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Harzianin HB I, an 11-residue peptaibol from Trichodermaharzianum: isolation, sequence, solution synthesis and membraneactivity |
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Journal of the Chemical Society, Perkin Transactions 1,
Volume 1,
Issue 10,
1997,
Page 1587-1594
Isabelle Augeven-Bour,
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摘要:
J. Chem. Soc. Perkin Trans. 1 1997 1587 Harzianin HB I an 11-residue peptaibol from Trichoderma harzianum isolation sequence solution synthesis and membrane activity Isabelle Augeven-Bour,a Sylvie Rebuffat,a Catherine Auvin,a Christophe Goulard,a Yann Prigent b and Bernard Bodo *,a a Laboratoire de Chimie URA 401 CNRS GDR 1153 CNRS Muséum National d’Histoire Naturelle 63 rue Buffon 75231 Paris Cedex 05 France b Laboratoire de RMN URA 464 CNRS Institut Fédératif de Recherche Multidisciplinaire sur les Peptides No 23 INSERM Université de Rouen 76821 Mont-Saint-Aignan Cedex France Harzianin HB I is a minor component of the peptaibol mixture biosynthesized by a Trichoderma harzianum strain. It is isolated by a multi-step chromatography procedure including HPLC; its sequence has been elucidated by LSIMS and two-dimensional 1H NMR experiments.This 11-residue peptaibol is an analogue of the 14-residue peptaibol harzianin HC IX resulting from the deletion of the tripeptide Aib- Pro-Ala. The CD and NMR data (NOE data and amide proton temperature coefficients) are similar to those of HC IX suggesting a right-handed helix-type conformation for the two peptides. Harzianin HB I was synthesized by the solution-phase method using BOP as coupling reagent. The membrane properties examined on liposomes are compared with those of other known peptaibols. Introduction Peptaibols biosynthesized by Trichoderma soil fungi are amphipathic linear peptides with a high proportion of a,adialkylated amino acids such as a-aminoisobutyric acid (Aib U) an N-terminal acetylated residue and a C-terminal amino alcohol.They can be classified into the long-sequence group containing 18 to 20 residues,1–5 the short-sequence group with 11 to 16 residues 6,7 and the lipopeptaibols with 7 to 11 residues and an N-terminal amino acid acylated by a lipid chain.8,9 The main interest in such peptides stems from their ability to form voltage-gated ion channels in planar lipid bilayer membranes 10,11 and therefore they can be viewed as a prototypic pore. In the absence of a voltage peptaibols induce permeabilization of liposomes;12,13 long-sequence ones being more effi- cient than short-sequence ones. Their membrane activity is modulated by both the length and hydrophobicity of the sequence. The known biological properties of these peptides are their antibiotic 14 and catecholamine secretion-inducing abilities 15,16 which have been suggested to be related to their membrane activity.Recently we isolated peptaibols organised in helical structures which still have significant membrane activity in spite of their short sequences such as the 11-residue lipopeptaibol GA IV and the proline-rich 14 residue peptaibols harzianins HC.7 From a Uruguayan strain of Trichoderma harzianum we previously isolated two groups of peptaibols trichorzins HA and harzianins HC containing 18 and 14 residues respectively. From the particularly complex mixture of the 14-residue peptides a minor component of the same polarity harzianin HB I was isolated. Its structure was determined from liquid secondary ion mass spectrometry (LSIMS) and NMR spectroscopy. It was synthesized in order to test its antibiotic and membrane properties.Harzianin HB I was shown to modify slightly the permeability of liposomes and induce voltage-gated macroscopic conductance in planar bilayers. Sequence of harzianin HB I Results and discussion Isolation and characterization of HB I 17 The culture broth extract of the Uruguayan M-903603 T. harzianum strain was fractionated by exclusion chromatography; the peptide fraction was further chromatographed over silica gel to yield two peptaibol groups of different polarity. The first group was composed of 18-residue peptaibols the trichorzins HA,5,14 and the second one mostly of the 14-residue harzianins HC.7 The HPLC chromatogram of the crude HC mixture was very complex showing more than 16 peaks one of them being assigned to a minor component HB I. Its isolation was undertaken by repetitive semi-preparative reversed-phase high-performance liquid chromatography (RP-HPLC) and its purity was checked by analytical HPLC (Fig.1). Further LSIMS and 1H NMR experiments confirmed HB I to be homogeneous. It reacted neither with diazomethane nor with ninhydrin indicating the presence of neither free carboxy nor amino groups. The presence of a sharp singlet at dH 2.02 in the 1D NMR spectrum suggested an acetylated N-terminal residue as usually found in peptaibols. The amino acid composition and absolute configuration of the residues resulted from GLC analysis of the total acid hydrolysate derivatives on a Chirasil L-Val capillary column Aib (3) L-Asn (1) L-Ile (1) D-Iva (1) L-Leu (2) L-Pro (2) L-Leuol (1). Sequence determination of HB I Positive liquid secondary-ion mass spectrometry [(1)-LSIMS].The amino acid sequence of HB I was examined by positive LSIMS. A relative molecular mass of 1160 was assigned to HB I from the sodium ion adduct [M 1 Na]1 observed at m/z 1183 in agreement with the postulated molecular mass arising from the amino acid and amino alcohol composition. The LSIMS spectrum exhibited several abundant b-type acylium ions 18 at m/z 947 553 468 355 242 and 128 and two lower-abundance ion regions between m/z 553 and 947 and between m/z 947 and 1183. This fragmentation pattern suggested the presence of two labile Aib–Pro bonds in HB I. The series of b-type ions ranging from m/z 128 to 553 gave the N-terminal pentapeptide sequence. The mass difference of 394 amu between m/z 947 and 553 was in agreement with the formation of the tetrapeptide ion observed at m/z 395 and 1588 J.Chem. Soc. Perkin Trans. 1 1997 Fig. 1 HPLC chromatogram of purified natural harzianin HB I (Spherisorb ODS2 5mm) 4.6 × 250 mm MeOH–water (83 17) flow rate 1 cm3 min21; absorption monitored at 220 nm obtained via cleavage of the Aib5–Pro6 amide bond.6,19 The Cterminal dipeptide resulting from cleavage of the Aib9–Pro10 amide bond was observed as a y2 ion at m/z 215. Furthermore a series of weak and unusual but significant [xn 1 Na]1 ions at m/z 1083 969 856 743 462 and 167 was observed and allowed the confirmation of the HB I sequence.18 Nevertheless the respective locations of the isomeric residues Leu/Ile remained to be determined. 1H NMR spectroscopy. The complete amino acid sequence of HB I was determined by two-dimensional 1H NMR spectroscopy.Assignments of 1H chemical shifts to specific protons of individual residues (Table 1) were obtained by 2D homonuclear (COSY) and phase-sensitive 2D total (TOCSY) chemical-shift correlation experiments showing complete spin systems of one Asn two Pro and one Leuol (Fig. 2) as well as those of one Ile and two Leu residues in agreement with 13C resonances of one Ile Ca at dC 64.1 and two Leu Ca at dC 53.2 and 54.1 (data not shown). The sequence-specific assignments of the backbone NH proton signals arose from the rotating-frame nuclear Overhauser effect (ROESY) spectrum and were completely carried out by using inter-residue connectivities dNN(i,i 1 1) and daN(i,i 1 1) [Fig. 3(a)]. The lowest-field singlet NH proton showing a cross-peak with the acetyl CbH3 protons was assigned to Aib1.Then dNN(i,i 1 1) connectivities extending from Asn2 to Aib5 and from Iva7 to Aib9 confirmed the location of Ile at position 4. Finally sequential assignment of Pro6 and Pro10 arose from dNd(i,i 1 1) connectivities between their d protons and the amide protons of Aib5 and Aib9 [Fig. 3(b)]. H3C C O NH C C O NH CH C NH2 O C O NH CH C O NH CH C O NH C C O N CH C O NH C C O NH CH C O NH C C O N CH C O NH CH CH2OH (x10 + Na)+ 1083 (6) (x9 + Na)+ 969 (1) (x8 + Na)+ 856 (2) (x7 + Na)+ 743 (2) (x4 + Na)+ 462 (2) (x1 + Na)+ 167 (2) (b) Mass fragmentation pattern of harzianin HB I (positive-ion LSIMS) exhibiting the bn ions (a) and the xn 1 Na ions (b) (relative intensities in parentheses) J. Chem. Soc. Perkin Trans. 1 1997 1589 Table 1 1H sequential and stereospecific assignments of harzianin HB I (500.13 MHz; CD3OH; 296 K).Chemical shifts (ppm) are given to the nearest three or two decimal places when obtained from 1D or 2D spectra respectively; 3JNH-CaH coupling constants (Hz) arise from the 1D spectrum and are given in parentheses Residue NH a-H b-H/b-Me Other groups Ac Me 2.027 s Aib1 8.679 s pro-R 1.46*/pro-S 1.450 Asn2 8.610 d (5.6) 4.38 2.76 e syn 7.04/e anti 7.77 Leu3 8.111 d (7.1) 4.25 1.93/1.56 g 1.74/d1 0.89/d2 0.96 Ile4 7.356 d (9.1) 4.22 1.95 g 1.55/g9 1.32/g-Me 0.95/d-Me 0.86 Aib5 7.806 s pro-R 1.498*/pro-S 1.501 Pro6 4.21 pro-R 1.78/pro-S 2.35 pro-R g 1.94/pro-S g 2.10/pro-R d 3.85/pro-S d 3.41 Iva7 7.484 s 2.45/1.77/b-Me 1.46 g 0.82 Leu8 7.627 d (8.5) 4.39 1.75/1.75 g 1.75/d1 0.85/d2 0.96 Aib9 7.836 s pro-R 1.450*/pro-S 1.501 Pro10 4.42 pro-R 1.78/pro-S 2.29 pro-R g 1.87/pro-S g 1.93/pro-R d 3.88/pro-S d 3.44 Leuol11 7.534 d (8.9) 3.96 1.60/1.35/3.53 g 1.64/d1 0.90/d2 0.95 * May be exchanged.Conformation of HB I The conformation of HB I in methanol solution was examined by CD and NMR spectroscopy using inter-residue NOE connectivities 3JNH-CaH coupling constants and amide temperature coefficients. The CD spectrum of HB I showed two transitions at 192 (1) and 205 (2) nm characteristic of a right-handed helix. Fig. 2 Expansion of the TOCSY spectrum of HB I in CD3OH (spin lock period 120 ms) (a) w2 = 0.6–4.5 ppm w1 = 7.3–8.7 ppm; (b) w2 = 4.1–4.6 ppm w1 = 1.7–4.6 ppm; spin-systems are labelled with the sequential residue positions A stretch of strong sequential dNN(i,i 1 1) accompanied by daN(i,i 1 1) and by a series of medium and strong daN(i,i 1 3) all along the sequence was observed in the ROESY spectrum (Fig.3) in agreement with a helical structure. The presence of daN(i,i 1 2) NOEs all along the sequence and the complete absence of daN(i,i 1 4) connectivities suggested a succession of turns stabilized by 4Æ1 intramolecular hydrogen bonds as observed for the 310-helix. This was in agreement with the amide protons’ thermal coefficients (Dd/DTNH) (Fig. 4). Little information was obtained from the 3JNH-CaH coupling constants as only half of the residues could give such data (Fig. 4). The Asn2 and Leu3 residues showed values <7 Hz whereas Ile4 Leu8 and Leuol11 had higher values around 9 Hz apparently inconsistent Fig. 3 Parts of the ROESY spectrum of HB I in CD3OH (mixing time 250 ms) (a) w2 = 7.3–8.8 ppm w1 = 7.1–8.7 ppm; (b) w2 = 7.3–8.0 w1 = 3.2–4.8 ppm 1590 J.Chem. Soc. Perkin Trans. 