Isolation and synthesis of falcitidin
B Somanadhan et al
263
by C18 VLC (14.5 ꢁ 10 cm) eluted with 0, 20, 30, 40, 50 and 100% MeOH in
10h at room temperature. After that, the reaction mixture was concentrated by
H2O (2 liter each fraction). The 100% MeOH fraction (B1 g) was active in the rotary evaporation. The residue obtained was dissolved in saturated NaHCO3
falcipain-2 assay and was further separated by C18 VLC (17ꢁ 4.5 cm) using a and the aqueous layer was washed with n-hexane. The aqueous bicarbonate
10% step-wise elution from 30% to 100% MeOH in H2O (400ml each layer was acidified with citric acid and then extracted with EtOAc. The organic
fraction). Falcipain-2 activity was found in the 60% to 80% MeOH in H2O layer was washed with brine, dried using anhydrous Na2SO4 and removed by
fractions, which were combined (80 mg) and separated using a Sephadex LH 20 rotary evaporation to give white solid, which was purified by silica column
column (50ꢁ 2 cm, eluent MeOH). The active fraction (8 mg) was separated by
chromatography (hexane/EtOAc, 2.5:1) to give 6 (2.5g, 7.5 mmol, 88%) as a
1
C18 preparative HPLC using the solvent A (0.1% HCO2H in H2O): solvent B white crystalline solid. H NMR (300MHz, DMSO-d6) d 0.77–0.83 (m, 6H),
(0.1% HCO2H in CH3CN), gradient elution from 100:0 to 95:5 for 2 min and 0.87 (m, 6H), 1.08 (m, 1H), 1.37 (s, 9H), 1.58 (m, 1H), 1.98 (m, 2H), 3.84
then to 70:30 over 40 min, flow rate 12 ml minꢀ1) to obtain falcitidin (1) (t, J ¼ 8.2Hz, 1H), 4.13 (dd, J ¼ 5.7, 8.4Hz, 1H), 6.73 (d, J ¼ 9.0 Hz, 1H), 7.74
(0.5mg, retention time 20min). The biomass was fractionated in a similar (d, J ¼ 8.4 Hz, 1H), 12.56 (s, 1H); 13C NMR (75 MHz, DMSO-d6) d 10.8, 15.3,
manner as the broth, which yielded B0.5 mg of peptide of B85% purity.
17.9, 18.9, 24.3, 28.1, 29.9, 36.3, 56.9, 58.7, 77.9, 155.8, 171.6, 172.7; ESI-MS m/z
330.9 (M þ H)þ , 353.1 (M þ Na) þ
.
Falcipain-2 biological assay
An in vitro enzymatic assay was performed using a 15mg ml-1 of the falcipain-2
derived from P. falciparum Gombak A strain9 to cleave 1 mM substrate (Z-Phe-
Arg-AMC) peptide (Bachem AG, I1160) in a 384-well black microtitre plate
(Griener, 781076) to identify potential inhibitors of falcipain-2. The buffer
system contained 0.1M sodium acetate buffer pH 5.6 (Merck, Singapore),
10 mM DTT (Boehringer Mannheim, 708992, Singapore), 0.1% 3-[(3-
cholamidopropyl)dimethylammonio]-1-propanesulfonate (CHAPS) (Sigma,
C3023, Singapore) and 20% trehalose (Calbiochem, 625625, Singapore).
