“Peptaibiomics”: Peptaibols from Mycosynthetix Fungus MSX … · 2015-07-14 · 3Natural...
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0.00 2.00 4.00 6.00 8.00 10.00 12.00 compound id Peptaibols IC 50 (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 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. Oberlies 1,* 1 Department of Chemistry and Biochemistry, University of North Carolina at Greensboro, Greensboro, NC 27402 2 Department of Pharmaceutical Sciences, BRITE, North Carolina Central University, Durham, NC 27707 3 Natural Products Laboratory, Research Triangle Institute, RTP, NC 27709 4 Mycosynthetix 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% CH 3 CN in H 2 O from 0-30 min, 15 mL/min, Phenomenex Synergi C 12 , 250×21.2mm, 4 μm, 205nm, 100 mg/injection, six injections, 600 mg total. Figure 5: CD 3 OH, 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-70741 2 was grown on solid rice media and extracted with 1:1 CHCl 3 /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: 1 H/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 C 91 H 149 N 23 O 24 2 Ac-Aib-Pro-Aib-Ala-Aib-Ser-Gln-Aib-Val-Aib-Gly-Leu-Aib-Pro-Val-Aib-Aib-Gln-Gln-Pheol 1979.12764 C 92 H 151 N 23 O 25 3 Ac-Aib-Pro-Aib-Ala-Aib-Ser-Gln-Aib-Val-Aib-Gly-Leu-Aib-Pro-Val-Aib-Iva*-Gln-Gln-Pheol 1993.14429 C 93 H 153 N 23 O 25 4 Ac-Aib-Pro-Aib-Ala-Aib-Gly-Gln-Aib-Val-Aib-Gly-Leu-Aib-Pro-Val-Aib-Aib-Gln-Gln-Pheol 1949.11357 C 92 H 151 N 23 O 25 5 Ac-Aib-Pro-Aib-Ser-Aib-Ala-Gln-Aib-Val-Aib-Gly-Leu-Aib-Pro-Val-Aib-Aib-Gln-Gln-Pheol 1979.12996 C 91 H 149 N 23 O 24 6 Ac-Aib-Pro-Aib-Ala-Aib-Ala-Gln-Aib-Val-Aib-Gly-Val-Aib-Pro-Val-Aib-Aib-Gln-Gln-Pheol 1949.11454 C 91 H 149 N 23 O 24 7 Ac-Aib-Ser-Val-Ile-Aib-Pro-Leu-Leu-Aib-Pro-Valol 1120.73083 C 55 H 97 N 11 O 13 8 Ac-Aib-Pro-Aib-Ala-Aib-Ala-Gln-Aib-Val-Aib-Gly-Leu-Aib-Pro-Val-Aib-Aib-Gln-Gln-Pheol 1963.13209 C 92 H 151 N 23 O 24 9 Ac-Aib-Pro-Aib-Ala-Aib-Ala-Gln-Aib-Val-Aib-Gly-Leu-Aib-Pro-Val-Aib-Iva*-Gln-Gln-Pheol 1977.14808 C 93 H 153 N 23 O 24 10 Ac-Aib-Pro-Aib-Ala-Aib-Aib-Gln-Aib-Val-Aib-Gly-Leu-Aib-Pro-Val-Aib-Aib-Gln-Gln-Pheol 1977.14682 C 93 H 153 N 23 O 24 11 Ac-Aib-Pro-Aib-Ala-Aib-Aib-Gln-Aib-Val-Aib-Gly-Leu-Aib-Pro-Val-Aib-Aib-Glu-Gln-Pheol 1978.13092 C 93 H 152 N 22 O 25 12 Ac-Aib-Pro-Aib-Ala-Aib-Aib-Gln-Aib-Val-Aib-Gly-Leu-Aib-Pro-Val-Aib-Iva*-Glu-Gln-Pheol 1992.14756 C 94 H 154 N 22 O 25 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-Serine D-Serine L-Glutamic acid L-Alanine L-Proline D-Glutamic acid D-Proline D-Alanine α-Aminoisobutyric acid L-Valine L-Isovaline D-Isovaline L-Phenylalaninol L-Leucine D-Leucine D-Valine D-Phenylalaninol Unreacted Marfey’s reagent + Glycine 01008-44-1_110419104241 # 287-302 RT: 2.12-2.19 AV: 4 NL: 5.71E7 F: 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 90 100 588.34096 920.51928 452.24907 1029.60876 724.43448 1950.12025 296.15996 1504.36283 809.48760 1820.26259 199.23971 1136.72913 1640.41245 01008-44-1_110419104241 # 287-302 RT: 2.12-2.19 AV: 4 NL: 3.20E5 F: 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 1950.12025 1951.12505 1952.12517 1953.13277 C 91 H 149 N 23 O 24 + H (-3.5 ppm) 01008-44-1_110419104241 # 288-300 RT: 2.12-2.17 AV: 3 NL: 6.72E6 F: 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 80 100 169.13283 393.21209 299.17063 373.18585 257.12360 478.26420 152.10630 512.31710 757.42274 342.17623 214.11797 427.22885 538.29634 606.32276 578.32827 101.07018 629.85765 809.00432 711.05333 682.91693 892.52350 860.34888 01008-44-1_110419104241 # 288-300 RT: 2.13-2.18 AV: 3 NL: 3.13E6 F: 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 80 100 538.29689 807.47075 774.40393 199.24014 1264.05151 273.66501 88.55112 469.27496 588.83038 711.01343 380.39304 153.37914 1022.81500 850.81995 412.75003 308.62500 888.42352 01008-44-1_110419104241 # 288-300 RT: 2.11-2.18 AV: 4 NL: 2.75E6 F: 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 80 100 254.14907 339.20177 183.11212 155.11718 268.16475 410.23834 285.15493 325.18633 200.10229 427.26471 129.10160 186.12304 311.20677 170.08038 356.19110 382.24375 101.07028 214.11791 242.14911 84.04359 441.24237 343.96100 142.93535 467.99933 488.90503 455.07306 372.02252 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 H 2 O (w/ 0.1% HCO 2 H) in 10 min, BEH C18, 50×2.1 mm, 1.7 μm, 40°C, 0.5 mL/min, 340 nm. -CO Q NH 2 ’s 2D-NOESY: NH Region (gives locations of valine vs. isovaline) G11 U13 U10 U8 L12 E18 Fol20 V9 U6 U5 Q19 Q7 J17 U3 U16 A4 V15 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.
Transcript of “Peptaibiomics”: Peptaibols from Mycosynthetix Fungus MSX … · 2015-07-14 · 3Natural...
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H460
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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
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4 5
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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
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D-S
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L-G
luta
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L-A
lanin
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α-A
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L-P
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lan
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Un
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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]
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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
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40
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60
70
80
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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
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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
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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.
