Exploring new activating groups for reactive cysteine NCAs

Article abstract of DOI:10.1016/j.tetlet.2016.01.104

Due to its ability to reversibly crosslink proteins, cysteine has a unique role as an amino acid in nature. For controlled, asymmetric formation of disulfides from two thiols, one thiol needs to be activated. While few activating groups for cysteine have been proposed, they are usually not stable against amines making them unsuitable for solid phase peptide synthesis or amine initiated polymerization of α-amino acid-N-carboxy-anhydrides (NCAs). In this Letter we describe a series of new thiol activated cysteines, as well as their NCAs and explore the link between electron deficiency of the leaving group and control over NCA polymerization.

Full text of DOI:10.1016/j.tetlet.2016.01.104

Tetrahedron Letters 57 (2016) 1138–1142
Contents lists available at ScienceDirect
Tetrahedron Letters
journal homepage: www.elsevier.com/locate/tetlet
Exploring new activating groups for reactive cysteine NCAs
⇑
David Huesmann, Olga Schäfer, Lydia Braun, Kristina Klinker, Thomas Reuter, Matthias Barz
Institute of Organic Chemistry, Johannes Gutenberg-Universität Mainz, Duesbergweg 10-14, 55128 Mainz, Germany
a r t i c l e i n f o
a b s t r a c t
Article history:
Due to its ability to reversibly crosslink proteins, cysteine has a unique role as an amino acid in nature.
For controlled, asymmetric formation of disulfides from two thiols, one thiol needs to be activated. While
few activating groups for cysteine have been proposed, they are usually not stable against amines making
Received 14 December 2015
Revised 27 January 2016
Accepted 28 January 2016
Available online 29 January 2016
them unsuitable for solid phase peptide synthesis or amine initiated polymerization of a-amino acid-N-
carboxy-anhydrides (NCAs). In this Letter we describe a series of new thiol activated cysteines, as well as
their NCAs and explore the link between electron deficiency of the leaving group and control over NCA
polymerization.
Keywords:
Leuchs anhydride
NCAs
Ó 2016 Elsevier Ltd. All rights reserved.
Cysteine
Reactive monomer
Thiosulfonates
Introduction
solid phase peptide coupling12 or as bifunctional linkers. Further,
a
series of cysteine NCAs (S-allyl-cysteine, S-benzyl-cysteine,
Since their discovery in 19061 Leuchs’ anhydrides also known as
-amino acid-N-carboxy-anhydrides (NCAs) have attracted consid-
S-benzyloxycarbonyl-cysteine, S-benzyloxycarbonyl-methylene-
cysteine, S-butyl-cysteine, S-tert-butylmecapto-cysteine, a range
of S-alkyl-cysteines (dodecyl, hexyl, methyl, ethyl, prolyl, iso-
propyl), S-(2-trimethylsilyl benzyl)-cysteine, S-(4-trimethylsilyl
benzyl)-cysteine, cystine (bisNCA), S-thiophenyl-cysteine) from
the early years of NCA polymerization have been collected.13
However none of them bear thiol reactive protecting groups, which
can be directly addressed after polymerization. Newer cysteine
NCAs have either not focused on post-polymerization modification
of polymers at all14–16 or have worked with deprotection and mod-
ification using thiol-ene chemistry, which leads to the formation of
biological non-reversible thioethers.17–19 Approaches of crosslink-
ing peptidic nanoparticles with disulfides have so far been focused
on oxidative disulfide formation20–24 or polymerization of cystine
bis-NCA,25 while the first method is time-consuming and incom-
plete the latter hinders control over polymerization and particle
formation.
