Solid-phase total synthesis of cherimolacyclopeptide e and discovery of more potent analogues by alanine screening

Article abstract of DOI:10.1021/np300266e

Cherimolacyclopeptide E (1) is a cyclic hexapeptide obtained from Annona cherimola, reported to be cytotoxic against the KB (human nasopharyngeal carcinoma) cell line. The solid-phase total syntheses of this cyclic peptide and its analogues were accomplished by employing FMOC/tert-butyl-protected amino acids and the Kenner sulfonamide safety-catch linker. The synthetic peptide 1 was found to be weakly cytotoxic against four cell lines (MOLT-4, Jurkat T lymphoma, MDA-MB-231, and KB). Analogues 3 and 7, where glycine at positions 2 and 6 of the parent compound was replaced by Ala, exhibited enhanced cytotoxicity against KB (3, IC₅₀ 6.3 μM; 7, IC₅₀ 7.8 μM) and MDA-MB-231 breast cancer cells (3, IC₅₀ 10.2 μM; 7, IC₅₀ 7.7 μM), thereby suggesting possible selective targeting of these cancer cells by these peptides. The spectral data of synthetic peptide 1 was found to be similar to that reported for the natural product. However, a striking difference in biological activity was noted, which warrants the re-evaluation of the original natural product for purity and the existence of conformational differences.

Full text of DOI:10.1021/np300266e

Article
pubs.acs.org/jnp
Solid-Phase Total Synthesis of Cherimolacyclopeptide E and
Discovery of More Potent Analogues by Alanine Screening
Farzana Shaheen,*,† Tania S. Rizvi,† Syed G. Musharraf,† A. Ganesan,‡ Kai Xiao,§ Jared B. Townsend,§
Kit S. Lam,§ and M. Iqbal Choudhary†,⊥
†H. E. J. Research Institute of Chemistry, International Center for Chemical and Biological Sciences, University of Karachi,
Karachi-75270, Pakistan
‡School of Pharmacy, University of East Anglia, Norwich NR4 7TJ, United Kingdom
§Department of Biochemistry and Molecular Medicine, University of California Davis, Sacramento, California 95817, United States
⊥Department of Biochemistry, Faculty of Science, King Abdulaziz University, Jeddah-21412, Saudi Arabia
S
* Supporting Information
ABSTRACT: Cherimolacyclopeptide E (1) is a cyclic hexapeptide obtained from Annona
cherimola, reported to be cytotoxic against the KB (human nasopharyngeal carcinoma) cell
line. The solid-phase total syntheses of this cyclic peptide and its analogues were
accomplished by employing FMOC/tert-butyl-protected amino acids and the Kenner
sulfonamide safety-catch linker. The synthetic peptide 1 was found to be weakly cytotoxic
against four cell lines (MOLT-4, Jurkat T lymphoma, MDA-MB-231, and KB). Analogues 3
and 7, where glycine at positions 2 and 6 of the parent compound was replaced by Ala,
exhibited enhanced cytotoxicity against KB (3, IC50 6.3 μM; 7, IC50 7.8 μM) and MDA-MB-
231 breast cancer cells (3, IC50 10.2 μM; 7, IC50 7.7 μM), thereby suggesting possible
selective targeting of these cancer cells by these peptides. The spectral data of synthetic
peptide 1 was found to be similar to that reported for the natural product. However, a
striking difference in biological activity was noted, which warrants the re-evaluation of the
original natural product for purity and the existence of conformational differences.
and release of the cyclic peptide by head-to-tail cyclization on a
solid phase. We have successfully used this methodology
previously for the total syntheses of kahalalide A11 and
phakellistatin 12.12
he fast-growing evergreen tree Annona cherimola Miller
T(Annonaceae) is native to the Andean mountain range in
South America and is cultivated widely for its edible fruits. The
plant is used locally in traditional medicines, and extracts have
been shown to have antimicrobial activity.1 A series of cytotoxic
cherimolacyclopeptides have been isolated from extracts of the
RESULTS AND DISCUSSION
■
́ ́
seeds of A. cherimola by Wele
et al.2−7 Among these
The total synthesis of cherimolacyclopeptide E (1) was
achieved by solid-phase chemistry, using FMOC/t-Bu-pro-
tected amino acids and 4-sulfamylbutyryl resin as the solid
support (Scheme 1). The starting FMOC-protected amino acid
was loaded on 4-sulfamylbutyryl AM resin using benzotriazole-
1-yloxy-tris-pyrrolidinophosphonium hexafluorophosphate
(PyBOP) as a coupling agent with N,N-diisopropylethylamine
(DIEA). Leucine was chosen as the site of resin attachment, as
it can undergo cyclization with glycine, and both are
unhindered amino acids. The resulting loading level was
determined by UV spectrophotometric analysis and found to be
0.517 mmol/g. The linear peptide was then synthesized by
following standard FMOC protocols in which all the FMOC-
protected amino acids were activated by hydroxybenzotriazole/
diisopropylcarbodiimide (HOBt/DIC) in N,N-dimethylforma-
mide (DMF). FMOC deprotection before each coupling step
was achieved by the treatment of the resin-bound peptide with
compounds, cherimolacyclopeptide E (1) was reported to
exhibit cytotoxic activity against the KB (human nasophar-
yngeal carcinoma) cell line with an IC50 value of 0.017 μM.6
Rajiv et al.8 reported the solution-phase synthesis of
cherimolacyclopeptide E (1) and its cytotoxicity against
Dalton’s lymphoma ascites (DLA) and Ehrlich’s ascites
carcinoma (EAC) cell lines with CTC50 values of 2.76 and
4.96 μM, respectively, and antimicrobial activity against the
pathogenic microbes Pseudomonas aeruginosa, Escherichia coli,
and Candida albicans, with MICs between 6 and 12.5 μg/mL.
