FULL PAPER

Structures of Peptaibol Antibiotics Hypomurocin A and B from the Ascomycetous Fungus Hypocrea muroiana Hino et Katsumoto Dieter Beckera, Michael Kiessb, and Hans Briickner*a,C

Institut fur Lebensmitteltechnologie der Universitat Hohenheim", Garbenstralje 25, D-70599 Stuttgart, Germany

Gesellschaft fur Biotechnologische Forschungb, Mascheroder Weg 1, D-38124 Braunschweig, Germany

Institut fur Ernahrungswissenschaft der Universitat Giessen", Sudanlage 6, D-35390 Giessen, Germany Fax: (internat.) +49(0)641/99-39149

Received October 10, 1996

Keywords: Peptide antibiotics I Mycotoxins I Peptaibols I Peptaibiotics I Sequence determination I Mass spectrometry I Non-proteinogenic amino acids I a-Aminoisobutyric acid I Isovaline

From a submerse culture of the fungus Hypocrea muroiana Hino et Katsumoto (Ascomycetes: Hypocreales) two major groups of peptides, designated hypomurocin (HM) A and B, belonging to the peptaibol family, could be characterized. Both groups showed antibiotic activity (Bacillus subtilis) and caused hemolysis of rat erythrocytes, HM A being less active than HM B. Peptides were isolated from the culture broth by chromatography on XAD-2 adsorber resin, octyl silica, and Sephadex LH 20. HM A and B were separated by preparative TLC, and individual peptides from each microheterogeneous group were isolated by preparative HPLC. Amino acid analy- sis and sequence determination by fast atom bombardment and electrospray tandem mass spectrometry revealed the composition and structures of six 11-mer peptides of the HM

A group and of six 18-mer peptides of the HM B group. Posi- tions of isomeric amino acids Leu/Ile and Val/Iva (present in some of the peptides) were determined by methanolytic cleavage of the pure peptides, followed by trifluoroacetyl- ation of the dipeptide methyl esters released and assignment of their structures by gas chromatography-selected ion moni- toring mass spectrometry. As examples, two sequences of HM A and HM B are presented (exchange positions in paren- theses). - HM A-l(2): Ac-Aibl(D-Ival)-Gln-Val-Val-Aib-Pro- Leu-Leu-Aib-Pro-Leuolll; HMB-l(2): Ac-Aibl-Ser-Ala-Leu- Aib-Gln-Aib-Val-Aib-Gly-Aib-Aib-Pro-Leu-Aib-Aib-Gln- Valo118 (Leuol18), (Ac = acetyl, Aib = a-aminoisobutyric acid, Iva = D-isovaline, Leuol = L-leucinol, Valol = L-valinol).

Introduction

Peptaibols['.21 are defined as a class of fungal peptide antibioticslmycotoxins which contain the characteristic non-proteinogenic amino acids a-aminoisobutyric acid (Aib) and, in many cases, D- or L-isovaline ( I v ~ ) [ ~ * ~ ] as well as proteinogenic amino acids. They have an acylated N-ter- minus and an L-amino alcohol forming the C-terminus. For peptaibols with phenylalaninol (Pheol) as the C-terminus, the name peptaibophols has been Since Aib- peptides lacking the amino as well as some with unusual N-terminiL7I or heterocyclic C-termini['] have been detected, the comprehensive name peptaibiotics[6] has been suggested. These are defined as fungal peptides which con- tain Aib and exert biological activity. Peptaibols such as alamethi~in[~I, parace l~ in[~~~I , trichorzianine['Ol, aibellin[' '1, trichovirinL6I, trichorozin[l21, harzianin[l31, and trichobra- chin[14] exhibit a wide spectrum of biological activities. They show antibiotic activities such as bactericidal[151, fungicidal[16], and insecticidal[8] action, and are capable of forming pores in biological membranes thus modifying their ion transport properties['7.'8]. Further, synergistic ef- fects of peptaibols against phytopathogenic microorgan- i s m ~ [ ' ~ ] and uncoupling of oxidative phosphorylation in mi- tochondria[201 have been reported. Here, we describe the se-

quence determination of microheterogeneous mixtures of 1 1-mer and 18-mer peptaibols, named hypomurocin A and B, respectively. The peptides were isolated from submerged cultures of the ascomycetous fungus Hypocrea muroiana Hino et Katsumoto. Hypocrea muroiana, a saprophytic fungus, is the most common of its genus in Japan and a parasite of the edible mushroom "Shiitake" (Lentinus edodes)[2'l.