A protective group stable during ROP or standard Fmoc solid
phase synthesis of peptides but selectively addressable in post-
polymerization modification reactions leading to the formation of
a bio-reversible disulfide bond in one step is simply absent. In
our search for a reactive cysteine-protecting group that is stable
under NCA polymerization conditions26 we have come across a
range of novel reactive cysteine NCAs, more or less suitable for
NCA polymerization. In this Letter we report the synthesis and
analysis of these NCAs.
a
erable attention.2,3 NCAs are mostly used as monomers for the
preparation of poly(amino acid)s through ring opening polymer-
ization, but they were also applied as synthons for the stepwise
synthesis of peptides.4,5 In recent years, poly(amino acids) proved
to be very successful as carriers or polymeric drugs in the field of
nanomedicine. While copaxone is an approved drug with 4.7 bil-
lion $ sales in 2013 clinical studies on multiple polypeptide based
polymer drug conjugates or drug formulations (OPAXIO, NC-6004,
NK 105) are underway.6 Additionally, polypetide based polymers
for drug delivery applications are emerging.6–9
However, while disulfides are already widely used in nanome-
dicine to reversibly attach drugs to carriers or to stabilize nanopar-
ticles, to our knowledge, poly(cysteine) has not been used for this
purpose. Neither protected or activated cysteine derivatives have
been explored. The reasons for this fact are on the one hand the
tendency of poly(cysteine) to adopt a b-sheet conformation which
leads to solubility issues and on the other hand the lack of reactive
cysteine protecting groups itself. While derivates of 3-nitro-2-
pyridinesulfenyl cysteine10 and 3-(2-pyridyldithio)propionate11
are frequently employed as thiol reactive moieties, both protecting
groups can only be utilized for endgroup modification in Fmoc
⇑
Corresponding author. Fax: +49 6131 39 24778.
E-mail address: barz@uni-mainz.de (M. Barz).
http://dx.doi.org/10.1016/j.tetlet.2016.01.104
0040-4039/Ó 2016 Elsevier Ltd. All rights reserved.

D. Huesmann et al. / Tetrahedron Letters 57 (2016) 1138–1142
1139
O
O
O
Results and discussion
O
O
O
O
S
HNO2
Diphosgene
S
R
S
O
O
HS
OH
R
S
OH
-
RSO2
HN
NH2
NH2
As starting point for the synthesis of cysteine NCAs with a thiol
reactive protecting group we synthesized thiol reactive groups
known in the literature starting with ortho-nitrothiophenol.27
The strong electron withdrawing effect from the nitro group favors
a thiol exchange with a more electron rich thiol. According to
Phocas et al. o-nitrothiophenol sulfenic acid chloride (1a), was
reacted with cysteine hydrochloride.27 The product (1b) was trans-
formed into the NCA by reaction with diphosgene in the absence of
water (1c, Scheme 1).
d
e
R =
6
Scheme 2. Synthesis of a thiosulfonyl-based NCA.
while its NCA (6c) was synthesized with 77% yield after 3 recrystal-
lization steps to achieve very high purity.
This reaction scheme was used for all disulfide based NCAs.
When the sulfenyl chloride was not commercially available, it
was synthesized from the thiophenol by chlorination with sulfuryl
chloride according to Shang et al.28 The modified cysteines 1–5b
were synthesized with yields ranging from 13% to 98%, while the
NCAs 1c, 3–4c were synthesized with 22–65% yields after 3 recrys-
tallization steps. The NCA of ((p-nitrophenyl)disulfanyl)cysteine
(2c) as well as S-(o-thiopyridinyl)cysteine (5c) could not be puri-
fied. While in the case of 2c cystine impurities could not be
removed, 5c is not even stable, since the pyridine can act as an ini-
tiator in the NCA polymerization2 which was additionally con-
firmed by polymerizing Lys(Z)NCA with pyridine under the
polymerization conditions applied in this study.
The suitability of the NCA 1c for amine initiated NCA polymer-
ization was investigated by polymerization with neopentylamine
in absolute DMF (see Supplementary data).29,30 The GPC plots cor-
responding to the molecular weight distribution of polymers were
multimodal or broad indicating the existence of multiple side reac-
tions. These side reactions can be attributed to protective group
cleavage by the amine chain end and reinitiation by the free thiol
(see Fig. S1). After 1c failed to yield defined polymers we lowered
the strength of the electron-withdrawing group (EWG) to decrease
the reactivity of the disulfide and prevent a reaction of the disulfide
with the primary amine initiator (neopentylamine) or the polymer
chain end. The reactivity was reduced to pentafluoro thiophenol
and ortho-fluoro thiophenol. With decreasing electron deficiency
of the thiol, the quality of the polymers increased (Ð = 2.1, 1.8
and 1.6 for 1c, 3c, and 4c respectively, see SI 2–4) but not to a point
which we considered satisfying.