1 1997 with a helical structure. However such values have frequently been observed for the two amino acids flanking Aib-Pro segments in a-helical peptaibols.4,5,20–22 The studied residues in HB I have such a location in the sequence. Comparison of the thermal coefficients and coupling constants of HB I with those of the longer analogue HC IX which contains an additional Aib-Pro-Ala tripeptide after Leu3 showed extensive similarity of the data.17 The results thus suggested a structure stabilized by 4Æ1-type intramolecular hydrogen bonds forming a ribbon of b-turns. Synthesis of HB I In order to make available a sufficient amount of harzianin HB I for bioassays it was synthesized by a solution-phase method in the presence of the (benzotriazol-1-yloxy)tris(dimethylamino) phosphonium hexafluorophosphate (BOP) coupling reagent in CH2Cl2 at room temperature according to the synthetic route shown in Scheme 1.The penta- and hexa-peptide fragments were built up in a stepwise manner using Boc/OMe Fig. 4 Amino acid sequence of harzianin HB I (the one-letter code of amino acid residues is used with U = Aib J = Iva Lol = Leuol) and a survey of the NOE connectivities involving NH and CaH (dad connectivities observed for prolines are indicated by white boxes) of the 3JNH-CaH coupling constants and of the temperature coefficients of the amide protons. The observed NOEs are classified as strong medium and weak (based on counting the cross-peak contour levels) and shown by thick medium and thin lines respectively.strategy. They were designed so that Aib was placed at the Cterminal position in order to avoid racemization during the deprotection and activation steps. The synthesis of HB I was achieved by reduction of the C-terminal methyl ester group into an alcohol function by NaBH4–EtOH. Synthetic HB I was finally purified by semi-preparative HPLC. The analytical HPLC retention time (tR) and the 1H NMR spectrum of synthetic HB I were identical to those of natural HB I. Antibacterial activity The antibacterial activity of HB I examined against S. aureus and E. coli showed it to be inactive against E. coli in agreement with previous observations on other peptaibols.4,5,7 However no antibacterial activity against S.aureus was detected even at 200 mg pit21 whereas growth inhibition induced by short-sequence peptaibols harzianins HC and trichogin GA IV could be detected up to 50 and 1.5 mg pit21 respectively.7,8 This result was in agreement with the absence of antibacterial activity noticed for C2-GA IV,23 the analogue of GA IV with an acetyl group instead of the lipid chain. The absence of antibacterial activity for this 11-residue peptaibol confirms the role of the Nterminal lipid chain in the lipopeptaibol activity. Membrane-modifying properties of HB I Long-sequence peptaibols have been previously shown to exhibit membrane-modifying properties by increasing the permeability of liposomes.5,11 Optimal membrane activity was observed for a hydrophobic neutral a-helix of 18–19 residues while the liposome permeabilization decreased for shortersequence peptaibols.5 The membrane-modifying activity of HB I was studied by fluorescence spectroscopy following the leakage of a carboxyfluorescein (CF) fluorescent probe previously entrapped at self-quenched concentration in small unilamellar vesicles.The results presented as a percentage of escaped CF at 20 min as a function of Ri 21 = [peptide]/[lipid] were compared with those of other short peptides such as the 14- residue HC IX and the 11-residue lipopeptaibol GA IV (Fig. 5). Comparison of Ri 21 values characteristic of 50% release of the entrapped material showed HB I (Ri 21 = 83 × 1023) to be less efficient than HC IX (Ri 21 = 12 × 1023) and GA IV Scheme 1 Scheme for the total synthesis of HB I J. Chem. Soc. Perkin Trans.1 1997 1591 (Ri 21 = 4 × 1023). This result points to the major role of the sequence length the presence of the N-terminal lipidic chain also favouring the liposome permeabilization. The voltage-dependent channel-forming properties of harzianin HB I were also examined by macroscopic current– voltage experiments (G. Molle H. Duclohier unpublished results). In such conditions HB I exhibited channel-forming activity for concentrations ranging between 1026 and 1025 M in the same way as the 11-residue peptaibol trichorozin TZ-IV,6 or the 14-residue harzianins HC.7 Experimental Isolation of harzianin HB I The T. harzianum strain (M-903603) collected in Uruguay was obtained from the ‘Collection de souches fongiques du Muséum National d’Histoire Naturelle’ (Paris); the strain was maintained and cultivated as previously described.7 The culture was incubated for 11 days at 27 8C.The filtered fermentation broth was extracted three times with butan-1-ol to give after removal of the solvent under reduced pressure 1.2 g of crude extract. The residue was submitted to gel filtration on Pharmacia Sephadex LH 20 with methanol as eluent. The crude peptide mixture (468 mg) was then chromatographed over silica gel (Kieselgel 60 H Merck Darmstadt) with CH2Cl2–MeOH (9 1 to 5 5) as eluent. The HC/HB mixture (130 mg) was eluted with CH2Cl2–MeOH (80 20). HPLC separation This was carried out with a Waters liquid chromatograph (6000 A and M45 pumps a 680 automated solvent programmer a WISP 712 automatic injector and a 481 UV–VIS detector) on a semipreparative C18 column (Spherisorb ODS2; 5 mm; 7.5 × 300 mm; AIT France); eluent methanol–water (83 17); flow rate 2 cm3 min21.The purity of HB I (3 mg) was confirmed on an analytical column (3.5 × 250 mm); eluent methanol–water (83 17); flow rate 1 cm3 min21; tR 16 min. Amino acid analysis Total hydrolysis of HB I was carried out according to the usual procedure for peptides (6 M HCl at 110 8C in sealed tubes for 24 h). Identification of the amino acids was accomplished by gas chromatography after derivatization.4 Retention times of the Ntrifluoroacetyl isopropyl ester derivatives were compared with those of standard samples. The GLC analyses were performed with a Girdel 3000 chromatograph on a Chirasil-L-Val (Npropionyl- L-valine tert-butylamide polysiloxane) quartz capillary column (Chrompack 25 m length 0.2 mm i.d.) with He (0.7 × 105 Pa) as carrier gas and a temperature programme 50–130 8C 3 8C min21; 130–190 8C 10 8C min21; tR (min) Aib Fig.5 Peptide-induced CF at t = 20 min for different [peptide]/[lipid] ratios (Ri 21) from egg PC/cholesterol 70/30 vesicles (a) HB I (b) HC IX (Ac-Aib-Asn-Leu-Aib-Pro-Ala-Ile-Aib-Pro-Iva-Leu-Aib-Pro-Leuol) and (c) GA IV (Oc-Aib-Gly-Leu-Aib-Gly-Gly-Leu-Aib-Gly-Ile-Leuol) (10.4) L-Asp (29.5) L-Ile (20.4) D-Iva (11.2) L-Leu (24.2) LLeuol (22.2). A special temperature programme was used for the separation of proline enantiomers 50–110 8C 3 8C min21; plateau at 110 8C for 10 min; 100–190 8C 10 8C min21; tR L-Pro (25.1). Secondary ion mass spectroscopy Positive LSIMS was recorded on a ZAB2-SEQ (VG Analytical Manchester UK) mass spectrometer equipped with a standard FAB source and a caesium ion-gun operating at 35 kV.Peptide methanolic solution was mixed with a-monothioglycerol as matrix. The resolution was 1000. Positive HR-LSIMS were recorded on a ZAB-HF spectrometer. The MS spectra were registered with either Li1 or Na1 added to the matrix. NMR Spectroscopy A 0.4 cm3 aliquot of 7 mM methanolic (CD3OH) solution of HB I peptide in a 5 mm tube (Wilmad) was used for all the NMR experiments. Proton NMR spectroscopy experiments were conducted at 296 K on a Bruker AC 300 equipped with an Aspect 3000 computer or on a Bruker Avance DMX 500 spectrometer equipped with a Bruker Station 1 computer and an indirect quadruple-resonance 1H–31P–13C–15N gradient probehead. Spectra were processed using UXNMR and AURELIA software (Bruker Inc).Chemical shifts were referenced to the central component of the quintet due to the CD2H resonance of methanol at dH 3.313 downfield from SiMe4. J Values are given in Hz. TOCSY24 experiments were run with the MLEV 17 sequence for spin locking and a mixing time of 120 ms (9 kHz). The ROESY25 experiment was carried out with a mixing time of 250 ms and a spin lock field of 2 kHz to reduce Hartmann–Hahn transfers. Two-dimensional spectra were obtained with quadrature detection in both dimensions using the hypercomplex method in the F1 dimension.26 The solvent signal was suppressed using the WATERGATE scheme27 included in the standard and ROESY pulse sequences. A total of 2048 data points were acquired in the F2 dimension and 512 complex points in the F1 dimension. For each complex data point in the F2 4 free induction decays were accumulated with a relaxation delay of 2 s.All spectra were apodized with p/2-shifted sine-bell functions in both dimensions. CD spectrum The spectrum of HB I was recorded with a Jobin-Yvon CD6 dichrograph with a 0.1 mm path cell at 22 8C (1 mmol cm23; CH3OH); l (nm) and [q]M (deg cm2 dmol21) 192 (56 000) and 205 (2110 000). Antimicrobial activity The antibacterial activity of HB I was examined against Staphylococcus aureus (strain 209P) and Escherichia coli (strain RL 65) by the agar diffusion test using the Mueller Hinton culture medium and 6 mm diameter pits. The peptide sample was dissolved in dimethyl sulfoxide (DMSO) such as to give a 4 mg cm23 solution. Eight other concentrations were obtained by successive dilutions and 50 mm3 of each solution was deposited into the pits (1.5 to 200 mg).Inhibition zones were measured after 24 h of incubation at 37 8C. Liposome permeabilization Egg phosphatidylcholine (egg PC) type V E and cholesterol were purchased from Sigma; egg PC was used without further purification and cholesterol was recrystallized from methanol. CF from Eastman Kodak was separated from hydrophobic contaminants and recrystallized from ethanol as previously described.11 Fluorescence spectra were measured at 20 8C on an Aminco SPF 500 spectrofluorometer. The peptide-induced release of intravesicular content was monitored by the method introduced by Weinstein,28 that uses the property of quenching relief upon dilution of an encapsulated fluorescent probe CF. 1592 J. Chem. Soc. Perkin Trans. 1 1997 CF-entrapped small unilamellar vesicles (SUV) were prepared as previously described,11 by sonication of an egg PC– cholesterol (7 3) mixture ([lip] = 0.6 mM).The SUV obtained by sonication were separated from unencapsulated CF by gel filtration (Sephadex G 75). Leakage kinetics were obtained for different peptide lipid molar ratios obtained by adding aliquots of methanolic solutions of peptides (methanol concentration kept below 0.5% by volume). Synthesis of HB I Diisopropylethylamine (DIEA) trifluoroacetic acid (TFA) ditert- butyl dicarbonate (Boc2O) and L-leucine were purchased from Sigma-Aldrich Chimie and D-isovaline [(R)-(2)-2-amino- 2-methylbutanoic acid] from Acros (France). All N-tertbutoxycarbonyl- protected L-amino acids and BOP were purchased from Propeptide (France) and used without subsequent purification.H-Leu-OMe was prepared according to Boissonnas et al.29 Boc-Iva-OH was prepared according to Bodanszky and Bodanszky.30 Column chromatography was carried out with 230–400 mesh Merck grade 60 silica gel. Analytical TLC was performed on aluminium sheets covered with Merck grade 60 silica gel. Gel filtration was carried out with Pharmacia Sephadex LH 20. General procedure A BOP-mediated peptide coupling. The Nprotected amino acid and BOP reagent were added to a solution of the TFA salt of the C-protected amino acid or peptide in CH2Cl2. The stirred solution was cooled in an ice-bath and DIEA was added. The mixture was stirred at room temperature until TLC analysis indicated that consumption of the amino component was no longer proceeding.It was then concentrated in vacuo to leave an oil which was dissolved in ethyl acetate and washed successively with 1 M HCl water 1 M NaOH and saturated aq. NaCl. The combined organics were dried over Na2SO4 filtered and concentrated in vacuo to leave an oil which was purified by chromatography on a silica gel column. General procedure B Removal of N-tert-butoxycarbonyl protection with 50% TFA solution in CH2Cl2. A stirred solution of the N-tert-butoxycarbonyl-protected peptide in CH2Cl2 was cooled in an ice-bath and TFA was added. The mixture was stirred at room temperature until TLC analysis indicated complete consumption of the starting material and was then evaporated in vacuo to leave an oil. The crude product was used without purification for the next coupling. General procedure C Removal of N-tert-butoxycarbonyl protection with pure TFA.The N-tert-butoxycarbonyl-protected peptide was treated with TFA (0.5 cm3 for 1 mmol of peptide). The mixture was stirred at room temperature until TLC analysis indicated that the totality of the peptide was deprotected and it was then evaporated in vacuo to give an oil. The crude product was used without purification for the next coupling. Boc-Pro-Leu-OMe. HCl H-Leu-OMe (0.92 g 5.07 mmol) and Boc-Pro-OH (1.20 g 5.58 mmol) were treated with BOP (2.47 g 5.58 mmol) and DIEA (2.7 cm3 15.71 mmol) via procedure A to yield the crude product which was used without purification. Boc-Aib-Pro-Leu-OMe. Boc-Pro-Leu-OMe (1.73 g 5.07 mmol) was deprotected according to procedure C. The crude TFA salt and Boc-Aib-OH (1.12 g 5.50 mmol) were treated with BOP (2.43 g 5.50 mmol) and DIEA (2.7 cm3 15.7 mmol) according to procedure A to yield after purification by chromatography on silica gel (ethyl acetate) 1 g of a powder; TLC Rf (ethyl acetate) 0.40; LSIMS m/z 434 [M 1 Li]1 (100) 378 (5) 334 (27) 332 (2) and 247 (9); HR-LSIMS [M 1 Li]1 434.2822 (Calc.for C21H37LiN3O6 m/z 434.2842); dH(300 MHz; CD3OH) 0.91 (d J 6.0 3 H d Leu) 0.94 (d J 6.0 3 H d Leu) 1.36 (s 3 H b Aib) 1.45 (s 12 H b Aib Boc) 1.81 (m 6 H 2 × b Leu g Leu b9 Pro 2 × g Pro) 2.19 (m 1 H b Pro) 3.59 (m 1 H d9 Pro) 3.68 (s 3 H ester) 3.81 (m 1 H d Pro) 4.37 (m 1 H a Leu) 4.45 (dd J 8.4 