HIBINO, MIKI AND NISHIUCHI
172.9.; ESI MS Calcd for [C30H29N3O6 + H]+ 528.21, found 528.2;
[a]D ꢂ20.0 (c 1.12, DMF).
Materials and Methods
All reagents and solvents were obtained from the Peptide Institute,
Inc. (Osaka, Japan), Wako Chemical (Osaka, Japan), Tokyo Chemical
Industry (Tokyo, Japan), and Watanabe Chemical Industries
(Hiroshima, Japan). Analytical HPLC was performed on a Shimadzu
liquid chromatograph Model LC-10AT (Kyoto, Japan) with a DAISO-
PAK SP-120-5-ODS-BIO (4.6 ꢀ 150 mm) using a flow rate of 1 ml/min
and the following solvent systems: 0.1% TFA in H2O (A) and 0.1%
TFA in MeCN (B). Purities are based upon area percent of the peaks
detected at 220 nm. High resolution mass spectra were measured
with a Bruker Esquire 200T (Billerica, MA, USA). Molecular weights
were measured with a MALDI-TOF MS (Voyager-DESTR, Applied
Fmoc-(2,4-DMBom)-OH (1b) and Fmoc-(3,4-DMBom)-OH (1c)
were prepared as described earlier for Fmoc-His(p-MBom)-OH
(1a) by using 3,4-DMBom-Cl and 2,4-DMBom-Cl, respectively.
Fmoc-His(2,4-DMBom)-OH (1b); 1H NMR (d6-DMSO) d = 7.89
(2H, d, J = 6.87 Hz), 7.75 (1H, d, J = 8.70 Hz), 7.66 (2H, d,
J = 7.79 Hz), 7.48 (2H, t, J = 7.20 Hz), 7.30 (2H, t, J = 7.20 Hz), 7.26
(2H, d, J = 8.80 Hz), 6.65 (1H, d, J = 2.70 Hz), 6.50 (1H, d, J = 8.80,
2.70 Hz), 4.73 (2H, s), 4.44 (2H, dd, J = 16.03, 10.99 Hz), 4.35–4.10
(4H, m), 3.71 (3H, s), 3.10–2.78 (2H, m).; 13C NMR (d6-DMSO)
d = 32.0, 46.6, 54.3, 55.0, 55.8, 65.8, 66.7, 72.7, 100.3, 106.2,
118.2, 120.1, 125.2, 125.3, 127.1, 127.6, 128.9, 137.3, 140.7, 143.8,
156.0, 158.8, 159.8, 172.3; HRMS Calcd for [C31H31N3O7H]+, m/z
558.2235, found 558.2238.
Biosystems) and an ESI MS (HP1100 LC/MSD). H and 13C NMR
1
spectra were recorded on a JEOL-ECX400 spectrometer (Tokyo,
Japan) in dimethyl sulfoxide-d6 (d6-DMSO) with the solvent residual
peak (d6-DMSO: 1H = 2.49 ppm, 13C = 39.52 ppm) as an internal
reference unless otherwise stated.
Fmoc-His(3,4-DMBom)-OH (1c); 1H NMR (d6-DMSO) d = 7.88
(2H, d, J = 6.87 Hz), 7.75 (1H, d, J = 8.70 Hz), 7.70 (2H, d, J = 7.79Hz),
7.40 (2H, t, J = 7.20Hz), 7.30 (2H, t, J = 7.20 Hz), 7.10 (2H, d,
J = 2.70 Hz), 6.87 (1H, d, J = 8.80, 2.70 Hz), 6.58 (1H, d, J = 8.80Hz),
4.73 (2H, s), 4.44 (2H, dd, J= 16.03, 10.99 Hz), 4.35–4.10 (4H, m),
3.71 (3H, s), 3.10–2.78 (2H, m); 13C NMR (d6-DMSO) d = 32.0, 46.6,
54.3, 55.0, 55.2, 65.8, 68.7, 72.7, 112.7, 114.6, 120.1, 121.9, 125.2,
125.3, 127.1, 127.6, 128.2, 128.9, 137.3, 140.7, 143.8, 148.8, 156.0,
158.8, 172.3; HRMS: Calcd for [C31H31N3O7H]+: m/z 558.2235, found
558.2237.
Synthesis of Fmoc-His(p-MBom)-OH (1a) and its Related
Compounds (1b, 1c)
Boc-His(p-MBom)-OH (4a)
A solution of Boc-His(t-Ac)-OMe (3a) (6.10 g, 19.6 mmol) and
4-methoxybenzyloxymethylchloride (MBom-Cl: 4.51g, 24.5mmol)
in CH2Cl2 (60ml) was stirred at room temperature for 4 h. After
removal of the solvent, the residue was triturated with diethyl ether
to give Boc-His(p-MBom)-OMe (HCl form, 10.6 g). A solution of
Boc-His(MBom)-OMe obtained previously in MeOH (40 ml) was
treated with 1 M NaOH aq. (40 ml) and stirred at room temperature
for 4 h. The reaction mixture was adjusted with 1 M HCl to pH 4.
After removal of the solvent, the residue was extracted with CHCl3.
