Neocarzinostatin (holo-NCS) is an antitumor antibiotic comprising a nonprotein chromophore component (1) and a 113-

amino acid carrier protein (apo-NCS). Goldberg and coworkers

first demonstrated that the reaction of the isolated

chromophore (1) with thiols in the presence of double-stranded DNA leads to DNA cleavage by a free-radical mechanism. The pathway shown in Scheme 1 was later proposed to account for this activity, a proposal that is now supported by a considerable body of evidence. In 1992, Saito and co-workers showed that the reaction of holo-NCS with small thiols, such as β-mercaptoethanol (BME), takes a different course, to form a product that is formally a 1:1:1 adduct of thiol, 1, and water. Structure 2 was proposed for this adduct, along with the mechanistic pathway shown in Scheme 2. Complicating the analysis was the fact that 2 was an inseparable mixture of two components, present in equal parts

Arvedson, Stephen P.

Lee, Robert W.

Myers, Andrew G.

Caltech Authors - Main

J. Am. Chem. Soc., 1996, 118(19), 4725-4726, DOI:10.1021/ja960335+
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Copyright © 1996 American Chemical Society
0 1996 American Chemical Society J. Am. Chem. Soc. VI 18 Page4725 Myers Supplemental Page 1
Preparation of Hydroxyisochromene 6
9 O
10
b OH
11
0
00 0
OH 13
4
0o
0
5 H
iC 
0
co&o--
12
)0
OKt
9H
C6O
4(a) C2H3MgBr, THF (60%). (b) OsO4, NMO, THF/H 2 0 (68%). (c) OzCO, Et3 N, CH2CI2, 0 *C (85%).
(d) Martin Sulfurane, CH2CI2, O C (86%). (e) OsO4. NMO, THF/H20 (67%). (f) NaIO 4 , THF/H20, 0 "C(89%). (g) HOAc, DMSO-d6 (92%).
-7,,I(,-l
0 1996 American Chemical Society J. Am. Chem. Soc. V1 18 Page4725 Myers Supplemental Page 2
Experimental Section: j
General Procedures. All reactions were performed in flame dried flasks under argon unless
otherwise noted. All commercial reagents were used as received with the following exceptions.
Tetrahydrofuran and ethyl ether were distilled from sodium benzophenone ketyl. Dichloromethane and
triethylamine were distilled from calcium hydride. Neocarzinostatin powder was obtained from Kayaku Ltd.
(Japan), and was used as received. Infrared spectra were obtained using a Perkin-Elmer 1600 FT-IR
spectrophotometer referenced to a polystyrene standard. Samples were analyzed as thin films on sodium
chloride plates. Absorptions are reported in wavenumbers (cm-') and intensities are designated as broad (b),
strong (s), medium (m), or weak (w). Proton magnetic resonance spectra ('H NMR), carbon magnetic
resonance spectra (13C NMR), and the DEPT-90 spectrum were recorded on a JEOL JX-400 (400 MHz)
spectrometer. The gradient-enhanced inverse detected HETCOR spectrum was recorded on a Bruker AMX-
500 (500 MHz) spectrometer. Chemical shifts are measured in parts per million (ppm) relative to
tetramethylsilane (TMS) and are referenced to residual protium in the NMR solvent. Coupling constants (J
values) are in hertz (Hz). Multiplicities are designated as singlet (s), doublet (d), triplet (t), quartet (q), or
multiplet (m). Peaks due to an additional diastereomer in a mixture are designated by a *. Melting points were
determined on a Buchi SMP-20, are reported in oC, and are uncorrected. Analytical thin layer chromatography
(TLC) was performed on Merck silica gel (60F-254) plates (0.25 mm) impregnated with a fluorescent indicator
