for C27
H
36
N
2
O
9 3
S
4
1
f
= 0.26 (40% ethyl
, 500 MHz) δ 8.13 (d, J = 7.5 Hz, 4H), δ 7.70 (t, J =
.5 Hz, 2H), δ 7.56 (t, J = 8.0 Hz, 4H), δ 7.16 (br, 2H), δ 5.59 (br, 2H), δ 4.43-4.50 (m,
H), δ 3.74 (s, 6H), δ 3.40 (br, 2H), δ 3.28 (dd, J = 13.5, 7.5 Hz, 2H), δ 2.11-2.20 (m,
1
acetate/hexanes). H NMR (CDCl
3
7
4
2
1
2
1
1
3
H), δ 1.45 (s, 18H), δ 0.90 (dd, J = 12.5, 7.0 Hz, 12H); C NMR (CDCl
3
, 125 MHz) δ
71.9, 169.7, 155.4, 135.6, 135.9, 132.0, 128.3, 99.9, 80.3, 57.2, 53.8, 52.1, 34.4, 31.1,
-1
8.1, 18.8, 17.6; IR (cm ) 3364, 2967, 2360, 1706, 1660, 1520, 1327, 1246, 1158, 1076,
+
018, 855, 732; HRMS (ESI-TOF, [M + H] ) m/z calcd for C41
H
60
N
4
O
14
S
4
: 961.3067,
found 961.3054.
.2.7. (S)-methyl
(ditosylmethyl)thio)propanamido)-3-methylbutanoate (2e)
Colorless oil (25 mg, 64%) R = 0.46 (30% ethyl acetate/hexanes). H NMR
, 500 MHz) δ 8.03 (d, J = 8.0 Hz, 2H), δ 7.91 (d, J = 8.0 Hz, 2H), δ 7.39 (d, J =
.0 Hz, 2H), δ 7.31 (d, J = 8.0 Hz, 2H), δ 7.24 (d, J = 8.5 Hz, 1H), δ 6.05 (s, 1H), δ 5.73
4
2-((R)-2-((tert-butoxycarbonyl)amino)-3-
(
1
f
(CDCl
3
8
24

(d, J = 6.5 Hz, 1H), δ 4.47 (dd, J = 8.5, 4.5 Hz, 1H), δ 4.39 (t, J = 6.5 Hz, 1H), δ 3.74 (s,
3
9
1
3
1
H), δ 2.64-2.71 (m, 2H), δ 2.48 (s, 3H), δ 2.45 (s, 3H), δ 2.21-2.28 (m, 1H), δ 1.49 (s,
13
H), δ 0.97 (dd, J = 11.0, 7.0 Hz, 6H); C NMR (CDCl
3
, 150 MHz) δ171.6, 169.6,
55.2, 146.0, 145.8, 134.5, 134.1, 130.3, 130.1, 129.6, 129.4, 85.6, 80.5, 57.8, 53.6, 52.1,
-
1
7.1, 30.54, 28.3, 21.8, 21.7, 19.1, 17.5; IR (cm ) 3412, 3370, 3052, 2970, 2929, 2872,
+
736, 1675, 1593, 1483, 1332, 1258, 1158, 1077, 734; HRMS (ESI-TOF, [M + H] ) m/z
calculated for C29
H
41
N
2
O
9
S
3
: 657.1974, found 657.1969.
