The Journal of Organic Chemistry
Article
of solvents was evaporated under reduced pressure to obtain the
were analyzed using a growth arrest assay. While the activities
of the ethyl and isopropyl ester analogs were similar to that of
the wild-type methyl ester, the benzyl ester analog was almost
100-fold less active suggesting that the C-terminal ester of a-
factor is an important structural element recognized by its
cognate receptor. The success of this simple method for the
synthesis of peptides containing C-terminal cysteine alkyl esters
using commercially available trityl resin opens the door to the
synthesis of a wide variety of C-terminal ester-modified
peptides functionalized with modified isoprenoids34−36 that
should be useful in studies of protein prenylation and other
structurally related biological processes.
product as a white solid (0.109 g, 99%). No further purification was
1
needed. Rf 0.7 (silica gel, hexane/EtOAc, 1:3, v/v). H NMR (300
MHz, CDCl3): δ 1.34 (m, 3H, SH), 3.01 (s (broad), 2H), 4.25 (m,
3H), 4.43 (d, 2H, J = 6.9), 4.65 (s (broad), 1H), 5.72 (s (broad), NH),
7.33 (t, 2H, J = 7.4), 7.41 (t, 2H, J = 7.4), 7.62 (d, 2H, J = 7.4), 7.78
(d, 2H, J = 7.4). 13C NMR (75.0 MHz, CDCl3): δ 14.3, 27.2, 47.2,
55.2, 62.1, 67.1, 120.0, 125.1, 127.1, 127.8, 141.3, 143.6, 155.6, 169.9.
HRMS (ESI-TOF) m/z: [M + Na]+; calcd for C20H21NO4S 394.1089;
found 394.1085.
Synthesis of Fmoc-Cys-OiPr (1d). A solution of Fmoc-Cys-OH·
H2O (0.1 g, 0.26 mmol) in iPrOH (∼10 mL) was prepared, and 12
drops of 37% HCl were added to this solution. The mixture was left
stirring overnight at reflux (83 °C). The material was dissolved in
acetone, and the mixture of solvents was evaporated under reduced
pressure to obtain a solution that was purified via column
chromatography (hexanes/EtOAc, 9:1). The product was obtained
as a white solid (57 mg, 51%). Rf 0.71 (silica gel, hexane/EtOAc, 7:3,
EXPERIMENTAL SECTION
■
General Methods. All reagents and solvents were used as received.
All the solvents were HPLC grade. N,N-Diisopropylethylamine
(DIEA) and TFA were of Sequalog/peptide synthesis grade. Standard
Fmoc/HCTU chemistry was used for SPPS of peptides. Fmoc-based
SPPS was performed on a Protein Technologies PS3 automated
peptide synthesizer. Additional steps performed on resin (after
coupling/deprotection of N-terminus) were carried out using a
polypropylene syringe equipped with a porous polypropylene disc at
the bottom. Peptide synthesis and other transformations were
performed at 25 °C unless otherwise indicated. Loading of first
amino acids on resin was determined using Fmoc quantitative
analysis,26 and the Fmoc solutions were analyzed at 301 nm on a
UV−vis spectrophotometer. The peptides were cleaved off the resin
using Reagent K. (Preparation of Reagent K: The specific amount of
compounds used to do the mixture were: 0.5 g phenol, 0.5 mL of
thioanisole, 0.5 mL of H2O, 0.25 mL ethanedithiol, and 8.25 mL of
TFA. Then N2(g) was bubbled into the solution to remove any oxygen
dissolved in the mixture.) The peptides were analyzed using reverse
phase high-performance liquid chromatography (RP-HPLC) and the
products were observed at 220 nm. The analytical column used was a
C18 Agilent Microsorb-MV 100-5 (4.6 × 250 mm), and the preparative
column was a Phenomenex C18 (10 μm, 10.00 × 250 mm). The buffer
solutions used for HPLC analysis and purifications were: Buffer A
(0.1% TFA in H2O) and Buffer B (0.1% TFA in CH3CN). The
retention times of the a-factor analogs are based on a flow rate of 1
mL/min and on a gradient increase of 1% Buffer B/min after
equilibration of the column with 1% Buffer B. The solutions
containing purified peptides were lyophilized and analyzed by ESI-
MS. The retention times of the model tripeptides are based on a flow
rate of 1 mL/min and a gradient increase of 1.6% Buffer B/min after
equilibration of the column with 5% Buffer B.
1
v/v). H NMR (300 MHz, CDCl3): δ 1.32 (m, 6H, SH), 3.00 (s
(broad), 2H), 4.23 (t, 1H, J = 3.4), 4.43 (d, 2H, J = 6.6), 4.61 (s
(broad), 1H), 5.79 (s (broad), NH), 7.32 (t, 2H, J = 7.5), 7.41 (t, 2H,
J = 7.5), 7.61 (d, 2H, J = 7.5), 7.77 (d, 2H, J = 7.5). 13C NMR (75.0
MHz, CDCl3): δ 21.8, 27.2, 47.2, 50.8, 55.2, 67.1, 120.0, 125.1, 127.1,
127.8, 141.3, 143.7, 155.7, 169.5. HRMS (ESI-TOF) m/z: [M + Na]+;
calcd for C21H23NO4S 408.1246; found 408.1258.
Synthesis of Fmoc-Cys-OBn (1e). A solution of Fmoc-Cys(Trt)-
OH (0.5 g, 0.85 mmol) in DMF (6 mL) was prepared, and benzyl
bromide (112 uL, 0.94 mmol) and DIEA (148 uL, 0.85 mmol) were
added to this solution. The mixture was left stirring for 2 h on a reflux
at 60 °C. The salt byproduct was removed by crystallization with Et2O.