This NCA performed better than the disulfide based NCAs in the
amine-initiated polymerization yielding polymers with narrower
molecular weight distribution (Ð = 1.16 for 6e, see SI5). The GPC
plots however display the presence of low molecular weight oligo-
mers, which indicates partial protective group cleavage. Although
we have developed new reactive cysteine NCA derivatives, further
investigation of the influence of the electron deficiency of the sub-
stituent with even less electron-deficient S-alkylthiosulfonyl-L-cys-
teine-NCAs seems necessary to achieve protective groups with
high stability against amines and combine it with high reactivity
toward thiols. Since the thiosulfonate group does only partially
engage in side reactions toward amines this class of protective
groups needs to be further investigated in future. The next gener-
ation of derivatives, however, can be synthesized using the herein
reported synthetic pathway.
Conclusion
We have reported the syntheses of a range of new, activated
cysteine amino acids and NCAs based on electron deficient disul-
fides and thiosulfonates. While especially the more reactive disul-
fides cannot be polymerized in a controlled manner using primary
amines as initiators, they might be polymerized more controlled
using organometallic catalyst38–40 or trimethylsilyl initiators.41,42
In contrast, the thiosulfonate based monomer can be polymerized
in a controlled fashion using primary amines, leading to a new
class of reactive polypeptides, showing that higher electron density
leads to higher stability against primary amines and thus decreases
side reactions during NCA polymerization.
Realizing, that weakening of the EWG would not be enough to
facilitate successful NCA polymerization and keep reactivity high
enough for postpolymerization modification reactions, we turned
our attention to different thiol activating groups. A group that
has been used to form asymmetrical disulfides are thiosul-
fonates.31,32 Of this class S-phenylsulfonates are known to react
readily with free thiols achieving complete conversions upon min-
utes,33,34 thus a cysteine NCA based on phenylthiosulfonate was
synthesized. To achieve this, cysteine was activated by transform-
ing it into S-nitroso cysteine, which was reacted with the corre-
sponding sulfinate without purification. The synthesis of
thiosulfonates was based on work of Hart et al.,35,36 which was
chosen over the more complex route of Weidner et al.37 After
purification the S-thiosulfonylcysteine was transformed into the
corresponding NCA using diphosgene (Scheme 2). The S-phenylth-
iosulfonylcysteine 6b was successfully synthesized with 48% yield,
Experimental
Materials and methods
All chemicals were purchased from Sigma Aldrich and used as
received unless otherwise noted. THF and hexane were distilled
from Na/K and ethyl acetate from CaH2. Cysteine was purchased
from OPREGEN and diphosgene from Alfa Aesar. Deuterated sol-
vents were purchased from Deutero.
1H, 19F and 13C NMR spectra were recorded on a Bruker AC 400
at a frequency of 400, 376 and 101 MHz, respectively. 1H and 19F
NMR spectra were also recorded on a Bruker Avance III HD 300
at 300 and 282 MHz, respectively. The spectra were recorded at
room temperature and calibrated using the solvent signals.43 Field
desorption mass spectrometry (FD-MS) was performed on a FD
Finnigan MAT90 spectrometer. Melting points were measured
using a Mettler FP62 melting point apparatus at a heating rate of
0.5 °C/min. Preparative reverse phase-HPLC was performed with
a Knauer HPLC-System (Berlin, Germany), consisting of two HPLC
pumps (Smartline 1000), an UV/vis-detector (Smartline 2500), a
RI-detector (Smartline 2400) and a Phenomenex (Torrance, U.S.
O
O
S
Cys*HCl
Diphosgene
SO2Cl2
S
SH
S
Ar
S
O
O
Ar
S
OH
Ar
Ar
Cl
HN
a
b
c
NH2
F
NO2
F
F
F
N
Ar =
O2N
F
A.) Luna-column (10
flow of 10.0 mL/min and loaded with a 2 mL injection loop. The
l
m, C18(2), 100A, 250 Â 30 mm2) run at a
F
3
1
4
2
5
Scheme 1. Synthesis of disulfide-based NCAs.
system was operated and samples analyzed with D-7000

1140
D. Huesmann et al. / Tetrahedron Letters 57 (2016) 1138–1142
HPLC-System-Manager software (version 4.1). The detector was
run at a wavelength of 214 nm.
