Chemoenzymatic Synthesis of N-Ras Lipopeptides
J. Am. Chem. Soc., Vol. 120, No. 28, 1998 6901
layers were dried over MgSO4, and the solvent was evaporated in vacuo.
The product HPhePheOAll (18a) was isolated from the residue by flash
chromatography (silica gel, ethyl acetate/methanol 4:1) to yield 12 mg
(69%) of 18a as a yellow oil. Rf ) 0.9 (ethyl acetate/methanol 4:1).
[R]20D ) -45° (c ) 0.3, CHCl3). 1H NMR (250 MHz, CDCl3): δ 2.6
(m, 1H, â-CH2 Phe); 3.0 (m, 3H, â-CH2 Phe); 3,6 (m, 1H, R-CH Phe);
4.5 (d, J ) 7 Hz, 2H, OCH2); 4.8 (m, 1H, R-CH Phe); 5.2 (m, 2H,
CH2 allyl); 5.7-5.9 (m, 1H, CH allyl); 6.9-7.3 (m, 10H, 2 × C6H5);
7.6 (d, J ) 10 Hz, 1H, NH amide). MS m/e: calcd for (M+)
C21H24O3N2 352.178, found 352.177.
Cleavage of the Allyl Ester Protecting Group from N-(4-
(Acetoxy)benzyloxycarbonyl)-dipeptide Allyl Esters. To a solution
of N-(4-(acetoxy)benzyloxycarbonyl)-dipeptide allyl ester (0.6 mmol)
(17) in THF (50 mL) under argon were added Pd(PPh3)4 (0.06 mmol)
and morpholine (0.72 mmol). The solution was stirred at room
temperature for 30 min, the solvent was evaporated in vacuo, and the
product was isolated from the residue by flash chromatography on silica
gel.
OMe (21) was isolated from the residue by flash chromatography (silica
gel, ethyl acetate/methanol 4:1) to yield 9.6 mg (65%) of 21 as a yellow
oil. Rf ) 0.16 (ethyl acetate/methanol 4:1). [R]20 ) -50° (c ) 1,
D
CHCl3). 1H NMR (400 MHz; CDCl3) δ 0.9 (m, 6H, 2 × CH3 Leu);
1.4 (m, 2H, γ-CH Leu); 1.6 (s, 6H, 2 × CH3 Far); 1.65 (s, 6H, 2 ×
CH3 Far); 1.7 (m, 1H, â-CH2 Leu); 1.8-2.2 (m, 13H, 4 CH2 Far, CH2
Pro (3H), NH2); 2.3 (m, 1H, CH2 Pro); 2.75 (dd, J1 ) 7 Hz, J2 ) 15
Hz, 1H, â-CH2a Cys); 2.95 (dd, J1 ) 5 Hz, J2 ) 14 Hz, 1H, â-CH2b
Cys); 3.0-3.2 (m, 2H, CH2 Far); 3.5-3.7 (m, 3H, δ-CH2 Pro, R-CH
Leu); 3.75 (s, 3H, CH3CO); 4.6-4.7 (m, 3H, 2 × R-CH); 5.1 (m, 2H,
2 × CH Far); 5.2 (t, J ) 7 Hz, CH Far); 7.5 (d, J ) 9 Hz, 1H, NH
amide). 13C NMR (100.5 MHz, CDCl3): δ 16.0 (CH3 Far); 16.1 (CH3
Far); 17.7 (CH3 Far); 21.4 (CH3 Leu); 23.6 (CH3 Leu); 24.6 (CH3 Far);
25.0 (γ-CH Leu); 25.7 (CH2 Pro); 26.4 (CH2 Far); 26.7 (CH2 Far);
27.0 (CH2 Pro); 29.6 (CH2 Far); 33.2 (CH2 Far); 39.6 (R-CH2 Far);
39.7 (â-CH2 Cys); 44.5 (â-CH2 Leu); 46.8 (CH2 Pro); 51.2 (R-CH Leu);
51.7 (R-CH Cys); 52.4 (OCH3); 59.7 (R-CH Pro); 119.6 (CH Far);
123.7 CH Far); 124.3 CH Far); 131.2 (Cq Far); 135.3 (Cq Far); 139.8
(Cq Far); 170.8 (CdO); 171.0 (CdO); 176.2 (CdO). MS m/e: calcd
for (M+) C30H51N3O4S 549.360, found 549.357.
