A. Chan et al. / Bioorg. Med. Chem. xxx (2016) xxx–xxx
3
described before.29 Sodium ethoxide solution was prepared by dis-
solving sodium in an ethanol solution, to which diethyl acetomido-
malonate and 1-bromo-tetradecane or 1-bromo-octadecane was
added to produce C16 or C20, respectively. The mixture was
refluxed for 4 days and the resultant precipitate was filtered and
washed with cold water. The solid was dried then dissolved in
DMF and fuming 37% HCl and refluxed again for 4 days. Ammonia
solution was added to yield precipitate, which was filtered and
washed with cold water and acetone. The final product was dried
until powdery.
Dde-OH (N-(4,4-dimethyl-2,6-dioxycyclohexylidene)ethyl alco-
hol) was synthesised following previously described methods.30
Then, Dde-OH (1.1 equiv) was dissolved in ethanol, to which tri-
ethylamine (3 equiv) and C16 (1 equiv), or C20 (1 equiv), was
added. The mixture was refluxed for 2 days and ethanol was evap-
orated. Ethyl acetate was added, and 5% HCl and brine solution
were used to wash the organic phase. The organic layers were col-
lected and filtered by manual short flash chromatography using a
silica column. The solution was evaporated and stored at À20 °C
to afford yellow crystals. The crystals were washed with cold
MeCN, filtered, and dried until powdery. The resulting Dde-C16
and Dde-C20 were analysed by 1H NMR (300 MHz, CDCl3) to pro-
duce spectra identical with those reported previously.30
Two serine moieties and a lysine residue were incorporated using
standard Fmoc-SPPS. Amino acid activation was achieved by dis-
solving Fmoc-amino acids (4.2 equiv) in 0.5 M HATU (4.0 equiv)
in DMF, followed by the addition of DIPEA (4.2 equiv) The coupling
cycle consisted of Fmoc deprotection with 20% piperidine in DMF
(twice, 10 min and 20 min), a 1 min DMF flow-wash, followed by
couplings with preactivated Fmoc-amino acids (2 Â 1 h). Pentynoic
acid (4.2 equiv) was coupled to the N-terminus of the sequence by
using HATU (4 equiv) and DIPEA (4.2 equiv) at room temperature
(2 Â 1 h), followed by washing with DMF, DCM, MeOH, and drying
under vacuum. The peptide was cleaved from the resin in a solu-
tion of neat TFA triisopropylsilane/water (95:2.5:2.5) for 4 h. The
cleaved peptide was precipitated, filtered, and washed with ice-
cold Et2O, dissolved in acetonitrile/water 90:10 and lyophilised.
The resulting crude product (9) was purified by RP-HPLC.
Peptide 9 yield: 24%. Purity: >95%. Molecular weight: 906.3. ESI-
MS: [M+H]+ m/z 906.9 (calcd: 907.3). Rt = 33 min (35–75% solvent
B, 60 min, C4-column).
3.4. Synthesis of peptides 10 and 11
The two azido derivative peptides 10 and 11 (Scheme 2) were
synthesised according to the general procedure by microwave-
assisted Fmoc-SPPS with 2 Â 5 min couplings for each Fmoc-amino
acid (4.2 equiv) using HATU (4.0 equiv) and DIPEA (4.2 equiv). The
temperature was set at 70 °C (20 W, 10 min) for all amino acids,
except for Cys and His, which were coupled at 50 °C (20 W,
10 min). For His, Cys, and Arg, initial coupling was performed at
rt for 3 min before microwave-assisted couplings.31 Each amino
acid coupling cycle consisted of Fmoc-deprotection with 20%
piperidine in DMF at 70 °C (twice, 2.5 min and 5 min), 2 min
DMF flow wash, followed by 10 min coupling with the pre-acti-
vated amino acid. In the case of Fmoc-deprotection for Asp, 0.1 M
HOBT in 20% piperidine/DMF was used. Upon completion of syn-
thesis, the resin was washed with DMF, DCM, MeOH, and vacuum
dried. The peptide was cleaved from the resin by stirring in a solu-
tion of TFA (99%)/triisopropylsilane/water (95:2.5:2.5) for 4 h. The
crude products (10 and 11) were purified by RP-HPLC.
3.2. Synthesis of LCP 1 and LCP 2
LCP 1 was synthesised by coupling Dde-C16 onto pMBHA resin
(0.45 mmol/g; 0.2 mmol scale) by using HBTU (4.0 equiv) and
DIPEA (6.2 equiv) in DMF (2 Â 1 h). Removal of the Dde-protecting
group was performed by treatments with 2% hydrazine in DMF
(2 Â 30 min). The remaining LCP sequence was synthesised by
microwave-assisted Boc-SPPS. Amino acid activation was achieved
by dissolving Boc-amino acids (4.2 equiv) in 0.5 M HBTU
(4.0 equiv) in DMF, followed by the addition of DIPEA (6.2 equiv).
The amino acid coupling cycle consisted of Boc-deprotection with
neat TFA at room temperature (2 Â 1 min), 2 min DMF flow wash,
followed by 10 min coupling with the pre-activated amino acid.
The temperature was set at 70 °C (35 W, 10 min) for all amino
acids, except for Cys and His, which were coupled at 50 °C (35 W,
10 min). N-terminal acetylation and final cleavage from resin was
performed as reported previously.29 The lyophilised products were
purified by RP-HPLC. The collected fractions were analysed by
ESI-MS and RP-HPLC. Where appropriate, fractions were combined
and lyophilised to yield pure product.
Peptide 10 yield: 6%. Purity: >95%. Molecular weight: 5446.2.
ESI-MS: [M+4H]4+ m/z 1362.1 (calcd: 1362.6), [M+5H]5+ m/z
1090.1 (calc: 1090.3), [M+6H]6+ m/z 908.8 (calcd: 908.7),
For the synthesis of LCP 2, Dde-C20 was coupled onto pMBHA
resin (0.45 mmol/g; 0.2 mmol scale). The remaining procedures
followed that of the synthesis of LCP 1, as mentioned above.
LCP 1 yield: 12.5%. Purity: 90%. Molecular weight: 6086.4. ESI-
MS: [M+4H]4+ m/z 1523.4 (calcd: 1522.6), [M+5H]5+ m/z 1218.4
(calcd: 1218.3), [M+6H]6+ m/z 1015.7 (calcd: 1015.3), [M+7H]7+
m/z 870.6 (calcd: 870.4), [M+9H]9+ m/z 677.5 (calcd: 677.3), [M
+10H]10+ m/z 608.8 (calcd: 609.5). Rt = 25.0 min (0–100% solvent
B, 40 min, C4-column).
LCP 2 Yield: 18.5%. Purity:>99%. Molecular weight: 6198.5. ESI-
MS: [M+5H]5+ m/z 1241.1 (calcd: 1240.7), [M+6H]6+ m/z 1034.7
(calcd: 1034.1), [M+7H]7+ m/z 886.6 (calcd: 886.5), [M+9H]9+ m/z
690.4 (calcd: 689.7). Rt = 29.0 min (0–100% solvent B, 40 min, C4-
column).
3.3. Synthesis of alkyne derivative 9
The synthesis of alkyne derivative 9 was performed with man-
ual Fmoc-SPPS (Scheme 1). Dde-C16 was coupled onto rink amide
MBHA resin (0.2 mmol scale) by using HATU (4.0 equiv) and DIPEA
(4.2 equiv) in DMF (2 Â 1 h). The Dde-protecting group was
removed by treatment with 2% hydrazine in DMF (8 Â 30 min).
Scheme 1. Synthesis of alkyne derivative 9.