Pentafluorophenylsulfenyl chloride (PFTPCl, 3a)
A solution of 5 mL pentafluorothiophenol (7.505 g, 37.5 mmol)
in 75 mL DCM was cooled to 0 °C. 4.56 mL sulfuryl chloride
(7.59 g, 56.3 mmol) was added and the solution was stirred over-
night. DCM and excess sulfuryl chloride were removed in vacuo
at 40 °C yielding 8.35 g (4.97 mL, 35 mmol, 93%) of an orange
liquid.
((o-Nitrophenyl)disulfanyl)-L-cysteine (Cys(oNTP), 1b)
To a suspension of 5.86 g (37.18 mmol) cysteine hydrochloride
in 150 mL acetic acid 10 g (52.74 mmol) (commercial) 2-nitroben-
zenesulfenyl chloride was added. The suspension was stirred until
it got solid, then 100 mL acetic acid was added and the mixture
was heated to 70 °C for 3.5 h. The solvent was removed in vacuo,
the residue was dissolved in 20 mL DMSO and precipitated with
250 mL CHCl3. After filtration the precipitate was suspended in
200 mL CHCl3, stirred for 30 min, and collected by filtration twice.
The product was dried in vacuo yielding 8.63 g (31.45 mmol, 85%)
of a yellow solid.
19F NMR (400 MHz, CDCl3) d [ppm] = À128.57 (m, 2F, o-F),
À145.49 (tt, 1F, p-F), À160.17 (m, 2F, m-F).
S-(Pentafluorothiophenyl)-L-cysteine (Cys(PFTP), 3b)
To a suspension of 1.35 g (8.59 mmol) cysteine hydrochloride in
35 mL acetic acid, 3.02 g (13 mmol) pentafluorophenylsulfenyl
chloride was added. The suspension solidified after 5 min and
another 23 mL of acetic acid was added, yielding a yellow solution,
which was stirred at 70 °C for 3.5 h. During the reaction the yellow
color of the solution got weaker. The solvent was removed in vacuo
and the slightly yellow residue was extracted twice with 58 mL
CHCl3, yielding 2.7 g (8.46 mmol, 98%) of a colorless solid.
1H NMR (400 MHz, DMSO-d6) d [ppm] = 8.85 (s, 3H, NH+3), 8.28
(dd, J = 9.6, 8.2 Hz, 2H, HAr), 7.89 (dd, J = 8.3, 7.2 Hz, 1H, HAr), 7.55
(dd, J = 8.3, 7.2 Hz, 1H, HAr), 4.20 (t, J = 5.8 Hz, 1H, CHNH3), 3.43–
3.26 (m, 2H, CHCH2S).
13C{1H} NMR (101 MHz, DMSO-d6) d [ppm] = 169.09 (COOÀ),
145.26 (CarNO2), 135.31 (CarS, Car), 127.42 (Car), 127.36 (Car),
126.44 (Car), 51.22 (C ), 37.14 (Cb).
1H NMR (400 MHz, CD3CN + TFA-d) d [ppm] = 4.43 (dd, J = 8.5,
a
FD-MS: 274.3 (M+H+).
4.3 Hz, 1H, C H), 3.51 (dd, J = 15.3, 4.3 Hz, 1H,CH2), 3.35 (dd,
a
J = 15.3, 8.6 Hz, 1H, CH2).
((o-Nitrophenyl)disulfanyl)-
L
-cystein-NCA (Cys(oNTP)NCA, 1c)
19F NMR (400 MHz, CD3CN + TFA-d) d [ppm] = À134.66 (m, 2F,
o-F), À153.01 (tt, J = 20.0, 3.9 Hz, 1F, p-F), À163.35 (m, 2F, m-F).