Results and Discussion

Production of Aib peptides and the antibiotic activity of extracts of Hypocrea muroiana have been reported[22]. In order to characterize the active principle, submerse fermen- tation of the fungus was carried out. Under the applied conditions, its anamorph, designated as Trichoderma harzi- anum according to Bi~se t t [~~] , was cultivated in shaken flasks. In the course of the fermentation on synthetic me- dium, aliquots of the culture broth were taken every day and the production of hypomurocins was monitored (Ex- perimental Section). The combined filtered culture broths were passed through an Amberlite XAD-2 column. Hydro- phobic components were eluted with a methanol gradient. Two groups of peptaibols designated hypomurocin (HM) A and B could be isolated and the sequences were determined

Liehigs Ann./Recueil1997,767- 772 0 VCH Verlagsgesellschaft mbH, D-69451 Weinheim, 1997 0947-3440/97/0404-0767 $ 17.50+.50/0 767

FULL PAPER D. Becker, M. Kiess, H. Briickner

Figure I . Sequences of and chiral amino acids

the microheterogeneous peptides of HM A and HM B; exchanged amino acids in bold characters; amino alcohols are of the L-configuration with the exception of D-(= R)-Iva. Valol = L-valinol, Leuol = L-leucinol, Ac = acetyl;

Iva = isovaline, Aib = a-aminoisobutyric acid

Ac-Aib-Gln-Val-Val-Aib-Pro-Leu-Leu-Aib-Pro-Leu01 HM A-1

Ac-Iva-Gln-Val-Val-Aib-Pro-Leu-Leu-Aib-Pro-Leuol HM A-2

Ac-Aib-Gln-Val-Leu-Aib-Pro-Leu-Ile-Aib-Pro-Leuo~ HM A-3

Ac-Aib-Zln-Ile-Val-Aib-Pro-Leu-Leu-Aib-Pro-Leuol HM A-4

A~-Aib-Glri-Ile-Ile-Aib-Pro-Leu-Leu-Aib-Pro-Leuol HM A-5

Ac-Aib-Gln-Ile-Leu-Aib-Pro-Leu-Ile-Aib-Pro-Leuol HM A-5a

lic-Aib-Ser-Ala-Leu-Aib-Gln-Aib-VaL-Aib-Gly-Aib-P.ib-Pro-Leu-Aib-Aib-Gln-Valo1 HM B-1

Ac-Aib-Ser-Ala-Le~~-Aib-Gln-Aib-Val-Aib-Gly-Aib-A~b-Pro-Leu-Aib-Aib-Gln-Leuol HM B-2

~.z-Aib-Ala-Ala-Leu-Aib-Gln-Aib-Val-Aib-Gly-Aib-Aib-Pro-Leu-Aib-Aib-Gln-Valol HM B-3a

Ac-Aib-Ser-Ala-Leu-Aib-Gln-Iva-Val-Aib-Gly-Aib-Aib-Pro-Leu-Aib-Aib-GLn-Valol HM B-3b

Ac-Aib-Ser-Aia-ieu-Aib-Gln-Aib-Val-Aib-Gly-Iva-Aib-Pr~-Leu-Aib-Aib-Gln-Valol HM B-4

~~.ii=-Aib-Ser-ALa-Leu-Aib-Gln-Aib-Val-Aib-Gly-Iva-Aib-Pro-Leu-Aib-Aib-Gln-Leuol HM B-5

(Figure I ) . Analytical RPHPLC revealed that HM A and B were microheterogeneous, each consisting of a mixture of four major and several minor components (Figure 2).