Furthermore, this compound possesses a moderate anthelmin-
tic activity against earthworms.8 The biological activities
reported for 1 have made it an attractive target for total
synthesis and SAR studies. During the current study, a solid-
phase approach, suitable for generating the natural product as
well as its analogues by amino acid substitution was employed.
For the synthesis, the Ellman variant of Kenner’s sulfonamide
“safety-catch” linker was used.9,10 This strategy permits resin
attachment through the backbone carboxylic acid of any residue
Received: April 11, 2012
Published: November 13, 2012
© 2012 American Chemical Society and
American Society of Pharmacognosy
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a
Scheme 1. Solid-Phase Synthesis of Cherimolacyclopeptide E (1)
a
Reagents and reaction conditions: PyBOP method (a) resin (1.1 mmol/g), FMOC-Leu (4 equiv), DIEA (8 equiv), PyBOP (4 equiv); (b) (i) 20%
piperidine/DMF; (ii) FMOC-L-Gly-OH, DIC, HOBt (3 equiv each); (iii) 20% piperidine/DMF; (iv) FMOC-L-Pro-OH, DIC, HOBt (3 equiv
each); (v) 20% piperidine/DMF; (vi) FMOC-L-Tyr (Ot-Bu)-OH, DIC, HOBt (3 equiv each); (vi) 20% piperidine/DMF; (vii) FMOC-L-Phe-OH,
DIC, HOBt (3 equiv each); (viii) 20% piperidine/DMF, (ix) FMOC-L-Gly-OH, DIC, HOBt (3 equiv each); (c) (i) 20% piperidine/DMF (ii) Trt-
Cl (4 equiv), DIEA (8 equiv); (iii) ICH2CN (10 equiv), DIEA (12 equiv); (d) 5% TFA/CH2Cl2, DIEA/THF; (e) TFA/CH2Cl2/TIS (1:1:0.01).
a 20% solution of piperidine in DMF, and the resin was
subjected to coupling−deprotection steps to build the linear
hexapeptide as the precursor for the cyclic peptide 1 (Scheme
1). After the construction of the linear peptide, the N-terminal
FMOC protecting group was removed and replaced by the
trityl group, followed by sulfonamide alkylation with
iodoacetonitrile, which activated the safety-catch linker. The
trityl deprotection to the free amine and subsequent treatment
with DIEA in THF resulted in macrocyclization and release of
the cyclic peptide 1 (Scheme 1) with the tert-butyl protecting
group still intact. Solution-phase treatment with TFA/CH2Cl2/
TIS (triisopropylsilane) yielded the crude peptide 1.
The crude peptide was concentrated, followed by precip-
itation in diethyl ether. The identity of the product was
validated by ESIMS, and its purity was checked by HPLC.
Crude peptide 1 was dissolved in CH3CN/H2O (1:2) and
purified by recycling preparative HPLC to obtain 1 in an overall
yield of 9.8%.
significantly, both in small peptides and in proteins.13 The 13C
NMR data of 1 indicated the presence of a trans-tyrosyl-proline
amide bond, as shown by the small 13C NMR chemical shift
difference of Pro, ΔδC −C = 4.6 ppm.13−15 The same value was
β
γ
reported for naturally isolated 1, indicating that the natural and
synthetic samples both have the trans-tyrosyl-proline amide
bond. However, slight broadening of signals was observed for
the alpha protons of the leucine and proline residues of 1,
indicating the possibility that some conformational equilibrium
exists for this compound. The overall NMR data for synthetic
peptide 1 showed a close resemblance with reported data of the
natural product (Table S1, Supporting Information). Moreover,
the NMR data differed from that reported for cherimolacyclo-
peptide E, as synthesized by Rajiv et al., and we attribute this to
use of different solvents (i.e., CDCl3 rather than pyridine-d6).
For example, Rajiv et al. reported the alpha protons for Leu in
the range δ 6.26−6.22 in CDCl3, whereas in pyridine, a
chemical shift of 5.26 ppm was observed.