(254 nm). Visualization was effected with ultraviolet light and immersion in an acidic staining solution of p-
anisaldehyde followed by heating on a hot plate. Flash column chromatography was performed as described
by Still et al., employing E. Merck silica gel 60 (240-400 mesh). Reverse phase high performance liquid
chromatography (HPLC) was performed using a Beckman SystemGold and Beckman Ultrasphere ODS C18
columns.
Isolation of NCS cyclization product (2,3). 2-mercaptoethanol (50 AL, 0.71 mmol, 68.6
equiv) was added to a solution of Neocarzinostatin (111.2 mg, 10.4 pmol, 1 equiv) and 2,5-dimethoxybenzyl
alcohol (10.0 gmol, 1.0 equiv1 internal standard for HPLC) in aqueous TRIS buffer (25 mM, pH 7.5, 10 mL).
The resulting solution was vortexed briefly and was allowed to stand in a cold room (4 C) for 14 hr. The
reaction was purified in 10 1.0-mL portions by reverse phase HPLC (Beckman Ultrasphere ODS C18) using a
gradient of 20% acetonitrile in aqueous ammonium acetate (pH 4.0) to 50% acetonitrile in aqueous ammonium
acetate (pH 4.0) over 40 min. The fractions containing 3 were combined, the organic phase was removed on a
01996 American Chemical Society J. Am. Chem. Soc. VI 18 Page4725 Myers Supplemental Page 3
rotary evaporator, and the aqueous phase was removed by lyophylization to give 3 (2.0 mg, 29%) as an off
white powder:
Alcohol 10. A solution of 2-indanol (9,758.2 mg, 5.74 mmol, 1 equiv) in tetrahydrofuran (6.0 mL)
was added dropwise via pressure equalizing dropping addition funnel to a solution of vinlymagnesium bromide
(1.0 M in THF, 8.0 mL, 8.0 mmol, 1.39 equiv) over 15 min. The addition was quantitated with additional
tetrahydrofuran (2.0 mL), and was stirred at 23 'C for 15 min. The reaction was cooled to 0 T and a saturated
aqueous solution of ammonium chloride (3.0 mL) was added dropwise. The resulting biphasic, heterogeneous
mixture was partitioned between water (40 mL) and ethyl ether (3 x 35 mL). The combined organic layers
were dried over sodium sulfate, were filtered, and were concentrated. The residue was purified by flash
column chromatography (17% ethyl acetate in hexanes) to give alcohol 10 (552.6 mg, 60%) as a white solid:
mp 49.5-50.5 'C; R,0.24 (17% ethyl acetate in hexanes); 'H NMR (400 MHz, CDCl3) 8 7.16-7.25 (in, 4H),
6.17 (dd, I, J=17.2, 10.6), 5.41 (dd, 1H, J=17.2, 1.1), 5.15 (dd, 1H, J=10.6, 1.1), 3.19 (d, 1H, J=16.5
), 2.98 (d, 1H, J=16.1), 1.87 (s, 1H); "C NMR (100 MHz, CDClI) 8 142.8, 141.0, 126.7, 124.9, 111.5,
82.2, 47.4; IR 3530 (b), 3380 (b), 3071 (in), 3022 (in), 2944 (in), 2901 (in), 2836 (in), 1749 (w), 1642 (w),
1586 (w), 1483 (s), 1460 (s), 1412 (s), 1302 (w), 1278 (m), 1247 (s), 1212 (s), 1104 (s), 1042 (s), 1023
(m), 1002 (s), 922 (s), 896 (m), 740 (s), 676 (w) cm-'.
Triol 11. 4-Methylmorpholine N-oxide (816.2 mg, 6.97 mmol, 2.1 equiv) and osmium tetroxide
(4% w/w in H20, 1.0 mL, 0.16 mmol, 4.8 mol %) were added to a solution of allylic alcohol 10 (529.8 mg,
3.31 mmol, 1 equiv) in a mixture of tetrahydrofuran and water (5:1, respectively, 40 mL), and the resulting
brown solution was stirred at 23 C for 21 hr. The volatiles were removed on a rotary evaporator, an aqueous
solution of sodium bisulfite (1.0 M, 50 mL) was added, and the resulting brown suspension was stirred at 23