Using repetitive preparative RPHPLC, homogeneous peptaibol sequences designated HM A-1 to A-5 and HM B-1 to B-5 could be isolated.

Figure 2. Analytical HPLC of the microheterogeneous components of (top) hypomurocin A and (bottom) hypomurocin B; for chroma- tographic conditions and abbreviations, see Experimental Section

Amino acid analysis of HM hydrolysates was performed by means of GClMS on Chirasil-L-Val. This method re- vealed the presence of L-Leuol in HM A and of both L- Leuol and L-Valol in HM B. Chiral amino acids were of the L-configuration with the exception of Iva, which was of the D-( = R)-config~ration[~]. The stoichiometry and con- figuration of components was also determined by HPLC, using pre-column derivatization with FMOC-Cl[241 and OPA/IBLC[251, respectively.

In hydrolysates of isolated peptides of the HM A group, the proteinogenic L-amino acids Glu, Pro, Leu, Ile, Ala, Val; the non-proteinogenic Aib, and D-(= R)-Iva, and the amino alcohol L-Leuol could be determined by HPLC.

Analogously, in the HM B group, the L-enantiomers of Ser, Ala, Glu, Val, Pro, Leu; the achiral Gly and Aib, and, in part, D-(= R)-Iva could be detected, as well as the amino alcohols L-Valol and L-Leuol (see Figure 1). Glu (generated from Gln on hydrolysis) was determined as Gln in all pep- tides using FABMS/MS and ES(IS)MS/MS (see below).

Sequence determinations of hypomurocins were per- formed using positive FABMS and ESMS (for differen- tiation of isomeric AAs LeulIle and VallIva, see below). Selected fragments were chosen for CID experiments to ob- tain complete sequence information. For nominal masses of sequence and molecular ions of peptides, see Tables 1 and 2. As an example, FABMS of the 1 1-mer peptaibol HM A- 3 is presented in Figure 3. Sodiated and protonated molecu- lar ions at m/z 1197 and 1175, respectively, as well as intense fragment ions b2, b3, b4, b5, and b9 are observed (nomencla- ture according to Roepstorff and Fohlman[261, modified by Biemann[*']). Sequence ions b6, b7, b8, and blo were of lower intensity (see Figure 3, top). The fragment at mlz 409, Pro6-Leu-Ile-Aib9 (PLIU) proved to be the internal frag- ment from AA positions 6 to 9. The fragment y2 at mlz 21 5 furnished intense product ions at mlz 70 and 118 in CID experiments, thus proving the C-terminal Pro''-Leuol' ' se- quence. Fragmentation of the precursor ion b5 yielded in-

168 Liebigs Ann.lRecueil 1991, 167-112

Peptaibol Antibiotics Hypomurocin A and B FULL PAPER tense abundant product ions bl-b4 at mlz 128, 256, 355, and 468, respectively (Figure 3, bottom), representing the N-terminus Ac-Aib'-Gln-Val-Leu-Aib5. In combination with AAA data and the assignments of positions of iso- meric AAs, the complete sequence of HM A-3 could be deduced. Analogously, the sequences of the other HM A peptaibols could be determined. Molecular ions and mass fragments of HM A-1 to A-5/5a are collected in Table 1.

Figure 3. (top) FABMS of HM A-3 (PLIU = Pro-Leu-Ile-Aib) and (bottom) FABMSIMS of selected precursor ion at mlz 553 (b,) of

HM A-3; for corresponding nominal masses, see Table 1

100 7 b9 I

(M+Naf

PLIU

100 300

100

500 m h

300 500 mla

The sequences of HM B were determined by means of ESMS and ESMS/MS. The most intense abundant sodiated molecular ion [M + 2 NaI2+ was doubly charged, but singly charged molecular ions were also formed. Cleavage of the AibI2-Prol3 bond generated intense abundant N-terminal b-series fragments b5, b,, b8, and bI2 and the C-terminal y- series (y6) counterpart in ESMS (Figure 4, top).