and 5.9 1 H a Pro) 7.29 (s 1 H NH Aib) and 8.23 (d J 7.7 1 H NH Leu). Boc-Leu-Aib-Pro-Leu-OMe. Boc-Aib-Pro-Leu-OMe (1.00 g 2.34 mmol) was deprotected according to procedure B.The crude TFA salt and Boc-Leu-OH (0.64 g 2,58 mmol) were treated with BOP (1.14 g 2.58 mmol) and DIEA (1.3 cm3 7.3 mmol) according to general procedure A. The mixture was purified by chromatography on silica gel eluted by CH2Cl2– MeOH (92 8) to yield 1.06 g (85%) of a powder; TLC Rf (CH2Cl2–MeOH 92 8) 0.50; LSIMS m/z 547 [M 1 Li]1 (100) 491 (8) 447 (17) 445 (3) and 247 (8); HR-LSIMS [M 1 Li]1 547.3657 (Calc. for C27H48LiN4O7 m/z 547.3683); [a]D 22 277 (c 0.1 MeOH); dH(300 MHz; CD3OH) 0.93 (m 12 H d Leu1 d Leu4) 1.44 (s 12 H b Aib Boc) 1.46 (s 3 H b Aib) 1.47 (m 2 H 2 × b Leu1) 1.60 (m 1 H b9 Leu4) 1.69 (m 2 H g Leu1 g Leu4) 1.75 (m 1 H b Leu4) 1.88 (m 3 H b9 Pro 2 × g Pro) 2.08 (m 1 H b Pro) 3.62 (m 2 H 2 × d Pro) 3.68 (s 3 H ester) 4.09 (m 1 H a Leu1) 4.34 (m 1 H a Leu4) 4.47 (m 1 H a Pro) 6.68 (d J 8.1 1 H NH Leu1) and 8.21 (br s 2 H NH Aib NH Leu4).Boc-Iva-Leu-Aib-Pro-Leu-OMe. Boc-Leu-Aib-Pro-Leu-OMe (150 mg 0.28 mmol) was deprotected according to procedure B. The crude TFA salt and Boc-Iva-OH (73 mg 0.34 mmol) were treated with BOP (149 mg 0.34 mmol) and DIEA (180 mm3 1 mmol) according to procedure A. The mixture was purified by chromatography on silica gel (CH2Cl2–MeOH 94 6) to yield 152 mg (85%) of the expected product; TLC Rf (CH2Cl2– MeOH 93 7) 0.43; LSIMS m/z 662 [M 1 Na]1 (15) 495 (3) 398 (44) 313 (4) and 243 (100); HR-LSIMS [M 1 Li]1 646.4385 (Calc. for C32H57LiN5O8 m/z 646.4367); dH(300 MHz; CD3OH) 0.84 (m 3 H Meg Iva) 0.95 (m 12 H d Leu2 d Leu5) 1.34 (s 3 H Meb Iva) 1.47 (s 15 H b Aib Boc) 1.62 (m 2 H b9 Leu2 b9 Leu5) 1.70 (m 2 H g Leu2 g Leu5) 1.77 (m 2 H b Leu2 b Leu5) 1.78 (m 1 H b9 Iva) 1.85 (m 1 H b9 Pro) 1.88 (m 2 H g Pro) 1.92 (m 1 H b Iva) 2.16 (m 1 H b Pro) 3.53 (m 1 H d9 Pro) 3.3 (m 1 H d Pro) 3.68 (s 3 H ester) 4.35 (m 2 H a Leu2 a Leu5) 4.48 (m 1 H a Pro) 6.93 (s 1 H NH Iva) 7.94 (d J 7.0 1 H NH Leu2) 8.14 (s 1 H NH Aib) and 8.16 (d J 8.3 1 H NH Leu5).Boc-Pro-Iva-Leu-Aib-Pro-Leu-OMe. Boc-Iva-Leu-Aib-Pro- Leu-OMe (140 mg 0.22 mmol) was deprotected according to procedure B. The crude TFA salt and Boc-Pro-OH (71 mg 0.33 mmol) were treated with BOP (146 mg 0.33 mmol) and DIEA (134 mm3 0.77 mmol) according to procedure A to yield after purification by silica gel chromatography (CH2Cl2–MeOH 95 5) 149 mg (92%) of a powder; TLC Rf (CH2Cl2–MeOH 94 6) 0.44; LSIMS m/z 743 [M 1 Li]1 (100) 643 (14) 641 (5) 473 (3) 388 (9) and 247 (5); HR-LSIMS [M 1 Li]1 743.4897 (Calc.for C37H64LiN6O9 m/z 743.4895); [a]D 22 233 (c 0.1 MeOH); dH(300 MHz; CD3OH) 0.82 (m 3 H Meg Iva) 0.92 (m 12 H d Leu3 d Leu6) 1.36 (s 3 H Meb Iva) 1.49 (s 3 H b Aib) 1.50 (s 12 H b Aib Boc) 1.62 (m 2 H b9 Leu3 b9 Leu6) 1.70 (m 2 H g Leu3 g Leu6) 1.80 (m 2 H b Leu3 b Leu6) 1.82 (m 1 H b9 Iva) 1.83 (m b9 Pro5) 1.86 (m 2 H g Pro5) 1.95 (m 1 H b9 Pro1) 1.98 (m 2 H g Pro1) 2.18 (m 1 H b Pro5) 2.23 (m 1 H b Iva) 4.31 (m 1 H b Pro1) 3.51 (m 2 H d9 Pro1 d9 Pro5) 3.60 (m 1 H d Pro1) 3.69 (s 3 H ester) 3.74 (m 1 H d Pro5) 4.08 (m 1 H a Pro1) 4.36 (m 2 H a Leu3 a Leu6) 4.49 (m 1 H a Pro5) 7.82 (d J 8.5 1 H NH Leu3) 7.87 (s 1 H NH Iva) 8.12 (s 1 H NH Aib) and 8.16 (d J 7.7 1 H NH Leu6). Boc-Ile-Aib-OMe. According to procedure A H-Aib-OMe (1.16 g 7.6 mmol) and Boc-Ile-OH (2.21 g 9.2 mmol) were treated with BOP (4.07 g 9.2 mmol) and DIEA (2.6 cm3 15.2 mmol).The crude product was used without purification for the next coupling. TLC Rf (ethyl acetate–cyclohexane 1 1) 0.61. Boc-Leu-Ile-Aib-OMe. Boc-Ile-Aib-OMe (2.51 g 7.6 mmol) was deprotected according to procedure B. The TFA salt and Boc-Leu-OH (2.27 g 9.1 mmol) were treated with BOP (4.03 g 9.1 mmol) and DIEA (4.2 cm3 24 mmol) according to procedure A. The mixture was purified by silica gel chromatography (ethyl acetate–cyclohexane 5 5) to yield 1.08 g (32% for the two steps) of a solid; TLC Rf (ethyl acetate–cyclohexane, J. Chem. Soc. Perkin Trans. 1 1997 1593 1 1) 0.53; LSIMS m/z 450 [M 1 Li]1 (100) 394 (12) 350 (24) 348 (3) 305 (2) and 192 (8); HR-LSIMS [M 1 Li]1 450.3159 (Calc.for C22H41LiN3O6 m/z 450.3155); dH(300 MHz; CD3OH) 0.93 (m 12 H d Leu Meg Ile Med Ile) 1.16 (m 1 H g9 Ile) 1.42 (s 3 H b Aib) 1.43 (s 9 H Boc) 1.45 (s 3 H b Aib) 1.53 (m 3 H g Ile 2 × b Leu) 1.66 (m 1 H g Leu) 1.77 (m 1 H b Ile) 3.64 (s 3 H ester) 4.07 (m 1 H a Leu) 4.18 (m 1 H a Ile) 6.86 (d J 7.7 1 H NH Leu) 7.71 (d J 8.6 1 H NH Ile) and 8.35 (s 1 H NH Aib). Boc-Asn-Leu-Ile-Aib-OMe. Boc-Leu-Ile-Aib-OMe (1.03 g 2.3 mmol) was deprotected according to procedure C. The crude TFA salt and Boc-Asn-OH (0.65 g 2.8 mmol) were treated with BOP (1.24 g 2.8 mmol) and DIEA (1.3 cm3 7.5 mmol) according to procedure A. After purification by silica gel chromatography (CH2Cl2–MeOH 95 5) it yielded 890 mg (69%) of a powder; TLC Rf (ethyl acetate) 0.17; LSIMS m/z 564 [M 1 Li]1 (100) 508 (4) 464 (31) 462 (3) 419 (4) and 306 (3); HR-LSIMS [M 1 Li]1 564.3583 (Calc.for C26H47LiN5O8 m/z 564.3585); dH(300 MHz; CD3OH) 0.89 (m 3 H Med Ile) 0.92 (m 6 H d Leu) 0.94 (m 3 H Meg Ile) 1.17 (m 1 H g9 Ile) 1.41 (s 3 H b Aib) 1.42 (s 3 H b Aib) 1.43 (s 9 H Boc) 1.43 (m 1 H b9 Leu) 1.55 (m 1 H g Ile) 1.62 (m 1 H b Leu) 1.73 (m 1 H g Leu) 1.90 (m 1 H b Ile) 2.66 (m 2 H 2 × Asn) 3.64 (s 3 H ester) 4.12 (m 1 H a Ile) 4.37 (m 1 H a Leu) 4.41 (m 1 H a Asn) 6.86 (d J 7.7 1 H NH Asn) 6.94 (s 1 H d syn Asn) 7.60 (s 1 H d anti Asn) 7.96 (d J 8.7 1 H NH Ile) 8.03 (s 1 H NH Aib) and 8.17 (d J 7.2 1 H NH Leu). Ac-Aib-Asn-Leu-Ile-Aib-OMe. Boc-Asn-Leu-Ile-Aib-OMe (0.54 g 0.97 mmol) was deprotected according to procedure C. The TFA salt and Ac-Aib-OH (0.27 g 1.84 mmol) were treated with BOP (0.81 g 1.84 mmol) and DIEA (0.5 cm3 3.0 mmol) according to procedure A.After purification by silica gel chromatography (CH2Cl2–MeOH 90 10) it yielded 190 mg of a powder; TLC Rf (CH2Cl2–MeOH 88 12) 0.30; LSIMS m/z 591 [M 1 Li]1 (100) 333 (6) 291 (16) 220 (11) 209 (40) 122 (7) and 106 (15); HR-LSIMS [M 1 Li]1 591.3678 (Calc. for C27H48LiN6O8 m/z 591.3694); dH(300 MHz; CD3OH) 0.90 (m 12 H d Leu Meg Ile Med Ile) 1.21 (m 1 H g9 Ile) 1.43 (m 12 H b Aib1 b Aib5) 1.55 (m 2 H b9 Leu g Ile) 1.69 (m 1 H b Leu) 1.87 (m 2 H g Leu b Ile) 1.99 (s 3 H Ac) 2.71 (ABX system J 7.6 and 15.4 1 H b9 Asn) 2.81 (ABX system J 6.3 and 15.4 1 H b Asn) 3.65 (s 3 H ester) 4.14 (m 1 H a Ile) 4.27 (m 1 H a Leu) 4.41 (m 1 H a Asn) 6.94 (s 1 H d syn Asn) 7.38 (d J 8.9 1 H NH Ile) 7.69 (s 1 H d anti Asn) 7.83 (s 1 H NH Aib5) 8.11 (d J 7.2 1 H NH Leu) 8.46 (d J 6.2 1 H NH Asn) and 8.54 (s 1 H NH Aib1).Ac-Aib-Asn-Leu-Ile-Aib-OH. A stirred solution of Ac-Aib- Asn-Leu-Ile-Aib-OMe (140 mg 0.24 mmol) in MeOH (1.4 cm3) was cooled in an ice-bath and 1.0 cm3 of 1 M NaOH was added. The mixture was stirred at room temperature until TLC analysis indicated complete consumption of the methyl ester. The cooled mixture was then neutralized with 1 M HCl and evaporated in vacuo. The crude product was purified using Sephadex LH-20 with MeOH as eluent to yield 116 mg (85%) of a solid; LSIMS m/z 593 [M 1 Na]1 (100) 468 (7) 355 (14) 242 (23) and 128 (18); HR-LSIMS [M 1 Na]1 593.3293 (Calc. for C26H46N6NaO8 m/z 593.3275); [a]D 22 210 (c 0.1 MeOH); dH(300 MHz; CD3OH) 0.87 (m 6 H Med Ile d Leu) 0.94 (d J 6.8 3 H Meg Ile) 0.95 (d J 6.5 3 H d Leu) 1.21 (m 1 H g9 Ile) 1.42 (s 3 H b Aib) 1.45 (s 3 H b Aib) 1.46 (s 3 H b Aib) 1.47 (s 3 H b Aib) 1.54 (m 1 H g Ile) 1.57 (m 1 H b9 Leu) 1.73 (m 1 H g Leu) 1.81 (m 1 H b Leu) 1.95 (m 1 H b Ile) 1.99 (s 3 H Ac) 2.73 (ABX system J 4.9 and 15.4 1 H b9 Asn) 2.81 (ABX system J 6.3 and 15.4 1 H b Asn) 4.16 (m 1 H a Ile) 4.28 (m 1 H a Leu) 4.45 (m 1 H a Asn) 6.93 (s 1 H d syn Asn) 7.45 (d J 9.1 1 H NH Ile) 7.69 (s 1 H d anti Asn) 7.78 (s 1 H NH Aib5) 8.12 (d J 7.5 1 H NH Leu) 8.45 (d J 6.5 1 H NH Asn) and 8.53 (s 1 H NH Aib1).Ac-Aib-Asn-Leu-Ile-Aib-Pro-Iva-Leu-Aib-Pro-Leu-OMe. Boc-Pro-Iva-Leu-Aib-Pro-Leu-OMe (100 mg 0.13 mmol) was deprotected according to procedure B. The TFA salt and Ac- Aib-Asn-Leu-Ile-Aib-OH (50 mg 0.087 mmol) were treated with BOP (38.5 mg 0.087 mmol) and DIEA (45 mm3 0.26 mmol) according to procedure A to yield after purification by silica gel chromatography (CH2Cl2–MeOH 85 15) 42 mg (41%) of a powder; TLC Rf (CH2Cl2–MeOH 85 15) 0.42; LSIMS m/z 1195 [M 1 Li]1 (100) 798 (6) 641 (27) 628 (6) 531 (13) 446 (16) 333 (9) and 291 (8); HR-LSIMS [M 1 Li]1 1195.7598 (Calc.for C58H100LiN12O14 m/z 1195.7642); dH(300 MHz; CD3OH) 0.83 (m 3 H Meg Iva) 0.87 (m 3 H Med Ile) 0.89 (m 18 H d Leu3 d Leu8 d Leu11) 0.95 (m 3 H Meg Ile) 1.30 (m 1 H g9 Ile) 1.45 (s 3 H Meb Iva) 1.46 (s 6 H b Aib) 1.49 (s 9 H b Aib) 1.50 (s 3 H b Aib) 1.55 (m 1 H g Ile) 1.76 (m 9 H b and g Leu3 b and g Leu8 b and g Leu11) 1.77 (m 1 H b9 Iva) 1.80 (m 1 H b9 Pro6) 1.83 (m 1 H b9 Pro10) 1.86 (m 2 H 2 × g Pro10) 1.92 (m 1 H g9 Pro6) 1.96 (m 1 H b Ile) 2.02 (s 3 H Ac) 2.12 (m 1 H g Pro6) 2.20 (m 1 H b Pro10) 2.32 (m 1 H b Pro6) 2.46 (m 1 H b Iva) 2.76 (d J 5.7 2 H b Asn) 3.42 (m 2 H d9 Pro6 d9 Pro10) 3.69 (s 3 H ester) 3.81 (m 2 H d Pro6 d Pro10) 4.20 (m 3 H a Leu3 a Ile4 a Pro6) 4.34 (m 3 H a Asn2 a Leu8 a Leu11) 4.50 (m 1 H a Pro10) 7.04 (s 1 H d syn Asn) 7.35 (d J 8.8 1 H NH Ile4) 7.49 (s 1 H NH Iva) 7.62 (d J 8.5 1 H NH Leu8) 7.76 (s 2 H NH Aib5 NH Aib9) 7.80 (s 1 H d anti Asn) 8.11 (d J 7.1 1 H NH Leu3) 8.16 (d J 7.8 1 H NH Leu11) 8.61 (d J 5.5 1 H NH Asn) and 8.67 (s 1 H NH Aib1).Ac-Aib-Asn-Leu-Ile-Aib-Pro-Iva-Leu-Aib-Pro-Leuol HB I. A solution of Ac-Aib-Asn-Leu-Ile-Aib-Pro-Iva-Leu-Aib-Pro- Leu-OMe (32 mg 0.027 mmol) in EtOH (1 cm3) was cooled in an ice-bath and NaBH4 (6 mg 0.162 mmol) was added. The mixture was stirred at 50 8C for 8 h and the solvent was evaporated off in vacuo.The residue was dissolved in ethyl acetate and washed with water. The organic layer was dried over Na2SO4 filtered and concentrated in vacuo to yield 25 mg (80%) of HB I; TLC Rf (CH2Cl2–MeOH 85 15) 0.37; LSIMS m/z 1167 [M 1 Li]1 (55) 947 (64) 553 (81) 468 (34) 395 (28) 355 (63) 310 (25) 242 (100) 197 (69) and 128 (70); HR-LSIMS [M 1 Na]1 1183.7333 (Calc. for C57H100N12NaO13 m/z 1183.7430); [a]D 22 17 (c 0.1 MeOH); 1 NMR data were identical with those described for natural HB I. Acknowledgements We are indebted to Dr M. F. Roquebert (Laboratoire de Cryptogamie du Muséum National d’Histoire Naturelle) who provided the T. harzianum strain and to Dr A. Galat (CEA Saclay France) for the dichrograph facility. We thank Dr M.Becchi and the Centre de Spectroscopie du CNRS (Lyon France) for LSIMS measurements and Dr Duclohier for macroscopic current–voltage experiments. The 500 MHz facilities used in this study were funded by the Région Haute- Normandie France. This work was supported in part by a grant from the Centre National de la Recherche Scientifique (GDR 1153). References 1 R. C. Pandey J. C. Cook Jr. and K. L. Rinehart J. Am. Chem. Soc. 1977 99 8469. 2 B. Bodo S. Rebuffat M. El Hajji and D. Davoust J. Am. Chem. Soc. 1985 107 6011. 3 A. Iida S. Uesato T. Shingu M. Okuda Y. Nagaoka Y. Kuroda and T. Fujita J. Chem. Soc. Perkin Trans. 1 1993 367. 4 S. Rebuffat L. Conraux M. Massias C. Auvin-Guette and B. Bodo Int. J. Pept. Protein Res. 1993 41 74. 5 C. Goulard S. Hlimi S. Rebuffat and B. Bodo J. Antibiot.1995 48 1248. 6 A. Iida M. Sanekata S. Wada T. Fujita H. Tanaka A. Enoki G. Fuse M. Kanai and K. Asami Chem. Pharm. Bull. 1995 43 392. 1594 J. Chem. Soc. Perkin Trans. 1 1997 7 S. Rebuffat C. Goulard and B. Bodo J. Chem. Soc. Perkin Trans. 1 1995 1849. 8 C. Auvin-Guette S. Rebuffat Y. Prigent and B. Bodo J. Am. Chem. Soc. 1992 114 2170. 9 T. Fujita S. Wada A. Iida T. Nishimura M. Kanai and N. Toyama Chem. Pharm. Bull. 1994 42 489. 10 M. S. P. Sansom Prog. Biophys. Mol. Biol. 1991 55 139. 11 S. Rebuffat H. Duclohier C. Auvin-Guette G. Molle G. Spach and B. Bodo FEMS Microbiol. Immunol. 1992 105 151. 12 T. Le Doan M. El Hajji S. Rebuffat M. R. Rajesvari and B. Bodo Biochim. Biophys. Acta 1986 858 1. 13 M. El Hajji S. Rebuffat T. Le Doan G. Klein M. Satre and B. Bodo Biochim.Biophys. Acta 1989 978 97. 14 S. Hlimi S. Rebuffat C. Goulard S. Duchamp and B. Bodo J. Antibiot. 1995 48 1254. 15 A. R. Artalejo C. Montiel P. Sanchez-Garcia G. Uceda J. M. Guantes and A. G. Garcia Biochem. Biophys. Res. Commun. 1990 1204. 16 A. Iida M. Okuda S. Uesato Y. Takaishi T. Shingu M. Morita and T. Fujita J. Chem. Soc. Perkin Trans. 