The Journal of Organic Chemistry
Article
General Procedure for Bioconjugation Reactions with
Glutathione (Table 4). To a 1.5 dram vial were added 0.625
mmol olefin, 0.25 mmol thiol, 0.63 μmol Ru(bpz)3(PF6)2, 0.125 mmol
p-toluidine and 1.25 mL Millipore water. The vial was sealed with a
Teflon cap and irradiated with blue LEDs. Unless otherwise indicated,
the crude reaction mixture was loaded directly onto a methanol-
charged Sephadex column and eluted with methanol at a flow rate of 1
mL/min. Fractions expected to contain the desired product were
(0.177 mmol, 71% yield). Spectral data were identical in all respects to
previously reported values.34
(2S)-2-Amino-5-(((2R)-1-((carboxymethyl)amino)-1-oxo-3-
((3-((4-((4S)-2-oxohexahydro-1H-thieno[3,4-d]imidazol-4-yl)-
butanoyl)oxy)propyl)thio)propan-2-yl)amino)-5-oxopentanoic
Acid (Table 4, Entry 4). To a 1.5 dram vial were added 171.2 mg
(0.602 mmol) allyl biotin,35 76.6 mg glutathione (0.250 mmol), 0.4
mg (0.462 μmol) Ru(bpz)3(PF6)2, 13.4 mg (0.125 mmol) p-toluidine,
and 1.25 mL Millipore water. The vial was sealed with a Teflon cap
and irradiated with blue LEDs for 2 h. Over the course of the reaction
a white solid crashed out of solution. This solid was isolated via
filtration; the solid was washed with water and dried in vacuo to give
analytically pure product. Obtained 63.8 mg (0.148 mmol, 59% yield)
of white solid. Experiment 2: 78.8 mg (0.257 mmol) glutathione, 174.9
mg (0.615 mmol) allyl biotin, 0.4 mg (0.462 μmol) Ru(bpz)3(PF6)2,
14.4 mg p-toluidine (0.134 mmol) and 1.25 mL water. Isolated 64.9
mg (0.150 mmol, 60% yield). IR (solution in DMF) 2325, 2089 cm−1;
1H NMR (500 MHz, DMSO-d6) δ 8.64 (t, J = 5.2 Hz, 2H), 8.42 (d, J
= 8.5 Hz, 1H), 4.42 (dt, J = 9.0, 4.5 Hz, 2H), 3.76−3.61 (m, 4H), 3.33
(dt, J = 13.8, 6.5 Hz, 4H), 2.88 (dd, J = 13.6, 4.3 Hz, 1H), 2.62 (dd, J =
13.5, 9.7 Hz, 1H), 2.55−2.44 (m, 8H), 2.33 (dddd, J = 15.5, 7.5 Hz,
3H), 1.90 (ddp, J = 20.8, 13.8, 7.3 Hz, 4H), 1.57−1.42 (m, 7H), 1.42−
1.24 (m, 6H); 13C NMR (126 MHz, DMSO) δ 172.2, 171.4, 171.2,
170.9, 53.6, 53.0, 51.0, 41.7, 33.9, 31.9, 31.6, 29.3, 28.6, 28.1, 27.3,
26.2; HRMS (ESI) calcd for [C16H28N6O6S]+ requires m/z 433.1864,
found m/z 433.1861.
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concentrated in vacuo and analyzed by H NMR in D2O. Fractions
containing >90% pure product were combined and used for calculating
the yield.
( S ) - 2 - A m i n o - 5 - ( ( ( R ) - 3 - ( ( 6 - a z i d o h e x y l ) t h i o ) - 1 -
((carboxymethyl)amino)-1-oxopropan-2-yl)amino)-5-oxopen-
tanoic Acid (Table 4, Entry 1). To a 1.5 dram vial were added 75.4
mg (0.602 mmol) azide,31 76.6 mg glutathione (0.250 mmol), 0.4 mg
(0.462 μmol) Ru(bpz)3(PF6)2, 13.4 mg (0.125 mmol) p-toluidine, and
1.25 mL Millipore water. The vial was sealed with a Teflon cap and
irradiated with blue LEDs for 2 h. Over the course of the reaction a
white solid crashed out of solution. This solid was isolated via
filtration; the solid was washed with water and dried in vacuo to give
analytically pure product. Obtained 63.8 mg (0.148 mmol, 59% yield)
of white solid. Experiment 2: 78.8 mg (0.257 mmol) glutathione, 77.1
mg (0.615 mmol) azide, 0.4 mg (0.462 μmol) Ru(bpz)3(PF6)2, 14.4
mg p-toluidine (0.134 mmol) and 1.25 mL water. Isolated 64.9 mg
(0.150 mmol, 60% yield). IR (ATR) 3348, 3042, 2856, 2093, 1673,
1
1645, 1516 cm−1; H NMR (500 MHz, DMSO-d6) δ 8.64 (t, J = 5.2
Hz, 1H), 8.42 (d, J = 8.5 Hz, 1H), 4.42 (dt, J = 9.0, 4.5 Hz, 1H), 3.76−
3.61 (m, 2H), 3.33 (dt, J = 13.8, 6.5 Hz, 1H and H2O), 2.88 (dd, J =
13.6, 4.3 Hz, 1H), 2.62 (dd, J = 13.5, 9.7 Hz, 1H), 2.50 (m, 4H, and
DMSO) 2.33 (dddd, J = 15.5, 7.5 Hz, 2H), 1.90 (ddp, J = 20.8, 13.8,
7.3 Hz, 2H), 1.57−1.42 (m, 4H), 1.42−1.24 (m, 4H); 13C NMR (126
MHz, DMSO) δ 172.2, 171.4, 171.2, 170.9, 53.6, 53.0, 51.0, 41.7, 33.9,
31.9, 31.6, 29.3, 28.6, 28.1, 27.3, 26.2; HRMS (ESI) calcd for
[C16H28N6O6S]+ requires m/z 433.1864, found m/z 433.1861.