N-(4-(Acetoxy)benzyloxycarbonyl)-L-phenylalanyl-phenylala-
nine (AcOZPhePheOH) (19a). Data for 19a: clear oil. Yield 40
mg (80%). Rf ) 0.5 (ethyl acetate/hexane 7:3 + 5% acetic acid). [R]20
N-(4-(Acetoxy)benzyloxycarbonyl)-L-methionyl-glycyl-L-leucyl-
L-prolyl-(S-farnesyl)-L-cysteine Methyl Ester (AcOZMetGlyLeu-
ProCys(Far)OMe) (22). To a solution of HLeuProCys(Far)OMe (21)
(27 mg, 50 µmol) and AcOZ-MetGlyOH (19c) (20 mg, 50 µmol) in
dichloromethane (10 mL) at 0 °C under argon was added a solution of
HOBt (13.6 mg, 100 µmol) and of EDC (11.5 mg, 60 µmol) in
dichloromethane (5 mL) and the mixture was stirred for 24 h at room
temperature. The solution was extracted with 1 M HCl, 1 M NaHCO3,
and distilled water and dried over MgSO4. The solvent was evaporated
in vacuo, and the product AcOZMetGlyLeuProCys(Far)OMe (22) was
isolated from the residue by flash chromatography (silica gel, ethyl
acetate/methanol 9:1) to yield 37 mg (80%) of 22 as a yellow oil. Rf )
D
) +28° (c ) 0.5, CHCl3). 1H NMR (250 MHz, CDCl3): δ 2.3 (s,
3H, CH3CO); 2.9-3.2 (m, 4H, 2 × â-CH2 Phe); 4.4 (m, 1H, R-CH
Phe); 4.5 (m, 1H, R-CH Phe); 5.0 (s, 2H, C6H4CH2); 5.5 (d, J ) 8 Hz,
1H, NH urethane); 6.5 (d, J ) 8.5 Hz, 1H, NH amide); 6.9-7.3 (m,
14H, CH aromatic). MS m/e calcd for (M+) C28H28O7N2 504.189, found
504.188.
N-(4-(Acetoxy)benzyloxycarbonyl)-L-leucyl-L-prolyl-(S-farnesyl)-
L-cysteine Methyl Ester (AcOZLeuProCys(Far)OMe) (20). To a
solution of AcOZ-LeuProOH (19b) (0.63 g, 1.5 mmol) and of HCys-
(Far)OMe34 (7) (0.5 g, 1.5 mmol) in dichloromethane (10 mL) under
argon was added a solution of HOBt (3 mmol, 0.4 g) and EDC (0.34
g, 1.8 mmol) in dichloromethane (10 mL). The solution was stirred
for 24 h at room temperature; and was extracted with 1 M HCl, 1 M
NaHCO3, and distilled water and dried over MgSO4. The solvent was
removed in vacuo, and the product AcOZLeuProCys(Far)OMe (20)
was isolated from the residue by flash chromatography (silica gel,
n-hexane/ethyl acetate 1:1) to yield 1 g (94%) of 20 as a yellow oil. Rf
) 0.3 (n-hexane/ethyl acetate 1:1). [R]20D ) -58° (c ) 0.5, CHCl3).