1 1990 3249. 17 S. Rebuffat P. Drognat-Landré C. Goulard I. Augeven-Bour C. Auvin and B. Bodo Peptides 1992 Proceedings of the 22nd European Peptide Symposium ed. C. H. Schneider and A. N. Eberle Escom Science Publishers Leiden 1993 pp. 427–428. 18 P. Roepstorff P. Höjrup and J. Möller Biomed. Mass Spectrom. 1985 12 181. 19 S. Wada A. Iida N. Akimoto M. Kanai N. Toyama and T. Fujita Chem. Pharm. Bull. 1995 43 910. 20 G. Esposito J. A. Carver J.Boyd and I. D. Campbell Biochemistry 1987 26 1043. 21 S. Rebuffat Y. Prigent C. Auvin-Guette and B. Bodo Eur. J. Biochem. 1991 201 661. 22 Y. Nagaoka A. Iida and T. Fujita Chem. Pharm. Bull. 1994 42 1258. 23 C. Toniolo M. Crisma F. Formaggio C. Peggion V. Monaco C. Goulard S. Rebuffat and B. Bodo J. Am. Chem. Soc. 1996 118 4952. 24 C. Griesinger G. Otting K. Wuthrich and R. R. Ernst J. Am. Chem. Soc. 1988 110 7870. 25 A. Bax and D. G. Davis J. Magn. Reson. 1985 63 207. 26 D. J. States R. A. Haberkorn and D. J. Ruben J. Magn. Reson. 1982 48 286. 27 M. Piotto U. Savdek and V. Sklenar J. Biomol. NMR 1992 2 661. 28 J. N. Weinstein S. Yoshikami P. Henkari R. Blumenthal and W. A. Hagins Science 1977 195 489. 29 R. A. Boissonnas S. Guttmann P. A. Jaquenoud and J. P. Waller Helv. Chim.Acta 1955 38 1491. 30 M. Bodanszky and A. Bodanszky The Practice of Peptide Synthesis Springer-Verlag Berlin Heidelberg New York and Tokyo 1984 p. 20. Paper 6/05629F Received 12th August 1996 Accepted 11th February 1997 © Copyright 1997 by the Royal Society of Chemistry J. Chem. Soc. Perkin Trans. 1 1997 1587 Harzianin HB I an 11-residue peptaibol from Trichoderma harzianum isolation sequence solution synthesis and membrane activity Isabelle Augeven-Bour,a Sylvie Rebuffat,a Catherine Auvin,a Christophe Goulard,a Yann Prigent b and Bernard Bodo *,a a Laboratoire de Chimie URA 401 CNRS GDR 1153 CNRS Muséum National d’Histoire Naturelle 63 rue Buffon 75231 Paris Cedex 05 France b Laboratoire de RMN URA 464 CNRS Institut Fédératif de Recherche Multidisciplinaire sur les Peptides No 23 INSERM Université de Rouen 76821 Mont-Saint-Aignan Cedex France Harzianin HB I is a minor component of the peptaibol mixture biosynthesized by a Trichoderma harzianum strain.It is isolated by a multi-step chromatography procedure including HPLC; its sequence has been elucidated by LSIMS and two-dimensional 1H NMR experiments. This 11-residue peptaibol is an analogue of the 14-residue peptaibol harzianin HC IX resulting from the deletion of the tripeptide Aib- Pro-Ala. The CD and NMR data (NOE data and amide proton temperature coefficients) are similar to those of HC IX suggesting a right-handed helix-type conformation for the two peptides. Harzianin HB I was synthesized by the solution-phase method using BOP as coupling reagent. The membrane properties examined on liposomes are compared with those of other known peptaibols.Introduction Peptaibols biosynthesized by Trichoderma soil fungi are amphipathic linear peptides with a high proportion of a,adialkylated amino acids such as a-aminoisobutyric acid (Aib U) an N-terminal acetylated residue and a C-terminal amino alcohol. They can be classified into the long-sequence group containing 18 to 20 residues,1–5 the short-sequence group with 11 to 16 residues 6,7 and the lipopeptaibols with 7 to 11 residues and an N-terminal amino acid acylated by a lipid chain.8,9 The main interest in such peptides stems from their ability to form voltage-gated ion channels in planar lipid bilayer membranes 10,11 and therefore they can be viewed as a prototypic pore. In the absence of a voltage peptaibols induce permeabilization of liposomes;12,13 long-sequence ones being more effi- cient than short-sequence ones.Their membrane activity is modulated by both the length and hydrophobicity of the sequence. The known biological properties of these peptides are their antibiotic 14 and catecholamine secretion-inducing abilities 15,16 which have been suggested to be related to their membrane activity. Recently we isolated peptaibols organised in helical structures which still have significant membrane activity in spite of their short sequences such as the 11-residue lipopeptaibol GA IV and the proline-rich 14 residue peptaibols harzianins HC.7 From a Uruguayan strain of Trichoderma harzianum we previously isolated two groups of peptaibols trichorzins HA and harzianins HC containing 18 and 14 residues respectively.From the particularly complex mixture of the 14-residue peptides a minor component of the same polarity harzianin HB I was isolated. Its structure was determined from liquid secondary ion mass spectrometry (LSIMS) and NMR spectroscopy. It was synthesized in order to test its antibiotic and membrane properties. Harzianin HB I was shown to modify slightly the permeability of liposomes and induce voltage-gated macroscopic conductance in planar bilayers. Sequence of harzianin HB I Results and discussion Isolation and characterization of HB I 17 The culture broth extract of the Uruguayan M-903603 T. harzianum strain was fractionated by exclusion chromatography; the peptide fraction was further chromatographed over silica gel to yield two peptaibol groups of different polarity.The first group was composed of 18-residue peptaibols the trichorzins HA,5,14 and the second one mostly of the 14-residue harzianins HC.7 The HPLC chromatogram of the crude HC mixture was very complex showing more than 16 peaks one of them being assigned to a minor component HB I. Its isolation was undertaken by repetitive semi-preparative reversed-phase high-performance liquid chromatography (RP-HPLC) and its purity was checked by analytical HPLC (Fig. 1). Further LSIMS and 1H NMR experiments confirmed HB I to be homogeneous. It reacted neither with diazomethane nor with ninhydrin indicating the presence of neither free carboxy nor amino groups. The presence of a sharp singlet at dH 2.02 in the 1D NMR spectrum suggested an acetylated N-terminal residue as usually found in peptaibols.The amino acid composition and absolute configuration of the residues resulted from GLC analysis of the total acid hydrolysate derivatives on a Chirasil L-Val capillary column Aib (3) L-Asn (1) L-Ile (1) D-Iva (1) L-Leu (2) L-Pro (2) L-Leuol (1). Sequence determination of HB I Positive liquid secondary-ion mass spectrometry [(1)-LSIMS]. The amino acid sequence of HB I was examined by positive LSIMS. A relative molecular mass of 1160 was assigned to HB I from the sodium ion adduct [M 1 Na]1 observed at m/z 1183 in agreement with the postulated molecular mass arising from the amino acid and amino alcohol composition. The LSIMS spectrum exhibited several abundant b-type acylium ions 18 at m/z 947 553 468 355 242 and 128 and two lower-abundance ion regions between m/z 553 and 947 and between m/z 947 and 1183.This fragmentation pattern suggested the presence of two labile Aib–Pro bonds in HB I. The series of b-type ions ranging from m/z 128 to 553 gave the N-terminal pentapeptide sequence. The mass difference of 394 amu between m/z 947 and 553 was in agreement with the formation of the tetrapeptide ion observed at m/z 395 and 1588 J. Chem. Soc. Perkin Trans. 1 1997 Fig. 1 HPLC chromatogram of purified natural harzianin HB I (Spherisorb ODS2 5mm) 4.6 × 250 mm MeOH–water (83 17) flow rate 1 cm3 min21; absorption monitored at 220 nm obtained via cleavage of the Aib5–Pro6 amide bond.6,19 The Cterminal dipeptide resulting from cleavage of the Aib9–Pro10 amide bond was observed as a y2 ion at m/z 215. Furthermore a series of weak and unusual but significant [xn 1 Na]1 ions at m/z 1083 969 856 743 462 and 167 was observed and allowed the confirmation of the HB I sequence.18 Nevertheless the respective locations of the isomeric residues Leu/Ile remained to be determined.1H NMR spectroscopy. The complete amino acid sequence of HB I was determined by two-dimensional 1H NMR spectroscopy. Assignments of 1H chemical shifts to specific protons of individual residues (Table 1) were obtained by 2D homonuclear (COSY) and phase-sensitive 2D total (TOCSY) chemical-shift correlation experiments showing complete spin systems of one Asn two Pro and one Leuol (Fig. 2) as well as those of one Ile and two Leu residues in agreement with 13C resonances of one Ile Ca at dC 64.1 and two Leu Ca at dC 53.2 and 54.1 (data not shown). The sequence-specific assignments of the backbone NH proton signals arose from the rotating-frame nuclear Overhauser effect (ROESY) spectrum and were completely carried out by using inter-residue connectivities dNN(i,i 1 1) and daN(i,i 1 1) [Fig.3(a)]. The lowest-field singlet NH proton showing a cross-peak with the acetyl CbH3 protons was assigned to Aib1. Then dNN(i,i 1 1) connectivities extending from Asn2 to Aib5 and from Iva7 to Aib9 confirmed the location of Ile at position 4. Finally sequential assignment of Pro6 and Pro10 arose from dNd(i,i 1 1) connectivities between their d protons and the amide protons of Aib5 and Aib9 [Fig. 3(b)]. H3C C O NH C C O NH CH C NH2 O C O NH CH C O NH CH C O NH C C O N CH C O NH C C O NH CH C O NH C C O N CH C O NH CH CH2OH (x10 + Na)+ 1083 (6) (x9 + Na)+ 969 (1) (x8 + Na)+ 856 (2) (x7 + Na)+ 743 (2) (x4 + Na)+ 462 (2) (x1 + Na)+ 167 (2) (b) Mass fragmentation pattern of harzianin HB I (positive-ion LSIMS) exhibiting the bn ions (a) and the xn 1 Na ions (b) (relative intensities in parentheses) J.Chem. Soc. Perkin Trans. 1 1997 1589 Table 1 1H sequential and stereospecific assignments of harzianin HB I (500.13 MHz; CD3OH; 296 K). Chemical shifts (ppm) are given to the nearest three or two decimal places when obtained from 1D or 2D spectra respectively; 3JNH-CaH coupling constants (Hz) arise from the 1D spectrum and are given in parentheses Residue NH a-H b-H/b-Me Other groups Ac Me 2.027 s Aib1 8.679 s pro-R 1.46*/pro-S 1.450 Asn2 8.610 d (5.6) 4.38 2.76 e syn 7.04/e anti 7.77 Leu3 8.111 d (7.1) 4.25 1.93/1.56 g 1.74/d1 0.89/d2 0.96 Ile4 7.356 d (9.1) 4.22 1.95 g 1.55/g9 1.32/g-Me 0.95/d-Me 0.86 Aib5 7.806 s pro-R 1.498*/pro-S 1.501 Pro6 4.21 pro-R 1.78/pro-S 2.35 pro-R g 1.94/pro-S g 2.10/pro-R d 3.85/pro-S d 3.41 Iva7 7.484 s 2.45/1.77/b-Me 1.46 g 0.82 Leu8 7.627 d (8.5) 4.39 1.75/1.75 g 1.75/d1 0.85/d2 0.96 Aib9 7.836 s pro-R 1.450*/pro-S 1.501 Pro10 4.42 pro-R 1.78/pro-S 2.29 pro-R g 1.87/pro-S g 1.93/pro-R d 3.88/pro-S d 3.44 Leuol11 7.534 d (8.9) 3.96 1.60/1.35/3.53 g 1.64/d1 0.90/d2 0.95 * May be exchanged.Conformation of HB I The conformation of HB I in methanol solution was examined by CD and NMR spectroscopy using inter-residue NOE connectivities 3JNH-CaH coupling constants and amide temperature coefficients. The CD spectrum of HB I showed two transitions at 192 (1) and 205 (2) nm characteristic of a right-handed helix.Fig. 2 Expansion of the TOCSY spectrum of HB I in CD3OH (spin lock period 120 ms) (a) w2 = 0.6–4.5 ppm w1 = 7.3–8.7 ppm; (b) w2 = 4.1–4.6 ppm w1 = 1.7–4.6 ppm; spin-systems are labelled with the sequential residue positions A stretch of strong sequential dNN(i,i 1 1) accompanied by daN(i,i 1 1) and by a series of medium and strong daN(i,i 1 3) all along the sequence was observed in the ROESY spectrum (Fig. 3) in agreement with a helical structure. The presence of daN(i,i 1 2) NOEs all along the sequence and the complete absence of daN(i,i 1 4) connectivities suggested a succession of turns stabilized by 4Æ1 intramolecular hydrogen bonds as observed for the 310-helix. This was in agreement with the amide protons’ thermal coefficients (Dd/DTNH) (Fig. 4). Little information was obtained from the 3JNH-CaH coupling constants as only half of the residues could give such data (Fig.4). The Asn2 and Leu3 residues showed values <7 Hz whereas Ile4 Leu8 and Leuol11 had higher values around 9 Hz apparently inconsistent Fig. 3 Parts of the ROESY spectrum of HB I in CD3OH (mixing time 250 ms) (a) w2 = 7.3–8.8 ppm w1 = 7.1–8.7 ppm; (b) w2 = 7.3–8.0 w1 = 3.2–4.8 ppm 1590 J. Chem. Soc. Perkin Trans. 