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compound id
Peptaibols IC50 (µM)
MCF-7wtk
H460
SF268
IMR90
AU
0.00
0.10
0.20
0.30
0.40
0.50
0.60
0.70
0.80
0.90
1.00
1.10
1.20
1.30
1.40
1.50
1.60
1.70
1.80
Minutes
0.00 0.50 1.00 1.50 2.00 2.50 3.00 3.50 4.00 4.50 5.00 5.50 6.00 6.50 7.00 7.50 8.00 8.50 9.00 9.50 10.00
FIGURE 2: PREPARATIVE-HPLC OF CRUDE PEPTAIBOLS
INTRODUCTION
ACKNOWLEDGMENTS
We thank the National Cancer Institute/National Institutes of Health
for research support via grant P01 CA125066, and Kevin Knagge of
the David H. Murdock Research Institute (Kannapolis, NC) for 950
MHz NMR data.
FIGURE 1: STRUCTURES OF ISOLATED PEPTAIBOLS 1-12
REFERENCES AND NOTES
FIGURE 6: UPLC of Marfey’s Products for 2
CONCLUSIONS
“Peptaibiomics”: Peptaibols from Mycosynthetix Fungus MSX-70741 (Trichoderma sp.)
Sloan Ayers,1 Brandie M. Ehrmann,1 Audrey F. Adcock,2 David J. Kroll,2 Mansukh C. Wani,3 Cedric J. Pearce,4 and Nicholas H. Oberlies1,*
1Department of Chemistry and Biochemistry, University of North Carolina at Greensboro, Greensboro, NC 27402
2Department of Pharmaceutical Sciences, BRITE, North Carolina Central University, Durham, NC 27707
3Natural Products Laboratory, Research Triangle Institute, RTP, NC 27709
4Mycosynthetix Inc., Hillsborough, NC 27278
FIGURE 4: MS/MS OF PEPTAIBOL 1 (HCD, single injection)
Figure 2: Preparative-HPLC of 100% MeOH wash from silica-gel flash chromatography:
40-100% CH3CN in H2O from 0-30 min, 15 mL/min, Phenomenex Synergi C12, 250×21.2mm,
4 µm, 205nm, 100 mg/injection, six injections, 600 mg total.
Figure 5: CD3OH, 950 MHz (with presaturation of -OH peak at 4.9 ppm)
• “Peptaibiomics” is a term coined by Brückner to represent the “analytical
methodology for structural characterization of all peptaibiotics in fungal
cells”.1
• “Peptaibiotics” or “peptaibols” are bioactive non-ribosomal peptides, usually
from the Trichoderma genus of filamentous fungi. The term “peptaibol”
originates from the peptidic nature of the compounds, containing a high
number of α-aminoisobutyric acid (Aib) residues, and terminating with an
amino alcohol at the C-terminus. The N-terminus is usually acetylated.
• Mycosynthetix fungus MSX-707412 was grown on solid rice media and
extracted with 1:1 CHCl3/MeOH. Flash silica-gel chromatography followed by
preparative RP-HPLC was used to purify the peptaibols.
• Twelve peptaibols were isolated. High-resolution MS/MS was utilized for
obtaining the amino acid sequence of the peptaibols on a Thermo LTQ-
Orbitrap using HCD (Higher-Energy Collisional Dissociation) for MS/MS.
• TOCSY and 2D-NOESY were required when a peptaibol contained both valine
and isovaline residues (as with 3, 9, and 12), or leucine and isoleucine (as with
7), and to confirm the sequences determined by MS/MS.
• Determination of the configuration of the amino acids was accomplished
from acid degradation of the peptaibols and subsequent derivatization of the
resulting free amino acids by Marfey’s reagent.3
• Compounds 1-12 were assayed for cytotoxic activity in vitro against three
cancer cell lines: H460, SF268, and MCF-7.
• The antibacterial activity (MRSA) of these compounds was also determined.
• Anthelmintic activity is being assessed in vitro using Haemonchus contortus.
• This class of compound is known to form ion channels in membranes, so 1-12
are being evaluated in a mitochondrial transmembrane potential assay (ΔΨ).
• Other cytotoxic compounds from MSX-70741 were the trichothecene
derivatives harzianum A and B.4
Figure 3: Conditions: Source Voltage = 4.5 kV, Tube Lens Voltage = 115 V, Capillary Voltage = 46 V.
1. Krause, C.; Kirschbaum, J.; Brückner, H. Chem. & Biodiv. 2007, 4, 1083-1102.
2. Mycosynthetix fungus MSX-70741: closest MIDI 16S DNA match =
Trichoderma minutsporium M8510; closest GenBank match = Trichoderma sp.
AF399255.