The b I 2 and y6 fragment ions served as precursor ions in CID experiments. The fragmentation of bI2 generated a complete series of product ions b3 to bll (Figure 4, center) providing the sequence Ala3-Aib12.

ESMS/MS of the b5 fragment ions yielded intense N-ter- minal acetylated fragment ions bl and b2 at m/z 128 and 215 furnishing the Ac-Aibl-Ser2 terminus (Table 2). Frag- mentation of the C-terminal y6 ion at m/z 626 produced a diagnostic series of singly protonated internal fragments at m/z 21 1, 296, 381, and 509 (Figure 4 bottom and Table 2 ) yielding the C-terminal hexapeptide (ProI3-Leu-Aib-Aib- Gln-Leuolls) of HM B-2. Molecular ions and all identified

Table 1. Summary of detected and identified nominal mass frag- ments and molecular ions of HM A peptaibols; sequences of HM A-1 to A-4 deduced from FAB ionization and HM A-5 deduced

from ES ionization; values in mlz

Fragment HM A-1 HMA-2 HMA-3 HMA-4 HMA-515a

bi b2 b3 b4 b5 b6 b7 b8 b9 b io Y 1 Y2 ( ~ 2 a 1 O)I PLLPLI PLLUPLIU IIUPLLULlLUPLIU

Y6

(M+Na)+ (M+2H)2+ (M+2Na)2+

(M+H)+

IM+K)+

128 256 355 454 539 636 749 862 947

1044 118 215

70

1161 1183

1199

142 270 369 468 553 650 763

961 1058 118 215

70

409

1175 1197

1213

128 256 355 468 553 650 763 876 96 1

1058 118 215

70

409

1175 1197

128 256 369 468 553 650

876 96 I

1058 118 215

70

409

1175 1197

1213

256 369 482 567

975 1072

118

215 70

324 409 719 623

1189 1211 595 617

mass fragments of peptaibols of the HM B group are sum- marized in Table 2.

Most of the microheterogeneous peptaibol sequences contain isomeric amino acids viz. Leu/Ile and Val/Iva (Fig- ure 1). The soft ionizing techniques employed did not allow these isomers to be distinguished[28]. In the solitary case of HM A-3, the position of Leu and Ile could be determined by applying automated Edman degradation directly to an acidic partial hydrolysate, thereby providing the internal tetrapeptide Pro6-Leu-Ile-Aib' as a result of cleavage of the acid-sensitive Aib-Pro bonds. The two other fragments re- leased, the N-acetylated pentapeptide and the C-terminal Pro-Leuol, did not interfere with the Edman sequencing. Since positions 7 and 8 were occupied by Leu and Ile, re- spectively, the remaining Leu (according to AAA) had to be placed in position 4 in HM A-3. Since Edman degra- dation could not be successfully applied to partial hydroly- sates of HM A-4 and A-5, a mass spectrometric method was developed. Methanolytic cleavage of peptaibols fol- lowed by trifluoroacetylation furnished TFA-dipeptide methyl esters which were subsequently analyzed by GC/ SIM-MS on Chirasil-L-Val.

Owing to the favourable selectivity of this column, didpeptides could be separated and their structures unam- biguously assigned by SIM-MS and comparison with au- thentic dipeptide standards.

Quantitative AAA of HM A-4 proved the presence of two Leu and one Ile, which had to be located as Xle (Leu or Ile) in positions 3, 7, and 8 of the peptide according to FABMS/MS (Figure 1). Detection of the dipeptides Ile-Val