1 1997 with a helical structure. However such values have frequently been observed for the two amino acids flanking Aib-Pro segments in a-helical peptaibols.4,5,20–22 The studied residues in HB I have such a location in the sequence. Comparison of the thermal coefficients and coupling constants of HB I with those of the longer analogue HC IX which contains an additional Aib-Pro-Ala tripeptide after Leu3 showed extensive similarity of the data.17 The results thus suggested a structure stabilized by 4Æ1-type intramolecular hydrogen bonds forming a ribbon of b-turns.Synthesis of HB I In order to make available a sufficient amount of harzianin HB I for bioassays it was synthesized by a solution-phase method in the presence of the (benzotriazol-1-yloxy)tris(dimethylamino) phosphonium hexafluorophosphate (BOP) coupling reagent in CH2Cl2 at room temperature according to the synthetic route shown in Scheme 1. The penta- and hexa-peptide fragments were built up in a stepwise manner using Boc/OMe Fig. 4 Amino acid sequence of harzianin HB I (the one-letter code of amino acid residues is used with U = Aib J = Iva Lol = Leuol) and a survey of the NOE connectivities involving NH and CaH (dad connectivities observed for prolines are indicated by white boxes) of the 3JNH-CaH coupling constants and of the temperature coefficients of the amide protons.The observed NOEs are classified as strong medium and weak (based on counting the cross-peak contour levels) and shown by thick medium and thin lines respectively. strategy. They were designed so that Aib was placed at the Cterminal position in order to avoid racemization during the deprotection and activation steps. The synthesis of HB I was achieved by reduction of the C-terminal methyl ester group into an alcohol function by NaBH4–EtOH. Synthetic HB I was finally purified by semi-preparative HPLC. The analytical HPLC retention time (tR) and the 1H NMR spectrum of synthetic HB I were identical to those of natural HB I.Antibacterial activity The antibacterial activity of HB I examined against S. aureus and E. coli showed it to be inactive against E. coli in agreement with previous observations on other peptaibols.4,5,7 However no antibacterial activity against S. aureus was detected even at 200 mg pit21 whereas growth inhibition induced by short-sequence peptaibols harzianins HC and trichogin GA IV could be detected up to 50 and 1.5 mg pit21 respectively.7,8 This result was in agreement with the absence of antibacterial activity noticed for C2-GA IV,23 the analogue of GA IV with an acetyl group instead of the lipid chain. The absence of antibacterial activity for this 11-residue peptaibol confirms the role of the Nterminal lipid chain in the lipopeptaibol activity.Membrane-modifying properties of HB I Long-sequence peptaibols have been previously shown to exhibit membrane-modifying properties by increasing the permeability of liposomes.5,11 Optimal membrane activity was observed for a hydrophobic neutral a-helix of 18–19 residues while the liposome permeabilization decreased for shortersequence peptaibols.5 The membrane-modifying activity of HB I was studied by fluorescence spectroscopy following the leakage of a carboxyfluorescein (CF) fluorescent probe previously entrapped at self-quenched concentration in small unilamellar vesicles. The results presented as a percentage of escaped CF at 20 min as a function of Ri 21 = [peptide]/[lipid] were compared with those of other short peptides such as the 14- residue HC IX and the 11-residue lipopeptaibol GA IV (Fig.5). Comparison of Ri 21 values characteristic of 50% release of the entrapped material showed HB I (Ri 21 = 83 × 1023) to be less efficient than HC IX (Ri 21 = 12 × 1023) and GA IV Scheme 1 Scheme for the total synthesis of HB I J. Chem. Soc. Perkin Trans. 1 1997 1591 (Ri 21 = 4 × 1023). This result points to the major role of the sequence length the presence of the N-terminal lipidic chain also favouring the liposome permeabilization. The voltage-dependent channel-forming properties of harzianin HB I were also examined by macroscopic current– voltage experiments (G. Molle H. Duclohier unpublished results). In such conditions HB I exhibited channel-forming activity for concentrations ranging between 1026 and 1025 M in the same way as the 11-residue peptaibol trichorozin TZ-IV,6 or the 14-residue harzianins HC.7 Experimental Isolation of harzianin HB I The T.harzianum strain (M-903603) collected in Uruguay was obtained from the ‘Collection de souches fongiques du Muséum National d’Histoire Naturelle’ (Paris); the strain was maintained and cultivated as previously described.7 The culture was incubated for 11 days at 27 8C. The filtered fermentation broth was extracted three times with butan-1-ol to give after removal of the solvent under reduced pressure 1.2 g of crude extract. The residue was submitted to gel filtration on Pharmacia Sephadex LH 20 with methanol as eluent. The crude peptide mixture (468 mg) was then chromatographed over silica gel (Kieselgel 60 H Merck Darmstadt) with CH2Cl2–MeOH (9 1 to 5 5) as eluent.The HC/HB mixture (130 mg) was eluted with CH2Cl2–MeOH (80 20). HPLC separation This was carried out with a Waters liquid chromatograph (6000 A and M45 pumps a 680 automated solvent programmer a WISP 712 automatic injector and a 481 UV–VIS detector) on a semipreparative C18 column (Spherisorb ODS2; 5 mm; 7.5 × 300 mm; AIT France); eluent methanol–water (83 17); flow rate 2 cm3 min21. The purity of HB I (3 mg) was confirmed on an analytical column (3.5 × 250 mm); eluent methanol–water (83 17); flow rate 1 cm3 min21; tR 16 min. Amino acid analysis Total hydrolysis of HB I was carried out according to the usual procedure for peptides (6 M HCl at 110 8C in sealed tubes for 24 h). Identification of the amino acids was accomplished by gas chromatography after derivatization.4 Retention times of the Ntrifluoroacetyl isopropyl ester derivatives were compared with those of standard samples.The GLC analyses were performed with a Girdel 3000 chromatograph on a Chirasil-L-Val (Npropionyl- L-valine tert-butylamide polysiloxane) quartz capillary column (Chrompack 25 m length 0.2 mm i.d.) with He (0.7 × 105 Pa) as carrier gas and a temperature programme 50–130 8C 3 8C min21; 130–190 8C 10 8C min21; tR (min) Aib Fig. 5 Peptide-induced CF at t = 20 min for different [peptide]/[lipid] ratios (Ri 21) from egg PC/cholesterol 70/30 vesicles (a) HB I (b) HC IX (Ac-Aib-Asn-Leu-Aib-Pro-Ala-Ile-Aib-Pro-Iva-Leu-Aib-Pro-Leuol) and (c) GA IV (Oc-Aib-Gly-Leu-Aib-Gly-Gly-Leu-Aib-Gly-Ile-Leuol) (10.4) L-Asp (29.5) L-Ile (20.4) D-Iva (11.2) L-Leu (24.2) LLeuol (22.2). A special temperature programme was used for the separation of proline enantiomers 50–110 8C 3 8C min21; plateau at 110 8C for 10 min; 100–190 8C 10 8C min21; tR L-Pro (25.1).Secondary ion mass spectroscopy Positive LSIMS was recorded on a ZAB2-SEQ (VG Analytical Manchester UK) mass spectrometer equipped with a standard FAB source and a caesium ion-gun operating at 35 kV. Peptide methanolic solution was mixed with a-monothioglycerol as matrix. The resolution was 1000. Positive HR-LSIMS were recorded on a ZAB-HF spectrometer. The MS spectra were registered with either Li1 or Na1 added to the matrix. NMR Spectroscopy A 0.4 cm3 aliquot of 7 mM methanolic (CD3OH) solution of HB I peptide in a 5 mm tube (Wilmad) was used for all the NMR experiments. Proton NMR spectroscopy experiments were conducted at 296 K on a Bruker AC 300 equipped with an Aspect 3000 computer or on a Bruker Avance DMX 500 spectrometer equipped with a Bruker Station 1 computer and an indirect quadruple-resonance 1H–31P–13C–15N gradient probehead.Spectra were processed using UXNMR and AURELIA software (Bruker Inc). Chemical shifts were referenced to the central component of the quintet due to the CD2H resonance of methanol at dH 3.313 downfield from SiMe4. J Values are given in Hz. TOCSY24 experiments were run with the MLEV 17 sequence for spin locking and a mixing time of 120 ms (9 kHz). The ROESY25 experiment was carried out with a mixing time of 250 ms and a spin lock field of 2 kHz to reduce Hartmann–Hahn transfers. Two-dimensional spectra were obtained with quadrature detection in both dimensions using the hypercomplex method in the F1 dimension.26 The solvent signal was suppressed using the WATERGATE scheme27 included in the standard and ROESY pulse sequences.A total of 2048 data points were acquired in the F2 dimension and 512 complex points in the F1 dimension. For each complex data point in the F2 4 free induction decays were accumulated with a relaxation delay of 2 s. All spectra were apodized with p/2-shifted sine-bell functions in both dimensions. CD spectrum The spectrum of HB I was recorded with a Jobin-Yvon CD6 dichrograph with a 0.1 mm path cell at 22 8C (1 mmol cm23; CH3OH); l (nm) and [q]M (deg cm2 dmol21) 192 (56 000) and 205 (2110 000). Antimicrobial activity The antibacterial activity of HB I was examined against Staphylococcus aureus (strain 209P) and Escherichia coli (strain RL 65) by the agar diffusion test using the Mueller Hinton culture medium and 6 mm diameter pits.The peptide sample was dissolved in dimethyl sulfoxide (DMSO) such as to give a 4 mg cm23 solution. Eight other concentrations were obtained by successive dilutions and 50 mm3 of each solution was deposited into the pits (1.5 to 200 mg). Inhibition zones were measured after 24 h of incubation at 37 8C. Liposome permeabilization Egg phosphatidylcholine (egg PC) type V E and cholesterol were purchased from Sigma; egg PC was used without further purification and cholesterol was recrystallized from methanol. CF from Eastman Kodak was separated from hydrophobic contaminants and recrystallized from ethanol as previously described.11 Fluorescence spectra were measured at 20 8C on an Aminco SPF 500 spectrofluorometer.The peptide-induced release of intravesicular content was monitored by the method introduced by Weinstein,28 that uses the property of quenching relief upon dilution of an encapsulated fluorescent probe CF. 1592 J. Chem. Soc. Perkin Trans. 1 1997 CF-entrapped small unilamellar vesicles (SUV) were prepared as previously described,11 by sonication of an egg PC– cholesterol (7 3) mixture ([lip] = 0.6 mM). The SUV obtained by sonication were separated from unencapsulated CF by gel filtration (Sephadex G 75). Leakage kinetics were obtained for different peptide lipid molar ratios obtained by adding aliquots of methanolic solutions of peptides (methanol concentration kept below 0.5% by volume). Synthesis of HB I Diisopropylethylamine (DIEA) trifluoroacetic acid (TFA) ditert- butyl dicarbonate (Boc2O) and L-leucine were purchased from Sigma-Aldrich Chimie and D-isovaline [(R)-(2)-2-amino- 2-methylbutanoic acid] from Acros (France).All N-tertbutoxycarbonyl- protected L-amino acids and BOP were purchased from Propeptide (France) and used without subsequent purification. H-Leu-OMe was prepared according to Boissonnas et al.29 Boc-Iva-OH was prepared according to Bodanszky and Bodanszky.30 Column chromatography was carried out with 230–400 mesh Merck grade 60 silica gel. Analytical TLC was performed on aluminium sheets covered with Merck grade 60 silica gel. Gel filtration was carried out with Pharmacia Sephadex LH 20. General procedure A BOP-mediated peptide coupling. The Nprotected amino acid and BOP reagent were added to a solution of the TFA salt of the C-protected amino acid or peptide in CH2Cl2.The stirred solution was cooled in an ice-bath and DIEA was added. The mixture was stirred at room temperature until TLC analysis indicated that consumption of the amino component was no longer proceeding. It was then concentrated in vacuo to leave an oil which was dissolved in ethyl acetate and washed successively with 1 M HCl water 1 M NaOH and saturated aq. NaCl. The combined organics were dried over Na2SO4 filtered and concentrated in vacuo to leave an oil which was purified by chromatography on a silica gel column. General procedure B Removal of N-tert-butoxycarbonyl protection with 50% TFA solution in CH2Cl2. A stirred solution of the N-tert-butoxycarbonyl-protected peptide in CH2Cl2 was cooled in an ice-bath and TFA was added.The mixture was stirred at room temperature until TLC analysis indicated complete consumption of the starting material and was then evaporated in vacuo to leave an oil. The crude product was used without purification for the next coupling. General procedure C Removal of N-tert-butoxycarbonyl protection with pure TFA. The N-tert-butoxycarbonyl-protected peptide was treated with TFA (0.5 cm3 for 1 mmol of peptide). The mixture was stirred at room temperature until TLC analysis indicated that the totality of the peptide was deprotected and it was then evaporated in vacuo to