A suspension of 2.015 g (7.35 mmol) of ((2-nitrophenyl)disul-
fanyl)cysteine and 1.55 mL (9.56 mmol) limonene in 40 mL dry
THF was heated to 70 °C. 0.8 mL (4.59 mmol) diphosgene was
added in 3 steps every 20 min. The yellow solution was heated
for 4 h. Nitrogen was bubbled though the solution for 2 h to
remove excess HCl and diphosgene and most of the solvent was
removed. The residue was redissolved in 11 mL THF and precipi-
tated by adding 100 mL of cyclohexane very slowly. The precipitate
was redissolved in 14 mL THF and insoluble solids were removed
by filtration. The product was recrystallized two more times from
THF/cyclohexane avoiding contact to air yielding 1.437 g
(4.78 mmol; 65%) of a pale yellow powder.
13C{1H} NMR (101 MHz, CD3CN + TFA-d)
d [ppm] = 168.95
(COO), 147.61 (ddd, J = 246.4, 11.6, 4.4 Hz, o-CAr), 144.73–140.78
(m, p-CAr), 139.72–135.61 (m, m-CAr), 110.06 (t, J = 20.6 Hz, CArS),
53.23 (CH2), 39.31 (C ).
a
FD-MS: 319.1 (M+H+).
S-(Pentafluorothiophenyl)-L-cysteine NCA (3c)
2.35 g (7.35 mmol) S-(pentafluorphenyl)cysteine was sus-
pended in 40 mL abs. THF and the suspension was heated to
70 °C. Then 0.8 mL (6.63 mmol) of diphosgene was added over
1 h in which the solid dissolved. The solution was stirred for 1.5
more hours. Nitrogen was bubbled through the solution overnight
to remove HCl and diphosgene. The slightly yellow solid was then
dissolved in 11 mL abs. THF and precipitated by slowly adding
130 mL abs. hexane. The precipitate was collected by filtration
without contact to air and recrystallized 3 times from THF/hexane
yielding 568 mg (1.64 mmol, 22%) of the S-(pentafluorphenyl)cys-
teine NCA.
1H NMR (300 MHz, DMSO-d6) d [ppm] = 9.33 (s, 1H, NH), 8.31
(dd, J = 8.2, 1.4 Hz, 1H, HAr), 8.23 (dd, J = 8.2, 1.2 Hz, 1H, HAr), 7.90
(ddd, J = 8.4, 7.2, 1.4 Hz, 1H, HAr), 7.56 (ddd, J = 8.4, 7.3, 1.3 Hz, 1H,
HAr), 4.77 (ddd, J = 6.0, 4.8, 1.3 Hz, 1H, C H), 3.38–3.16 (m, 2H, CH2).
a
1H NMR (400 MHz, THF-d8) d [ppm] = 8.47 (s, 1H, NH), 8.30 (td,
J = 8.0, 1.2 Hz, 2H, HAr), 7.81 (ddd, J = 8.4, 7.2, 1.4 Hz, 1H, HAr), 7.47
(ddd, J = 8.1, 7.1, 1.1 Hz, 1H, HAr), 4.68 (dd, J = 7.8, 3.7 Hz, 1H, C H),
a
3.26 (dd, J = 14.0, 3.9 Hz, 2H, CH2), 3.17 (dd, J = 14.0, 7.8 Hz, 1H,
CH2).
1H NMR (400 MHz, THF-d8) d [ppm] = 4.77 (dd, J = 7.8, 3.6 Hz,
1H, C H), 3.41 (dd, J = 14.4, 3.6 Hz, 1H, CH2), 3.22 (dd, J = 14.3,
a
13C{1H} NMR (101 MHz, THF-d8) d [ppm] = 170.27 (C (CO)O),
7.6 Hz, 1H, CH2).
a
152.57 (NH(CO)O), 146.98 (CArNO2), 136.85 (CArS), 135.53 (CAr),
128.21 (CAr), 127.88 (CAr), 127.14 (CAr), 57.77 (C ), 40.34 (CH2).