The structure of 1 was deduced by mass spectrometry and
1D- and 2D-NMR spectroscopy. The molecular mass of 1 was
determined by HRESIMS, as m/z 635.3157 [M + H]+. The 1H
NMR data were recorded in d5-pyridine, as this was the solvent
used for the NMR studies of the naturally isolated
With the solid-phase synthesis of 1 successfully completed,
several alanine-substituted analogues of 1 were synthesized.
The constituent amino acids of 1 were sequentially substituted
by alanine, one at a time through the “alanine scanning”
technique.16 All of the amino acids of the natural product were
found to be of the L-series, and therefore L-alanine was used for
the replacement of different amino acids in the parent
molecule.
The alanine-substituted linear precursors were synthesized
and cyclized to produce analogues 2−7. In addition, analogues
8 and 9 were prepared in which Pro was replaced by the more
polar acidic amino acid glutamic acid and the less hindered
glycine, respectively, to observe the effect of proline
substitution by these amino acids on the resultant cytotoxicity.
All analogues were subjected to product identification by
1
cherimolacyclopeptide E (1). The H NMR data of 1 showed
the five amide protons at δ 11.24 (2H, s, Gly-NHCO), 9.98
(1H, brs, Phe5-NHCO), 9.89 (1H, brs, Leu1-NHCO), and 8.80
(1H, s, Tyr4-NHCO), whereas the alpha protons were observed
at δ 5.64 (1H, brs, Phe5-CH), 5.64 (1H, m, Tyr4-CH), 5.26
(1H, m, Leu1-CH), and 4.47 (1H, brs, Pro3-CH) (Table S1,
Supporting Information).
13C NMR spectroscopy provides useful information about
cis−trans isomerism in proline-containing peptides. 13C NMR
chemical shifts for Pro in cis and trans configurations vary
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Table 1. Cherimolacyolpeptide E (1) and Alanine-Substituted Analogues 2−9
compound
substituted residue
m/z [M + H]+ (mol. formula, calcd)
sequence of compound
1
2
3
4
5
6
7
8
9
none
Leu
Gly
Pro
Tyr
Phe
Gly
Pro
Pro
635.3157 (C33H43N6O7, 635.3193)
593.2776 (C30H37N6O7, 593.2724)
649.3379 (C34H45N6O7, 649.3350)
609.3102 (C31H41N6O7, 609.3037)
543.2981 (C27H39N6O6, 543.2931)
559.2815 (C27H39N6O7, 559.2880)
649.3311 (C34H45N6O7, 649.3350)
667.3119 (C34H45N6O7, 667.3086)
595.2819 (C30H39N6O7, 595.2874)
cyclo (Leu-Gly-Pro-Tyr-Phe-Gly)
cyclo (Ala-Gly-Pro-Tyr-Phe-Gly)
cyclo (Leu-Ala-Pro-Tyr-Phe-Gly)
cyclo (Leu-Gly-Ala-Tyr-Phe-Gly)
cyclo (Leu-Gly-Pro-Ala-Phe-Gly)
cyclo (Leu-Gly-Pro-Tyr-Ala-Gly)
cyclo (Leu-Gly-Pro-Tyr-Phe-Ala)
cyclo (Leu-Gly-Glu-Tyr-Phe-Gly)
cyclo (Leu-Gly-Gly-Tyr-Phe-Gly)
Peptide Synthesis. To the 4-sulfamylbutyryl AM resin (Nova-
biochem, 1.1 mmol/g) preswollen in DMF were added FMOC-amino
acid (4 equiv), PyBOP (2.28 g, 4 equiv), and DIEA (1.5 mL, 8 equiv)
in 5 mL of DMF. The reaction mixture was left overnight, and the
coupling repeated twice more. The efficiency of loading was
determined by FMOC removal analysis.
ESIQTOFMS and MS/MS analysis. Results are summarized in
Tables 1 and 2.
The cytotoxic effects of cherimolacyclopeptide E (1) and its
analogues on the KB, MOLT-4, Jurkat T lymphoma, and
MDA-MB-231 cancer cell lines were evaluated by employing a
cell viability assay (Figure S3, Supporting Information).
Cherimolacyclopeptide E (1), as isolated from the natural
source, was reported to have potent cytotoxic activity against
the KB (human nasopharyngeal carcinoma) cell line, with an
IC50 value of 0.017 μM.5 However, 1 when synthesized in our
laboratory was found to elicit no cytotoxic effect (IC50 >10
μM) against the KB, MOLT-4, Jurkat T, and MDA-MB231
cancer cell lines (Table 3).