give an oil. The crude product was used without purification for the next coupling. Boc-Pro-Leu-OMe. HCl H-Leu-OMe (0.92 g 5.07 mmol) and Boc-Pro-OH (1.20 g 5.58 mmol) were treated with BOP (2.47 g 5.58 mmol) and DIEA (2.7 cm3 15.71 mmol) via procedure A to yield the crude product which was used without purification.Boc-Aib-Pro-Leu-OMe. Boc-Pro-Leu-OMe (1.73 g 5.07 mmol) was deprotected according to procedure C. The crude TFA salt and Boc-Aib-OH (1.12 g 5.50 mmol) were treated with BOP (2.43 g 5.50 mmol) and DIEA (2.7 cm3 15.7 mmol) according to procedure A to yield after purification by chromatography on silica gel (ethyl acetate) 1 g of a powder; TLC Rf (ethyl acetate) 0.40; LSIMS m/z 434 [M 1 Li]1 (100) 378 (5) 334 (27) 332 (2) and 247 (9); HR-LSIMS [M 1 Li]1 434.2822 (Calc. for C21H37LiN3O6 m/z 434.2842); dH(300 MHz; CD3OH) 0.91 (d J 6.0 3 H d Leu) 0.94 (d J 6.0 3 H d Leu) 1.36 (s 3 H b Aib) 1.45 (s 12 H b Aib Boc) 1.81 (m 6 H 2 × b Leu g Leu b9 Pro 2 × g Pro) 2.19 (m 1 H b Pro) 3.59 (m 1 H d9 Pro) 3.68 (s 3 H ester) 3.81 (m 1 H d Pro) 4.37 (m 1 H a Leu) 4.45 (dd J 8.4 and 5.9 1 H a Pro) 7.29 (s 1 H NH Aib) and 8.23 (d J 7.7 1 H NH Leu).Boc-Leu-Aib-Pro-Leu-OMe. Boc-Aib-Pro-Leu-OMe (1.00 g 2.34 mmol) was deprotected according to procedure B. The crude TFA salt and Boc-Leu-OH (0.64 g 2,58 mmol) were treated with BOP (1.14 g 2.58 mmol) and DIEA (1.3 cm3 7.3 mmol) according to general procedure A. The mixture was purified by chromatography on silica gel eluted by CH2Cl2– MeOH (92 8) to yield 1.06 g (85%) of a powder; TLC Rf (CH2Cl2–MeOH 92 8) 0.50; LSIMS m/z 547 [M 1 Li]1 (100) 491 (8) 447 (17) 445 (3) and 247 (8); HR-LSIMS [M 1 Li]1 547.3657 (Calc. for C27H48LiN4O7 m/z 547.3683); [a]D 22 277 (c 0.1 MeOH); dH(300 MHz; CD3OH) 0.93 (m 12 H d Leu1 d Leu4) 1.44 (s 12 H b Aib Boc) 1.46 (s 3 H b Aib) 1.47 (m 2 H 2 × b Leu1) 1.60 (m 1 H b9 Leu4) 1.69 (m 2 H g Leu1 g Leu4) 1.75 (m 1 H b Leu4) 1.88 (m 3 H b9 Pro 2 × g Pro) 2.08 (m 1 H b Pro) 3.62 (m 2 H 2 × d Pro) 3.68 (s 3 H ester) 4.09 (m 1 H a Leu1) 4.34 (m 1 H a Leu4) 4.47 (m 1 H a Pro) 6.68 (d J 8.1 1 H NH Leu1) and 8.21 (br s 2 H NH Aib NH Leu4).Boc-Iva-Leu-Aib-Pro-Leu-OMe. Boc-Leu-Aib-Pro-Leu-OMe (150 mg 0.28 mmol) was deprotected according to procedure B. The crude TFA salt and Boc-Iva-OH (73 mg 0.34 mmol) were treated with BOP (149 mg 0.34 mmol) and DIEA (180 mm3 1 mmol) according to procedure A. The mixture was purified by chromatography on silica gel (CH2Cl2–MeOH 94 6) to yield 152 mg (85%) of the expected product; TLC Rf (CH2Cl2– MeOH 93 7) 0.43; LSIMS m/z 662 [M 1 Na]1 (15) 495 (3) 398 (44) 313 (4) and 243 (100); HR-LSIMS [M 1 Li]1 646.4385 (Calc.for C32H57LiN5O8 m/z 646.4367); dH(300 MHz; CD3OH) 0.84 (m 3 H Meg Iva) 0.95 (m 12 H d Leu2 d Leu5) 1.34 (s 3 H Meb Iva) 1.47 (s 15 H b Aib Boc) 1.62 (m 2 H b9 Leu2 b9 Leu5) 1.70 (m 2 H g Leu2 g Leu5) 1.77 (m 2 H b Leu2 b Leu5) 1.78 (m 1 H b9 Iva) 1.85 (m 1 H b9 Pro) 1.88 (m 2 H g Pro) 1.92 (m 1 H b Iva) 2.16 (m 1 H b Pro) 3.53 (m 1 H d9 Pro) 3.3 (m 1 H d Pro) 3.68 (s 3 H ester) 4.35 (m 2 H a Leu2 a Leu5) 4.48 (m 1 H a Pro) 6.93 (s 1 H NH Iva) 7.94 (d J 7.0 1 H NH Leu2) 8.14 (s 1 H NH Aib) and 8.16 (d J 8.3 1 H NH Leu5). Boc-Pro-Iva-Leu-Aib-Pro-Leu-OMe. Boc-Iva-Leu-Aib-Pro- Leu-OMe (140 mg 0.22 mmol) was deprotected according to procedure B. The crude TFA salt and Boc-Pro-OH (71 mg 0.33 mmol) were treated with BOP (146 mg 0.33 mmol) and DIEA (134 mm3 0.77 mmol) according to procedure A to yield after purification by silica gel chromatography (CH2Cl2–MeOH 95 5) 149 mg (92%) of a powder; TLC Rf (CH2Cl2–MeOH 94 6) 0.44; LSIMS m/z 743 [M 1 Li]1 (100) 643 (14) 641 (5) 473 (3) 388 (9) and 247 (5); HR-LSIMS [M 1 Li]1 743.4897 (Calc.for C37H64LiN6O9 m/z 743.4895); [a]D 22 233 (c 0.1 MeOH); dH(300 MHz; CD3OH) 0.82 (m 3 H Meg Iva) 0.92 (m 12 H d Leu3 d Leu6) 1.36 (s 3 H Meb Iva) 1.49 (s 3 H b Aib) 1.50 (s 12 H b Aib Boc) 1.62 (m 2 H b9 Leu3 b9 Leu6) 1.70 (m 2 H g Leu3 g Leu6) 1.80 (m 2 H b Leu3 b Leu6) 1.82 (m 1 H b9 Iva) 1.83 (m b9 Pro5) 1.86 (m 2 H g Pro5) 1.95 (m 1 H b9 Pro1) 1.98 (m 2 H g Pro1) 2.18 (m 1 H b Pro5) 2.23 (m 1 H b Iva) 4.31 (m 1 H b Pro1) 3.51 (m 2 H d9 Pro1 d9 Pro5) 3.60 (m 1 H d Pro1) 3.69 (s 3 H ester) 3.74 (m 1 H d Pro5) 4.08 (m 1 H a Pro1) 4.36 (m 2 H a Leu3 a Leu6) 4.49 (m 1 H a Pro5) 7.82 (d J 8.5 1 H NH Leu3) 7.87 (s 1 H NH Iva) 8.12 (s 1 H NH Aib) and 8.16 (d J 7.7 1 H NH Leu6).Boc-Ile-Aib-OMe. According to procedure A H-Aib-OMe (1.16 g 7.6 mmol) and Boc-Ile-OH (2.21 g 9.2 mmol) were treated with BOP (4.07 g 9.2 mmol) and DIEA (2.6 cm3 15.2 mmol). The crude product was used without purification for the next coupling. TLC Rf (ethyl acetate–cyclohexane 1 1) 0.61. Boc-Leu-Ile-Aib-OMe. Boc-Ile-Aib-OMe (2.51 g 7.6 mmol) was deprotected according to procedure B. The TFA salt and Boc-Leu-OH (2.27 g 9.1 mmol) were treated with BOP (4.03 g 9.1 mmol) and DIEA (4.2 cm3 24 mmol) according to procedure A. The mixture was purified by silica gel chromatography (ethyl acetate–cyclohexane 5 5) to yield 1.08 g (32% for the two steps) of a solid; TLC Rf (ethyl acetate–cyclohexane, J.Chem. Soc. Perkin Trans. 1 1997 1593 1 1) 0.53; LSIMS m/z 450 [M 1 Li]1 (100) 394 (12) 350 (24) 348 (3) 305 (2) and 192 (8); HR-LSIMS [M 1 Li]1 450.3159 (Calc. for C22H41LiN3O6 m/z 450.3155); dH(300 MHz; CD3OH) 0.93 (m 12 H d Leu Meg Ile Med Ile) 1.16 (m 1 H g9 Ile) 1.42 (s 3 H b Aib) 1.43 (s 9 H Boc) 1.45 (s 3 H b Aib) 1.53 (m 3 H g Ile 2 × b Leu) 1.66 (m 1 H g Leu) 1.77 (m 1 H b Ile) 3.64 (s 3 H ester) 4.07 (m 1 H a Leu) 4.18 (m 1 H a Ile) 6.86 (d J 7.7 1 H NH Leu) 7.71 (d J 8.6 1 H NH Ile) and 8.35 (s 1 H NH Aib). Boc-Asn-Leu-Ile-Aib-OMe. Boc-Leu-Ile-Aib-OMe (1.03 g 2.3 mmol) was deprotected according to procedure C. The crude TFA salt and Boc-Asn-OH (0.65 g 2.8 mmol) were treated with BOP (1.24 g 2.8 mmol) and DIEA (1.3 cm3 7.5 mmol) according to procedure A.After purification by silica gel chromatography (CH2Cl2–MeOH 95 5) it yielded 890 mg (69%) of a powder; TLC Rf (ethyl acetate) 0.17; LSIMS m/z 564 [M 1 Li]1 (100) 508 (4) 464 (31) 462 (3) 419 (4) and 306 (3); HR-LSIMS [M 1 Li]1 564.3583 (Calc. for C26H47LiN5O8 m/z 564.3585); dH(300 MHz; CD3OH) 0.89 (m 3 H Med Ile) 0.92 (m 6 H d Leu) 0.94 (m 3 H Meg Ile) 1.17 (m 1 H g9 Ile) 1.41 (s 3 H b Aib) 1.42 (s 3 H b Aib) 1.43 (s 9 H Boc) 1.43 (m 1 H b9 Leu) 1.55 (m 1 H g Ile) 1.62 (m 1 H b Leu) 1.73 (m 1 H g Leu) 1.90 (m 1 H b Ile) 2.66 (m 2 H 2 × Asn) 3.64 (s 3 H ester) 4.12 (m 1 H a Ile) 4.37 (m 1 H a Leu) 4.41 (m 1 H a Asn) 6.86 (d J 7.7 1 H NH Asn) 6.94 (s 1 H d syn Asn) 7.60 (s 1 H d anti Asn) 7.96 (d J 8.7 1 H NH Ile) 8.03 (s 1 H NH Aib) and 8.17 (d J 7.2 1 H NH Leu).Ac-Aib-Asn-Leu-Ile-Aib-OMe. Boc-Asn-Leu-Ile-Aib-OMe (0.54 g 0.97 mmol) was deprotected according to procedure C. The TFA salt and Ac-Aib-OH (0.27 g 1.84 mmol) were treated with BOP (0.81 g 1.84 mmol) and DIEA (0.5 cm3 3.0 mmol) according to procedure A. After purification by silica gel chromatography (CH2Cl2–MeOH 90 10) it yielded 190 mg of a powder; TLC Rf (CH2Cl2–MeOH 88 12) 0.30; LSIMS m/z 591 [M 1 Li]1 (100) 333 (6) 291 (16) 220 (11) 209 (40) 122 (7) and 106 (15); HR-LSIMS [M 1 Li]1 591.3678 (Calc. for C27H48LiN6O8 m/z 591.3694); dH(300 MHz; CD3OH) 0.90 (m 12 H d Leu Meg Ile Med Ile) 1.21 (m 1 H g9 Ile) 1.43 (m 12 H b Aib1 b Aib5) 1.55 (m 2 H b9 Leu g Ile) 1.69 (m 1 H b Leu) 1.87 (m 2 H g Leu b Ile) 1.99 (s 3 H Ac) 2.71 (ABX system J 7.6 and 15.4 1 H b9 Asn) 2.81 (ABX system J 6.3 and 15.4 1 H b Asn) 3.65 (s 3 H ester) 4.14 (m 1 H a Ile) 4.27 (m 1 H a Leu) 4.41 (m 1 H a Asn) 6.94 (s 1 H d syn Asn) 7.38 (d J 8.9 1 H NH Ile) 7.69 (s 1 H d anti Asn) 7.83 (s 1 H NH Aib5) 8.11 (d J 7.2 1 H NH Leu) 8.46 (d J 6.2 1 H NH Asn) and 8.54 (s 1 H NH Aib1).Ac-Aib-Asn-Leu-Ile-Aib-OH. A stirred solution of Ac-Aib- Asn-Leu-Ile-Aib-OMe (140 mg 0.24 mmol) in MeOH (1.4 cm3) was cooled in an ice-bath and 1.0 cm3 of 1 M NaOH was added. The mixture was stirred at room temperature until TLC analysis indicated complete consumption of the methyl ester. The cooled mixture was then neutralized with 1 M HCl and evaporated in vacuo. The crude product was purified using Sephadex LH-20 with MeOH as eluent to yield 116 mg (85%) of a solid; LSIMS m/z 593 [M 1 Na]1 (100) 468 (7) 355 (14) 242 (23) and 128 (18); HR-LSIMS [M 1 Na]1 593.3293 (Calc.for C26H46N6NaO8 m/z 593.3275); [a]D 22 210 (c 0.1 MeOH); dH(300 MHz; CD3OH) 0.87 (m 6 H Med Ile d Leu) 0.94 (d J 6.8 3 H Meg Ile) 0.95 (d J 6.5 3 H d Leu) 1.21 (m 1 H g9 Ile) 1.42 (s 3 H b Aib) 1.45 (s 3 H b Aib) 1.46 (s 3 H b Aib) 1.47 (s 3 H b Aib) 1.54 (m 1 H g Ile) 1.57 (m 1 H b9 Leu) 1.73 (m 1 H g Leu) 1.81 (m 1 H b Leu) 1.95 (m 1 H b Ile) 1.99 (s 3 H Ac) 2.73 (ABX system J 4.9 and 15.4 1 H b9 Asn) 2.81 (ABX system J 6.3 and 15.4 1 H b Asn) 4.16 (m 1 H a Ile) 4.28 (m 1 H a Leu) 4.45 (m 1 H a Asn) 6.93 (s 1 H d syn Asn) 7.45 (d J 9.1 1 H NH Ile) 7.69 (s 1 H d anti Asn) 7.78 (s 1 H NH Aib5) 8.12 (d J 7.5 1 H NH Leu) 8.45 (d J 6.5 1 H NH Asn) and 8.53 (s 1 H NH Aib1).Ac-Aib-Asn-Leu-Ile-Aib-Pro-Iva-Leu-Aib-Pro-Leu-OMe. Boc-Pro-Iva-Leu-Aib-Pro-Leu-OMe (100 mg 0.13 mmol) was deprotected according to procedure B. The TFA salt and Ac- Aib-Asn-Leu-Ile-Aib-OH (50 mg 0.087 mmol) were treated with BOP (38.5 mg 0.087 mmol) and DIEA (45 mm3 0.26 mmol) according to procedure A to yield after purification by silica gel chromatography (CH2Cl2–MeOH 85 15) 42 mg (41%) of a powder; TLC Rf (CH2Cl2–MeOH 85 15) 0.42; LSIMS m/z 1195 [M 1 Li]1 (100) 798 (6) 641 (27) 628 (6) 531 (13) 446 (16) 333 (9) and 291 (8); HR-LSIMS [M 1 Li]1 1195.7598 (Calc. for C58H100LiN12O14 m/z 1195.7642); dH(300 MHz; CD3OH) 0.83 (m 3 H Meg Iva) 0.87 (m 3 H Med Ile) 0.89 (m 18 H d Leu3 d Leu8 d Leu11) 0.95 (m 3 H Meg Ile) 1.30 (m 1 H g9 Ile) 1.45 (s 3 H Meb Iva) 1.46 (s 6 H b Aib) 1.49 (s 9 H b Aib) 1.50 (s 3 H b Aib) 1.55 (m 1 H g Ile) 1.76 (m 9 H b and g Leu3 b and g Leu8 b and g Leu11) 1.77 (m 1 H b9 Iva) 1.80 (m 1 H b9 Pro6) 1.83 (m 1 H b9 Pro10) 1.86 (m 2 H 2 × g Pro10) 1.92 (m 1 H g9 Pro6) 1.96 (m 1 H b Ile) 2.02 (s 3 H Ac) 2.12 (m 1 H g Pro6) 2.20 (m 1 H b Pro10) 2.32 (m 1 H b Pro6) 2.46 (m 1 H b Iva) 2.76 (d J 5.7 2 H b Asn) 3.42 (m 2 H d9 Pro6 d9 Pro10) 3.69 (s 3 H ester) 3.81 (m 2 H d Pro6 d Pro10) 4.20 (m 3 H a Leu3 a Ile4 a Pro6) 4.34 (m 3 H a Asn2 a Leu8 a Leu11) 4.50 (m 1 H a Pro10) 7.04 (s 1 H d syn Asn) 7.35 (d J 8.8 1 H NH Ile4) 7.49 (s 1 H NH Iva) 7.62 (d J 8.5 1 H NH Leu8) 7.76 (s 2 H NH Aib5 NH Aib9) 7.80 (s 1 H d anti Asn) 8.11 (d J 7.1 1 H NH Leu3) 8.16 (d J 7.8 1 H NH Leu11) 8.61 (d J 5.5 1 H NH Asn) and 8.67 (s 1 H NH Aib1).Ac-Aib-Asn-Leu-Ile-Aib-Pro-Iva-Leu-Aib-Pro-Leuol HB I. A solution of Ac-Aib-Asn-Leu-Ile-Aib-Pro-Iva-Leu-Aib-Pro- Leu-OMe (32 mg 0.027 mmol) in EtOH (1 cm3) was cooled in an ice-bath and NaBH4 (6 mg 0.162 mmol) was added. The mixture was stirred at 50 8C for 8 h and the solvent was evaporated off in vacuo. The residue was dissolved in ethyl acetate and washed with water. The organic layer was dried over Na2SO4 filtered and concentrated in vacuo to yield 25 mg (80%) of HB I; TLC Rf (CH2Cl2–MeOH 85 15) 0.37; LSIMS m/z 1167 [M 1 Li]1 (55) 947 (64) 553 (81) 468 (34) 395 (28) 355 (63) 310 (25) 242 (100) 197 (69) and 128 (70); HR-LSIMS [M 1 Na]1 1183.7333 (Calc. for C57H100N12NaO13 m/z 1183.7430); [a]D 22 17 (c 0.1 MeOH); 1 NMR data were identical with those described for natural HB I.Acknowledgements We are indebted to Dr M. F. Roquebert (Laboratoire de Cryptogamie du Muséum National d’Histoire Naturelle) who provided the T. harzianum strain and to Dr A. Galat (CEA Saclay France) for the dichrograph facility. We thank Dr M. Becchi and the Centre de Spectroscopie du CNRS (Lyon France) for LSIMS measurements and Dr Duclohier for macroscopic current–voltage experiments. The 500 MHz facilities used in this study were funded by the Région Haute- Normandie France. This work was supported in part by a grant from the Centre National de la Recherche Scientifique (GDR 1153). References 1 R. C. Pandey J. C. Cook Jr. and K. L. Rinehart J. Am. Chem. Soc. 1977 99 8469. 2 B. Bodo S. Rebuffat M. El Hajji and D. Davoust J. Am. Chem.Soc. 1985 107 6011. 3 A. Iida S. Uesato T. Shingu M. Okuda Y. Nagaoka Y. Kuroda and T. Fujita J. Chem. Soc. Perkin Trans. 1 1993 367. 4 S. Rebuffat L. Conraux M. Massias C. Auvin-Guette and B. Bodo Int. J. Pept. Protein Res. 1993 41 74. 5 C. Goulard S. Hlimi S. Rebuffat and B. Bodo J. Antibiot. 1995 48 1248. 