Intermolecular â-Sheet Stabilization with Aminopyrazoles
J. Am. Chem. Soc., Vol. 119, No. 50, 1997 12067
dried over powdered molecular sieve 4Å and freshly distilled for each
measurement. Optical rotations were measured on a Perkin Elmer 241
MC digital polarimeter equipped with a 1 dm cell operating at 589 nm
(sodium D line). Thin layer chromatographic (TLC) analyses were
performed on silica gel 60 F-254 with a 0.2 mm layer thickness.
Preparative chromatography columns were packed with Kieselgel 60
(70-230 mesh). L-Amino acids, N-acetylamino acids, amino acid
methyl ester hydrochlorides, 3-amino-4-carbethoxypyrazole 1, and
3-amino-5-methylpyrazole 2 were purchased from Aldrich Chemical
Co. Pyrazole was purchased from Fluka Chemical Co. All solvents
were dried and freshly distilled before use.
1H NMR Titrations. A solution of the binding site molecule was
added in portions via microsyringe to a solution of a peptide
(concentration equal to the estimated dissociation constant) in a septum-
capped NMR tube. Volume and concentration changes were taken into
account during analysis.26
Job Plots. Equimolar solutions (0.2 M) of binding site molecule
and peptide were prepared and mixed in various amounts. 1H NMR
spectra of the mixtures were recorded, and the chemical shifts were
analyzed by Job’s method modified for NMR results.25
Temperature-Dependent NMR Spectra. Variable-temperature
(VT) NMR studies were carried out at 0.02 M (concentration of peptide)
in the range of 21-55 °C. After temperature change the sample was
equilibrated for 10 min before shimming, and then measurements were
made.
precipitate was removed again, the filtrate was evaporated in vacuo,
and the residue was purified by silica gel chromatography (ethyl acetate/
ethanol 3:1; Rf ) 0.53). 1H NMR (CDCl3) δ 0.92 + 0.95 (2 d, 6H, J
) 6.9, CH(CH3)2), 2.05 (s, 3H, C(O)CH3), 2.11-2.22 (m, 1H,
CH(CH3)2), 3.74 (s, 3H, OCH3), 4.02 (d, 2H, J ) 5.22, CRH2), 4.50 +
4.55 (dd, 1H, J ) 5.0, 8.7, CRH), 6.55 (br, 1H, NH(2)), 6.88 (d, 1H, J
) 8.3, NH(1)); 13C NMR (CDCl3) δ 17.72, 18.98, 22.91, 31.04, 43.32,
589
32
52.23, 57.36, 169.10, 170.75, 172.16; [R]
(MeOH) ) -27.4°.
Anal. Calcd for C10H18N2O4 (230.3): C, 52.16; H, 7.88; N, 12.17.
Found: C, 52.13; H, 8.01; N, 12.17.
3-(Methacryloylamino)-5-methylpyrazole (MAMP) 3. A solution
of 3-amino-5-methylpyrazole 2 (10 mmol) and triethylamine (10 mmol)
in dioxane (25 mL) was cooled to 10 °C. Under vigorous stirring
methacryloylchloride (10 mmol) was added very slowly. The reaction
mixture was stirred for 1 h at 10 °C and for another 1 h at room
temperature. The precipitated triethylamine hydrochloride was removed
by filtration, and the solvent was evaporated to dryness. The residue
was resolved in water and washed three times with dichloromethane.
The organic layer was dried over magnesium sulfate, and the solvent
was evaporated in vacuo. The mixture of regioisomers was purified
1
by recrystallization from ethyl acetate. Yield: 40%; mp 162 °C; H
NMR (CDCl3) δ 2.05 (s, 3H, ring CH3), 2.29 (s, 3H, CH3), 5.47 +
5.83 (2s, 2H, CH2), 6.50 (s, 1H, Harom), 8.81 (s, 1H, C(O)NH); 13C
NMR (DMSO) δ 10.77, 18.69, 96.06, 120.13, 138.43, 139.54, 146.88,
165.605; MS m/z 165 (M+, 18%), 164 (19), 150 (3), 137 (9), 120 (10),
110 (9), 97 (12), 96 (12), 69 (21), 41 (100). Anal. Calcd for C8H11N3O
(165.2) C, 58.17; H, 6.71; N, 25.44. Found: C, 58.05; H, 6.79; N,
25.50.