(18R,23S)-23-Amino-18-((carboxymethyl)carbamoyl)-1-hy-
droxy-20-oxo-3,6,9,12-tetraoxa-16-thia-19-azatetracosan-24-
oic Acid (Table 4, Entry 2). To a 1.5 dram vial were added 137.4 mg
(0.587 mmol) allyl(tetra)ethylene glycol,32 77.9 mg glutathione (0.253
mmol), 0.4 mg (0.462 μmol) Ru(bpz)3(PF6)2, 13.7 mg (0.128 mmol)
p-toluidine, and 1.25 mL Millipore water. The vial was sealed with a
Teflon cap and irradiated with blue LEDs for 2 h. Upon completion of
the reaction, the mixture was directly loaded onto a column of
Sephadex LH-20 for purification (100% methanol). Obtained 96.6 mg
(0.178 mmol, 71% yield) of white solid. Experiment 2: 143.1 mg
(0.611 mmol) allyl(tetra)ethylene glycol, 76.4 mg glutathione (0.249
mmol), 0.4 mg (0.462 μmol) Ru(bpz)3(PF6)2, 13.3 mg (0.124 mmol)
p-toluidine, and 1.25 mL Millipore water. Isolated 98.1 mg (0.181
mmol, 71% yield). IR (ATR) 2667, 1639, 1539, 1449, 1402 cm−1;1H
NMR (500 MHz, D2O) δ 4.46 (dd, J = 8.7, 5.1 Hz, 1H), 3.86 (s, 2H),
3.71 (t, J = 6.3 Hz, 1H), 3.65−3.49 (m, 18H), 2.96 (dd, J = 14.1, 5.1
Hz, 1H), 2.78 (dd, J = 14.1, 8.8 Hz, 1H), 2.55 (tdd, J = 7.2, 2.4, 1.2 Hz,
2H), 2.48−2.38 (m, 2H), 2.06 (q, J = 7.1 Hz, 2H), 1.76 (p, J = 6.7 Hz,
2H); 13C NMR (126 MHz, D2O) δ 174.7, 173.5, 173.5, 172.7, 71.7,
69.6, 69.5, 69.5, 69.5, 69.40, 69.2, 60.3, 53.7, 53.1, 41.5, 32.7, 31.1,
28.4, 28.0, 26.0 (one aliphatic carbon signal is absent due to accidental
equivalence); HRMS (ESI) calcd for [C21H39N3O11S]+ requires m/z
542.2379, found m/z 542.2386.
(S)-2-Amino-5-(((R)-1-((carboxymethyl)amino)-1-oxo-3-((3-
((2R,3S,4R,5S,6R)-3,4,5-triacetoxy-6-(acetoxymethyl)-
tetrahydro-2H-pyran-2-yl)propyl)thio)propan-2-yl)amino)-5-
oxopentanoic Acid (Table 4, Entry 3). To a 1.5 dram vial were
added 232.5 mg (0.624 mmol) allylated tetraacetate galactose,33 76.6
mg glutathione (0.250 mmol), 0.4 mg (0.462 μmol) Ru(bpz)3(PF6)2,
12.8 mg (0.120 mmol) p-toluidine, and 1.25 mL Millipore water. The
vial was sealed with a Teflon cap and irradiated with blue LEDs for 18
h. Upon completion of the reaction, the mixture was directly loaded
onto a column of Sephadex LH-20 for purification (100% methanol).
Obtained 119.0 mg (0.175 mmol, 70% yield) of white solid.