6 A. Iida M. Sanekata S. Wada T. Fujita H. Tanaka A. Enoki G. Fuse M. Kanai and K. Asami Chem. Pharm. Bull. 1995 43 392. 1594 J. Chem. Soc. Perkin Trans. 1 1997 7 S. Rebuffat C. Goulard and B. Bodo J. Chem. Soc. Perkin Trans. 1 1995 1849. 8 C. Auvin-Guette S. Rebuffat Y. Prigent and B. Bodo J. Am. Chem. Soc. 1992 114 2170. 9 T. Fujita S. Wada A. Iida T. Nishimura M. Kanai and N. Toyama Chem. Pharm. Bull. 1994 42 489. 10 M. S. P. Sansom Prog. Biophys. Mol. Biol. 1991 55 139.11 S. Rebuffat H. Duclohier C. Auvin-Guette G. Molle G. Spach and B. Bodo FEMS Microbiol. Immunol. 1992 105 151. 12 T. Le Doan M. El Hajji S. Rebuffat M. R. Rajesvari and B. Bodo Biochim. Biophys. Acta 1986 858 1. 13 M. El Hajji S. Rebuffat T. Le Doan G. Klein M. Satre and B. Bodo Biochim. Biophys. Acta 1989 978 97. 14 S. Hlimi S. Rebuffat C. Goulard S. Duchamp and B. Bodo J. Antibiot. 1995 48 1254. 15 A. R. Artalejo C. Montiel P. Sanchez-Garcia G. Uceda J. M. Guantes and A. G. Garcia Biochem. Biophys. Res. Commun. 1990 1204. 16 A. Iida M. Okuda S. Uesato Y. Takaishi T. Shingu M. Morita and T. Fujita J. Chem. Soc. Perkin Trans. 1 1990 3249. 17 S. Rebuffat P. Drognat-Landré C. Goulard I. Augeven-Bour C. Auvin and B. Bodo Peptides 1992 Proceedings of the 22nd European Peptide Symposium ed.C. H. Schneider and A. N. Eberle Escom Science Publishers Leiden 1993 pp. 427–428. 18 P. Roepstorff P. Höjrup and J. Möller Biomed. Mass Spectrom. 1985 12 181. 19 S. Wada A. Iida N. Akimoto M. Kanai N. Toyama and T. Fujita Chem. Pharm. Bull. 1995 43 910. 20 G. Esposito J. A. Carver J. Boyd and I. D. Campbell Biochemistry 1987 26 1043. 21 S. Rebuffat Y. Prigent C. Auvin-Guette and B. Bodo Eur. J. Biochem. 1991 201 661. 22 Y. Nagaoka A. Iida and T. Fujita Chem. Pharm. Bull. 1994 42 1258. 23 C. Toniolo M. Crisma F. Formaggio C. Peggion V. Monaco C. Goulard S. Rebuffat and B. Bodo J. Am. Chem. Soc. 1996 118 4952. 24 C. Griesinger G. Otting K. Wuthrich and R. R. Ernst J. Am. Chem. Soc. 1988 110 7870. 25 A. Bax and D. G. Davis J. Magn. Reson. 1985 63 207. 26 D. J. States R. A.Haberkorn and D. J. Ruben J. Magn. Reson. 1982 48 286. 27 M. Piotto U. Savdek and V. Sklenar J. Biomol. NMR 1992 2 661. 28 J. N. Weinstein S. Yoshikami P. Henkari R. Blumenthal and W. A. Hagins Science 1977 195 489. 29 R. A. Boissonnas S. Guttmann P. A. Jaquenoud and J. P. Waller Helv. Chim. Acta 1955 38 1491. 30 M. Bodanszky and A. Bodanszky The Practice of Peptide Synthesis Springer-Verlag Berlin Heidelberg New York and Tokyo 1984 p. 20. Paper 6/05629F Received 12th August 1996 Accepted 11th February 1997 © Copyright 1997 by the Royal Society of Chemistry J. Chem. Soc. Perkin Trans. 1 1997 1587 Harzianin HB I an 11-residue peptaibol from Trichoderma harzianum isolation sequence solution synthesis and membrane activity Isabelle Augeven-Bour,a Sylvie Rebuffat,a Catherine Auvin,a Christophe Goulard,a Yann Prigent b and Bernard Bodo *,a a Laboratoire de Chimie URA 401 CNRS GDR 1153 CNRS Muséum National d’Histoire Naturelle 63 rue Buffon 75231 Paris Cedex 05 France b Laboratoire de RMN URA 464 CNRS Institut Fédératif de Recherche Multidisciplinaire sur les Peptides No 23 INSERM Université de Rouen 76821 Mont-Saint-Aignan Cedex France Harzianin HB I is a minor component of the peptaibol mixture biosynthesized by a Trichoderma harzianum strain.It is isolated by a multi-step chromatography procedure including HPLC; its sequence has been elucidated by LSIMS and two-dimensional 1H NMR experiments. This 11-residue peptaibol is an analogue of the 14-residue peptaibol harzianin HC IX resulting from the deletion of the tripeptide Aib- Pro-Ala. The CD and NMR data (NOE data and amide proton temperature coefficients) are similar to those of HC IX suggesting a right-handed helix-type conformation for the two peptides.Harzianin HB I was synthesized by the solution-phase method using BOP as coupling reagent. The membrane properties examined on liposomes are compared with those of other known peptaibols. Introduction Peptaibols biosynthesized by Trichoderma soil fungi are amphipathic linear peptides with a high proportion of a,adialkylated amino acids such as a-aminoisobutyric acid (Aib U) an N-terminal acetylated residue and a C-terminal amino alcohol. They can be classified into the long-sequence group containing 18 to 20 residues,1–5 the short-sequence group with 11 to 16 residues 6,7 and the lipopeptaibols with 7 to 11 residues and an N-terminal amino acid acylated by a lipid chain.8,9 The main interest in such peptides stems from their ability to form voltage-gated ion channels in planar lipid bilayer membranes 10,11 and therefore they can be viewed as a prototypic pore.In the absence of a voltage peptaibols induce permeabilization of liposomes;12,13 long-sequence ones being more effi- cient than short-sequence ones. Their membrane activity is modulated by both the length and hydrophobicity of the sequence. The known biological properties of these peptides are their antibiotic 14 and catecholamine secretion-inducing abilities 15,16 which have been suggested to be related to their membrane activity. Recently we isolated peptaibols organised in helical structures which still have significant membrane activity in spite of their short sequences such as the 11-residue lipopeptaibol GA IV and the proline-rich 14 residue peptaibols harzianins HC.7 From a Uruguayan strain of Trichoderma harzianum we previously isolated two groups of peptaibols trichorzins HA and harzianins HC containing 18 and 14 residues respectively.From the particularly complex mixture of the 14-residue peptides a minor component of the same polarity harzianin HB I was isolated. Its structure was determined from liquid secondary ion mass spectrometry (LSIMS) and NMR spectroscopy. It was synthesized in order to test its antibiotic and membrane properties. Harzianin HB I was shown to modify slightly the permeability of liposomes and induce voltage-gated macroscopic conductance in planar bilayers. Sequence of harzianin HB I Results and discussion Isolation and characterization of HB I 17 The culture broth extract of the Uruguayan M-903603 T.harzianum strain was fractionated by exclusion chromatography; the peptide fraction was further chromatographed over silica gel to yield two peptaibol groups of different polarity. The first group was composed of 18-residue peptaibols the trichorzins HA,5,14 and the second one mostly of the 14-residue harzianins HC.7 The HPLC chromatogram of the crude HC mixture was very complex showing more than 16 peaks one of them being assigned to a minor component HB I. Its isolation was undertaken by repetitive semi-preparative reversed-phase high-performance liquid chromatography (RP-HPLC) and its purity was checked by analytical HPLC (Fig. 1). Further LSIMS and 1H NMR experiments confirmed HB I to be homogeneous.It reacted neither with diazomethane nor with ninhydrin indicating the presence of neither free carboxy nor amino groups. The presence of a sharp singlet at dH 2.02 in the 1D NMR spectrum suggested an acetylated N-terminal residue as usually found in peptaibols. The amino acid composition and absolute configuration of the residues resulted from GLC analysis of the total acid hydrolysate derivatives on a Chirasil L-Val capillary column Aib (3) L-Asn (1) L-Ile (1) D-Iva (1) L-Leu (2) L-Pro (2) L-Leuol (1). Sequence determination of HB I Positive liquid secondary-ion mass spectrometry [(1)-LSIMS]. The amino acid sequence of HB I was examined by positive LSIMS. A relative molecular mass of 1160 was assigned to HB I from the sodium ion adduct [M 1 Na]1 observed at m/z 1183 in agreement with the postulated molecular mass arising from the amino acid and amino alcohol composition.The LSIMS spectrum exhibited several abundant b-type acylium ions 18 at m/z 947 553 468 355 242 and 128 and two lower-abundance ion regions between m/z 553 and 947 and between m/z 947 and 1183. This fragmentation pattern suggested the presence of two labile Aib–Pro bonds in HB I. The series of b-type ions ranging from m/z 128 to 553 gave the N-terminal pentapeptide sequence. The mass difference of 394 amu between m/z 947 and 553 was in agreement with the formation of the tetrapeptide ion observed at m/z 395 and 1588 J. Chem. Soc. Perkin Trans. 1 1997 Fig. 1 HPLC chromatogram of purified natural harzianin HB I (Spherisorb ODS2 5mm) 4.6 × 250 mm MeOH–water (83 17) flow rate 1 cm3 min21; absorption monitored at 220 nm obtained via cleavage of the Aib5–Pro6 amide bond.6,19 The Cterminal dipeptide resulting from cleavage of the Aib9–Pro10 amide bond was observed as a y2 ion at m/z 215.Furthermore a series of weak and unusual but significant [xn 1 Na]1 ions at m/z 1083 969 856 743 462 and 167 was observed and allowed the confirmation of the HB I sequence.18 Nevertheless the respective locations of the isomeric residues Leu/Ile remained to be determined. 1H NMR spectroscopy. The complete amino acid sequence of HB I was determined by two-dimensional 1H NMR spectroscopy. Assignments of 1H chemical shifts to specific protons of individual residues (Table 1) were obtained by 2D homonuclear (COSY) and phase-sensitive 2D total (TOCSY) chemical-shift correlation experiments showing complete spin systems of one Asn two Pro and one Leuol (Fig.2) as well as those of one Ile and two Leu residues in agreement with 13C resonances of one Ile Ca at dC 64.1 and two Leu Ca at dC 53.2 and 54.1 (data not shown). The sequence-specific assignments of the backbone NH proton signals arose from the rotating-frame nuclear Overhauser effect (ROESY) spectrum and were completely carried out by using inter-residue connectivities dNN(i,i 1 1) and daN(i,i 1 1) [Fig. 3(a)]. The lowest-field singlet NH proton showing a cross-peak with the acetyl CbH3 protons was assigned to Aib1. Then dNN(i,i 1 1) connectivities extending from Asn2 to Aib5 and from Iva7 to Aib9 confirmed the location of Ile at position 4. Finally sequential assignment of Pro6 and Pro10 arose from dNd(i,i 1 1) connectivities between their d protons and the amide protons of Aib5 and Aib9 [Fig.3(b)]. H3C C O NH C C O NH CH C NH2 O C O NH CH C O NH CH C O NH C C O N CH C O NH C C O NH CH C O NH C C O N CH C O NH CH CH2OH (x10 + Na)+ 1083 (6) (x9 + Na)+ 969 (1) (x8 + Na)+ 856 (2) (x7 + Na)+ 743 (2) (x4 + Na)+ 462 (2) (x1 + Na)+ 167 (2) (b) Mass fragmentation pattern of harzianin HB I (positive-ion LSIMS) exhibiting the bn ions (a) and the xn 1 Na ions (b) (relative intensities in parentheses) J. Chem. Soc. Perkin Trans. 1 1997 1589 Table 1 1H sequential and stereospecific assignments of harzianin HB I (500.13 MHz; CD3OH; 296 K). Chemical shifts (ppm) are given to the nearest three or two decimal places when obtained from 1D or 2D spectra respectively; 3JNH-CaH coupling constants (Hz) arise from the 1D spectrum and are given in parentheses Residue NH a-H b-H/b-Me Other groups Ac Me 2.027 s Aib1 8.679 s pro-R 1.46*/pro-S 1.450 Asn2 8.610 d (5.6) 4.38 2.76 e syn 7.04/e anti 7.77 Leu3 8.111 d (7.1) 4.25 1.93/1.56 g 1.74/d1 0.89/d2 0.96 Ile4 7.356 d (9.1) 4.22 1.95 g 1.55/g9 1.32/g-Me 0.95/d-Me 0.86 Aib5 7.806 s pro-R 1.498*/pro-S 1.501 Pro6 4.21 pro-R 1.78/pro-S 2.35 pro-R g 1.94/pro-S g 2.10/pro-R d 3.85/pro-S d 3.41 Iva7 7.484 s 2.45/1.77/b-Me 1.46 g 0.82 Leu8 7.627 d (8.5) 4.39 1.75/1.75 g 1.75/d1 0.85/d2 0.96 Aib9 7.836 s pro-R 1.450*/pro-S 1.501 Pro10 4.42 pro-R 1.78/pro-S 2.29 pro-R g 1.87/pro-S g 1.93/pro-R d 3.88/pro-S d 3.44 Leuol11 7.534 d (8.9) 3.96 1.60/1.35/3.53 g 1.64/d1 0.90/d2 0.95 * May be exchanged.Conformation of HB I The conformation of HB I in methanol solution was examined by CD and NMR spectroscopy using inter-residue NOE connectivities 3JNH-CaH coupling constants and amide temperature coefficients.The CD spectrum of HB I showed two transitions at 192 (1) and 205 (2) nm characteristic of a right-handed helix. Fig. 2 Expansion of the TOCSY spectrum of HB I in CD3OH (spin lock period 120 ms) (a) w2 = 0.6–4.5 ppm w1 = 7.3–8.7 ppm; (b) w2 = 4.1–4.6 ppm w1 = 1.7–4.6 ppm; spin-systems are labelled with the sequential residue positions A stretch of strong sequential dNN(i,i 1 1) accompanied by daN(i,i 1 1) and by a series of medium and strong daN(i,i 1 3) all along the sequence was observed in the ROESY spectrum (Fig. 3) in agreement with a helical structure. The presence of daN(i,i 1 2) NOEs all along the sequence and the complete absence of daN(i,i 1 4) connectivities suggested a succession of turns stabilized by 4Æ1 intramolecular hydrogen bonds as observed for the 310-helix.This was in agreement with the amide protons’ thermal coefficients (Dd/DTNH) (Fig. 4). Little information was obtained from the 3JNH-CaH coupling constants as only half of the residues could give such data (Fig. 4). The Asn2 and Leu3 residues showed values <7 Hz whereas Ile4 Leu8 and Leuol11 had higher values around 9 Hz apparently inconsistent Fig. 3 Parts of the ROESY spectrum of HB I in CD3OH (mixing time 250 ms) (a) w2 = 7.3–8.8 ppm w1 = 7.1–8.7 ppm; (b) w2 = 7.3–8.0 w1 = 3.2–4.8 ppm 1590 J. Chem. Soc. Perkin Trans. 