Journal of Natural Products
ARTICLE
(d, J = 6.8 Hz, 3H), 0.85 (d, J = 6.8 Hz, 3H); HRESIMS m/z 497.1640
[M þ Na]þ (calcd for C21H3579BrN2O5Na, 497.1627), m/z [M þ
Na]þ 499.1616 (calcd for C21H3581Br N2O5Na, 499.1607) (100:100
[M þ Na]þ ion cluster).
Advanced Marfey’s Analysis of β-Amino Acid Unit in
Dolastatin 12 and Dolastatin 16. Samples of dolastatin 12 and
dolastatin 16 (0.2 mg each) were hydrolyzed (200 μL of 6 N HCl,
110 °C, 20 h) to liberate the β-amino acids Map and Dml, respectively.
Another portion of dolastatin 12 and dolastatin 16 (0.2 mg each) was
first heated with 3 N NaOH (80 °C, 3 h), and the resulting product
mixture was acidified with 5% (v/v) HCl, dried under N2, and hydro-
lyzed (200 μL of 6 N HCl, 110 °C, 20 h) to liberate the partially C-2-
epimerized β-amino acid mixture. The reaction products were deriva-
tized using L-FDLA and DL-FDLA.28 The order of elution of the β-amino
acids Dml and Map liberated from dolastatin 16 and dolastatin 12,
respectively, was determined by reversed-phase HPLC-MS [column,
Phenomenex Synergi Hydro-RP (150 ꢁ 4.6 mm) using a linear gradient
of 0.1% HCOOH in MeOHꢀ 0.1% aqueous HCOOH (25ꢀ80% for
50 min and then 100% MeOH for 5 min); flow rate, 0.5 mL/min;
detection by ESIMS in negative ion mode (MRM scan)] of the FDLA
adducts. The enantiomers bearing the 3R configuration consistently
eluted later than those with the 3S configuration. The retention times
(tR, min; MRM ion pair) of the Dml-L-FDLA adducts were as follows:
(2S,3S)-Dml [(29.3; 438.2f310.3); (= D-FDLA-derivatized (2R,3R)-
Dml)], (2R,3S)-Dml [(30.8); (= D-FDLA-derivatized (2S,3R)-Dml)],
(2R,3R)-Dml (33.7), (2S,3R)-Dml (35.6). The retention times (tR, min;
MRM ion pair) of the Map-L-FDLA adducts were as follows: (2R,3S)-
Map [(27.5; 424.5f310.4); (= D-FDLA-derivatized (2S,3R)-Map)],
(2S,3S)-
Map [(29.6); (= D-FDLA-derivatized (2R,3R)-Dml)], (2R,3R)-Dml
(31.0), (2S,3R)-Dml (32.5). Compound-dependent parameters used were
as follows: DP ꢀ95.0, EP ꢀ6.0, CE ꢀ31.0, CXP ꢀ2.0, CEP ꢀ26.0.
Source gas parameters used were as follows: CUR 40, CAD Medium, IS
4500, TEM 450, GS1 40, GS2 40.
Base Hydrolysis of Dolastatin 16. Dolastatin 16 (4.0 mg) was
hydrolyzed using 2 N KOHꢀMeOH (1:1) for 3 h at 80 °C. The base
hydrolysate was neutralized with 2 N HCl and partitioned between
EtOAc and H2O, and the organic layer was collected and dried under N2.
The crude base hydrolysate product was purified by semipreparative
HPLC (Phenomenex Synergi Hydro-RP, 250 ꢁ 10 mm, 4 μm; flow rate,
2.0 mL/min) using a linear gradient of ACNꢀH2O (40ꢀ100% MeOH
in 45 min) to yield 12 (tR 22.7 min, 1.0 mg).
