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Shen F, et al. Sci China Chem January (2011) Vol.54 No.1
2.4 Ligation efficiency between amino acid thioester
and L-cysteine to form dipeptide in 60% HFIP/8 M urea
Synthesis of Cbz-protected amino acid thioester
The synthesis is exemplified by 1a (Scheme 1): 4-Methyl-
benzenethiol (186 mg, 1.5 mmol) and 1-hydroxybenzotriazole
(203 mg, 1.50 mmol) were added to a solution of the
Cbz-L-alanine (223 mg, 1.0 mmol) in dry ethyl acetate
(2 mL/mmol) at 0 °C followed by N,N′-dicyclohexylcar-
bodiimide (206 mg, 1.0 mmol). The mixture was stirred
overnight at room temperature and the reaction progress was
monitored by TLC. At the end of the reaction a few drops of
50% acetic acid in ethyl acetate were added. The mixture
was filtered through CeliteTM and the organic phase was
washed with NaHCO3 solution, and brine, dried over MgSO4,
filtered, and concentrated in vacuo. The crude product was
purified by flash chromatography (silica gel, ethyl acetate in
petroleum ether) affording the desired product. Yield: 0.283 g
Scheme 1 Synthesis of Cbz-protected amino acid thioesters.
8 M urea, and 5 L benzamide (internal standard) were
added. The total volume of the reaction mixture was 0.5 mL.
The pH value was adjusted to 6.0, 7.0, 8.0 and 9.0 respec-
tively. Subsequently, 8.2 mg 1a and 3.0 mg L-cysteine were
dissolved in 0.5 mL acetonitrile and water to form 0.05 M
solutions separately. 40 L of L-cysteine solution and 20 L
1a were added to the solution prepared before. RP-HPLC
was used to monitor reactions so as to find out the best liga-
tion pH. The HPLC results were obtained with a C18 RP
column at 45 °C employing a linear gradient between 0.1%
TFA in acetonitrile (A) and 0.1% TFA in water (B) from
20% to 70% B in 20 min, then to 100% B in 5min, at a flow
rate of 1 mL/min.
1
(90%). H NMR (300 MHz, CDCl3): 1.47 (d, 3H, J = 7.2
Hz), 1.55 (s, 1H), 2.37 (s, 3H), 4.60 (q, 1H, J = 7.6 Hz),
5.16 (s, 2H), 5.24 (d, 1H, J = 7.2 Hz), 7.23–7.38 (m, 9H).
13C NMR (75 MHz, CDCl3): 21.4, 38.5, 61.4, 67.3, 127.3,
128.1, 128.3, 128.6, 128.8, 129.5, 130.2, 134.6, 140.0.
1b: 1H NMR (300 MHz, CDCl3): 2.38 (s, 3H), 4.22 (d,
2H, J = 6.2 Hz), 5.16 (s, 3H), 5.34 (s, 1H), 7.26–7.38 (m,
9H). 13C NMR (75 MHz, CDCl3): 21.5, 50.6, 67.5, 122.8,
128.3, 128.4, 128.7, 130.3, 134.8, 140.2, 156.3, 196.5.
1c: 1H-NMR (300 MHz, CDCl3): 2.38 (s, 3H), 3.87 (q,
1H, J = 4.1 Hz), 3.90 (q, 1H, J=3.8 Hz), 4.62 (t, 1H, J=8.6
Hz), 5.20 (s, 2H), 5.80 (d, 1H, J = 8.3 Hz), 7.24–7.39 (m,
9H). 13C NMR (75 MHz, CDCl3): 21.4, 62.4, 67.6, 76.7,
123.4, 128.2, 128.4, 128.7, 130.2, 134.6, 136.1, 140.1,
156.3, 199.1.
2.5 60% HFIP/8M urea ligation condition used for
Trifolitoxin analog synthesis
Synthesis of MDNKVAKNVEVKKGSIKATFKA-thioester
(segment 1)
Peptide thioester (segment 1) was synthesized using the
standard protocol described by Kent et al. [24]. On HSCH2-
CH2CO-Leu-OCH2-Pam-resin, side-chain protection for
amino acids was as follows: Lys(2-Cl-Z), Thr(Bzl), Ser(Bzl),
Glu(OBzl), Asn(Xan), Asp(OBzl). After the chain assembly
was completed, peptides were deprotected and cleaved from
the resin support by treatment with anhydrous HF contain-
ing p-cresol (90:10, v/v) for 1 h at 0 °C. After evaporation
of the HF under reduced pressure, crude products were pre-
cipitated with chilled diethyl ether. The crude products were
dissolved in 50% aqueous acetonitrile containing 0.1%
TFA and injected into the preparative HPLC. The product
was analyzed by MALDI-TOF/MS: [M+H]+ calcd for
C115H200N31O33S2 2607.4, found 2607.2.
1d: 1H NMR (300 MHz, CDCl3): 2.38 (s, 3H), 3.16 (d,
2H, J = 5.5 Hz), 4.83 (t, 2H, J = 8.3 Hz), 5.12 (s, 2H), 5.20
(d, 1H), 7.22–7.33 (m, 14H). 13C NMR (75 MHz, CDCl3):
21.4, 38.5, 67.3, 127.3, 128.1, 128.3, 128.6, 128.8, 129.5,
130.2, 134.6, 140.0, 155.8, 199.2.
1
1e: H NMR (300 MHz, CDCl3): 0.92 (d, 3H, J = 6.5
Hz), 1.03 (d, 3H, J = 6.9 Hz), 1.26 (m, 1H), 2.38 (s, 3H),
4.51 (t, 1H, J = 4.5 Hz), 5.17 (s, 2H), 5.27 (d, 1H, J = 9.9
Hz). 13C NMR (75 MHz, CDCl3): 17.0, 19.5, 21.4, 31.4,
65.8, 67.4, 123.6, 128.3, 128.4, 128.7, 130.2, 134.7, 136.2,
140.0, 156.3, 199.2.
Synthesis of AVC*GQRSGGIDVKTKSKLVC-NH2 (segment
2) following the Fmoc strategy
Synthesis of the N-terminal peptide segment (segment 2)
followed the standard Fmoc strategy [22, 23]. Wang resin
was initially swelled with DCM/DMF (1/1) for 3 h. To
pre-activate the first protected amino acid, 3.6 equiv HBTU,
4 equiv HOBt, 8 equiv DIEA and 0.1 equiv DMAP were
added to a solution of 4 equiv protected amino acid (0.4 M)
in DMF. After preactivation for 2 min, the mixture was
added to the resin. After 10 h the resin was washed with
DMF and DCM and capped with acetic anhydride/DIEA/
DMF (1:1:8) (2 × 5 min). After treatment with 20%
Condition optimization for ligation between Cbz-L-alanine
thioester with L-cysteine
The transmembrane peptide dissolves the best in 60% HFIP/
8 M urea. Therefore, we performed NCL in this solvent
system to optimize the reaction pH. The reactions were car-
ried out with standard procedure in literature except that the
solvent was replaced by 60% HFIP/8M urea. In detail, 2.86
mg TCEP and 11.9 mg HEPES were mixed together in a
2 mL Eppendorf reaction tube. Then 0.3 mL HFIP, 0.2 mL