Liebigs Ann.lRecueil1997, 161- 112 169

FULL PAPER- D. Becker, M. Kiess, H. Briickner

Figure 4. (top) ESMS of HM B-2, (center) ESMSIMS of selected presursor ion at nil: 1 I08 (b12) from HM B-2, and (bottom) ESMSI MS of selected precursor ion at mlz 626 (y6) from HM B-2; PL = Pro-Leu; PLU = Pro-Leu-Aib; PLUU = Pro-Leu-Aib-Aib; PLUUQ = Pro-Leu-Aib-Aib-Gin; for corresponding nominal mas-

ses, see Table 2

(M+2Na12+

400 800 1200 1600 miz

loo 1 - I $ -1

200 600 1000 mlz

PLU

.A a

20

100 Joo SO0

and Leu-Leu in hydrolysates by GC/SIM-MS proved that lie had to be placed in position 3 and the two Leu in posi- tions 7 and 8. Similarly, AAA of HM B-4 revealed the pres- ence of one Val and one Iva, which had to be placed as Xva (Val or Iva) in positions 8 and 11 according to ESI-MS/ MS. Detection of the dipeptide Gly-Iva in hydrolysates by GC/SIM-MS proved the position I1 of Iva. Consequently, Val had to be placed at position 8 of the peptide. Anal- ogously, the isomeric positions of sequences of HM A-4, HM A-5, H M B-3b, HM B-4, and HM B-5 could be deter- mined.

Furthermore, this method made possible the detection of traces of HM A-Sa, which co-eluted with HM A-5 in HPLC. AAA revealed the presence of two Leu and two Ile in HM A-5, which could be placed as Xle in positions 3 and 4 and 7 and 8 by ESMS/MS. GCISIM-MS indicated

Table 2. Summary of detected and identified nominal mass frag- ments and molecular ions of HM B peptaibols; sequences deduced from ES ionization except the fragment at mlz (b, 1037 from HM

B-5 which was deduced by FAB ionization; values in rnlz

Fragment HM B-1 HM B-2 HM B-3a HM B-3b HM B-4 HM B-5

bl b2

b3

b4 b5

b7 b8 b9 b10 b l l b12

b2-H20

b3-H20

b6

Y6 PL PLU PLUU PLUUQ (M+H)+ (M+2H)2+ (M+Na)+ (M+2Na)2i (M+K)+

128 215 197 286 268 399 484 612 697 796 88 1 938

1023 1108 612 21 I 296 381 509

1719 860 - -

-

128 215 197 286 268 399 484 612 697 796 88 1

93 8 1023 1108 626 21 1 296 381 509 -

-

1755 889 -

128 199 -

270 268 383 468 596 68 1 780 865 922

1007 1092 612 21 1 296 381 509

1703 -

1725 874 -

128 215 197 286 268 399 484 612 71 1 810 895 952

1037 1122 612 211 296 381 509

1733 -

-

-

1771

128 215 197 286 268 399 484 612 697 796 881 938

1037 1122 612 21 1 296 381 509

1733 -

1755 889 -

128 215 197 286 268 399 484 612 697 796 881 938

1037 1122 626 21 1 296 381 509

1747 -

1769 896 -

the release of the dipeptides Ile-Ile and Leu-Leu on meth- anolysis. However, minor sequences Ile-Leu and Leu-Ile were also detected (ca. 3% according to their peak intensi- ties in GC/SIM-MS). These minor components were only detectable as a result of the high sensitivity and specificity of GC/SIM-MS. Since Pro6-Leu' was detectable by GC/ SIM-MS in HM A-5 and 5a, it was logical to place Leu- Leu as well as Leu-Ile at positions 7 and 8. Consequently, in HM A-5 Ile-Ile were placed at positions 3 and 4, and in HM A-5a Ile-Leu were placed in positions 3 and 4.

In HM A-2, the presence of one Iva and two Val was determined by AAA and located as Xva at positions 1, 3, and 4 by FAB-MS/MS. Methanolysis of the peptide yielded directly Ac-1va'-OMe which could be assigned by GC/SIM- MS and comparison with synthetic material. Consequently, the two Val had to be placed at positions 3 and 4.

In the microtiter test, Bacillus subtilis was inhibited by HM B-1 and HM B-2 at concentrations of 75 pg/ml, whereas HM A-4 required 600 pg/ml for full inhibition. Thus, HM B-1 and HM B-2 are approximately 12 times more active than HM A-4, based on molecular weights.