The extract was washed with water and dried over Na2SO4. After
removal of the solvent, the residue was triturated with AcOEt to
give Boc-His(p-MBom)-OH (4.92 g, 62% from Boc-His(t-Ac)-OMe).
1H NMR (DMSO-d6) 1.34 (s, 9H), 2.82–3.12 (m, 2H), 3.72 (s, 3H),
4.20 (br s, 1H), 4.33 (s, 2H), 5.37 (br s, 2H), 6.75 (s, 1H), 6.88
(d, J = 8.70 Hz, 2H), 7.15–7.25 (m, 3H), 7.73 (s, 1H).; 13C NMR
(DMSO-d6) 25.3, 28.2, 52.8, 55.1, 69.0, 73.2, 78.1, 113.7, 127.6,
127.9, 129.0, 129.5, 138.3, 155.4, 158.9, 173.3; ESI MS Calcd for
[C20H27N3O6 + H]+ 406.198, found 406.1.
Examination of His Racemization during Synthesis of the
Model Peptide, Z-Ala-His-Pro-OH
Starting with H-Pro-Trt(2-Cl) resin (0.29 g, 0.69 mmol/g), manual
peptide chain assembly was carried out using the protocol of
30-min coupling with Fmoc-amino acid/HCTU/6-Cl-HOBt/DIEA
(4/4/4/8 equiv with respect to the peptide resin, 0.2 M, 0–5 min
preactivation) in DMF. MW heating was performed in a 25-ml
polypropylene open vessel placed into the MW cavity of a
300 W single-mode manual MW peptide synthesizer (CEM,
Discover SPS) set at 50 or 80 ꢁC; power pulsing sequences of
30 W were used for His coupling steps (5 min). Fmoc deprotection
was carried out with 20% piperidine in DMF, followed by washing
with DMF (5 ꢀ 2 min). The final release of peptides was achieved
with TFA/triisopropylsilane/H2O (95/5/5) containing MeONH2.HCl
(5 equiv with respect to the peptide resin) at room temperature
for 1 h. The crude peptides were directly analyzed by HPLC and
ESI MS analyses.
Fmoc-His(p-MBom)-OH (1a)
To a vigorously stirred solution of Boc-His(p-MBom)-OH (10.0 g,
23.8 mmol) and 2,6-lutidine (33.3 ml, 191 mmol) in CH2Cl2
(75 ml) at 0 ꢁC was added TMSOTf (34.6 ml, 191 mmol). After
10 min, the ice bath was removed, and the mixture was stirred
at room temperature for 18 h. The reaction mixture was placed
again in an ice bath, and ice-cold water (50 ml) was added.
Subsequently, the mixture was washed with CHCl3 (2 ꢀ 10 ml).
The aqueous layer was added to DMF (50 ml) and treated with
Fmoc-OSu (8.03 g, 23.8 mmol) and stirred at room temperature
for 4 h. The solution was adjusted with Na2CO3 aq. to pH 7–8.
After removal of the solvent, the residue was washed with H2O.
The residue was triturated with CHCl3/MeOH and Et2O to give
Fmoc-His(p-MBom)-OH (11.8 g, 84% yield): 1H NMR (DMSO-d6)
2.90–3.17 (m, 2H), 3.71 (s, 3H), 4.15–4.38 (m, 6H), 5.38 (q, 2H,
J = 10.0 Hz) 6.74–6.87 (m, 3H), 7.15–7.42 (m, 6H) 7.60–7.94
(m, 6H); 13C NMR (DMSO-d6) 25.2, 46.6, 50.1, 53.2, 55.0, 63.8,
65.7, 69.0, 73.2, 113.7, 119.8, 120.1, 125.2, 126.8, 127.0, 127.1,
127.6, 127.8, 128.9, 129.5, 138.3, 140.6, 143.7, 145.2, 155.9, 158.8,
Loading of the Fmoc-His-OH Derivatives onto Wang Resin
Wang resin (4-methoxybenzylalcohol resin) (185mg, 1.00 mmol)
was left to swell in THF/DMF (3:1, 10 ml) for 1 h before adding the
Fmoc-His-OH derivative [Fmoc-His(p-MBom)-OH (1a) or Fmoc-His
(t-Trt)-OH (1.50 mmol)] and DCC (1.50mmol). The suspensions were
cooled to ꢂ15 ꢁC, and then, 0.06 eq of DMAP was added. The
reaction mixture was kept at ꢂ15 ꢁC with slight shaking for 4 h
and left in an ice bath overnight. Fmoc deprotection was carried
out with 20% piperidine in DMF, followed by washing with DMF
(5ꢀ 2 min). The resin was then filtered off and washed with DMF
and DCM. After an aliquot (ca. 50 mg) of the resin had been treated
with 6 ml of TFA/water (v/v, 95:5) for 1 h, the filtrate was evaporated
to give the residue, which was checked for optical purity by using
Merfey’s method [9].
wileyonlinelibrary.com/journal/jpepsci Copyright © 2012 European Peptide Society and John Wiley & Sons, Ltd. J. Pept. Sci. 2012; 18: 763–769