1H{1H}-Nuclear Overhauser Effect Measurements. All samples
were prepared under a dry argon atmosphere, exhaustively degassed
by the freeze-thaw method, and sealed under vacuum. Nuclear
Overhauser enhancements were obtained by saturation of the desired
resonance during a preacquisition time (set to 5 times the longest T1
of the sample). Percent NOEs were calculated by setting the integral
for the saturated resonance equal to -100 (inverted signal). The percent
NOEs are reported as percentages of this inverted signal.
3-Trifluoroacetylamino-5-methylpyrazole (TriFlAMP) 4. Prepa-
ration follows the procedure described above. When the oily residue
is treated with water, pure product is obtained by precipitation. Yield:
99% mp 203 °C; 1H NMR (DMSO) δ 2.25 (s, 3H, CH3), 6.32 (s, 1H,
CHarom), 11.87 (s, 1H, C(O)NH), 12.42 (s, 1H, NHarom); 13C NMR
(DMSO) δ 10.60, 96.60, 115.91, 139.14, 144.88, 153.98; MS m/z 193
(M+, 72%), 124 (61), 96 (40), 81 (5), 69 (39), 41 (100). Anal. Calcd
for C6H6F3N3O (193.13): C, 37.31; H, 3.13; N, 21.76. Found: C,
37.48; H, 3.11; N, 21.69.
N-Acetyl-L-valyl-L-valine Methyl Ester Ac-L-Val-L-Val-OMe 8.33
A suspension of Ac-L-Val (10 mmol), L-Val-OMe‚HCl (10 mmol),
N-ethylmorpholine (10 mmol), and 3-hydroxybenzotriazole (HOBT)
(20 mmol) in tetrahydrofuran (30 mL) was cooled to 0 °C, and DCC
(10 mmol) was added. The reaction mixture was stirred vigorously
for 1 h and afterwards for another 1 h at room temperature. The mixture
was filtered, and the solvent was removed under reduced pressure. The
residue was resolved in ethyl acetate and washed with saturated aqueous
sodium bicarbonate, 2 N citric acid, sodium bicarbonate and water.
After drying over magnesium sulfate the solvent was evaporated and
the residue was purified by silica gel chromatography (ethyl acetate;
Rf ) 0.35). 1H NMR (CDCl3) δ 0.90-0.98 (4 d, 12H, J ) 6.7, CH-
(CH3)2), 2.02 (s, 3H, C(O)CH3), 2.04-2.20 (m, 2H, CH(CH3)2, 3.74
(s, 3H, OCH3), 4.43-4.51 (2dd, 2H, CRH), 6.75 (d, 1H, J ) 8.5, NH-
(2)), 7.14 (d, 1H, J ) 8.2, NH(1)); 13C NMR (CDCl3) δ 17.91, 18.45,
3-(p-Vinylphenyl)-3-oxopropanenitrile 5. A solution of cyanoace-
tic acid (80 mmol) in tetrahydrofuran (400 mL) is cooled to -78 °C
with stirring under an argon atmosphere. The mixture is titrated with
n-butyllithium (1.6 M in hexane, 160 mmol), while the reaction
temperature slowly rises to 0 °C. When the red color persists at 0 °C,
the slurry is again cooled to -78 °C, and a solution of p-vinylbenzoyl
chloride24 (40 mmol) in tetrahydrofuran is added dropwise. The slurry
is stirred at -78 °C for 1 h and then allowed to gradually warm to
room temperature over a period of 1 h. Hydrochloride acid (1 M, 200
mL) is added to the mixture. The resulting solution is extracted twice
with chloroform (300 mL and 100 mL), and the combined organic layers
are washed with saturated aqueous sodium bicarbonate (200 mL) and
then with saturated aqueous sodium chloride (200 mL). The organic
layer is dried over magnesium sulfate, filtered, and evaporated in vacuo
(bath temperature 30 °C). The residual crude product is used without
further purification. Yield 6.6 g (97%); mp 99 °C; 1H NMR (CDCl3)
δ 4.12 (s, 2H, CH2), 5.46 (d, 1H, J ) 10.7, CH2,cis), 5.91 (d, 1H, J )
17.6, CH2,trans), 7.51 ( 7.86 (AB pattern, 4H, JAB ) 8.5, Harom); 13C
NMR (CDCl3) δ 29.45, 114.12, 126.73, 128.89, 133.28, 135.46, 143.62,
186.76; MS m/z 171 (M+, 59%), 131 (100), 103 (62), 77 (65), 51 (80).