Even though the synthetic peptide 1 has the same trans-
tyrosyl-proline amide bond as present in the natural product, it
did not show the same in vitro cytotoxicity against various
cancer cell lines including the KB cell line. Several investigators
have reported that in some cases synthetic proline-containing
cyclic peptides are chemically equivalent to their natural
counterparts, while deviating in their biological activities.17−20
This has been attributed to conformational differences or the
presence of biologically active impurities in the naturally
isolated material. The same reasons may explain the variation in
biological activity of synthetic and natural cherimolacyclopep-
tide E (1). This also suggests the need for a careful re-
evaluation of the highly purified natural product against the KB
cell line under identical experimental conditions.
Peptide Coupling. The FMOC-peptidyl resin, swollen in DMF,
was deprotected by 20% piperidine in DMF for 2 min, and the
deprotection repeated for 20 min. The resin was washed (3 × DMF)
and the presence of the amino group was checked with the Kaiser test.
At the same time, the next FMOC amino acid (3 equiv) was activated
in 5 mL of DMF by DIC (3 equiv, 514 μL) and HOBt (3 equiv, 505.2
mg). The solution was filtered in case of precipitation before addition
to the deprotected peptidyl resin. Completion of reaction was checked
by a ninhydrin colorimetric test. Elongation of the linear peptide was
continued, until the FMOC group on the last residue was removed.
“Safety-Catch” Activation and Cyclative Cleavage of Cyclic
Peptides. The resin was treated with trityl chloride (1.227 g, 4 equiv)
and DIEA (1.53 mL, 8 equiv) for 2 h. After DMF washing, this step
was repeated under the same conditions. The sulfonamide linker was
activated by iodoacetonitrile (0.8 mL, 10 equiv) and DIEA (2.3 mL, 12
equiv) in 5.0 mL of N-methylpyrrolidinone for 12 h. This activation
step was repeated under the same conditions. After resin washing (N-
methylpyrrolidinone, CH2Cl2), the trityl group was removed by 5%
TFA in CH2Cl2 treatment for 2 min. The deprotection was repeated
for 2 h, followed by a Kaiser test. The linear peptidyl resin was then
washed (3 × CH2Cl2, 3 × THF, once by 1% DIEA in THF).
Immediately afterward, the resin was swollen in THF, and DIEA
(565.5 μL, 3 equiv) added. The cyclization proceeded overnight,
releasing the cyclic peptide 1 with a tert-butyl group, which was
deprotected by TFA/CH2Cl2/TIS (1:1:0.01).
Mass Spectrometric Analysis of Cherimolacyclopeptide E (1)
and Its Analogues. Matrix-assisted laser desorption/ionization
(MALDI) was carried out on an Ultraflex III TOF/TOF (Bruker
Daltonics, Bremen, Germany) mass spectrometer. All cyclic peptides
(5 pmol in MeOH/H2O, 1:1, with 0.1% TFA) were mixed with 0.5 μL
of the matrix (DHB) solution (saturated solution in 0.1% TFA/
CH3CN, 2:1) and deposited on a MALDI plate. Mass was recorded
with the ion source 1 (IS1) set to 25.00 kV and ion source 2 (IS2) set
to 21.50 kV and without delay extraction (DE). These experiments
were performed with the laser energies of 70−80%. The LIFT
experiment (MS/MS) was performed on the same instrument with
IS1 set to 8.00 kV and IS2 set to 7.15 kV, and LIFT 1 set to 19.00 kV
and without DE. The laser energies used were 75−80%. The validation
of data obtained, including baseline subtraction of the TOF data,
external calibration using peptide calibration standard (Bruker
Daltonics, Bremen, Germany), and all further data processing was
carried out by using Flex analysis 2.0 postanalysis software and for data
acquisition by Flex control 2.0.
The screening of derivatives of 1 against the KB cell line
provided interesting results. Analogues 3 and 7, where the Gly
residues were replaced with alanine, displayed a significant
antiproliferative activity against the MDA-MB231 and KB cell
lines (Table 3). As both of these compounds are Gly
replacements, and Gly is the most flexible amino acid, this
further suggests that cherimolacyclopeptide E is susceptible to
conformational equilibration, which is minimized by Ala
substitution in 3 and 7.
EXPERIMENTAL SECTION
■
General Experimental Procedures. All FMOC amino acids,
coupling reagents, and resins were purchased from Novabiochem, and
other chemicals were from Aldrich. Dichloromethane and triethyl-
amine were distilled over CaH2, and THF was distilled over Na in the
presence of benzophenone. Optical rotations were measured on a
JASCO DIP 360 polarimeter. UV spectra were recorded on a Hitachi
3200 spectrophotometer. The 1H and 13C NMR spectra were
recorded on Bruker NMR spectrometers, operating at 600 MHz
(125 MHz for 13C). HRFABMS was recorded on JEOL JMS HX 110
mass spectrometers. Chemical shifts are reported in ppm. A LC-908W
recycling preparative HPLC with a Jaigel-polyamine PBMIN-5-A
column and an analytical HPLC Waters 996 with photodiode array
detector was used.