5,11-bis((tert-butoxycarbonyl)amino)-2,14-
4
.2.8. (2S,5R,11R,14S)-dimethyl
diisopropyl-4,12-dioxo-8,8-ditosyl-7,9-dithia-3,13-diazapentadecane-1,15-dioate (3e)
1
Colorless oil (8.5 mg, 29%) R
f
= 0.31 (30% ethyl acetate/hexanes). H NMR
3
(CDCl , 500 MHz) δ 7.99 (d, J = 8.5 Hz, 2H), δ 7.35 (d, J = 8.5 Hz, 2H), δ 7.17 (br, 1H),
δ 5.64 (br, 1H), δ 4.43-4.49 (m, 2H), δ 3.74 (s, 3H), δ 3.40-3.42 (m, 1H), δ 3.28 (dd, J =
1
1
1
3
7
9
4
3.5, 7.5 Hz, 1H), δ 2.46 (s, 3H), δ 2.12-2.19 (m, 1H), δ 1.45 (s, 9H), δ 0.91 (dd, J =
1
3
3
2.0, 6.5 Hz, 6H); C NMR (CDCl , 125 MHz) δ 171.9, 169.9, 155.6, 146.4, 132.7,
-1
32.2, 129.2, 80.45, 57.4, 54.0, 52.2, 34.6, 31.3, 28.3, 21.8, 18.9, 17.7; IR (cm ) 3359,
050, 2973, 2929, 2359, 2333, 1733, 1718, 1697, 1682, 1504, 1337, 1262, 1152, 737,
+
01; HRMS (ESI-TOF, [M + H] ) m/z calculated for C43
65 4 14 4
H N O S : 989.3362, found
89.3358.
.2.9. (2S)-methyl
2-((2R)-2-((tert-butoxycarbonyl)amino)-3-
((cyano(phenylsulfonyl)methyl)thio)propanamido)-3-methylbutanoate (2f)
Inseparable mixture of two isomers in 1:1 ratio. Colorless oil (7 mg, 22%) R =
f
1
0
7
.47 (30% ethyl acetate/hexanes). H NMR (CDCl
3
, 600 MHz) δ 8.06-8.09 (m, 2H), δ
.77-7.80 (m, 1H), δ 7.64-7.68 (m, 2H), δ 7.029 (d, J = 7.0 Hz, 0.5H), δ 6.86 (d, J = 8.5
25

Hz, 0.5H), δ 5.61 (d, J = 6.5 Hz, 0.5H), δ 5.42-5.49 (m, 1H), δ 4.48-4.54 (m, 2H), δ 3.76
s, 1.5H), δ 3.75 (s, 1.5H), δ 3.50 (dd, J = 15.0, 5.0 Hz, 0.5H), δ 3.22-3.23 (m, 1H), δ
.13-3.16 (m, 0.5H), δ 2.17-2.27 (m, 1H), δ 1.48 (s, 4.5H), δ 1.47 (s, 4.5H), δ 0.91-0.99
(
3
1
3
(
m, 6H); C NMR (CDCl
34.8, 134.7, 132.2, 132.1, 130.4, 130.3, 129.7, 129.6, 129.5, 112.2, 111.9, 81.4, 81.1,
7.9, 57.7, 57.6, 57.4, 56.9, 54.3, 53.6, 52.5, 52.4, 36.1, 35.6, 31.1, 30.9, 28.4, 28.3, 19.2,
3
, 150 MHz) δ 171.8, 171.7, 170.1, 169.9, 155.7, 135.7, 135.6,
1
5
1
7
-
1
9.1, 17.8, 17.7; IR (cm ) 3420, 3018, 2971, 2251, 1735, 1675, 1485, 1211, 1155, 905,
+
58, 734, 650; HRMS (ESI-TOF, [M + H] ) m/z calculated for C22
H
31 3
N O
7
S
2
: 514.1682,
found 514.1688.
4
2
1
.2.10. (2S,5R,11R,14S)-dimethyl
5,11-bis((tert-butoxycarbonyl)amino)-8-cyano-
,14-diisopropyl-4,12-dioxo-8-(phenylsulfonyl)-7,9-dithia-3,13-diazapentadecane-
,15-dioate (3f)
White solid (18 mg, 70%). Mp: 45-48 °C. R
H NMR (CDCl , 500 MHz) δ 8.05 (d, J = 7.5 Hz, 2H), δ 7.77 (t, J = 7.5 Hz, 1H), δ 7.62
t, J = 7.5 Hz, 2H), δ 7.14 (br, 2H), δ 5.59-5.56 (m, 2H), δ 4.59 (br, 1H), δ 4.52-4.56 (m,
H), δ 3.76 (s, 3H), δ 3.75 (s, 3H), δ 3.35-3.40 (m, 4H), δ 2.13-2.23 (m, 2H), δ 1.46 (s,
f
= 0.22 (30% ethyl acetate/hexanes).