19F NMR (400 MHz, THF-d8) d [ppm] = À134.64 (m, 2F, o-F),
À152.94 (tt, J = 20.7, 3.5 Hz, 1F, p-F), À163.28 (m, 2F, m-F).
a
13C{1H} NMR (101 MHz, THF-d8) d [ppm] = 168.99 (C (CO)O),
a
((p-Nitrophenyl)disulfanyl)-
L
-cysteine (Cys(pNTP), 2b)
151.50 (NH(CO)O), 147.47 (ddq, J = 247.0, 11.8, 4.2 Hz, o-CAr),
142.75 (dtt, J = 256.2, 13.8, 5.3 Hz, p-CAr),139.08–136.55 (m-CAr),
To a suspension of 277 mg (1.76 mmol) cysteine hydrochloride
in 2 mL DMF was added 6 mL acetic acid followed by 500 mg
(2.64 mmol) commercial 4-nitrobenzenesulfenyl chloride. The sus-
pension was stirred at room temperature for 2 days during which it
lost most of its yellow color. The AcOH was removed in vacuo. Then
30 mL CHCl3 was added slowly to precipitate the product. After fil-
tration the precipitate was suspended in 30 mL CHCl3, stirred for
30 min, and collected by filtration three times. The product was
dried in vacuo yielding 394 mg (1.44 mmol, 82%) of a pale yellow
solid.
111.68–110.23 (m, CArS), 56.59 (CH2), 40.94 (C ).
a
o-Fluorophenylsulfenyl chloride (oFTPCl, 4a)
5 mL (46 mmol) o-fluorothiophenol was dissolved in 92 mL
DCM and cooled to 0 °C. 5.65 mL (70 mmol) sulfurylchloride was
added and the solution was stirred for 16 h at room temperature.
DCM and excess sulfuryl chloride were removed in vacuo, yielding
7.39 g (45 mmol, 98%) of an orange–red liquid. That was used
without further purification.
1H NMR (300 MHz, DMSO-d6) d [ppm] = 10.02–8.36 (br, 3H,
1H NMR (400 MHz, CDCl3) d [ppm] = 7.74 (td, 1H, J = 1.7, 5.8 Hz,
1H, HAr), 7.51–7.46 (m, 1H, HAr), 7.26–7.17 (m, 2H, HAr).
19F NMR (400 MHz, CDCl3) d [ppm] = À105.82 (m, 1F, FAr).
NH+3), 8.24 (d, J = 8.9 Hz, 2H, HAr), 7.84 (d, J = 9.0 Hz, 2H, HAr), 4.15
(t, J = 5.9 Hz, 1H, C H), 3.36 (dd, J = 6.0, 2.4 Hz, 2H, CH2).
a

D. Huesmann et al. / Tetrahedron Letters 57 (2016) 1138–1142
1141
S-(o-Fluorothiophenyl)-L-cysteine (Cys(oFTP), 4b)
ESI-MS: 231.0 (M+H+).
The S-(o-thiopyridinyl)cysteine NCA (5c) could not be com-
pletely purified. It is not stable, as the pyridine can act as an initia-
tor in the NCA polymerization, which was confirmed by
polymerizing Lys(Z)NCA with pyridine.
7.94 g (49 mmol) o-fluorophenylsulfenyl chloride was mixed
with 3 mL TFA and cooled in an ice-bath. 3.68 g (23 mmol) cysteine
hydrochloride was dissolved in 40 mL TFA and added to the solution
at a rate of 15 mL/h under constant stirring. The TFA was removed in
vacuo at 45 °C and 100 mL CHCl3/MeOH (1:1) was added to the
resulting solid to extract the product (residue: cystine, side pro-
duct). The residue was filtered off and the filtrate was concentrated
in vacuo. 150 mL Et2O was added und the product was collected by
filtration. This was repeated one more time yielding 0.99 g
(2.88 mmol, 13%) a slightly yellow/brownish solid (TFA salt).