After mixing the enzyme, substrate and test sample in buffer, the protease
reaction was allowed to proceed at 4 1C overnight (16 h). The amount of
released AMC was quantified by measuring its fluorescence using an Ultra
Preparation of tripeptide (7a)
Tert-butyl (2S,3S)-1-((S)-1-((S)-2-carbamoylpyrrolidin-1-yl)-3-methyl-1-oxo-
butan-2-ylamino)-3-methyl-1-oxopentan-2-ylcarbamate: Boc-L-proline amide
4 (350mg, 1.6 mmol) was coupled to the dipeptide 6 (647mg, 1.95mmol)
according to the general procedure for peptide coupling using HATU/HOAt to
give the desired compound 7a (368mg, 0.86mmol, 53%) after silica column
purification with CHCl3/MeOH (25:1). 1H NMR (500MHz, DMSO-d6) d 0.77
(m, 6H), 0.85 (m, 6H), 1.04 (m, 1H), 1.37 (s, 9H), 1.65 (m, 1H), 1.75 (m, 2H),
1.82 (m, 4H), 3.54 (m, 1H), 3.70 (m, 1H), 3.81 (t, 1H), 4.20 (1H, m), 4.33
(t, 1H) 6.82 (brs, 2H), 7.24 (s, 1H), 7.72 (d, J ¼ 8.8 Hz, 1H); 13C NMR (125
MHz, DMSO-d6) d 10.8, 15.3, 18.1, 19.1, 24.4, 28.1, 29.2, 30.1, 36.5, 46.9, 58.8,
78.0, 79.1, 155.2, 169.6, 171.2, 173.3; ESI-MS m/z 427.0 (Mþ H)þ , 449.2
¨
(Tecan, Mannedorf, Switzerland) with excitation at 360 nm and emission at
(Mþ Na)þ
.
465nm. The percentage of (%) enzyme inhibition was calculated using the
following formula: ((Sample fluorescenceꢀlow fluorescence control)/(high
fluorescence controlꢀlow fluorescence control)) ꢁ 100. This assay had an
overall signal to background ratio of 2.7 0.3 and a Z0 value of 0.66 0.13, as
no standard inhibitor was available when the assay was originally run.
Preparation of Fmoc–tetrapeptide (8)
(9H-fluoren-9-yl)methyl(R)-1-((2S,3S)-1-((S)-1-((S)-2-carbamoylpyrrolidin-1-
yl)-3-methyl-1-oxobutan-2-ylamino)-3-methyl-1-oxopentan-2-ylamino)-1-oxo-
3-(1-trityl-1H-imidazol-4-yl)propan-2-ylcarbamate (8): Tripeptide 7 (160 mg,
0.37 mmol) was coupled to Na-fmoc-N(im)-trityl-D-histidine (256 mg,
0.41 mmol) using the general procedure for HATU coupling. The desired
compound tetrapeptide 8 (262mg, 0.28mmol, 75%) was obtained after silica
column purification using chloroform/methanol (25:1). 1H NMR (300 MHz,
CDCl3) d 0.75 (m, 12H), 1.02 (m, 1H), 1.18 (m, 1H), 1.41 (m, 1H), 1.87 (6H,
m), 3.0 (d, J ¼ 6.2 Hz, 2H), 3.61 (m, 1H), 3.77 (m, 1H), 4.19 (m, 3H), 4.51(m,
3H), 6.54 (brs, 1H), 6.66 (s, 1H), 7.06 (m, 7H), 7.14 (brs, 1H), 7.28 (m, 14H),
7.56 (m, 2H), 7.64 (brd, 1H), 7.72 (m, 2H), 7.79p.p.m. (brd, 1H); 13C NMR
(75 MHz, CDCl3) 11.3, 15.8, 17.9, 19.2, 24.4, 25.0, 27.7, 31.2, 47.0, 47.7, 55.1,
55.8, 57.8, 59.3, 67.2, 75.3, 119.3, 119.8, 125.1, 125.2, 127.0, 127.6, 128.0, 129.6,
138.5, 141.1, 141.2, 142.2, 143.7, 143.9, 171.2, 171.3, 171.7, 173.5; ESI-MS m/z
Plasmepsin-2 and tryptase biological assays
The fluorescence resonance energy transfer and fluorescence polarization assay
formats used for the plasmepsin-2 assays27,28 and the fluorescence quenching
format used for the tryptase assay29 were performed as reported previously.