ESIQTOFMS spectra were recorded on a Q-STAR XL mass
spectrometer (Applied Biosystems). Each cyclic peptide (5 pmol in
MeOH/H2O, 1:1, with 0.1% TFA) was infused directly into the mass
spectrometer at a flow rate of 3 μL/min to acquire full scan and
product ion mass spectra. The electrospray voltage at the spraying
needle was optimized at 5200 V. Low-energy collision-induced
dissociation (CID) experiments were performed using nitrogen
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Table 2. Mass Spectrometric Analysis of 1 and Its Analogues
MS/MS frag. of [M +
H]+
compound
structure
[M + H]+ (mol,. formula, calcd)
635.3157 (C33H43N6O7, 635.3193)
proposed fragment structure
ab
,
1
607
522
472
444
368
325
297
268
240
175
155
136
[M − CO]+
Leu1-Gly2-Pro3-Tyr4-Phe5-Gly6
a
[H-Gly2-Pro3-Tyr4-Phe5-Gly6]+
[H-Pro3-Gly2-Leu1-Gly6-Phe5]+
[(H-Pro3-Gly2-Leu1-Gly6-Phe5) − CO]+
[H-Tyr4-Phe5-Gly6]+
ab
,
ab
,
a
ab
,
[H-Pro3-Gly2-Leu1-Gly6]+
[(H-Pro3-Gly2-Leu1-Gly6) − CO]+
[H-Leu1-Gly2-Pro3]+
b
ab
,
ab
,
[(H-Leu1-Gly2-Pro3) − CO]+
a
[H-Phe5-Gly6- CO]+
ab
,
[H-Gly2-Pro3]+
a
immonium ion tyrosine
immonium ion proline
[M − CO]+
a
70
2
593.2776 (C30H37N6O7, 593.2724)
565
430
402
368
333
283
226
592
558
486
339
136
70
Ala1-Gly2-Pro3-Tyr4-Phe5-Gly6
[H-Pro3-Gly2-Ala1-Gly6-Phe5]+
[(H-Pro3-Gly2-Ala1-Gly6-Phe5)-CO]+
[H-Tyr4-Phe5-Gly6]+
[H-Gly2-Ala1-Gly6-Phe5]+
[H-Pro3-Gly2-Ala1-Gly6]+
[H-Ala1-Gly2-Pro3]+
[H-Leu1-Ala2-Pro3-Tyr4-Phe5]+
[H-Gly6-Leu1-Ala2-Pro3-Tyr4]+
[H-Phe5-Gly6-Leu1-Ala2-Pro3]+
[H-Gly6-Leu1-Ala2-Pro3]+
immonium ion tyrosine
immonium ion proline
[M − CO]+
3
4
649.3379 (C34H45N6O7, 649.3350)
Leu1-Ala2-Pro3-Tyr4-Phe5-Gly6
609.3102 (C31H41N6O7, 609.3037)
581
552
495
446
375
299
136
515
472
430
396
325
268
226
171
155
136
70
Leu1-Gly2-Ala3-Tyr4-Phe5-Gly6
[H -Leu1-Gly6-Phe5-Tyr4-Ala3]+
[H-Gly6-Phe5-Tyr4-Ala3-Gly2]+
[H- Ala3-Gly2-Leu1-Gly6-Phe5]+
[H-Gly2-Leu1-Gly6-Phe5]+
[H-Ala3-Gly2-Leu1-Gly6]+
immonium ion tyrosine
[M − CO]+
5
543.2981 (C27H39N6O6, 543.2931)
Leu1-Gly2-Pro3-Ala4-Phe5-Gly6
[H-Phe5-Gly6-Leu1-Gly2-Pro3 ]+
[H-Gly2-Pro3-Ala4-Phe5-Gly6]+
[H-Gly6-Leu1-Gly2-Pro3-Ala4]+
[H- Gly6-Leu1-Gly2-Pro3]+
[H-Leu1-Gly2-Pro3]+
[H-Gly2-Pro3-Ala4]+
[H-Leu1-Gly2]+
[H-Gly2-Pro3]+
immonium ion tyrosine
immonium ion proline
[M − CO]+
6
559.2815 (C27H39N6O7, 559.2880)
531
502
Leu1-Gly2-Pro3-Tyr4-Ala5-Gly6
[H-Pro3-Tyr4-Ala5‑Gly6-Leu1]+/[H-Ala5-Tyr4-Pro3-
Gly2-Leu1]+
445
396
325
268
155
621
592
486
339
268
240
155
136
[H-Gly2-Pro3-Tyr4-Ala5-Gly6]+
[H-Pro3-Gly2-Leu1-Gly6-Ala5 ]+
[H-Pro3-Gly2-Leu1-Gly6]+
[H-Leu1-Gly2-Pro3]+
[H-Gly2-Pro3]+
7
649.3311 (C34H45N6O7, 649.3350)
[M − CO]+
Leu1-Gly2-Pro3-Tyr4- Phe5-Ala6
[H-Leu1-Ala6-Phe5-Tyr4-Pro3]+
[H-Pro3-Gly2-Leu1-Ala6-Phe5]+
[H-Pro3-Gly2-Leu1-Ala6 ]+
[H-Pro3-Gly2-Leu1]+
[(H-Pro3-Gly2-Leu1)− CO]+
[H-Gly2-Pro3]+
immonium ion tyrosine
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Table 2. continued
MS/MS frag. of [M +
H]+
compound
structure
[M + H]+ (mol,. formula, calcd)
