8
L.J. Alcock et al. / Tetrahedron xxx (2017) 1e9
was not detected.
1.58e1.50 (2H, contains COCHCHAHBCH and COCH), 1.45 (1H,
CHCHAHBCH); 13C NMR (150 MHz, CDCl3):
135.3, 47.1, 46.4, 41.8, 40.3, 31.0, 25.6.
d
¼ 171.6, 169.3, 138.5,
4.9. Oxidation of N-acetylcysteine to its disulfide using hydrogen
peroxide at pD 5.0 (Fig. 7B)
4.13. Norbornene probe 22
N-acetylcysteine (9.8 mg, 0.06 mmol 9), D2O (0.25 mL) and
NaOH (2.4 mg, 0.06 mmol) were added to a vial and stirred or
shaken until fully dissolved. Next, pD 5.0 sodium acetate buffer
(400 mM in D2O, 0.25 mL) was added and the solution was stirred.
Norbornene-NHS 21 (359 mg, 1.53 mmol) was dissolved in
anhydrous DCM (8 mL) with stirring at room temperature and then
1-amino-4-butyne (125
mL, 1.53 mmol) and DIPEA (650 mL,
A solution of H2O2 (7
m
L, 30 wt% in H2O) was added and left to stir
3.73 mmol) were then added successively. After 1 h a white pre-
cipitate had formed (N-hydroxysuccinimide). The crude material
was concentrated under reduced pressure and purified by column
chromatography (40% EtOAc in hexane) to give the product 22 as a
white solid (257 mg, 89%). m.p. 86e88 ꢂC; Rf (40% EtOAc in hexane)
0.47; IR (nmax, ATR) 3300, 3267, 3058, 2963, 2869, 1633, 1547, 1442,
1359, 1330, 1243, 1221, 1149, 1070, 1018, 901, 864, 721, 680,
for 20 min. The reaction was then analysed directly by 1H NMR. 60%
conversion to disulfide 10 and 40% unreacted 9 were observed.
4.10. Synthesis of alkyne 19 (Fig. 8A)
1-amino-3-butyne (59.2 mL, 0.723 mmol) was added to a stirred
solution of cis-5-norbornene-endo-2,3-dicarboxylic anhydride
(239 mg, 1.46 mmol) in acetonitrile (1 mL) and stirred at room
temperature for 20 min, over which time a white precipitate
formed. The resulting mixture was transferred into a centrifuge
tube and pelleted by centrifugation for 10 min. The supernatant
was decanted and the remaining pellet washed with EtOAc. The
final product was isolated by filtration without further purification
to give the product 19 as a white solid (154 mg, 91% yield): m.p.
129 ꢂC; IR (nmax, ATR): 3359, 2987, 1716, 1622, 1550, 1321, 1267,
1229, 1074, 846, 759, 679, 625; 1H NMR (600 MHz, DMSO-d6):
625 cmꢀ1
;
1H NMR (600 MHz, CDCl3):
d
¼ 6.15 (1H, dd, J ¼ 5.7,
3.0 Hz, CH¼CH), 6.11 (1H, dd, J ¼ 5.7, 3.0 Hz, CH¼CH), 5.81 (1H, br-s,
CONH), 3.42 (2H, m, CONHCH2CH2), 2.93 (2H, m, CHCH¼CHCH),
2.42 (2H, tt, J ¼ 6.1, 2.1 Hz, CONHCH2CH2), 2.01 (2H, contains
CH2C≡CH and CH¼CHCHCHCONH), 1.92 (1H, m, CH¼CHCHCHAHB),
1.70 (1H, d, J ¼ 8.1 Hz, CHCHAHBCH), 1.35 (2H, contains CHCHAHBCH
and CH¼CHCHCHAHB); 13C NMR (150 MHz, CDCl3):
¼ 175.7, 138.3,
d
136.0, 81.7, 69.9, 47.2, 46.4, 44.8, 41.6, 38.0, 30.5, 19.5; HRMS (ESI):
[MþH]þ, found 190.1228. C12H16NOþ requires 190.1226.
d
¼ 11.54 (1H, br-s, COOH), 7.86 (1H, t, J ¼ 5.9 Hz, NH), 6.15 (1H, dd,
4.14. Norbornene amine derivative 24
J ¼ 5.5, 2.9 Hz, HC¼CH), 5.95 (1H, dd, J ¼ 5.4, HC¼CH), 3.14 (1H, dd,
J ¼ 10.3, 3.3 Hz, CH¼CHCHCHCOOH or CH¼CHCHCHCONH), 3.08
(3H, contains CONHCH2CH2 and CH¼CHCHCHCOOH or
CH¼CHCHCHCONH), 2.93 (2H, m, CHCH¼CHCH), 2.80 (1H, t, J ¼ 2.6,
C≡CH), 2.28e2.14 (2H, m, CH2C≡CH), 1.25 (1H, d, J ¼ 8.1 Hz,
CHCHAHBCH), 1.21 (1H, d, J ¼ 8.2 Hz, CHCHAHBCH); 13C NMR
(150 MHz, DMSO-d6): 173.5, 171.2, 134.9, 133.7, 82.4, 71.9, 48.4, 48.1,
48.1, 46.7, 45.3, 37.8, 18.7; LRMS (ESI): [M-H]-: found 232.0,
1,2-bis(2-aminoethoxy)ethane (624
mL, 4.2 mmol) and DIPEA
(163 L, 0.92 mmol) were dissolved in anhydrous DCM (5 mL) with
m
stirring at room temperature. A solution of norbornene-NHS 21
(99 mg, 0.42 mmol) was dissolved in anhydrous DCM (1 mL) and
added dropwise to the 1,2-bis(2-aminoethoxy)ethane solution and
then the reaction mixture was stirred at room temperature for
30 min. After this time, the crude mixture was concentrated under
reduced pressure. The resulting oil was purified by column chro-
matography (10% MeOH in DCM with 1% NEt3) to give the product
24 as a yellow oil (103 mg, 91%). Rf (10% MeOH in DCM with 1%
NEt3) 0.20; IR (nmax, ATR): 3294, 3057, 2936, 2868, 1645, 1541, 1448,
C
13H14NO2 requires 232.1.