1
3
(
3
1
1
3
1
8
4
1
3
0H), δ 1.45 (s, 8H), δ 0.90-0.94 (m, 12 H); C NMR (CDCl
3
, 125 MHz) δ 172.2, 169.6,
55.5, 135.8, 133.0, 131.8, 129.3, 112.3, 80.8, 72.5, 57.6, 53.5, 53.4, 52.5, 52.4, 36.6,
-1
6.1, 31.4, 28.4, 19.0, 17.8; IR (cm ) 3323, 3048, 2973, 2133, 1718, 1660, 1514, 1400,
+
240, 1146, 1070, 861; HRMS (ESI-TOF, [M + H] ) m/z calcd for C36
55
H N
5
O
S
12 3
46.3071, found 846.3088.
.2.11. (2S)-methyl
2-((2R)-2-((tert-butoxycarbonyl)amino)-3-
((cyano(tosyl)methyl)thio)propanamido)-3-methylbutanoate (2g)
26

1
Colorless oil (11 mg, 34%) R
, 600 MHz) δ 7.96 (d, J = 8.5 Hz, 1H), δ 7.93 (d, J = 8.5 Hz, 1H), δ 7.43 (t, J =
.0 Hz, 2H), δ 7.05 (d, J = 8.0 Hz, 1H), δ 6.87 (d, J = 8.0 Hz, 1H), δ 5.62 (d, J = 7.0 Hz,
H), δ 5.45 (s, 1H), δ 4.48-4.54 (m, 2H), δ 3.76 (s, 1.5H), δ 3.75 (s, 1.5H), δ 3.49 (dd, J
15.0, 5.0 Hz, 0.5H), δ 3.22 (d, J = 6.0 Hz, 1H), δ 3.14 (dd, J = 15.0, 6.0 Hz, 0.5H), δ
.50 (s, 1.5H), δ 2.49 (s, 1.5H), δ 2.17-2.27 (m, 1H), δ 1.48 (s, 4H), δ 1.47 (s, 5H), δ
f
= 0.42 (30% ethyl acetate/hexanes). H NMR
(
CDCl
3
8
1
=
2
0
1
5
1
1
13
3
.91-0.99 (m, 6H); C NMR (CDCl , 150 MHz) δ 171.9, 171.8, 170.1, 169.9, 155.7,
47.3, 147.2, 131.7, 131.6, 130.5, 130.4, 130.3, 130.2, 112.4, 112.1, 81.3, 81.0, 57.9,
7.7, 57.4, 57.0, 54.2, 53.6, 52.5, 52.4, 36.1, 35.5, 31.1, 31.0, 28.4, 28.3, 22.0, 19.2, 19.1,
-1
7.8, 17.7; IR (cm ) 3347, 3052, 2965, 2929, 2871, 2241, 1738, 1677, 1594, 1518, 1363,
+
334, 1262, 1151, 1083, 1018, 816, 737, 701; HRMS (ESI-TOF, [M + H] ) m/z
calculated for C23
H
34
N
3
O
7
S
2
:528.1838, found 528.1841.