1H NMR (400 MHz, CDCl3): δ 0.9 (d, J ) 7 Hz, 3H, CH3 Leu); 1.0
(d, J ) 7 Hz, 3H, CH3 Leu); 1.5 (m, 2H, Leu γ-CH); 1.6 (s, 6H, 2 ×
CH3 Far); 1.65 (s, 6H, 2 × Far CH3); 1.7 (m, 1H, Leu â-CH2); 1.8-
2.1 (m, 11H, 4 × CH2 Far, CH2 Pro (3H)); 2.3 (s, 3H CH3CO); 2.35
(m, 1H, CH2 Pro); 2.75 (dd, J1 ) 6 Hz, J2 ) 13.8 Hz, 1H, â-CH2
Cys); 2.95 (dd, J1 ) 6.7 Hz, J2 ) 13.7 Hz, 1H, Cys â-CH2); 3.0-3.2
(m, 2H, CH2 Far); 3.6 (m, 1H, δ-CH2a Pro); 3.7 (m, 1H, δ-CH2b Pro);
3.75 (s, 3H, CH3CO); 4.55 (m, 1H, R-CH); 4.6 (m, 1H, R-CH); 4.65
(m, 1H, R-CH); 5.0 (s, 2H, C6H4CH2O); 5.1 (m, 2H, 2 × CH Far); 5.2
(t, J ) 7 Hz, CH Far); 5.7 (d, J ) 8.5 Hz, 1H, NH urethane); 7.1 (d,
J ) 8 Hz, 2H, 2 × CHCCH2 aromatic); 7.3 (d, J ) 8 Hz, 2H, 2 ×
OCCH aromatic); 7.4 (d, J ) 9 Hz, 1H, NH amide). 13C NMR (100.6
MHz, CDCl3): δ 15.95 (C(CH3)2 Far); 16.07 (C(CH3)2 Far); 17.6 (Far
CH3); 21.0 (CH3CO); 21.58 (CH3 Leu); 23.3 (CH3 Leu); 24.5 (Leu
γ-CH); 24.9 (CH3 Far); 25.6 (CH2 Pro); 26.4 (CH2 Far); 26.4 (CH2
Far); 26.6 (CH2 Far); 27.2 (CH2 Pro); 29.6 (CH2 Far); 33.1 (â-CH2
Cys); 39.6 (2 × CH2 Far); 42.2 (Leu â-CH2); 47.1 (Pro δ-CH2); 50.8
(OCH3); 51.7 (R-CH Leu); 52.1 (R-CH Cys); 59.64 (R-CH Pro); 66.0
(C6H5CH2O); 119.6 (CH Far); 121.6 (2 × CH aromatic); 123.7 (CH
Far); 124.3 (CH Far); 129.1 (2 × CH aromatic); 131.1(Cq Far); 133.9
(Cq aromatic); 135.8 (Cq Far); 139.6 (Cq Far); 150.3 (Cq aromatic);
156.1 (OCONH); 169.3 (CO); 170.9 (CO); 171.06 (CO); 172.7 (CO).
MS m/e: calcd for (M+) C40H59O8N3S 741.402, found 741.401. Anal.
Calcd: C, 64.75; H, 8.01; N, 5.66. Found: C, 64.30; H, 7.83; N, 5.48.
L-Leucyl-L-prolyl-(S-farnesyl)-L-cysteine Methyl Ester (HLeu-
ProCys(Far)OMe) (21). AcOZLeuProCys(Far)OMe (20) (20 mg, 27
µmol) was dissolved with ultrasonication in a mixture of 0.2 M KJ
solution (160 mL) (pH 5) and methanol (40 mL). Next, 25 units of
lipase from M. miehei was added, and the mixture was stirred at 37 °C
and at pH 5 for 12 h. The solution was extracted three times with
dichloromethane, and the organic layer was dried over MgSO4. The
solvent was evaporated in vacuo, and the product HLeuProCys(Far)-
0.58 (ethyl acetate/methanol 9:1). [R]20 ) -56° (c ) 0.6, CHCl3).