Experiment 2: 78.8 mg (0.257 mmol) glutathione, 231.1 mg (0.624
mmol) allylated galactose, 0.4 mg (0.462 μmol) Ru(bpz)3(PF6)2, 14.4
mg p-toluidine (0.134 mmol), and 1.25 mL water. Isolated 120.2 mg
(2S,11R,16S)-16-Amino-11-((carboxymethyl)carbamoyl)-2-
isopropyl-4,13-dioxo-5-oxa-9-thia-3,12-diazaheptadecane-
1,17-dioic Acid (Table 4, Entry 5). To a 1.5 dram vial were added
124.4 mg (0.618 mmol) Alloc-Val-OH, 77.0 mg glutathione (0.251
mmol), 0.4 mg (0.462 μmol) Ru(bpz)3(PF6)2, 12.7 mg (0.118 mmol)
p-toluidine, and 1.25 mL Millipore water. The vial was sealed with a
Teflon cap and irradiated with blue LEDs for 2 h. Upon completion of
the reaction, the mixture was directly loaded onto a column of
Sephadex LH-20 for purification (100% methanol). Obtained 105.8
mg (0.208 mmol, 83% yield) of white solid. Experiment 2: 123.6 mg
(0.614 mmol) Alloc-Val-OH, 77.0 mg glutathione (0.251 mmol), 0.4
mg (0.462 μmol) Ru(bpz)3(PF6)2, 13.0 mg (0.121 mmol) p-toluidine
and 1.25 mL Millipore water. Isolated 99.2 mg (0.195 mmol, 78%
1
yield). IR (ATR) 3301, 3076, 2925, 1701, 1654.cm−1; H NMR (500
MHz, D2O) δ 4.47 (dd, J = 8.5, 5.2 Hz, 1H), 4.05 (t, J = 5.8 Hz, 2H),
3.94 (d, J = 5.3 Hz, 1H), 3.88 (s, 2H), 3.74 (t, J = 6.3 Hz, 1H), 2.96
(dd, J = 14.1, 5.0 Hz, 1H), 2.78 (dd, J = 14.0, 8.8 Hz, 1H), 2.60−2.51
(m, 1H), 2.43 (m, 2H), 2.07 (q, J = 7.3 Hz, 3H), 1.82 (m, 2H), 0.85
(dd, J = 22.8, 6.8 Hz, 6H); 13C NMR (126 MHz, D2O) δ 176.4, 174.7,
173.3, 173.3, 172.7, 158.5, 64.0, 59.9, 53.6, 53.0, 41.3, 32.7, 31.1, 29.8,
28.1, 27.9, 25.9, 18.4, 16.9; HRMS (ESI) calcd for [C19H32N4O10S]+
requires m/z 509.1912, found m/z 509.1936.
( 2 S , 1 1 R , 1 6 S ) - 1 6 - A m i n o - 2 - ( 4 - a m i n o b u t y l ) - 1 1 -
((carboxymethyl)carbamoyl)-4,13-dioxo-5-oxa-9-thia-3,12-dia-
zaheptadecane-1,17-dioic Acid, TFA Salt (Table 4, Entry 6). To
a 1.5 dram vial were added 77.1 mg (0.251 mmol) glutathione, 215.0
mg (0.625 mmol) Alloc-Lys-OH.TFA salt, 0.5 mg (0.63 μmol)
Ru(bpz)3(PF6)2, 13.4 mg (0.125 mmol) p-toluidine,and 1.25 mL
Millipore water. The vial was sealed with a Teflon cap and irradiated
with blue LEDs for 2 h. The reaction mixture was then loaded directly
onto a Sephadex column and eluted with MeOH to afford the product
as a yellow solid (155.4 mg, 0.239 mmol, 96%). Experiment 2: 77.6 mg
(0.253 mmol) glutathione, 215 mg (0.625 mmol) Alloc-Lys-OH.TFA
salt, 0.5 mg (0.63 μmol) Ru(bpz)3(PF6)2, 13.4 mg (0.125 mmol) p-
toluidine, and 1.25 mL Millipore water. Obtained 162.6 mg product
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(0.250 mmol, 99%). IR (ATR) 3088, 2952, 1697, 1529 cm−1; H
NMR (500 MHz, D2O) δ 4.55 (dd, J = 8.5, 5.2 Hz, 1H), 4.18−4.06
(m, 3H), 3.98 (s, 2H), 3.87 (t, J = 6.4 Hz, 1H), 3.04 (dd, J = 14.1, 5.1
Hz, 1H), 2.98 (t, J = 7.6 Hz, 2H), 2.86 (dd, J = 14.0, 8.7 Hz, 1H), 2.64
(t, J = 5.9 Hz, 2H), 2.61−2.45 (m, 2H), 2.17 (q, J = 7.4 Hz, 2H),
1.95−1.82 (m, 3H), 1.79−1.59 (m, 3H), 1.53−1.38 (m, 2H); 13C
NMR (126 MHz, D2O) δ 176.7, 174.7, 173.2, 173.1, 172.9, 163.0 (q,
1
2JCF = 35.4 Hz, TFA), 158.4, 116.4 (q, JCF = 291.7 Hz, TFA), 64.1,
54.0, 53.4, 53.1, 41.3, 39.2, 32.8, 31.1, 30.2, 28.2, 27.9, 26.2, 25.9, 22.1;
H
dx.doi.org/10.1021/jo500031g | J. Org. Chem. XXXX, XXX, XXX−XXX