667.3119 (C33H43N6O9, 667.3086)
proposed fragment structure
70
immonium ion proline
8
649
639
610
554
504
486
458
339
282
136
577
567
482
448
425
391
368
285
278
221
205
136
[M + H − H2O]+
Leu1-Gly2-Glu3-*Tyr4-Phe5-Gly6
[M + H − CO]+
[H-Phe5-Tyr4-Glu3-Gly2-Leu1]+
[H-Gly6-Phe5-Tyr4-Glu3-Gly2]+
[H-Glu3-Gly2-Leu1-Gly6-Phe5]+
[H-Glu3-Gly2-Leu1-Gly6-Phe5 − H2O]+
[H-Glu3-Gly2-Leu1-Gly6-Phe5 − H2O − CO]+
[H-Glu3-Gly2-Leu1-Gly6 − H2O]+
[H-Glu3-Gly2-Leu1 − H2O]+
immonium ion tyrosine
[M + H − H2O]+
9
595.2819 (C30H39N6O7, 595.2874)
Leu1-Gly2-Gly3-Tyr4-Phe5-Gly6
[M + H − CO]+
[H-Gly6-Phe5-Tyr4-Gly3-Gly2]+
[H-Tyr4-Gly3-Gly2-Leu1-Gly6]+
[H-Gly6-Phe5-Tyr4-Gly3]+
[H-Tyr4-Gly3-Gly2-Leu1]+
[H-Gly6-Phe5-Tyr4]+
[H-Gly3-Gly2-Leu1-Gly6]+
[H-Tyr4-Gly3-Gly2]+
[H-Tyr4-Gly3]+
[H-Gly6-Phe5]+
immonium ion tyrosine
a
b
Obtained from MALDI-TOF-TOF-MS. Obtained from ESIQTOFMS.
which was then purified on silica gel preparative TLC plates (5%
MeOH/CHCl3) to obtain compound 3 as an amorphous solid. Yield
0.8%; [α]23D −20.3 (c 0.03, MeOH); UV(MeOH) λmax 285, 270, 261,
223, nm; IR (KBr) νmax 3255, 2922, 2856, 1681, 1562, 1417 cm−1;
FABMS m/z 649 [M + H]+; MS/MS (ESI+), see Table 2.
Table 3. IC50 Values of Peptide 1 and Alanine-Substituted
Analogues 2−9
cell line (IC50 μM)
compound
KB
MDA-MB-231
cyclo-Leu1-Gly2-Ala3-Tyr(OH)4-Phe5-Gly6 (4). The protected cyclic
peptide 4a (5 mg) was dissolved in a mixture of TFA/DCM/TIS
(1:1:0.01) and reacted for 1 h at room temperature. The reaction
mixture was concentrated in vacuo and purified on silica gel
preparative TLC plates (5% MeOH/CHCl3) to obtain compound 4
1
2
3
4
5
6
7
8
9
>10
>10
>10
>10
10.2
>10
>10
>10
7.7
6.3
>10
>10
>10
as an amorphous solid. Yield 4.6%; [α]23 −4.8 (c 0.1, MeOH);
D
UV(MeOH) λmax 270, 265, 240, nm; IR (KBr) νmax 3745, 2925, 2858,
1678, 1624, 1454 cm−1; FABMS m/z 609 [M + H]+; MS/MS (ESI+),
see Table 2.
7.8
>10
>10
>10
>10
0.04
cyclo-Leu1-Gly2-Pro3-Ala4-Phe5-Gly6 (5). Yield 4.9%; [α]23D −8.9 (c
0.06, MeOH); UV(MeOH) λmax 288, 275, 253, 222, nm; IR (KBr)
νmax 3622, 2921, 2862, 1681, 1461 cm−1; FABMS m/z 543 [M + H]+;
MS/MS (ESI+), see Table 2.
doxorubicin (positive control)
0.4
(CID gas valve set to 4) as collision gas, and a collision energy of 35
eV was used.
cyclo-Leu1-Gly2-Pro3-Tyr (OH)4-Ala5-Gly6 (6). The protected cyclic
peptide 6a (5.5 mg) was dissolved in a mixture of TFA/DCM/TIS
(1:1:0.01) and reacted for 1 h at room temperature. The reaction
mixture was concentrated in vacuo and purified on silica gel
preparative TLC plates (5% MeOH/CHCl3) to obtain compound 6
cyclo-Leu1-Gly2-Pro3-Tyr (OH)4-Phe5-Gly6 (Synthetic Cherimola-
cyclopeptide E, 1). Yield 9.8%; [α]23D −46.0 (c 0.3, MeOH, lit. [α]23
D
−56.0 (c 0.005, MeOH); UV (MeOH) λmax 267, 254, 226, nm; IR
1
(KBr) νmax 3310, 2922, 1693, 1562, 1514, 1413 cm−1; H NMR and
as a gummy material. Yield 2.2%; [α]23 −11.9 (c 0.04, MeOH);
13C NMR, see Table S1; HRMS (ESI+) m/z 635.3157 [M + H]+
(C33H43N6O7, calcd for 635.3193).