Preparation of MTPA Esters. The methanolysis product 9 was
dissolved in 50 μL of CDCl3 and was divided into two equal portions; to
each was added 0.75 mL of triethylamine. To one portion was added 10
μL of (R)-MTPA-Cl, and to the other was added 10 μL of (S)-MTPA-Cl
to give the (S)-MTPA ester (10) and (R)-MTPA ester (11), respec-
tively. Each reaction was allowed to stir for 24 h, and 10 μL of N,N-
dimethylaminopropylamine was added to quench the reactions. The
reaction products were dried under N2 and applied onto silica SPE
eluting with EtOAcꢀhexanes (1:1). The semipure product was further
purified by semipreparative HPLC (Phenomenex Phenyl-Hexyl, 250 ꢁ
10 mm, 4 μm; flow rate, 2.0 mL/min) using a linear gradient of
MeOHꢀH2O (70ꢀ100% MeOH in 45 min and then 100% MeOH
for 10 min) to yield 10 (tR 38.0 min, 0.1 mg) or 11 (tR 37.8 min, 0.1 mg).
10: colorless, amorphous solid; 1H NMR (CDCl3) δ 7.57 (dd, J = 6.4,
2.7 Hz, 2H), 7.41 (m, 3H), 6.14 (d, J = 9.2 Hz, 1H), 5.28 (q, J = 6.9 Hz,
1H), 4.93 (d, J = 10.6 Hz, 1H), 4.68 (dd, J = 9.1, 7.6 Hz, 1H), 3.69 (s,
3H), 3.58 (s, 3H), 3.06 (s, 3H), 2.46 (quintet, J = 7.1 Hz, 1H), 2.21 (m,
1H), 2.15 (t, J = 6.8 Hz, 2H), 1.95 (m, 1H), 1.68 (m, 1H), 1.47 (m, 2H),
1.09 (d, J = 7.0 Hz, 3H), 1.01 (d, J = 6.6 Hz, 3H), 0.92 (d, J = 6.9 Hz, 3H),
0.86 (d, J = 6.9 Hz, 3H), 0.81 (d, J = 6.9 Hz, 3H); HRESIMS m/z
729.1746 [M þ K]þ (calcd for C31H4279BrF3N2O7K, 729.1759), m/z
[M þ K]þ 731.1747 (calcd for C31H4281BrF3N2O7K, 731.1755)
(100:100 [M þ K]þ ion cluster); LRESIMS m/z 691/693 (100:100
[M þ H]þ ion cluster), 713/715 (100:100 [M þ Na]þ ion cluster).
11: colorless, amorphous solid; 1H NMR (CDCl3) δ 7.56 (dd, J = 4.3,
3.6 Hz, 2H), 7.41 (m, 3H), 6.27 (d, J = 8.3 Hz, 1H), 5.27 (q, J = 6.0 Hz,
1H), 4.94 (d, J = 10.4 Hz, 1H), 4.73 (dd, J = 9.1, 7.5 Hz, 1H), 3.69 (s,
3H), 3.57 (s, 3H), 3.07 (s, 3H), 2.54 (quintet, J = 6.7 Hz, 1H), 2.22 (m,
1H), 2.08 (td, J = 7.2, 2.9 Hz, 2H), 2.00 (m, 1H), 1.63 (m, 1H), 1.31 (m,
2H), 1.19 (d, J = 6.7 Hz, 3H), 1.01 (d, J = 6.3 Hz, 3H), 0.95 (d, J = 6.8 Hz,
3H), 0.90 (d, J = 7.0 Hz, 3H), 0.83 (d, J = 6.5 Hz, 3H); HRESI/APCIMS
m/z 691.2202 [M þ H]þ (calcd for C31H4379BrF3N2O7, 691.2206), m/
z [M þ H]þ 693.2186 (calcd for C31H4381BrF3N2O7, 693.2186)
(100:100 [M þ H]þ ion cluster).
12: colorless, amorphous solid; 1H NMR (CDCl3) δ 7.13ꢀ7.26 (m),
6.81 (d, J = 9.0 Hz, 1H), 6.65 (d, J = 10.4 Hz, 1H), 4.83 (d, J = 10.6 Hz,
1H), 4.73 (dd, J = 8.7, 3.8 Hz, 1H), 4.59 (m, 2H), 4.48 (m, 1H), 4.28 (m,
1H), 3.79 (m, 1H), 3.56ꢀ3.69 (m, 2H), 3.50 (m, 1H), 3.36 (m, 1H),
3.16 (s, 3H), 2.70 (m, 2H), 2.24ꢀ2.48 (m, 6H), 1.94ꢀ2.19 (m, 2H),
1.77ꢀ1.94 (m), 1.14 (d, J = 6.8 Hz, 3H), 1.03 (d, J = 7.3 Hz, 3H), 0.96
(m, 6H), 0.90 (m, 6H), 0.80 (m, 6H); HRESI/APCIMS m/z 750.4407
[M þ Na]þ (calcd for C39H61N5O8Na, 750.4418).