The product remained in the filtrate, and the DMF was removed by
lyophilization. The material was used without further purification.
Fmoc-Cys(Trt)-OBn (0.3 g, 0.44 mmol, 1 equiv) was dissolved in 25
mL of 1% TFA solution in CH2Cl2. Triisopropylsilane (0.9 mL, 4.4
mmol, 10 equiv) was added to this solution resulting in no immediate
color change. TLC showed 95% conversion after 2 h of constant
stirring. A solution of 1% TEA in CH2Cl2 (28 mL) was added slowly
to the reaction mixture to neutralize the TFA. Twenty mL of water
was added to the mixture, and the CH2Cl2 was evaporated under
reduced pressure. CH2Cl2 (25 mL) was added to the solution and an
extraction with water (20 mL, 2× ) was performed. The product
(CH2Cl2 solution) was dried with MgSO4 and filtered. The solvents
were removed under reduced pressure, and the product was purified
by flash chromatography (hexanes/EtOAc, 9.5:5). Rf 0.48 (silica gel,
hexane/EtOAc, 7:3, v/v). 1H NMR (300 MHz, CDCl3): δ 1.27 (t, 1H,
SH, J = 8.9 Hz), 3.01 (s (broad), 2H), 4.23 (t, 1H, J = 7.0), 4.43 (d,
2H, J = 6.8), 4.72 (s (broad), 1H), 5.23 (q, 2H, J = 12.8), 5.74 (s
(broad), NH), 7.37 (m (arom), 9H), 7.61 (d, 2H, J = 7.6), 7.78 (d,
2H, J = 7.6). 13C NMR (75.0 MHz, CDCl3): δ 27.2, 47.2, 55.2, 67.2,
67.8, 120.0, 125.1, 127.1, 127.8, 128.6, 128.7, 129.5, 135.0, 141.3,
143.78, 155.6, 169.9. HRMS (ESI-TOF) m/z: [M + Na]+ Calcd for
C25H23NO4S 456.1246; Found 456.1248.
General Procedure for the Loading of Fmoc-Cys-OR (R = Me,
Et, iPr) to Trt-Cl Resin. Trityl chloride (Trt-Cl) resin, with a loading
of 1.40 mmol/g, (1 equiv) was placed into a fritted syringe and was
washed with DMF (3×). The Fmoc-Cys-OR (2 equiv) was added into
a separate test tube and dissolved with CH2Cl2 (3 mL). This solution
was added to the syringe containing the resin followed by the addition
of DIEA (4 equiv). The mixture was left to react overnight while
mixing with a rotisserie. The next morning, CH3OH (50 μL) was
added to the solution and allowed to react for 5 min in order to cap
any unreacted Trt-Cl groups on the resin. Then, the reagents were
drained from the syringe, and the resin was washed with DMF (3×)
and CH2Cl2 (3×). Loading of Fmoc-Cys-OR on the resin was
calculated via Fmoc-quantitative analysis (see Table 1).26
Synthesis of Fmoc-L-Cys-OMe (1a). A solution of Fmoc-Cys-OH·
H2O (0.5 g, 1.38 mmol) in CH3OH (∼10 mL) was prepared, and 6
drops of 37% HCl were added to this solution. The mixture was left
stirring overnight at rt. A wet white solid (with excess of CH3OH) was
obtained. The material was dissolved in acetone, and the mixture of
solvents was evaporated under reduced pressure to obtain the product
as a white solid (0.470 g, 97%). No further purification was needed. Rf
0.5 (silica gel, hexane/EtOAc, 1:1, v/v). 1H NMR (300 MHz, CDCl3):
δ 1.37 (t, 1H, SH, J = 8.6 Hz), 3.01 (s (broad), 2H), 3.81 (s, 3H), 4.24
(t, 1H, J = 7.0), 4.44 (d, 2H, J = 6.3), 4.68 (s (broad), 1H), 5.69 (s
(broad), NH), 7.33 (t, 2H, J = 7.3), 7.42 (t, 2H, J = 7.3), 7.62 (d, 2H, J
= 7.3), 7.78 (d, 2H, J = 7.3). 13C NMR (75.0 MHz, CDCl3): δ 27.2,
47.2, 52.9, 55.2, 67.1, 120.0, 125.1, 127.1, 127.8, 141.3, 143.6, 155.6,
170.4. HRMS (ESI-TOF) m/z: [M + Na]+; calcd for C19H19NO4S
380.0933; found 380.0905.
Synthesis of Fmoc-D-Cys-OMe (1b). Compound 1b was prepared
via the same procedure described above for 1a using Fmoc-D-Cys-OH
as starting material.
Procedure for the Loading of Fmoc-Cys-OBn to Trt-Cl Resin.
The trityl chloride (Trt-Cl) resin, with a loading of 1.40 mmol/g, (1
equiv, 40 mg) was placed into a round-bottom flask and was
suspended in CH2Cl2. Fmoc-Cys-OBn (6 equiv, 144 mg, 0.333 mmol)
was added into a separate test tube and dissolved with CH2Cl2 (3 mL).
Synthesis of Fmoc-Cys-OEt (1c). A solution of Fmoc-Cys-OH·H2O
(0.10 g, 0.26 mmol) in EtOH (∼10 mL) was prepared, and 6 drops of
37% HCl were added to this solution. The mixture was left stirring
overnight at reflux (80 °C). A wet white solid (with excess of EtOH)
was obtained. The material was dissolved in acetone, and the mixture
G
J. Org. Chem. XXXX, XXX, XXX−XXX