' for 20 min. The suspension was added to water (25 mL), and the mixture was saturated with sodium
chloride. The aqueous phase was extracted with ethyl acetate (8 x 75 mL). The combined organic layers were
dried over sodium sulfate, were filtered, and were concentrated. The solid residue was triturated with
dichloromethane to give triol 11 (437.9 mg, 68%) as a beige solid: mp 161.0-162.0 oC; Rf 0.07 (67% ethyl
acetate in hexanes), 'H NMR (400 MHz, DMSO-d) 8 7.13-7.17 (in, 2H), 7.06-7.10 (in, 2H), 4.73 (d, 1H,
J=5.2), 4.51 (s, 1H), 4.42 (t, 1H, J=5.5), 3.68 (ddd, 1H, J-9.5, 5.9, 3.3), 3.51 (ddd, 1H, J=7.7, 5.2, 3.3),
3.38 (ddd, 1H, J=11.4, 7.4, 5.2), 3.20 (d, 1H, J=16.1), 3.10 (d, 1H, J=16.1), 2.65 (d, 1H, J=16.1); ' 3C
NMR (100 MHz, DMSO-d6 ) d 142.0, 141.9, 125.8, 125.8, 124.6, 124.5, 82.8, 75.7, 62.8, 43.8, 42.1; IR
0 1996 American Chemical Society J. Am. Chem. Soc. VI 18 Page4725 Myers Supplemental Page 4
3325 (b), 2932 (m), 2896 (m), 1487 (in), 1455 (m), 1434 (in), 1414 (in), 1226 (w), 1117 (m), 1092 (in),
1067 (s), 1038 (s), 996 (m), 876 (in), 818 (m), 742 (s) cm-n.
Carbonate 12. Phosgene (20% w/w in H.0, 1.7 mL, 3.28 mmol, 1.5 equiv) was added dropwise
over 7 min to a solution of triol 11 (431.5 mg, 2.22 mmol, I equiv) and triethylamine (3.1 mL, 22.2 mmol,
10.0 equiv) in dichloromethane (30 mL) at 0 oC. The resulting solution was stirred at 0 C for 30 min. The
reaction was partitioned between a saturated aqueous solution of sodium bicarbonate (35 mL) and ethyl acetate
(4 x 40 mL). The combined organic layers were dried over sodium sulfate, were filtered, and were
concentrated. The residue was purified by flash column chromatography (40% ethyl acetate in hexanes) to give
carbonate 12 (413.5 mg, 85%) as a white solid, along with small amounts of olefin 13 (29.8 mg, 7%).
Carbonate 12 was recrystallized from a mixture of hexane and ethyl acetate (2:1, respectively): mp 162-163 'C;
RfO.55 (25% hexanes in ethyl acetate); 'H NMR (400 MHz, DMSO-d.) 8 7.13-7.23 (m, 4H), 5.45 (s, 1H),
4.83 (dd, 1H, J=8.4, 5.8), 4.57 (dd, 1H, J=8.4, 8.4), 4.43 (dd, 1H, J=8.4, 5.8), 3.11 (d, 1H, J=16.5), 2.96
(d, IH, J=16.4), 2.92 (d, 1H, J=14.7), 2.81 (d, 1H, J=16.4); "C NMR (100 MHz, DMSO-d) 8 155.0,
140.7, 140.3, 126.4, 126.4, 124.7, 124.7, 80.4, 80.0, 65.7, 43.1, 42.0; IR 3439 (b), 3025 (w), 2908 (w),
1781 (s), 1482 (m), 1399 (m), 1328 (m), 1255 (m), 1127 (s), 1088 (s), 1073 (s), 1020 (m), 979 (m), 773
(m), 745 (s) cm-'.
Vinyl carbonate 13. A solution of the Martin sulfurane dehydrating agent (1.12 g, 1.67 mmol, 1.3
equiv) in dichloromethane (10 mL) was added via canula to a solution of carbonate alcohol 12 (289.9 mg, 1.32
mmol, 1 equiv) in dichloromethane (10 mL) at 0 oC. The resulting yellow solution was stirred at 0 C for 45
min. The reaction mixture was concentrated in vacuo, and the residue was purified by flash column
chromatography (dichloromethane) to give indene 13 (229.9 mg, 86%) as a white solid: mp 98-99 aC- Rf 0.44
(50% ethyl acetate in hexanes); 'H NMR (400 MHz, CDCl3) 8 7.48 (d, 1H, J=7.3), 7.41 (d, 1H, J=7.3), 7.31
(ddd, 1H, J=7.3, 7.3, 1.5), 7.26 (ddd, 1H, J=7.3, 7.3, 1.5), 6.98 (s, 1H), 5.68 (dd, IH, J=8.1, 7.7), 4.71
(dd, IH, J=8.8, 8.1), 4.40 (dd, 1H, J=8.8, 7.4), 3.50 (s, 2H); 13C NMR (100 MHz, CDCl3) 8 154.7, 143.0,