General procedure for cleavage of N-Boc group and amide coupling
using HATU
The Boc protected amino acid or peptide compound was dissolved in CH2Cl2,
and TFA was added slowly at 0 1C and the reaction was left stirring at room
temperature for 30 min. After completion of the reaction, excess TFA was
removed using rotary evaporation and diethylether was added to precipitate
the product. The supernatant diethylether layer was removed and the solid was
dissolved in CH2Cl2. The a-amino protected amino acid or peptide with
free carboxy group was then added to the reaction flask at 0 1C, followed by the
addition of DIPEA, HATU and HOAt and a catalytic amount of DMAP. The
reaction was stirred for 5 h at room temperature. After completion of the
reaction, the reaction mixture was evaporated in vacuo and the residue
dissolved in EtOAc. The organic layer was washed with saturated NaHCO3
solution, followed by brine solution. The organic layer was finally dried using
Na2SO4 and evaporated in vacuo to give a solid residue that was purified by
column chromatography.
928.2 (Mþ H)þ , 950.4 (Mþ Na)þ
.
Preparation of tetrapeptide (9)
(S)-1-((S)-2-((2S,3S)-2-((R)-2-amino-3-(1-trityl-1H-imidazol-4-yl)propa-
namido)-3-methylpentanamido)-3-methylbutanoyl)pyrrolidine-2-carbox-
amide (9): Fmoc–tetrapeptide 8 (140 mg, 0.15mmol) was cleaved using
piperidine to give the desired compound 9 (77 mg, 0.11mmol, 70%). 1H
NMR (300MHz, CDCl3) d 0.83 (m, 12H), 1.10 (m, 1H), 1.40 (m, 1H), 1.93
(m, 6H), 2.82 (m, 2H), 3.62 (m, 1H), 3.85 (m, 1H), 4.00 (m, 1H), 4.30 (m,
1H), 4.53 (m, 2H), 5.86 (s, 1H), 6.63 (s, 1H), 7.03 (brs, 1H), 7.10 (m, 6H), 7.16
(d, 1H), 7.33 (m, 9H), 7.46 (s, 1H), 7.65 (d, J ¼ 8.2 Hz, 1H), 8.28 (d, J ¼ 8.2
Hz, 1H); 13C NMR (75 MHz, CDCl3) d 11.2, 15.7, 18.1, 19.1, 24.6, 24.9, 28.2,
30.6, 32.2, 36.5, 47.6, 54.5, 55.9, 58.4, 59.5, 75.1, 119.5, 127.9, 129.5, 136.7,
138.6, 142.0, 171.3, 171.5, 173.4, 173.7; ESI-MS m/z 706.1 (M þ H)þ , 728.3
Preparation of dipeptide (6)
(S)-2-((2S,3S)-2-(tert-butoxycarbonylamino)-3-methylpentanamido)-3-methyl
butanoic acid: bis-TMS-valine (5) was separately prepared by dissolving
L-valine (1.6g, 13.6mmol) in 60ml CH2Cl2. Triethyl amine (6.7ml, 48 mmol)
and trimethylsilylchloride (6.1ml, 48mmol) were then added (2.0g,
8.65 mmol). The mixture was refluxed for 2 h. The mixture was then used
in situ for coupling to the anhydride. Thus, Boc-L-Isoleucine 2 (2.0g,
(Mþ Na)þ
.
Preparation of Trt-falcitidin (10)
(S)-1-((S)-3-methyl-2-((2S,3S)-3-methyl-2-((R)-2-(3-methylbutanamido)-
8.65 mmol, 1 eq) was dissolved in THF and the solution was maintained at 3-(1-trityl-1H-imidazol-4-yl)propanamido)pentanamido)butanoyl)pyrrolidine-
ꢀ201C using an ice-salt bath. Then, N-methyl morpholine (1.2ml, 10.3mmol, 2-carboxamide (10): Tetrapeptide 9 (74 mg, 0.1mmol) was coupled to
1.2eq) was added followed by the addition of ethyl chlorofomate (1.0ml, isovaleric acid (10ml, 0.1 mmol) using the general procedure for HATU
10.3 mmol, 1.0 eq). The mixture was stirred at ꢀ20 1C for 15 to 20min. At this coupling. The desired compound 10 (48 mg, 0.06 mmol, 58%) was obtained
point, bis-TMS-valine (5) was added to the anhydride solution and stirred for after silica column purification using chloroform/methanol (25:1). 1H NMR
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