3. Kjer, J.; Debbab A.; Proksch, P. Nat. Protocols 2010, 5, 479-490.
4. Jin, H.-Z.; Lee J.-H.; Zhang, W.-D., Lee, H.-B.; Hong, Y.-S.; Kim, Y.-H.; Lee, J.-
J. J. Asian Nat. Prod. 2007, 9, 203-207.
1: 1.13 mg 7: 012.39 mg
2: 3.28 mg 8: 242.46 mg
3: 0.72 mg 9: 077.56 mg
4: 2.00 mg 10: 52.64 mg
5: 2.62 mg 11: 28.78 mg
6: 6.66 mg 12: 04.11 mg
• HCD is an effective technique for high-resolution MS/MS
fragmentation of peptaibols to obtain the amino acid sequence.
High-resolution is useful for distinguishing leucine/isoleucine from
hydroxyproline, which have the same nominal mass but different
formulae. HCD also is not bound by the so-called “Rule of One-
Third” which is a limitation of CID.
• NMR was necessary to distinguish between leucine and isoleucine
as well as valine and isovaline. NMR was also required when a
peptaibol contained both valine and isovaline (3, 9, 12), or both
leucine and isoleucine (7), to determine their locations in the
sequence.
• The bioassay data for peptaibols revealed some preliminary
structure-activity relationships.
For cytotoxicity: the only peptaibol that was inactive contained
Ala1 instead of Aib1.
For anti-MRSA activity: the peptaibols with Iva17 were slightly
more active.
Other peptaibols isolated in our laboratory containing Ala2
instead of Pro2 showed no significant difference in activity.
FIGURE 7: Cytotoxicity Results for 1-12
FIGURE 3: HRMS OF PEPTAIBOL 1 FIGURE 5: 1H/NOESY NMR OF PEPTAIBOL 12
2
Compound Sequence Exp. [M + H]+ Formula
1 Ac-Ala-Pro-Aib-Ala-Aib-Ala-Gln-Aib-Val-Aib-Gly-Leu-Aib-Pro-Val-Aib-Aib-Gln-Gln-Pheol 1949.11558 C91H149N23O24
2 Ac-Aib-Pro-Aib-Ala-Aib-Ser-Gln-Aib-Val-Aib-Gly-Leu-Aib-Pro-Val-Aib-Aib-Gln-Gln-Pheol 1979.12764 C92H151N23O25
3 Ac-Aib-Pro-Aib-Ala-Aib-Ser-Gln-Aib-Val-Aib-Gly-Leu-Aib-Pro-Val-Aib-Iva*-Gln-Gln-Pheol 1993.14429 C93H153N23O25
4 Ac-Aib-Pro-Aib-Ala-Aib-Gly-Gln-Aib-Val-Aib-Gly-Leu-Aib-Pro-Val-Aib-Aib-Gln-Gln-Pheol 1949.11357 C92H151N23O25
5 Ac-Aib-Pro-Aib-Ser-Aib-Ala-Gln-Aib-Val-Aib-Gly-Leu-Aib-Pro-Val-Aib-Aib-Gln-Gln-Pheol 1979.12996 C91H149N23O24
6 Ac-Aib-Pro-Aib-Ala-Aib-Ala-Gln-Aib-Val-Aib-Gly-Val-Aib-Pro-Val-Aib-Aib-Gln-Gln-Pheol 1949.11454 C91H149N23O24
7 Ac-Aib-Ser-Val-Ile-Aib-Pro-Leu-Leu-Aib-Pro-Valol 1120.73083 C55H97N11O13
8 Ac-Aib-Pro-Aib-Ala-Aib-Ala-Gln-Aib-Val-Aib-Gly-Leu-Aib-Pro-Val-Aib-Aib-Gln-Gln-Pheol 1963.13209 C92H151N23O24
9 Ac-Aib-Pro-Aib-Ala-Aib-Ala-Gln-Aib-Val-Aib-Gly-Leu-Aib-Pro-Val-Aib-Iva*-Gln-Gln-Pheol 1977.14808 C93H153N23O24
10 Ac-Aib-Pro-Aib-Ala-Aib-Aib-Gln-Aib-Val-Aib-Gly-Leu-Aib-Pro-Val-Aib-Aib-Gln-Gln-Pheol 1977.14682 C93H153N23O24
11 Ac-Aib-Pro-Aib-Ala-Aib-Aib-Gln-Aib-Val-Aib-Gly-Leu-Aib-Pro-Val-Aib-Aib-Glu-Gln-Pheol 1978.13092 C93H152N22O25
12 Ac-Aib-Pro-Aib-Ala-Aib-Aib-Gln-Aib-Val-Aib-Gly-Leu-Aib-Pro-Val-Aib-Iva*-Glu-Gln-Pheol 1992.14756 C94H154N22O25
1
2
3
4 5
6
7
8
9
10
11
12
Figure 4: a) MS/MS of 774 peak, CE = 22. b) MS/MS of 1175 peak, CE = 15. c) MS/MS of 1175
peak, CE = 35. Fragmentation by HCD. CE = Collision Energy.