1H NMR (400 MHz, MeOD) d [ppm] = 7.73 (td, J = 7.6, 1.7 Hz, 1H,
HAr), 7.50–7.38 (m, 1H, HAr), 7.3–7.13 (m, 2H, HAr), 4.40 (dd, J = 8.3,
S-Phenylthiosulfonyl-L-cysteine (Cys(SO2Ph), 6d)
3 g (17.08 mmol) cysteine hydrochloride monohydrate was dis-
solved in 17 mL 2 M hydrochloric acid and cooled to 5 °C in an ice
bath. 1.18 g (17.08 mmol) Sodium nitrite was dissolved in 10 mL
milli-Q water, cooled and added to the cysteine solution via a drop-
ping funnel. The solution turned red. After stirring for 80 min at
5 °C, 5.61 g (34.16 mmol) sodium benzylsulfinate was added. After
2 h discoloration of the solution was observed and a precipitate
formed. Another 1.40 g (8.54 mmol) sodium benzylsulfinate was
added and the mixture was stirred for 2 more hours and stored
at 4 °C overnight. The solid was collected by filtration and washed
two times with ice-cold milli-Q water. The brown solid was recrys-
tallized from water, washed with ethanol and diethyl ether and
dried in vacuo to yield 2.14 g (8.20 mmol, 48%) of a slightly yellow-
ish solid.
4.2 Hz, 1H, C H), 3.39 (dd, J = 14.9, 4.2 Hz, 1H, CH2), 3.21 (dd,
a
J = 14.8, 8.3 Hz, 1H, CH2).
19F NMR (400 MHz, MeOD) d [ppm] = À112.16 (ddd, J = 9.4, 7.5,
5.1 Hz, 1F, FAr).
S-(o-Fluorothiophenyl)-L-cysteine NCA (Cys(oFTP)NCA, 4c)
0.99 g (2.88 mmol) S-(o-fluorphenyl)cysteine was suspended in
30 mL abs. THF and the suspension was heated to 70 °C. Then
0.35 mL (2.88 mmol) of diphosgene was added over 1 h in which
the solid dissolved. The solution was stirred for 1.5 more hours.
Nitrogen was bubbled through the solution overnight to remove
HCl and diphosgene. The slightly yellow solid was then dissolved
in 11 mL abs. AcOEt and precipitated by slowly adding 60 mL
abs. hexane. The precipitate was collected by filtration without
contact to air and recrystallized 3 times from AcOEt/hexane yield-
ing 332 mg (1.21 mmol, 42%) of the S-(o-fluorphenyl)cysteine
NCA.
1H NMR (400 MHz, D2O + TFA-d) d [ppm] = 7.90–7.81 (m, 2H,
HAr), 7.69–7.61 (m, 1H), 7.58–7.48 (m, 2H, HAr), 4.27 (dd, J = 7.3,
4.6 Hz, 1H, C H), 3.48 (dd, J = 15.8, 4.6 Hz, 1H, CH2), 3.39 (dd,
a
J = 15.8, 7.3 Hz, 1H, CH2).
13C{1H} NMR (101 MHz, D2O) d [ppm] = 171.52 (COOH), 144.52
(CArS), 137.88 (CAr), 132.51 (CAr), 129.69 (CAr), 54.71 (CH2), 36.97
(C ).
a
S-Phenylthiosulfonyl-L-cysteine-NCA (Cys(SO2Ph)NCA, 6e)
1H NMR (400 MHz, DMSO-d6) d [ppm] = 9.31 (s, 1H, NH), 7.73
(td, J = 7.8, 1.8 Hz, 1H, HAr), 7.45 (dddd, J = 8.7, 7.2, 5.3, 1.7 Hz,
1H, HAr), 7.37–7.21 (m, 2H, HAr), 4.82 (ddd, J = 6.0, 4.6, 1.3 Hz, 1H,
In an inert gas atmosphere 5 g (19.13 mmol) phenyl cysteine
thiosulfonate was suspended in 50 mL abs. THF and heated to
70 °C. Diphosgene (2.08 mL, 17.22 mmol) was added in three steps
every 20 min and the suspension was stirred for another 2 h, in
which all solid dissolved. The solution was allowed to cool down
and THF was removed, by passing dry nitrogen through the solu-
tion overnight. The crude product was dried in vacuo and recrystal-
lized 3 times by dissolving in THF and precipitating with hexane,
yielding 4.20 g (14.73 mmol, 77%) of a slightly yellow powder.