Anal. Calcd for C11H9NO (171.2): C, 77.17; H, 5.30; N, 8.18.
Found: C, 77.23; H, 5.21; N, 7.92.
18.96, 19.06, 23.03, 30.77, 31.40, 52.01, 57.50, 58.36, 170.33, 172.05,
589
32
172.16; [R]
(MeOH) ) -59.6°. Anal. Calcd for C13H24N2O4
(272.3): C, 57.33; H, 8.88; N, 10.29. Found: C, 57.45; H, 8.84; N,
10.37.
N-Acetyl-L-phenylalanyl-L-phenylalanine Methyl Ester (Ac-L-
Phe-L-Phe-OMe) 10. 10 was synthesized from L-Phe-OMe‚HCl (10
mmol) and N-Ac-L-Phe (10 mmol) as in the foregoing experiment. 1H
NMR (CDCl3) δ 1.91 (s, 3H, C(O)CH3), 2.94-3.06 (m, 4H, CH2),
3.66 (s, 3H, OCH3), 4.68-4.76 (m, 2H, CRH), 6.37 (d, 1H, J ) 7.9,
NH(2)), 6.54 (d, 1H, J ) 7.6, NH(1)); 13C NMR (CDCl3) δ 22.99,
37.89, 38.23, 52.26, 53.50, 54.27, 126.90, 127.07, 128.52, 129.19,
589
20
129.31, 135.71, 136.47, 170.09, 170.86, 171.34; [R] (CHCl3) )
3-Amino-5-(p-vinylphenyl)pyrazole 6. Oxonitrile 5 (30 mmol) and
hydrazine hydrate (90 mmol) dissolved in ethanol (150 mL) are heated
under reflux for 2.5 h. The reaction mixture is filtered, and the solvent
is removed under reduced pressure. The crude product is recrystallized
+24.3°; Anal. Calcd for C21H24N2O4 (368.4): C, 68.46; H, 6.57; N,
7.60. Found: C, 68.30; H, 6.43; N, 7.52.
N-Acetylglycyl-L-Valine Methyl Ester (Ac-Gly-L-Val-OMe) 9.34
A solution of acetylglycine (10 mmol) and triethylamine (10 mmol) in
acetonitrile (40 mL) was cooled to -10 °C and treated with isobutyl
chloroformate (10 mmol). After 10 min a suspension of L-valine methyl
ester hydrocyloride (10 mmol) and triethylamine (10 mmol) in
acetonitrile (10 mL) was added. The mixture was stirred for 1 h at
-10 °C to 0 °C and then for 1 h at room temperature. The precipitated
triethylamine hydrochloride was removed by filtration, and the solvent
was replaced by 50 mL of hot tetrahydrofuran. The tetrahydrofuran
solution was allowed to stand in a refrigerator overnight. The
1
from toluene. Yield 90%. mp 159 °C; H NMR (CDCl3) δ 5.25 (d,
1H, J ) 11.0, CH2,cis), 5.76 (d, 1H, J ) 17.7, CH2,trans), 5.88 (s, 1H,
CHarom), 6.70 (dd, 1H, J ) 11.0, 17.7, CH), 7.41 + 7.60 (AB pattern,
4H, JAB ) 8.3, Harom); 13C NMR (CDCl3) δ 89.14, 113.91, 125.33,
126.50, 130.36, 136.28, 136.90, 145.32, 153.60; MS m/z 185 (M+,
100%), 156 (30), 143 (11), 128 (29), 115 (11), 103 (18), 93 (25), 77
(28). Anal. Calcd for C11H11N3 (185.2): C, 71.33; H, 5.99; N, 22.69.
Found: C, 71.16; H, 5.85; N, 22.59.