D
UV(MeOH) λmax 275, 246, 223, nm; IR (KBr) νmax 3722, 2925, 2862,
1732, 1683, 1458 cm−1; FABMS m/z 559 [M + H]+; MS/MS (ESI+),
see Table 2.
cyclo-Ala1-Gly2-Pro3-Tyr(OH)4-Phe5-Gly6 (2). The protected cyclic
peptide 2a (4.5 mg) was dissolved in a mixture of TFA/DCM/TIS
(1:1:0.01) and reacted for 1 h at room temperature. The reaction
mixture was concentrated in vacuo and purified on silica gel
preparative TLC plates (5% MeOH/CHCl3) to obtain compound 2
cyclo-Leu1-Gly2-Pro3-Tyr (OH)4-Phe5-Ala6 (7). The protected cyclic
peptide 7a (10 mg) was dissolved in a mixture of TFA/DCM/TIS
(1:1:0.01) and reacted for 2 h at room temperature. The reaction
mixture was concentrated in vacuo and purified on silica gel
preparative TLC plates (5% MeOH/CHCl3) to obtain compound 7
as an amorphous solid. Yield 1.4%; [α]23 −10.8 (c 0.06, MeOH);
D
UV(MeOH) λmax 295, 286, 275, 269, 225, nm; IR (KBr) νmax 3292,
2923, 2858, 1678, 1560, 1452 cm−1; FABMS m/z 593 [M + H]+; MS/
MS (ESI+), see Table 2.
as a gummy material. Yield 1.7%; [α]23 −5.1 (c 0.04, MeOH);
D
UV(MeOH) λmax 286, 252, 250, nm; IR (KBr) νmax 3300, 2923, 2858,
1733, 1674, 1560, 1456 cm−1; FABMS m/z 649; MS/MS (ESI+), see
Table 2.
cyclo-Leu1-Ala2-Pro3-Tyr (OH)4-Phe5-Gly6 (3). The protected cyclic
peptide 3a (5.0 mg) was dissolved in a mixture of TFA/DCM/TIS
(1:1:0.01) and reacted for 1 h at room temperature. The reaction
mixture was concentrated in vacuo to obtain the crude cyclic peptide,
cyclo-Leu1-Gly2-Glu3-Tyr (OH)4-Phe5-Gly6 (8). The protected cyclic
peptide (4 mg) was dissolved in a 2 mL mixture of TFA/DCM/TIS
1886
dx.doi.org/10.1021/np300266e | J. Nat. Prod. 2012, 75, 1882−1887

Journal of Natural Products
Article
(1:1:0.01) and reacted for 1 h at room temperature. The reaction
mixture was concentrated in vacuo and purified on silica gel
preparative TLC plates (5% MeOH/CHCl3) to obtain compound 8
(5) Wel
́
e,
́
A.; Zhang, Y.; Ndoye, I.; Brouard, J. P.; Pousset, J. L.;
Bodo, B. Phytochemistry 2005, 66, 693−696.
(6) Wele, A.; Zhang, Y.; Brouard, J-. P.; Pousset, J. L.; Bodo, B.
Phytochemistry 2005, 66, 2376−2380.
(7) Wele, A.; Zhang, Y.; Dubost, L.; Pousset, J. L.; Bodo, B. Nigerian
́
́
as a gummy material. Yield 4.2%; [α]23 −10.7 (c 0.05, MeOH);
D
UV(MeOH) λmax 299, 276, 264, 247, nm; IR (KBr) νmax 2925, 2860,
1733, 1552, 1510, 1460 cm−1; FABMS m/z 667 [M + H]+; MS/MS
(ESI+), see Table 2.
́
́
J. Nat. Prod. Med. 2005, 9, 68−72.
(8) Dahiya, R. J. Chil. Chem. Soc. 2007, 52, 1224−1229.
(9) (a) Backes, B. J.; Virgilio, A. A.; Ellman, J. A. J. Am. Chem. Soc.
1996, 118, 3055−3056. (b) Yang, L.; Morriello, G. Tetrahedron Lett.