Hemolytic activity[29] was tested using a semi-quantita- tive agar diffusion assay (see Experimental Section). HM A-4, HM B-1, and HM B-2 were investigated as representa- tives and compared to the hemolytically active 20-mer pep- taibol paracelsin['] and the saponin digitonin. Based on their molecular weights, peptaibols of the HM B group are comparable in activity to paracelsin and approximately 6 times more active compared to the HM A group.

770 Liebigs Ann.lRecuei1 1991, 767-772

FULL PAPER Peptaibol Antibiotics Hypomurocin A and B

Experimental Section Abbreviations: AA, amino acid(s); AAA, amino acid analysis;

GC, gas chromatography; MS, mass spectrometry; SIM, selected ion monitoring; FAB, fast atom bombardment; ES, electrospray; IS, ion spray; CID, collision induced dissociation; MS/MS, tandem mass spectrometry; FID, flame ionization detector; TLC, thin-layer chromatography; RPHPLC, reversed phase high performance liquid chromatography; MPLC, medium pressure liquid chroma- tography; Ac, acetyl; Aib, a-aminoisobutyric acid (2-methylala- nine); Iva, isovaline (2-ethylalanine); Leuol, leucinol; Valol, valinol; FMOC-C1, 9-fluorenylmethyloxycarbonyl chloride; OPA, o-phthal- dialdehyde; IBLC, N-isobutyryl-L-cysteine; TFA, trifluoroacetic acid; TFAA, trifluoroacetic acid anhydride; PFPAA, pentafluoro- propionic acid anhydride; iPrOH, 2-propanol; MeOH, methanol; MeCN, acetonitrile; DCM, dichloromethane; TDM (reagent), chlorine/4,4'-bis(dimethylamino)diphenylmethane.

TLC: Pre-coated plates (Merck, Darmstadt), silica gel 60 F254, thickness 0.25 mm (analytical) and 2 mm (preparative). - Solvent system I: chloroform/MeOH/AcOH/water (65:25:3 :4). - Detec- tion of components: spraying with water (preparative) and TDM reagent. - Fermentation: Model G25 incubation shaker (Bruns- wick Scientific, Edison, NJ, USA).

Fermentation and Isolation Procedures: H. muroiuna Hino et Kat- sumoto[21] was obtained as lyophilised culture ( IF0 31288) from the Institute for Fermentation, Osaka, Japan. Lyophilisate was sus- pended in sterile sodium chloride (0.825%), soaked for 15 minutes, and transferred to Petri dishes with Raulin-Thom medium (RTM)[']]. After 13 days at room temperature, vigorous growth of the mould was observed. A spore suspension was used for the in- oculation of 18 Erlenmayer flasks (2 1) each containing 400 ml of RTM. The flasks were shaken at 100 rpm on a rotatory shaker at ambient temperature. Aliquots (20 ml) of filtered culture broth were passed through Sep-Pak@ C-18 (Waters) cartridges. Pro- duction of peptaibols was monitored by TLC and on total hy- drolysates using Aib as a marker component for GC analysis[30]. - After 11 days, the filtered culture broth ( 5 1) was passed through a column (38 X 3 cm) packed with Amberlite XAD-2 adsorber resin (particle size 20-50 pm, Serva, Heidelberg), the resin was washed with water (1 l), 40% MeOH (0.5 I), and the components adsorbed were eluted with a linear gradient from 40-100% MeOH (1.7 1); flow rate 1.7 ml/min. Elution of peptides was monitored by TLC and GC of derivatized total hydrolysates. Appropriate fractions were combined and concentrated to dryness, yielding 500 mg of crude peptide mixture. The residue was dissolved in MeOH (9 ml) and subjected to MPLC on LiChroprep@ RP 8 (particle size 40-63 p, 38 X 1.5 cm, Merck). The column was washed with 50% MeOH (400 ml) and peptides were eluted with a gradient 50- 100% MeOH (620 ml), flow rate 1 ml/min. A total of 62 fractions, each of 12 ml were collected and the presence of peptides was monitored by TLC. HM A [R,(I) = 0.531 was eluted in fractions 40-48 and HM B [RLI) = 0.391 in fractions 51 -58. Appropriate fractions were com- bined and concentrated to dryness, yielding crude HM A (84 mg) and HM B (61 mg). Gel filtration of these crude peptides was per- formed on Sephadex LH-20 columns (37 X 4 cm), with MeOH (250 ml). The elution of peptides was monitored by TLC (system I) and appropriate fractions were combined and concentrated to dryness, yielding HM A (66 mg) and HM B (55 mg).