.2.12. (2S,5R,11R,14S)-dimethyl 5,11-bis((tert-butoxycarbonyl)amino)-8-cyano-
,14-diisopropyl-4,12-dioxo-8-tosyl-7,9-dithia-3,13-diazapentadecane-1,15-dioate
4
2
(3g)
1
Colorless oil (14 mg, 53%) R
3
CDCl , 500 MHz) δ 7.92 (d, J = 8.0 Hz, 2H), δ 7.40 (d, J = 8.0 Hz, 2H), δ 7.13 (br, 1H),
f
= 0.42 (30% ethyl acetate/hexanes). H NMR
(
δ 5.58-5.62 (m, 2H), δ 4.58 (br, 1H), δ 4.51-4.55 (m, 2H), δ 3.76 (s, 3H), δ 3.75 (s, 3H), δ
3
1
1
.33-3.39 (m, 4H), δ 2.49 (s, 3H), δ 2.14-2.21 (m, 2H), δ 1.46 (s, 18H), δ 0.90-0.94 (m,
1
3
2H); C NMR (CDCl
3
, 125 MHz) δ 172.2, 169.7, 155.5, 147.5, 131.8, 130.0, 129.9,
12.5, 80.8, 72.6, 57.6, 53.6, 53.4, 52.5, 52.4, 36.6, 36.2, 31.4, 28.4, 22.1, 19.1, 17.9; IR
-1
(
cm ) 3318, 2965, 2929, 2871, 2360, 1741, 1659, 1515, 1367, 1270, 1248, 1151, 1018,
27

+
7
34, 650; HRMS (ESI-TOF, [M + H] ) m/z calculated for C37
found 860.3231.
.2.13. (2S)-methyl
(tert-butoxycarbonyl)amino)propanamido)-3-methylbutanoate (2h)
Colorless oil (6.5 mg, 18%) R = 0.51 (30% ethyl acetate/hexanes). H NMR
, 600 MHz) δ 7.91-7.96 (m, 2H), δ 7.78-7.81 (m, 2H), δ 6.99 (d, J = 8.0 Hz, 1H),
58 5 12 3
H N O S :860.3244,
4
2-((2R)-3-((((4-bromophenyl)sulfonyl)(cyano)methyl)thio)-2-
(
1
f
(CDCl
3
δ 6.86 (d, J = 8.5 Hz, 1H), δ 5.55-5.60 (m, 1H), δ 5.46 (br, 1H), δ 4.48-4.54 (m, 2H), δ
3
1
.76 (s, 1.5H), δ 3.75 (s, 1.5H), δ 3.54 (dd, J = 15.0, 4.0 Hz, 0.5H), δ 3.13-3.26 (m,
13
.5H), δ 2.17-2.27 (m, 1H), δ 1.48 (s, 4.5H), δ 1.47 (s, 4.5H), δ 0.91-0.98 (m, 6H); C
, 150 MHz) δ 171.7, 171.6, 170.0, 169.8, 155.6, 155.5, 133.4, 133.3, 133.2,
33.0, 132.9, 132.8, 131.7, 131.6, 131.5, 112.0, 111.8, 81.3, 81.0, 57.7, 57.6, 57.5, 57.3,
6.8, 54.1, 53.5, 52.4, 52.3, 36.1, 35.6, 30.9, 30.8, 28.3, 28.2, 19.1, 19.0, 18.9, 17.6, 17.5;
NMR (CDCl
3
1
5
-
1
IR (cm ) 3404, 3048, 2969, 2929, 2872, 1734, 1677, 1569, 1500, 1389, 1342, 1259,
+
1
158, 1064, 1007, 823, 737, 701; HRMS (ESI-TOF, [M + H] ) m/z calculated for
C
22
H31BrN
3
O
7
S
2
: 592.0787, found 592.0793.