Advanced Marfey’s Analysis of Dpv Unit in Dolastatin 16.
To liberate the authentic (2S,3R)-Dpv standard, pitiprolamide (500 μg)
was hydrolyzed (400 μL of 6 N HCl, 110 °C, 20 h). The mixture was
dried and reconstituted in 400 μL of H2O. Half of this sample was used
to epimerize the R-amino acids in this mixture (40 μL of Et3N and 40 μL
of Ac2O, heating at 60 °C for 1 h)27 to yield a mixture of (2S,3R)-Dpv
and (2R,3R)-Dpv standards. These standards were derivatized with 1%
(w/v) Marfey’s reagent (L-FDLA and DL-FDLA).28 Dolastatin 16 was
hydrolyzed and derivatized with 1% (w/v) L-FDLA according to the
procedure stated above. The absolute configuration of the Dpv unit
was determined by reversed-phase HPLC-MS [column, Phenomenex
Synergi Hydro-RP (150 ꢁ 4.6 mm) using a linear gradient of 0.1%
HCOOH in MeOHꢀ0.1% aqueous HCOOH (40%ꢀ100% for 45 min
and then 100% MeOH for 10 min); flow rate, 0.5 mL/min; detection by
ESIMS in negative ion mode (MRM scan)] of the FDLA adducts. The
acid hydrolysate of dolastatin 16 showed a peak at 36.1 min, correspond-
ing to (2S,3R)-Dpv. Co-injection of the acid hydrolysate with the DL-
FDLA-derivatized epimerization product showed enrichment of the peak
corresponding to (2S,3R)-Dpv. The retention times (tR, min; MRM ion
pair) of the authentic amino acid L-FDLA adducts were as follows:
(2S,3R)-Dpv (36.1; 486f191), (2S,3S)-Dpv [(36.6); (= D-FDLA-deri-
vatized (2R,3R)-Dpv)], (2R,3S)-Dpv [(43.5); (= D-FDLA-derivatized
(2S,3R)-Dpv)], (2R,3R)-Dpv (44.3). Compound-dependent parameters
used were as follows: DP ꢀ95.0, EP ꢀ6.0, CE ꢀ31.0, CXP ꢀ2.0,
CEP ꢀ26.0. Source gas parameters used were as follows: CUR 40,
CAD High, IS 4500, TEM 450, GS1 40, GS2 40.
Modified Mosher’s Analysis Using PGME. The base hydro-
lysate product was dissolved in 600 μL of DMF-d7 and divided into two
portions. To one portion was added 0.7 mg of (S)-PGME, and the
solution cooled and stirred at 0 °C before the successive addition of
PyBop (1.8 mg), HOBt (0.5 mg), and N-Me morpholine (5.0 μL). The
reaction was left to stir for another 3 h at room temperature and
quenched with the addition of EtOAc. The resulting solution was
successively washed with 5% HCl, saturated NaHCO3, and brine. The
organic layer was collected and dried over anhydrous MgSO4 before
drying under N2 to yield 13 (0.8 mg). The same procedure was used to
1
prepare the (R)-PGME-derivatized product 14 (0.6 mg). H NMR
chemical shifts of the Dml spin system were assigned by COSY and
TOCSY analyses.
13: colorless, amorphous solid; 1H NMR (CDCl3) δ 7.27ꢀ7.38 (m,
12H, incl. NH Dml at 7.27), 5.57 (dd, J = 14.0, 7.4 Hz, 1H), 5.02 (d, J =
10.2 Hz, 1H), 4.42 (m, 2H), 4.24 (d, J = 2.6 Hz, 1H), 3.74 (s, 3H),
3.56ꢀ3.65 (m, 3H, incl. H-3 Dml at 3.62), 3.45 (m, 2H), 3.29 (m, 1H),
2.99 (s, 3H), 2.75 (m, 2H, incl. H-2 Dml), 2.30ꢀ2.40 (m, 5H),
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dx.doi.org/10.1021/np200076t |J. Nat. Prod. 2011, 74, 917–927