142.8, 141.1, 132.3, 126.9, 126.1, 124.1, 121.8, 75.3, 69.0, 37.0; IR 3072 (w), 3025 (w), 2978 (w), 2923
(w), 1790 (s), 1556 (w), 1480 (w), 1462 (m), 1391 (s), 1360 (m), 1334 (in), 1308 (w), 1218 (w), 1169 (s),
1073 (s), 1053 (s), 961 (w), 916 (s), 864 (s), 778 (s), 754 (s), 717 (s), 552 (in) cm-'.
Diol carbonate 4. 4-Methylmorpholine N-oxide (227.6 mg, 1.94 mmol, 2.1 equiv) and osmium
tetroxide (4% w/w in H2 0, 0.3 mL, 0.048 mmol, 5.1 mol %) were added to a solution of indene 13 (188.9
0 1996 American Chemical Society J. Am. Chem. Soc. VI 18 Page4725 Myers Supplemental Page 5
mg, 0.93 mmol, 1 equiv) in a mixture of tetrahydrofuran and water (5:1, respectively, 12 mL), and the
resulting pale yellow solution was stirred at 23 oC for 15 hr. An aqueous solution of sodium sulfite buffer (pH
7, 0.5 M in sodium bisulfite and 1.0 M in sodium sulfite, 10 mL) was added, and the resulting brown biphasic
mixture was stirred vigorously at 23 oC for 35 min.. The mixture was added to a saturated aqueous solution of
sodium chloride (20 mL), and was extracted with ethyl acetate (4 x 40 mL). The combined organic layers were
dried over sodium sulfate, were filtered, and were concentrated. The residue was purified by flash column
chromatography (45% hexanes in ethyl acetate) to give diol carbonate 4 (147.5 mg, 67%, 1.2:1 mixture of
diastereomers) as a white solid: mp 138-144 'C; Rf0.2 0 (50% ethyl acetate in hexanes); 'H NMR (400 MHz,
DMSO-d6 ) 8 7.18-7.35 (m, 8H), 5.74 (d, 1H, J=6.9 ), 5.57* (d, 1H, J=8.1 Hz), 5.07 (s, 1H), 4.96* (s,
1H), 4.88-4.94 (m, 2H), 4.77-4.81 (m, 2H), 4.64 (dd, 1H, J=8.0, 5.9), 4.50-4.59 (in, SH), 2.98 (d, 1H,
J= 16.4), 2.93 (d, 1H, J=16.5), 2.85* (d, 1H, J=16.1), 2.8 1* (d, IH, J=16.4); C NMR (100 MHz, DMSO-
d,) 8 155.6, 143.9, 139.2, 128.6, 128.3*, 127.3, 127.1*, 125.5, 125.4*, 125.4, 125.3*, 125.0, 81.1,
80.7*, 80.3, 80.0*, 76.1, 66.2, 65.9*; IR 3448 (b), 3049 (w), 2931 (w), 1785 (s), 1479 (m), 1461 (w),
1399 (m), 1328 (m), 1302 (w), 1182 (s), 1078 (s), 971 (m), 748 (s), 618 (w) cm-.
Keto aIdehyde 5 and hydroxyisochromene 6. Sodium meta-periodate (187.3 mg, 0.88 mmol,
1.3 equiv) was added to a solution of indandiol 4 (162.6 mg, 0.69 mmol, 1 equiv) in a mixture of
tetrahydrofuran and water (1:1, 10 mL) at 0 oC. The resulting solution was stirred at 0 'C for 10 min. The
reaction was partitioned between a saturated aqueous solution of sodium bicarbonate (40 mL) and ethyl acetate
(4 x 40 mL). The combined organic layers were dried over sodium sulfate, were filtered, and were
concentrated. The resulting white solid (143.5 mg, 89%) consisted almost entirely of keto aldehyde 5: Rf 0.31
(50% ethyl acetate in hexanes); 'H NMR (400 MHz, DMSO-d) 8 9.97 (s, 1H), 7.95 (dd, 1H, J=7.7, 1.4),
7.65 (ddd, IH, J=7.7, 7.3, 1.4), 7.56 (ddd, 1H, J=7.7, 7.3, 1.1), 7.36 (d, 1H,.J=7.3), 5.52 (dd, 1H, J=9.5,
5.5), 4.79 (dd, 1H, J=9.5, 8.4), 4.65 (dd, 1H, J=8.4, 5.5), 4.41 (d, 1H, J=18.0), 4.29 (d, 1H, J=17.9); '3C
NMR (100 MHz, DMSO-d) 8 202.6, 194.3, 154.3, 134.6, 134.2, 133.9, 132.8, 127.9, 77.8, 66.4, 43.0.
The crude keto aldehyde was purified by flash column chromatography (50% ethyl acetate in hexanes) to give
an equimolar mixture of 5 and isochromene 6 (95.3 mg, 59%) as a white solid: IR 3433 (b), 2924 (w), 2849
(w), 2755 (w), 1790 (s), 1732 (s), 1694 (s), 1603 (m), 1577 (m), 1486 (in), 1392 (m), 1303 (in), 1173 (s),