1H NMR (400 MHz, DMSO-d6) d [ppm] = 9.23 (s, 1H, NH), 7.95
(d, J = 7.6 Hz, 2H, HAr), 7.81 (t, J = 7.4 Hz, 1H, HAr), 7.71 (t,
C H), 3.32–3.15 (m, 2H, CH2).
a
19F NMR (376 MHz, DMSO-d6) d [ppm] = À111.70 (ddd, J = 10.2,
7.8, 5.2 Hz, 1F, FAr).
13C{1H} NMR (101 MHz, DMSO-d6) d [ppm] = 170.48 (C (CO)O),
a
160.53 (d, J = 244.3 Hz, CArF), 152.21 (NH(CO)O), 131.93 (CAr),
131.16 (d, J = 7.9 Hz, CAr), 125.95 (d, J = 3.5 Hz, CAr), 122.96 (d,
J = 17.0 Hz, CAr), 116.62 (d, J = 21.5 Hz, CAr), 57.14 (C ), 39.51 (CH2).
a
o-Pyridinylsulfenyl chloride hydrochloride (oTPCl, 5a)
J = 7.7 Hz, 2H, HAr), 4.73 (t, J = 5.6 Hz, 1H, C H), 3.49 (d, J = 5.6 Hz,
a
2H, CH2).
See o-fluorophenylsulfenyl chloride. Yield: 97%.
1H NMR (300 MHz, DMSO-d6) d [ppm] = 14.50 (s, 1H, NArH+),
8.49 (ddd, J = 4.9, 1.9, 0.9 Hz, 1H, HAr), 7.84 (ddd, J = 8.1, 7.5,
1.8 Hz, 1H, HAr), 7.65 (dt, J = 8.1, 1.0 Hz, 1H, HAr), 7.31 (ddd,
J = 7.5, 4.9, 1.0 Hz, 1H, HAr).
13C{1H} NMR (101 MHz, DMSO-d6) d [ppm] = 169.29 (C (CO)O),
a
151.47 (NH(CO)O), 143.59 (CArS), 134.69 (CAr), 130.02 (CAr), 126.69
(CAr), 56.52 (CH2), 36.04 (C ).
a
FD-MS: 287.0 (M+).
S-(o-Thiopyridinyl)-L-cysteine (Cys(oTP), 5b)
Acknowledgments
7.90 g (54.25 mmol) o-pyridinylsulfenyl chloride and 5.70 g
(36.17 mmol) cysteine hydrochloride were heated in 50 mL acetic
acid for 30 min. The solution turned bright yellow. The reaction
mixture was kept in the fridge overnight. The solid was collected
by filtration and washed with acetic acid. The solid was further
washed by suspending it in chloroform, stirring for 1 h and filtering
of the chloroform 3 times, yielding a bright yellow powder. The
powder was further purified by HPLC yielding 4.01 g (17.41 mmol,
48%) of a yellow powder.
We would like to thank Prof. K. W. Klinkhammer and
Prof. H. Kunz for stimulating discussions. M.B. acknowledges finan-
cial support by the SFB 1066-1 and the NMFZ Mainz. D.H. would
like to acknowledge the support by the ‘Verband der Chemischen
Industrie’ (VCI) and the ‘Max Planck Graduate Center with the
Johannes Gutenberg-Universität Mainz’ (MPGC).
Supplementary data
1H NMR (300 MHz, DMSO-d6) d [ppm] = 8.52 (ddd, J = 4.8, 1.8,
0.9 Hz, 1H, HAr), 7.84 (ddd, J = 8.1, 7.4, 1.9 Hz, 1H, HAr), 7.73 (dt,
J = 8.1, 1.0 Hz, 1H, HAr), 7.32 (ddd, J = 7.4, 4.9, 1.1 Hz, 1H, HAr),
Supplementary data associated with this article can be found, in
the online version, at http://dx.doi.org/10.1016/j.tetlet.2016.01.
104.
4.21 (dd, J = 7.5, 5.0 Hz, 1H, C H), 3.48–3.22 (m, 2H, CH2).
a

1142
D. Huesmann et al. / Tetrahedron Letters 57 (2016) 1138–1142
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