4
.2.14. (2S,5R,11R,14S)-dimethyl 8-((4-bromophenyl)sulfonyl)-5,11-bis((tert-
butoxycarbonyl)amino)-8-cyano-2,14-diisopropyl-4,12-dioxo-7,9-dithia-3,13-
diazapentadecane-1,15-dioate (3h)
1
Colorless oil (20.5 mg, 73%) R
f
= 0.48 (30% ethyl acetate/hexanes). H NMR
3
(CDCl , 500 MHz) δ 7.90 (d, J = 8.0 Hz, 2H), δ 7.76 (d, J = 8.0 Hz, 2H), δ 7.13 (br, 1H),
δ 5.58 (br, 2H), δ 4.60 (br, 2H), δ 4.52-4.56 (m, 2H), δ 3.77 (s, 3H), δ 3.76 (s, 3H), δ
3
1
.34-3.39 (m, 4H), δ 2.15-2.21 (m, 2H), δ 1.46 (s, 18H), δ 0.92 (dd, J = 14.0, 7.0 Hz,
1
3
3
2H); C NMR (CDCl , 125 MHz) δ 172.0, 169.5, 169.4, 155.4, 132.9, 132.5, 131.8,
28

131.7, 112.0, 80.7, 72.5, 57.4, 53.4, 53.2, 52.4, 52.3, 36.5, 36.0, 31.2, 28.3, 18.9, 17.7; IR
-1
(
cm ) 3401, 3340, 3055, 2965, 2929, 2868, 2303, 1738, 1677, 1572, 1518, 1389, 1367,
+
1
262, 1158, 1064, 737, 704; HRMS (ESI-TOF, [M + H] ) m/z calculated for
C
36
H54BrN
5
O
12
S
3
: 924.2193, found 924.2184.
4
.2.15. (2S)-methyl 2-((2R)-2-((tert-butoxycarbonyl)amino)-3-((((4-
chlorophenyl)sulfonyl)(cyano)methyl)thio)propanamido)-3-methylbutanoate (2i)
1
Colorless oil (5 mg, 15%) R
f
= 0.30 (20% ethyl acetate/hexanes). H NMR
3
(CDCl , 500 MHz) δ 8.03 (d, J = 9.0 Hz, 1H), δ 7.95 (d, J = 8.5 Hz, 1H), δ 7.61-7.64 (m,
2
4
3
6
1
5
H), δ 6.99 (d, J = 7.5 Hz, 0.5H), δ 6.86 (d, J = 8.0 Hz, 0.5H), δ 5.46-5.57 (m, 2H), δ
.47-4.54 (m, 2H), δ 3.76 (s, 2H), δ 3.75 (s, 1H), δ 3.54 (dd, J = 14.5, 4.0 Hz, 0.5H), δ
.13-3.26 (m, 1.5H), δ 2.17-2.27 (m, 1H), δ 1.48 (s, 4H), δ 1.47 (s, 5H), δ 0.91-0.99 (m,
1
3
3
H); C NMR (CDCl , 125 MHz) δ 171.7, 171.6, 170.0, 169.8, 155.6, 155.5, 142.8,
42.7, 132.9, 132.8, 131.7, 131.6, 130.0, 129.9, 112.0, 111.8, 81.3, 81.0, 57.7, 57.6, 57.4,
6.8, 54.1, 53.5, 52.4, 52.3, 36.1, 35.6, 31.0, 30.8, 28.3, 28.2, 19.0, 18.9, 17.6, 17.5; IR
-1
(
cm ) 3416, 3032, 2982, 2945, 2862, 2304, 1741, 1662, 1581, 1372, 1351, 1260, 1152,
+
1
5
4
069, 823, 734, 708; HRMS (ESI-TOF, [M + Na] ) m/z calculated for C22
3 7 2
H30ClN O S :
70.1111, found 570.1104.
.2.16. (2S,5R,11R,14S)-dimethyl
5,11-bis((tert-butoxycarbonyl)amino)-8-((4-
chlorophenyl)sulfonyl)-8-cyano-2,14-diisopropyl-4,12-dioxo-7,9-dithia-3,13-
diazapentadecane-1,15-dioate (3i)
1
Colorless oil (18 mg, 78%) R
, 600 MHz) δ 7.98 (d, J = 7.0 Hz, 2H), δ 7.59 (dt, J = 7.5, 2.0 Hz, 2H), δ 7.16 (br,
H), δ 5.59 (br, 2H), δ 4.60 (br, 2H), δ 4.52-4.56 (m, 2H), δ 3.77 (s, 4H), δ 3.76 (s, 2H), δ
f
= 0.28 (20% ethyl acetate/hexanes). H NMR
(
CDCl
3
1
29