In a second approach, cultur broth (2.4 1) was subjected to XAD- 2 chromatography as described above and the resulting crude pep- tide mixture (150 mg) was dissolved in MeOH (750 pl). Aliquots (250 pl) were subjected to preparative TLC; separation was moni- tored by spraying with water and zones containing HM A and HM B were collected. Peptides were eluted from the silica by treatment

with MeOH (2 ml), and the silica was removed by centrifugation (2000 X g). This procedure was repeated three times, yielding HM A (24 mg) and HM B (18 mg).

Instruments: MPLC for XAD and LiChrospheP chromatogra- phy: MD 80/100 pump, controller PS 1 (Labomatic, Sinsheim), Model FRAC-100 fraction collector (Pharmacia, Freiburg).

Analytical RPHPLC: Model 2200 pump, Lambda 100 UV/Vis detector (Bischoff, Leonberg), column 250 mm X 4 mm, Nucleo- silo 100 RP-18 (particle size 5 pm). Eluent for HM A: MeCN/ water/TFA, 55:45:0.1 (v/v); for HM B: MeCN/MeOH/water, 20:60:17 (v/v); flow rate 1 ml/min, ambient temperature.

Preparative RPHPLC: Series 3B instrument, LC 75 UVNis de- tector (Perkin-Elmer, Uberlingen), column 250 mm X 16 mm, Nu- cleosila 100 RP-18 (particle size 3 pm). Eluent for HM A: MeCN/ MeOH/water, 20: 18:60 (v/v), for HM B: MeCN/MeOH/water, 20: 11 :60 (v/v); flow rate 5 ml/min, 10-ml fractions collected. Data acquisition: Merck-Hitachi D-2500 integrator.

GC-FID: Shimadzu GC-l4A, Chirasil-L-Valine column (25 m X

0.25 mm; Chrompack, Middelburg, The Netherlands).

GC-MSfor Analysis of Dipeptides: Shimadzu 17A/QP 5000; de- tector gain: 1.5 kV, scan time 0.2 s, split or splitless mode; Chirasil- L-Valine column. Conditions: synthetic dipeptides: Ile-Ile, Leu-Ile, Ile-Leu, Leu-Leu, Ile-Val (Bachem, Heidelberg); Pro-Leu (Sigma); Val-Aib, Gly-DL-ha-OMe were synthesized in our laboratory using standard procedure^[^'].

Ile-Val (HM A-4): temperature program: 170°C - 2"C/min - 190°C - 10 min; pressure program: 5 kPa - 1 kPa/min - 15 kPa - 10 min; selected mass fragments in SIM-mode: 7 rnin to 9.8 min, m/z 154, 183, 267, 281.

Leu-Leu, Ile-Leu, Leu-Ile, Ile-Ile (HM A-4, HM A-5/5a): tem- perature program: 170°C - 2"Clmin - 190°C - 10 min; pressure program: 5 kPa - 1 kPa/min - 15 kPa - 10 min; selected mass fragments (SIM-mode): 9 rnin to 12 min, m/z 140, 154, 168, 183.

Pro-Leu (HM A-5/5a): temperature program: 170°C - 2"C/min - 190°C - 10 min; pressure program: 5 kPa - 1 kPa/min - 15 kPa - 10 min; selected mass fragments (SIM-mode): 5 rnin to 12 min. m/z 139, 166, 194, 279.

