Macromolecules
Article
Synthesis of Ethanesulfinic Acid Sodium Salt. A solution of
sodium sulfite (391.98 g, 3.11 mol) in 800 mL of water was heated to
80 °C. Ethanesulfonyl chloride (147.40 mL, 199.30 g, 1.55 mol) and
sodium carbonate (329.63 g, 3.11 mol) were added simultaneously
while significant quantities of CO2 evolved. The reaction mixture was
stirred for 1 h at 80 °C, and afterward the water was removed in vacuo
at 60 °C. The resulting solid was suspended in methanol (degassed)
and filtrated. Evaporation of methanol gave ethanesulfinic acid sodium
thiol protective group. Only recently we were able to realize S-
alkylsulfonyl protecting groups for cysteine-based polypeptides,
which fulfill the requirements mentioned above.31,32 It has
been shown that the thiol in the side chain is activated toward
the reaction with other thiols under formation of asymmetric
disulfides and is simultaniously deactivated against the reaction
with amines, which ensures stability not only during NCA
synthesis, but also under polymerization conditions or solid
phase peptide synthesis (SPPS).31,32
Because of the balanced reactivity profile of the S-
ethylsulfonyl protecting group, well-defined homopolymers
could be synthesized with dispersity indices lower than 1.2 and
chain lengths of up to 50.31 The limitation regarding the
maximum achievable molecular weight is attributed to the
formation of β-sheets, which causes aggregation and
precipitation of the growing polycysteine chains.35 By using
L-homocysteine instead of L-cysteine, this limitation may be
resolved, since polyhomocysteine is known to form α-helical
secondary structures, which prevents the growing chains from
precipitation.36 Therefore, polymerization may be carried out
to higher degrees of polymerization without a loss in
polymerization control.
This work describes the synthesis of S-ethylsulfonyl-L-
homocysteine NCA (Hcy(SO2Et)-NCA) out of L-methionine,
its nucleophilic ring-opening polymerization, and the post-
polymerization modifications of the corresponding polypep-
tides. We report on the polymer properties, as well as the
reaction kinetics under various conditions, and relate the
results to the analogue polycysteine derivatives. Finally, the
PPM of the synthesized polymers will be presented, which
displays chemoselective formation of asymmetric disulfides.
1
salt (153.54 g, 1.32 mol, 85%) as a colorless solid. H NMR (400
MHz, D2O/[D1]TFA) δ [ppm] = 2.67 (q, 3J = 7.6 Hz, 2H, −CH2−),
3
1.15 (t, J = 7.6 Hz, 3H, −CH3).
Synthesis of L-Homocysteine (Hcy). L-Methionine (5.62 g,
37.67 mmol) was placed in a three-necked round-bottom flask and
cooled to −60 °C (acetone/dry ice). Ammonia gas was condensed
into the reaction flask through a gas injection apparatus to solve the L-
methionine. With the addition of sodium (2.69 g, 116.78 mmol) in
small portions the colorless solution turned dark blue. The blue color
vanished after a view minutes, and a colorless precipitate formed.
After complete addition of the sodium the solution stayed dark-blue
and was stirred for an additional 30 min. After this, ammonium
chloride was added in small portions until the blue color disappeared
completely.38 The cooling bath (acetone/dry ice) was removed, and a
slight stream of nitrogen was passed through the apparatus to remove
the ammonia. The dry residue was taken up with degassed water.
Concentrated hydrochloric acid was added slowly until a pH of 5.5
was reached. The solution was degassed via freeze−pump−thaw and
filtrated afterward. The filtrate was stored at 4 °C until the product
crystallized to yield 4.76 g (34.98 mmol, 93%) of L-homocysteine. 1H
3
NMR (400 MHz, D2O) δ [ppm] = 4.02 (dd, J = 7.2, 5.6 Hz, 1H,
−CH−), 2.84−2.72 (m, 2H, HS-CH2−), 2.36−2.20 (m, 2H, HS−
CH2−CH2−). 13C NMR (101 MHz, D2O): δ [ppm] = 174.41
(COOH), 53.74 (NH2−CH−COOH), 34.69 (HS−CH2−CH2−),
20.06 (HS−CH2). ESI-MS: Hcy (calcd. 135.18): 136.06 ([M+H]+).
Synthesis of S-Ethylsulfonyl-L-homocysteine (Hcy(SO2Et)).