1072 (s), 914 (m), 700 (s), 666 (w) cm-'. Acetic acid (10 pL, 0.17 mmol, 0.4 equiv) was added to a solution
of keto aldehyde 5 and isochromene 6 (95.3 mg, 0.41 mmol, I equiv) in perdeuterodimethyl sulfoxide (0.6
0 1996 American Chemical Society J. Am. Chem. Soc. VI 18 Page4725 Myers Supplemental Page 6
mL) in an NMR tube. After 75 min, 'H NMR showed the mixture to be 12:1 6:5, where 6 exists as an
equimolar mixture of two diastereomers: R1 0.31 (50% ethyl acetate in hexanes); 'H NMR (400 MHz, DMSO-
d) 8 7.48 (d, 1H, J=5.8), 7.47* (d, 1H, J=6.2), 7.19-7.35 (m, 8H), 6.38 (d, 1H, J=6.6), 6.36* (d, 1H,
J=6.2), 6.34 (s, 1H), 6.31* (s, 1H), 5.46 (dd, lH, J=8.4, 5.5), 5.46* (dd, 1H, J=8.4, 5.5), 4.72 (dd, 1H,
J=8.4, 8.0), 4.69* (dd, 1H, J=8.4, 8.0), 4.45 (dd, 1H, J=8.0, 5.8), 4.44* (dd, 1H, J=8.0, 5.8); 13C NMR
(100 MHz, DMSO-d6) 8 154.8, 146.2, 145.6*, 129.6, 128.9, 128.2, 128.1*,', 127.6, 127.4*, 125.7, 124.4,
124.3*, 105.2, 104.0*, 92.8, 92.5*, 75.3, 75.1*, 66.6, 66.5*.
Deuterium exchange on hydroxyisochromene 6. Deuterium oxide (20 pL, 1.11 mmol, 10
equiv) was added to a solution of isochromene 6 (approx. 0.11 mmol, 1 equiv) in perdeuterodimethyl
sulfoxide (0.6 mL) that had been prepared in a drybox in an NMR tube. The resulting solution was analyzed
by 'H NMR periodically to monitor the exchange of the C3 and CS protons (NCS numbering). The CS proton
was observed to exchange completely within 5 min, whereas the C3 proton exchanged more slowly (Graph 1).
Graph 1
% Deuterium Incorporation at C3 vs Time
for 6 with D.0 in DMSO-d
60l
50
40
.30
20
10
-o5 10 15 20
time (hr)
0 1996 American Chemical Society J. Am. Chem. Soc. VI 18 Page4725 Myers Supplemental Page 7
Comparison of 13C (100 MHz) NMR Data for 3 in DMSO-4 Saito et. aL and this wokk
Assignment Saito (Tet. Lett. 1992)
1 135.5
2 136.4
3 139.7
4 132.8
5 88.2, 89.4*
6 155.0
7 118.8
8 122.2, 127.5*
9 139.0
10 823
11 845
12 50.6
13 76.1, 76.7*
14 68.7
15 1543, 154.5*
16 34.9, 35.1*
17 61.0
1' 963
2' 58.2
3' 69.0
4' 71.0
5' 66.7
6' 16.8
2' NCH3  34.0
1" 107.5
2" 161.0
3" 1153
4" 1293
4a" 121.5
5" 138.6
6" 116.5
7" 160.1
8" 100.7
8a" 136.0
5" CH3  19.4
7" OCH3  54.8
1" CO2 172.9
* Peaks due to diastereomers at CS
Saito (Tetrahedron 1994)
128.4, 129.5*
134.5, 1353*
139.2, 139.9*
132.9
88.1, 89.0*
156.2, 1563*
117.7, 117.8*
127.2, 1273*
138.2, 138.9*
82.1
84.2, 84.4*
503,50.4*
76.1, 76.6*
68.6
154.6, 154.7*
34.6,34.8*
61.0
96.2, 963*
58.1
68.9
70.8
66.8
16.8
33.9
110.9, 111.1*
168.2
1153
1293
120.1
136.8
116.6
158.4
1003
133.4
193
54.6,54.8*
172.6
This Work
127.7, 127.8*
140.2
119.7, 122.6*
133.3
88.6, 89.5*
156.6
118.1, 118.2*
127.5, 127.6*
139.6
82.5
84.7, 84.8*
50.7, 50.8*
76.5, 77.0*
69.0, 69.3*
155.0
34.6, 34.7*
61-5
96.7, 96.8*
58.6
69-5
713
67.2
17.2
34.2
111.8, 111.9*
168.5
115.7
129.6, 129.6*
122-5
137.2
117.0, 117.0*
158.7
100.9, 100.9*
133.8
19.6
55.0,55.2*
172.5
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A New and Unusual Pathway for the Reaction of Neocarzinostatin Chromophore with Thiols. Revised Structure of the Protein-Directed Thiol Adduct

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A New and Unusual Pathway for the Reaction of Neocarzinostatin Chromophore with Thiols. Revised Structure of the Protein-Directed Thiol Adduct

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