4.11. Hydrolysis study of alkyne probe 19 (Fig. 8B)
Alkyne probe 19 (24 mg, 0.10 mmol) was added to a vial and
dissolved in 1.25 mL of D2O. To this solution was added sodium
acetate buffer in D2O (1.25 mL, pD 5.0, 400 mM). After 20 min and
24 h, this solution was analysed directly by 1H NMR and LC-MS,
indicating hydrolysis to 8 and 18. After 20 min, 50% hydrolysis
was observed by 1H NMR and complete hydrolysis was observed
after 24 h (as indicated by integration of the alkene signals and
comparison to an authentic sample of 8, 19, and 18).
1351, 1247, 1105, 905, 808, 724 cmꢀ1 1H NMR (600 MHz, CDCl3):
;
d
¼ 6.29 (1H, br-s, CONH), 6.13 (1H, dd, J ¼ 5.7, 2.9 Hz, CH¼CH), 6.09
(1H, dd, J ¼ 5.8, 3.1 Hz, CH¼CH), 3.63 (4H, m, 2 ꢁ CH2 PEG), 3.56
(4H, dt, J ¼ 18.5, 5.1 Hz, 2 ꢁ CH2 PEG), 3.47 (2H, q, J ¼ 5.2 Hz,
CONHCH2), 2.91 (4H, contains CH2 PEG and CHCH¼CHCH), 2.03
(1H, m, CHCHCONH), 1.91 (1H, dt,
J
¼
11.5, 4.0 Hz,
CH¼CHCHCHAHB), 1.72 (1H, d, J ¼ 8.3 Hz, CHCHAHBCH), 1.31 (2H,
contains CH¼CHCHCHAHB and CHCHAHBCH); 13C NMR (150 MHz,
CDCl3):
d
¼ 175.8, 138.2, 136.0, 70.2, 70.1, 70.0, 47.2, 46.3, 44.6, 41.6,
4.12. Norbornene NHS derivative 21
39.3, 30.5; HRMS (ESI): [MþH]þ, found 269.1867. C14H25N2Oþ3 re-
quires 269.1865.
Exo-5-norbornenecarboxylic acid (455 mg, 3.30 mmol), N-
hydroxysuccinimide (357 mg, 3.40 mmol), and N,N0-dicyclohex-
ylcarbodiimide (679 mg, 3.30 mmol) were dissolved in anhydrous
THF (15 mL) and stirred at room temperature overnight, over which
time a white precipitate formed (N,N0-dicyclohexylurea). The pre-
cipitate was removed by filtration and washed with THF and the
filtrate was collected and concentrated under reduced pressure.
The crude solid was purified by column chromatography (40%
EtOAc in hexane) to give the product 21 as a white solid (537 mg,
69%). m.p. 84e86 ꢂC; Rf (40% EtOAc:hexane) 0.37; IR (nmax, ATR):
2983, 2949,1735,1428, 1361, 1200,1047, 947, 841, 711, 644 cmꢀ1; 1H
4.15. Biotin NHS ester 26
Biotin (498 mg, 2 mmol) was dissolved in anhydrous DMF
(10 mL) by heating to approximately 70 ꢂC for 10 min or until fully
dissolved. The reaction mixture was allowed to cool to room tem-
perature before adding N-hydroxysuccinimide (240 mg, 2.1 mmol)
with stirring at room temperature. A solution of N,N0-dicyclohex-
ylcarbodiimide (438 mg, 2.13 mmol) in anhydrous DMF (2 mL) was
added dropwise to the stirred solution. The reaction was then
stirred overnight at room temperature over which time a white
precipitate formed (N,N0-dicyclohexylurea). The precipitate was
removed by filtration and washed with DMF. The filtrate was
diluted with EtO2 until a white precipitate formed. The precipitate
was collected by filtration and rinsed with EtO2 then dried to give
NMR (600 MHz, CDCl3):
d
¼ 6.20 (1H, dd, J ¼ 5.7, 3.0 Hz,
CH2CHCH¼CH), 6.15 (1H, dd, J ¼ 5.7, 3.0 Hz, CHCH¼CH), 3.28 (1H,
m, CH¼CHCHCH), 3.00 (1H, m, CH¼CHCHCH2), 2.83 (4H, m,
COCH2CH2CO), 2.51 (1H, m, CHCHAHB), 2.05 (1H, m, COCHCHAHB),
Please cite this article in press as: Alcock LJ, et al., Norbornene probes for the study of cysteine oxidation, Tetrahedron (2017), https://doi.org/
10.1016/j.tet.2017.11.011