* Iva = isovaline
L-S
erine
D-S
erine
L-G
luta
mic
acid
L-A
lanin
e
L-P
rolin
e
D-G
luta
mic
acid
D
-Pro
line
D-A
lanin
e
α-A
min
ois
ob
uty
ric a
cid
L-V
alin
e
L-I
so
va
line
D-I
so
va
line
L-P
he
nyla
lan
ino
l
L-L
eu
cin
e
D-L
eu
cin
e
D-V
alin
e
D-P
he
nyla
lan
ino
l
Un
rea
cte
d M
arf
ey’s
re
ag
en
t +
Gly
cin
e
01008-44-1_110419104241 #287-302 RT: 2.12-2.19 AV: 4 NL: 5.71E7F: FTMS + p ESI Full ms [75.00-2000.00]
100 200 300 400 500 600 700 800 900 1000 1100 1200 1300 1400 1500 1600 1700 1800 1900 2000
m/z
0
10
20
30
40
50
60
70
80
90
100
Re
lative
Ab
un
da
nce
774.44883
1175.67515
975.56485
650.71126
588.34096
1300.41884920.51928452.24907 1029.60876
1205.68796
724.43448 1950.12025296.15996 1504.36283
809.48760
1820.26259199.23971
1136.72913
1640.41245
01008-44-1_110419104241 #287-302 RT: 2.12-2.19 AV: 4 NL: 3.20E5F: FTMS + p ESI Full ms [75.00-2000.00]
1946 1948 1950 1952 1954 1956
m/z
0
10
20
30
40
50
60
70
80
90
100
Rela
tive A
bundance
1950.12025
1949.11558
1951.12505
1952.12517
1953.13277
C91H149N23O24 + H
(-3.5 ppm)
01008-44-1_110419104241 #288-300 RT: 2.12-2.17 AV: 3 NL: 6.72E6F: FTMS + c ESI Full ms2 [email protected] [80.00-900.00]
100 150 200 250 300 350 400 450 500 550 600 650 700 750 800 850 900
m/z
0
20
40
60
80
100
Rela
tive A
bundance
282.18024
367.23280
495.29047197.12774
774.44965169.13283 623.34914393.21209299.17063
373.18585
257.12360 478.26420152.10630 512.31710 757.42274342.17623214.11797 427.22885 538.29634 606.32276578.32827101.07018 629.85765 809.00432711.05333682.91693 892.52350860.34888
01008-44-1_110419104241 #288-300 RT: 2.13-2.18 AV: 3 NL: 3.13E6F: FTMS + c ESI Full ms2 [email protected] [80.00-1300.00]
100 150 200 250 300 350 400 450 500 550 600 650 700 750 800 850 900 950 1000 1050 1100 1150 1200 1250 1300
m/z
0
20
40
60
80
100
Rela
tive A
bundance
920.51861
736.39671
835.46569
523.28526452.24857 977.54128 1090.62242 1175.67553538.29689 807.47075774.40393651.34611199.24014 1264.05151273.6650188.55112 469.27496 588.83038
928.66107
711.01343380.39304153.37914 1022.81500850.81995412.75003308.62500
888.42352
01008-44-1_110419104241 #288-300 RT: 2.11-2.18 AV: 4 NL: 2.75E6F: FTMS + c ESI Full ms2 [email protected] [80.00-1300.00]
80 100 120 140 160 180 200 220 240 260 280 300 320 340 360 380 400 420 440 460 480
m/z
0
20
40
60
80
100
Rela
tive A
bundance
254.14907
296.15961
211.10701367.19653
339.20177183.11212
155.11718 452.24878268.16475
410.23834285.15493 325.18633200.10229 427.26471129.10160
186.12304
311.20677170.08038 356.19110 382.24375101.07028 214.11791 242.1491184.04359 441.24237
343.96100
142.93535 467.99933 488.90503
455.07306372.02252
300.42007
a)
b)
c)
774.44883
1175.67515
975.56485 650.71126
1949.11558
774.44965 623.34914 495.29047
367.23280 282.18024
197.12774
1175.67553
452.24878
452.24857 1090.62242
977.54128
920.51861
835.46569 736.39671
523.28526
367.19653
296.15961 211.10701
651.34611
Pheol Gln Gln
Aib
Aib
Aib Leu
Gly
in-source fragmentation
(double-charged) (triple-charged)
(triple-charged dimer)
1300.41884
Aib
Val Aib Gln Ala
Aib Ala
Aib
(To 0)
Pro+Val
Ac+Ala+Pro -CO -CO -CO
(To 0)
Standards
Compound 2
Acid Hydrolysate
Figure 6: 10-70% MeOH in H2O (w/ 0.1% HCO2H) in 10 min,
BEH C18, 50×2.1 mm, 1.7 µm, 40°C, 0.5 mL/min, 340 nm.
-CO
Q NH2’s
2D-NOESY: NH Region (gives locations of valine vs. isovaline)
G11
U13
U10
U8
L
12
E1
8
Fol2
0
V9
U6
U5
Q
19
Q7
J17
U3
U16
A4
V
15
U = α-aminoisobutyric acid
G = glycine
L = leucine
E = glutamic acid
Q = glutamine
J = isovaline
V = valine
A = alanine
Fol = phenylalaninol U1
-CO
* Positive control.
** These are peptaibols isolated from Mycosynthetix fungus MSX-57715. These compounds have Ala2 instead of Pro2.