1999, 40, 8197−8200.
cyclo-Leu1-Gly2-Gly3-Tyr (OH)4-Phe5-Gly6 (9). The protected cyclic
peptide (4 mg) was dissolved in a 2 mL mixture of TFA/DCM/TIS
(1:1:0.01) and reacted for 1 h at room temperature. The reaction
mixture was concentrated in vacuo and purified on silica gel
preparative TLC plates (5% MeOH/CHCl3) to obtain compound 9
(10) (a) Heidler, P.; Link, A. Bioorg. Med. Chem. 2005, 13, 585−599.
(b) Ganesan, A. In Linker Strategies in Solid-phase Organic Synthesis;
Scott, P., Ed.; Wiley: New York, 2009; pp 135−149. (c) Lebreton, S.;
Pateek, M. In Linker Strategies in Solid-phase Organic Synthesis; Scott,
P., Ed.; Wiley: New York, 2009; pp 195−219. (d) White, J. C.; Yudin,
A. K. Nat. Chem. 2011, 3, 509−524.
as a gummy material. Yield 3.1%; [α]23 −37.7 (c 0.01, MeOH);
D
UV(MeOH) λmax 298, 277, 265, 248 nm; IR (KBr) νmax 3255, 2922,
2856, 1681, 1562, 1417 cm−1; FABMS m/z 595 [M + H]+ ; MS/MS
(ESI+), see Table 2.
Cytotoxicity Assays. Human KB cells, human MDA-MB-231
breast cancer cells, MOLT-4, and Jurkat leukemia cells were seeded in
a 96-well plate at the cell densities of 4 × 103 cells/well. After
overnight incubation, the cells were treated with increasing
concentrations (ranging between 0.064 and 40 μM) of parent
cherimolacyclopeptide E (1) and its corresponding alanine-substituted
analogues 2−9. The compounds were first dissolved in DMSO and
then diluted to the desired concentration with culture medium. After
72 h incubation, CellTiter 96 Aqueous Cell Proliferation Reagent
(Promega, Madison, WI, USA), which is composed of MTS [3-(4,5-
dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)-
2H-tetrazolium] and the electron coupling reagent PMS (phenazine
methosulfate), was added to each well, according to the manufacturer’s
instructions. The cell viability was determined by measuring the
absorbance at 490 nm by using a microplate ELISA reader
(SpectraMax M2, Molecular Devices, Sunnyvale, CA, USA). Results
were shown as the average cell viability [(ODtreat − ODblank)/(ODcontrol
− ODblank) × 100%] of triplicate wells. Untreated cells (DMSO +
medium) served as the positive control.
(11) Bourel-Bonnet, L.; Rao, K. V.; Hamann, M. T.; Ganesan, A. J.
Med. Chem. 2005, 48, 1330−1335.
(12) Ali, L.; Musharraf, S. G.; Shaheen, F. J. Nat. Prod. 2008, 71,
1059−1062.
(13) Siemion, I. Z.; Wieland, T.; Pook, K. H. Angew. Chem., Int., Ed.
Engl. 1975, 14, 702−703.
(14) Auvin-Guette, C.; Baraguey, C.; Blond, A.; Xavier, H. S.;
Pousset, J. L.; Bodo, B. Tetrahedron 1999, 55, 11495−11510.
(15) Tabudravu, J.; Morris, L. A.; Kettenes-van den Bosch, J. J.;
Jaspars, M. Tetrahedron Lett. 2001, 42, 9273−9276.
(16) Morrison, K. L.; Weiss, G. A. Curr. Opin. Chem. Biol. 2001, 5,
302−307.
(17) Napolitano, A.; Rodriquez, M.; Bruno, I.; Marzocco, S.; Autore,
G.; Riccio, R.; Gomez-Paloma, L. Tetrahedron 2003, 59, 10203−
10211.
(18) Napolitano, A.; Bruno, I.; Riccio, R.; Gomez-Paloma, L.
Tetrahedron 2005, 61, 6808−6815.
(19) Pettit, G. R.; Toki, B. E.; Xu, J.-P.; Brune, D. C. J. Nat. Prod.
2000, 63, 22−28.
(20) Fairweather, K. A.; Sayyadi, N.; Roussakis, C.; Jolliffe, K. A.
Tetrahedron 2010, 66, 935−939.
ASSOCIATED CONTENT
* Supporting Information
Complete NMR data of synthetic and natural peptide 1 and
cytotoxicity data of synthetic peptides 1−9; H spectra of
compound 1. This material is available free of charge via the
■
S
1
Internet at http://pubs.acs.org.
AUTHOR INFORMATION
Corresponding Author
*Phone: (92-213) 4824925. Fax: (92-213) 4819018. E-mail:
afnan.iccs@gmail.com.
■
Notes
The authors declare no competing financial interest.
ACKNOWLEDGMENTS
■
This work was supported by a grant (L/ICA/ICB/157800/10)
from the Organization for the Prohibition of Chemical
Weapons (OPCW), The Netherlands.
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dx.doi.org/10.1021/np300266e | J. Nat. Prod. 2012, 75, 1882−1887