An ice-cold solution of sodium nitrite (1.62 g, 23.5 mmol) in degassed
water (8 mL) was added slowly to a cooled solution of L-
homocysteine (3.2 g, 23.5 mmol) in 25 mL of degassed water and
22.5 mL of hydrochloric acid (2 M). It is crucial to keep the solution
temperature around 0 °C during the whole addition to prevent the
formation of nitrous gases. After complete addition, the deep-red
solution was stirred for an additional 30 min at 0 °C. A solution of
ethanesulfinic acid sodium salt (6.8 g, 58.75 mmol) in 10 mL of
degassed water and 22.5 mL of hydrochloric acid (2 M) was added,
and the reaction mixture was stirred overnight at 0 °C with a gentle
flow of nitrogen over the reaction mixture. The colorless solution was
stored at 4 °C for several days, and eventually S-ethylsulfonyl-L-
homocysteine (3.6 g, 15.6 mmol, 66%) precipitated. If no
precipitation occurred, the water was removed in vacuo, and the
residue was suspended in degassed ethanol and filtered. Ethanol was
removed from the filtrate in vacuo, resulting a slightly yellow oil, which
contains the product as well as byproducts. The product was isolated
via semipreparative HPLC (Column: Phenomenex Luna C18(2);
size: 250 × 30 mm2; eluent: water/MeOH with the following
gradient: 1 min: 90/10; 16 min: 40/60; 17 min: 0/100; 20.5 min: 0/
100; 21 min: 90/10). The product peak was collected in several runs,
methanol was removed in vacuo, and the solution was freeze-dried.
After lyophilization 546 mg (2.4 mmol, 10%) of S-ethylsulfonyl-L-
homocysteine was obtained as a sticky, colorless solid. For further
characterization, a small amount of the product was recrystallized
from water and analyzed via X-ray crystallography. Crystal structure:
CCDC-1858027. 1H NMR (400 MHz, D2O) δ [ppm] = 3.87 (t, 1H,
EXPERIMENTAL SECTION
■
THF, n-hexane, and diethyl ether were distilled from Na. DMF
(99.8%, extra dry over molecular sieve (4 Å) with AcroSeal) was
purchased from Acros. Prior to use, DMF was degassed in vacuo to
remove traces of dimethylamine. HFIP was purchased from
Fluorochem. Millipore water was prepared by a MILLI-Q Reference
A+ System. Neopentylamine was purchased from TCI Europe, dried
over NaOH for several days, and fractionally distilled before use. L-
Methionine was purchased from OPREGEN and used as received.
Diphosgene was purchased from Alfa Aesar and deuterated solvents
from Deutero GmbH. Other chemicals were purchased from Sigma-
Aldrich and used as received unless otherwise stated. H and 13C
1
NMR spectra were recoreded on a Bruker AC 400 at a frequency of
400 and 101 MHz, respectively. 1H NMR spectra were also recorded
on a Bruker Avance III HD 300 at 300 MHz. Two-dimensional NMR
spectra such as DOSY, COSY, HSQC, and HMBC were recorded on
a Bruker Avance III HD 400 at 400 MHz and 101 MHz. All spectra
were recorded at room temperature (25 °C) and calibrated using the
solvent signals.37 Melting points were measured using a Mettler FP62
melting point apparatus at a heating rate of 1 °C min−1. Gel
permeation chromatography (GPC) was performed with hexafluoro-
isopropanol (HFIP) containing 3 g L−1 potassium trifluoroacetate
(KTFA) as eluent at 40 °C and a flow rate of 0.8 mL min−1. The
columns were packed with modified silica (PFG columns particle size:
7 μm; porosity: 100 and 1000 Å). Poly(methyl methacrylate)
standards (PMMA, Polymer Standards Services GmbH) were used
for calibration, and toluene was used as the internal standard. A
refractive index detector (G1362A RID) and an UV/vis detector (at
230 nm unless otherwise stated, Jasco UV-2075 Plus) were used for
polymer detection. Infrared (IR) spectroscopy was performed on a
Jasco FT/IR-4100 with an ATR sampling accessory (MIRacle, Pike
Technologies), and Spectra Manager 2.0 (Jasco) was used for
integration.
3
−CH−), 3.62 (q, J = 7.28 Hz, 2H, CH3−CH2−SO2−), 3.40−3.27
(m, 2H, −S−CH2−), 2.42−2.30 (m, 2H, −S−CH2−CH2−), 1.44 (t,
3J = 7.28 Hz, 3H, CH3−CH2−SO2−). 13C NMR (101 MHz, D2O): δ
[ppm] = 173.33 (−COO), 56.42 (−CH2−SO2−), 53.24 (−CH−),
31.42 (−S−CH2−), 31.12 (−CH−CH2−CH2−), 7.58 (−CH3). ESI-
MS: Hcy(SO2Et) (calcd. 227.03): 228.04 (M + H+), 250.02 ([M
+Na]+).
Synthesis of S-Ethylsulfonyl-L-homocysteine N-Carboxyan-
hydride (Hcy(SO2Et)-NCA). Dried S-ethylsulfonyl-L-homocysteine
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Macromolecules XXXX, XXX, XXX−XXX