D
1H NMR (400 MHz, CDCl3): δ 0.9 (m, 6H, 2 × CH3 Leu); 1.2 (m,
2H, γ-CH2 Leu); 1.6 (s, 6H, 2 × CH3 Far); 1.65 (s, 6H, 2 CH3 Far);
1.7 (m, 1H, â-CH2 Leu); 1.8-2.2 (m, 17H, 4 × CH2 Far, 2 × CH2
Pro, γ-CH2 Met, SCH3); 2.3 (s, 3H, CH3CO); 2.6 (m, 2H, â-CH2 Met);
2.75 (m, 1H, â-CH2 Cys); 2.95 (m, 1H, â-CH2 Cys); 3.0-3.2 (m, 2H,
CH2 Far); 3.5-3.7 (m, 2H, CH2 Pro); 3.75 (s, 3H, OCH3); 4.0 (m, 2H,
CH2 Gly); 4.4 (m, 1H, R-CH); 4.8 (m, 1H, R-CH); 5.1-5.2 (m, 4H,
CH2O, 2 × CH Far); 5.2 (t, J ) 7 Hz, 1H, CH Far); 5.8 (d, J ) 8 Hz,
1H, NH urethane); 6.8 (d, J ) 8 Hz, 1H, NH amide); 7.1 (d, J ) 8 Hz,
2H, 2 aromatic CHCCH2); 7.15 (d, J ) 8 Hz, 1H, NH amide); 7.2 (d,
J ) 8 Hz, 2H, 2 × OCCH aromatic); 7.5 (d, J ) 8 Hz, 1H, NH amide).
13C NMR (100.6 MHz, CDCl3): δ 15.2 (SCH3); 16.0 (Far C(CH3)2);
16.1 (Far C(CH3)2); 17.7 (Far CH3); 21.1 (CH3CO); 21.8 (CH3 Leu);
21.9 (CH3 Leu); 24.7 (γ-CH Leu); 24.9 (CH3 Far); 25.7 (CH2 Pro);
26.5 (CH2 Far); 26.7 (CH2 Far); 27.7 (CH2 Pro); 29.6 (CH2 Met); 30.0
(CH2 Met); 32.6 (â-CH2 Cys); 39.7 (CH2 Far); 41.9 (CH2 Gly); 42.9
(â-CH2 Leu); 47.3 (δ-CH2 Pro); 51.9 (R-CH Leu); 52.5 (R-CH Cys);
53.9 (R-CH Met); 59.8 (R-CH Pro); 66.4 (CH2O); 119.6 (CH Far);
121.7 (2 × CH aromatic); 123.7 (CH Far); 124.3 (CH Far); 129.4 (2
× CH aromatic); 131.3 (Cq Far); 133.8 (Cq aromatic); 135.3 (Cq Far);
139.9 (Cq Far); 150.5 (Cq aromatic); 156.9 (OCONH); 171.1 (CO);
171.5 (CO); 171.6 (CO); 171.9 (CO); 172.0 (CO); 172.8 (CO). MS
m/e: calcd for (M+) C47H71O10N5S2 929.464, found 929.4510. Anal.
Calcd (dihydrate): C, 58.42; H, 7.82; N, 7.25. Found: C, 58.47; H,
7.39; N, 7.19.
L-Methionyl-glycyl-L-leucyl-L-prolyl-(S-farnesyl)-L-cysteine Meth-
yl Ester (HMetGlyLeuProCys(Far)OMe) (23). AcOZMetGlyLeu-
ProCys(Far)OMe (22) (20 mg, 27 µmol) was dissolved with ultrason-
ication in a mixture of 0.2 M KJ solution (160 mL) (pH5) and methanol
(40 mL). Next, 25 units of lipase from M. miehei was added, and the
mixture was stirred at 37 °C and at pH 5 for 12 h. The solution was
extracted three times with dichloromethane and the organic layer was
dried over MgSO4. The solvent was evaporated in vacuo, and the
product HMetGlyLeuProCys(Far)OMe (23) was isolated from the
residue by flash chromatography (silica gel, ethyl acetate/methanol 4:1)
to yield 9.5 mg (48%) of 23 as a yellow oil. Rf ) 0.25 (ethyl acetate/
methanol 4:1); [R]20D ) -78° (c ) 0.5, CHCL3). 1H NMR (400 MHz,
CDCl3): δ 0.9 (m, 6H, 2 × CH3 Leu); 1.4 (m, 2H, γ-CH Leu); 1.55