Val-Aib (HM B-3b): temperature program: 140°C - 3"C/min - 200°C - 10 min; pressure program: 5 kPa - 0.6 kPa/min - 17 kPa - 10 min; selected mass fragments (SIM-mode): 8 min to 12 min, m/z 100, 116, 154, 169, 253.

Gly-DL-Iva (HM B-4 and B-5): temperature program: 140°C - 3'C/min - 200°C - 10 min; pressure program: 5 kPa ~ 0.6 kPa/ rnin - 17 kPa - 10 min; selected mass fragments (SIM-mode): 11.5 rnin to 14 min, m/z 126, 127, 130, 225.

FAB-MS analysis and CID experiments of HM A I , 2, 3, and 4: JMS-HWHX llOA (JEOL, Tokyo, Japan) tandem mass spec- trometer; 3-nitrobenzyl alcohol; 6 kV Xenon beams, collision gas: He, collision energy: 3 kV.

ESMS/MS of HM A-5, A-5a and HM B-I, 2,4, and 5 : TSQ 700 triple stage quadrupole mass spectrometer (Finnigan MAT, Bre- men); peptides in MeOH (10 pmollpl) were infused, flow rate: 2 pl/ min; electrospray voltage: 5.5 kV; collision gas: argon.

HM B-3a and B-3b: API 300 triple stage quadrupole mass spec- trometer (Perkin-Elmer, Langen, Germany); flow rate: 5 pl/min; electrospray voltage: 5 kV; orifice voltage: 80 eV collision gas: NZ; collision energy: 40-50 eV.

Amino Acid Analysis: Hydrolysis, 6 N HCl (24 h, 1 10 "C); deriva- tization for GC/MS: 2.5 N HC1 in 2-PrpOH, followed by acylation with PFPAA.

HPLC analysis: derivatization with FMOC-Cl[241 or with OPA and IBLC[251.

Liebigs Ann./Recueil 1991, 767- 772 771

FULL PAPER D. Becker, M. Kiess, H. Briickner

Dc~trrminarion of' Positions of' Isomeric Amino Acids: Edman degradation of a partial hydrolysate of HM A-3 (ca. 10 pg, 100 p1 TFA, 30°C. 3.5 h); ABI 471A/03 proteinlpeptide-sequencer (con- ditions: standard protocol of Applied Biosystems).

Dipepride Partial hydrolysis of peptaibols (ca. 1 pg) in 4 N HCI in MeOH (500 pl) at l l0"C for 5 h (HM A) or 9 h (HM B). trifluoroacetylation with TFAA. TFA-dipeptide[methyl]- ester analyzed on GC/SIM-MS.

Detection of Ac-D-Iva-OMe by GCISIM-MS: partial hydrolysis of peptides: 21 h (lOO°C), 2.5 N HC1 in MeOH; temperature pro- gram: 70°C - 2.5"Clmin - 90°C - 7"C/min - 19O0C/-1O min; m a s s scan: m/z 102, 114, 210.

Anfihiofic. i ind Hemolytic Activity. - Microtiter Test: peptaibols dissolved in MeOH were tested against Bacillus subtilis DSM 3561 in dilution rows. well volume: 150 pl, control without antibiotics or pure MeOH, incubation: 8 h, 30°C, measurement of optical density: 450 Micro Plate Reader (BioRad, Miinchen, Germany).

Hcwio/ytic Activity; Rat full blood (10 ml) was centrifuged (780 X g, 3 min), (Labofuge 400, Heraeus, Hanau, Germany) and the precipitate of erythrocytes was suspended in blood agar basis (4%, w/v) (Merck). Peptaibols (0.2 mg for HM B-I, B-2, digitonin, and paracelsin; 0.7 mg for HM A-4) in MeOH were exposed to the medium (20 ml per Petri dish) on filter disks (0 9 mm). After 24 h at 4"C, hemolysis zones were measured and the diameter of disks subtracted: HM A-4 and B-1 (13 mm), HM B-2, paracelsin and digitonin ( 1 I mm), blank MeOH (0 mm).

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