1 1997 with a helical structure. However such values have frequently been observed for the two amino acids flanking Aib-Pro segments in a-helical peptaibols.4,5,20–22 The studied residues in HB I have such a location in the sequence.Comparison of the thermal coefficients and coupling constants of HB I with those of the longer analogue HC IX which contains an additional Aib-Pro-Ala tripeptide after Leu3 showed extensive similarity of the data.17 The results thus suggested a structure stabilized by 4Æ1-type intramolecular hydrogen bonds forming a ribbon of b-turns. Synthesis of HB I In order to make available a sufficient amount of harzianin HB I for bioassays it was synthesized by a solution-phase method in the presence of the (benzotriazol-1-yloxy)tris(dimethylamino) phosphonium hexafluorophosphate (BOP) coupling reagent in CH2Cl2 at room temperature according to the synthetic route shown in Scheme 1.The penta- and hexa-peptide fragments were built up in a stepwise manner using Boc/OMe Fig. 4 Amino acid sequence of harzianin HB I (the one-letter code of amino acid residues is used with U = Aib J = Iva Lol = Leuol) and a survey of the NOE connectivities involving NH and CaH (dad connectivities observed for prolines are indicated by white boxes) of the 3JNH-CaH coupling constants and of the temperature coefficients of the amide protons. The observed NOEs are classified as strong medium and weak (based on counting the cross-peak contour levels) and shown by thick medium and thin lines respectively. strategy. They were designed so that Aib was placed at the Cterminal position in order to avoid racemization during the deprotection and activation steps.The synthesis of HB I was achieved by reduction of the C-terminal methyl ester group into an alcohol function by NaBH4–EtOH. Synthetic HB I was finally purified by semi-preparative HPLC. The analytical HPLC retention time (tR) and the 1H NMR spectrum of synthetic HB I were identical to those of natural HB I. Antibacterial activity The antibacterial activity of HB I examined against S. aureus and E. coli showed it to be inactive against E. coli in agreement with previous observations on other peptaibols.4,5,7 However no antibacterial activity against S. aureus was detected even at 200 mg pit21 whereas growth inhibition induced by short-sequence peptaibols harzianins HC and trichogin GA IV could be detected up to 50 and 1.5 mg pit21 respectively.7,8 This result was in agreement with the absence of antibacterial activity noticed for C2-GA IV,23 the analogue of GA IV with an acetyl group instead of the lipid chain.The absence of antibacterial activity for this 11-residue peptaibol confirms the role of the Nterminal lipid chain in the lipopeptaibol activity. Membrane-modifying properties of HB I Long-sequence peptaibols have been previously shown to exhibit membrane-modifying properties by increasing the permeability of liposomes.5,11 Optimal membrane activity was observed for a hydrophobic neutral a-helix of 18–19 residues while the liposome permeabilization decreased for shortersequence peptaibols.5 The membrane-modifying activity of HB I was studied by fluorescence spectroscopy following the leakage of a carboxyfluorescein (CF) fluorescent probe previously entrapped at self-quenched concentration in small unilamellar vesicles.The results presented as a percentage of escaped CF at 20 min as a function of Ri 21 = [peptide]/[lipid] were compared with those of other short peptides such as the 14- residue HC IX and the 11-residue lipopeptaibol GA IV (Fig. 5). Comparison of Ri 21 values characteristic of 50% release of the entrapped material showed HB I (Ri 21 = 83 × 1023) to be less efficient than HC IX (Ri 21 = 12 × 1023) and GA IV Scheme 1 Scheme for the total synthesis of HB I J. Chem. Soc. Perkin Trans. 1 1997 1591 (Ri 21 = 4 × 1023). This result points to the major role of the sequence length the presence of the N-terminal lipidic chain also favouring the liposome permeabilization. The voltage-dependent channel-forming properties of harzianin HB I were also examined by macroscopic current– voltage experiments (G.Molle H. Duclohier unpublished results). In such conditions HB I exhibited channel-forming activity for concentrations ranging between 1026 and 1025 M in the same way as the 11-residue peptaibol trichorozin TZ-IV,6 or the 14-residue harzianins HC.7 Experimental Isolation of harzianin HB I The T. harzianum strain (M-903603) collected in Uruguay was obtained from the ‘Collection de souches fongiques du Muséum National d’Histoire Naturelle’ (Paris); the strain was maintained and cultivated as previously described.7 The culture was incubated for 11 days at 27 8C. The filtered fermentation broth was extracted three times with butan-1-ol to give after removal of the solvent under reduced pressure 1.2 g of crude extract.The residue was submitted to gel filtration on Pharmacia Sephadex LH 20 with methanol as eluent. The crude peptide mixture (468 mg) was then chromatographed over silica gel (Kieselgel 60 H Merck Darmstadt) with CH2Cl2–MeOH (9 1 to 5 5) as eluent. The HC/HB mixture (130 mg) was eluted with CH2Cl2–MeOH (80 20). HPLC separation This was carried out with a Waters liquid chromatograph (6000 A and M45 pumps a 680 automated solvent programmer a WISP 712 automatic injector and a 481 UV–VIS detector) on a semipreparative C18 column (Spherisorb ODS2; 5 mm; 7.5 × 300 mm; AIT France); eluent methanol–water (83 17); flow rate 2 cm3 min21. The purity of HB I (3 mg) was confirmed on an analytical column (3.5 × 250 mm); eluent methanol–water (83 17); flow rate 1 cm3 min21; tR 16 min.Amino acid analysis Total hydrolysis of HB I was carried out according to the usual procedure for peptides (6 M HCl at 110 8C in sealed tubes for 24 h). Identification of the amino acids was accomplished by gas chromatography after derivatization.4 Retention times of the Ntrifluoroacetyl isopropyl ester derivatives were compared with those of standard samples. The GLC analyses were performed with a Girdel 3000 chromatograph on a Chirasil-L-Val (Npropionyl- L-valine tert-butylamide polysiloxane) quartz capillary column (Chrompack 25 m length 0.2 mm i.d.) with He (0.7 × 105 Pa) as carrier gas and a temperature programme 50–130 8C 3 8C min21; 130–190 8C 10 8C min21; tR (min) Aib Fig. 5 Peptide-induced CF at t = 20 min for different [peptide]/[lipid] ratios (Ri 21) from egg PC/cholesterol 70/30 vesicles (a) HB I (b) HC IX (Ac-Aib-Asn-Leu-Aib-Pro-Ala-Ile-Aib-Pro-Iva-Leu-Aib-Pro-Leuol) and (c) GA IV (Oc-Aib-Gly-Leu-Aib-Gly-Gly-Leu-Aib-Gly-Ile-Leuol) (10.4) L-Asp (29.5) L-Ile (20.4) D-Iva (11.2) L-Leu (24.2) LLeuol (22.2).A special temperature programme was used for the separation of proline enantiomers 50–110 8C 3 8C min21; plateau at 110 8C for 10 min; 100–190 8C 10 8C min21; tR L-Pro (25.1). Secondary ion mass spectroscopy Positive LSIMS was recorded on a ZAB2-SEQ (VG Analytical Manchester UK) mass spectrometer equipped with a standard FAB source and a caesium ion-gun operating at 35 kV. Peptide methanolic solution was mixed with a-monothioglycerol as matrix. The resolution was 1000. Positive HR-LSIMS were recorded on a ZAB-HF spectrometer.The MS spectra were registered with either Li1 or Na1 added to the matrix. NMR Spectroscopy A 0.4 cm3 aliquot of 7 mM methanolic (CD3OH) solution of HB I peptide in a 5 mm tube (Wilmad) was used for all the NMR experiments. Proton NMR spectroscopy experiments were conducted at 296 K on a Bruker AC 300 equipped with an Aspect 3000 computer or on a Bruker Avance DMX 500 spectrometer equipped with a Bruker Station 1 computer and an indirect quadruple-resonance 1H–31P–13C–15N gradient probehead. Spectra were processed using UXNMR and AURELIA software (Bruker Inc). Chemical shifts were referenced to the central component of the quintet due to the CD2H resonance of methanol at dH 3.313 downfield from SiMe4. J Values are given in Hz. TOCSY24 experiments were run with the MLEV 17 sequence for spin locking and a mixing time of 120 ms (9 kHz).The ROESY25 experiment was carried out with a mixing time of 250 ms and a spin lock field of 2 kHz to reduce Hartmann–Hahn transfers. Two-dimensional spectra were obtained with quadrature detection in both dimensions using the hypercomplex method in the F1 dimension.26 The solvent signal was suppressed using the WATERGATE scheme27 included in the standard and ROESY pulse sequences. A total of 2048 data points were acquired in the F2 dimension and 512 complex points in the F1 dimension. For each complex data point in the F2 4 free induction decays were accumulated with a relaxation delay of 2 s. All spectra were apodized with p/2-shifted sine-bell functions in both dimensions. CD spectrum The spectrum of HB I was recorded with a Jobin-Yvon CD6 dichrograph with a 0.1 mm path cell at 22 8C (1 mmol cm23; CH3OH); l (nm) and [q]M (deg cm2 dmol21) 192 (56 000) and 205 (2110 000).Antimicrobial activity The antibacterial activity of HB I was examined against Staphylococcus aureus (strain 209P) and Escherichia coli (strain RL 65) by the agar diffusion test using the Mueller Hinton culture medium and 6 mm diameter pits. The peptide sample was dissolved in dimethyl sulfoxide (DMSO) such as to give a 4 mg cm23 solution. Eight other concentrations were obtained by successive dilutions and 50 mm3 of each solution was deposited into the pits (1.5 to 200 mg). Inhibition zones were measured after 24 h of incubation at 37 8C. Liposome permeabilization Egg phosphatidylcholine (egg PC) type V E and cholesterol were purchased from Sigma; egg PC was used without further purification and cholesterol was recrystallized from methanol.CF from Eastman Kodak was separated from hydrophobic contaminants and recrystallized from ethanol as previously described.11 Fluorescence spectra were measured at 20 8C on an Aminco SPF 500 spectrofluorometer. The peptide-induced release of intravesicular content was monitored by the method introduced by Weinstein,28 that uses the property of quenching relief upon dilution of an encapsulated fluorescent probe CF. 1592 J. Chem. Soc. Perkin Trans. 1 1997 CF-entrapped small unilamellar vesicles (SUV) were prepared as previously described,11 by sonication of an egg PC– cholesterol (7 3) mixture ([lip] = 0.6 mM). The SUV obtained by sonication were separated from unencapsulated CF by gel filtration (Sephadex G 75).Leakage kinetics were obtained for different peptide lipid molar ratios obtained by adding aliquots of methanolic solutions of peptides (methanol concentration kept below 0.5% by volume). Synthesis of HB I Diisopropylethylamine (DIEA) trifluoroacetic acid (TFA) ditert- butyl dicarbonate (Boc2O) and L-leucine were purchased from Sigma-Aldrich Chimie and D-isovaline [(R)-(2)-2-amino- 2-methylbutanoic acid] from Acros (France). All N-tertbutoxycarbonyl- protected L-amino acids and BOP were purchased from Propeptide (France) and used without subsequent purification. H-Leu-OMe was prepared according to Boissonnas et al.29 Boc-Iva-OH was prepared according to Bodanszky and Bodanszky.30 Column chromatography was carried out with 230–400 mesh Merck grade 60 silica gel.Analytical TLC was performed on aluminium sheets covered with Merck grade 60 silica gel. Gel filtration was carried out with Pharmacia Sephadex LH 20. General procedure A BOP-mediated peptide coupling. The Nprotected amino acid and BOP reagent were added to a solution of the TFA salt of the C-protected amino acid or peptide in CH2Cl2. The stirred solution was cooled in an ice-bath and DIEA was added. The mixture was stirred at room temperature until TLC analysis indicated that consumption of the amino component was no longer proceeding. It was then concentrated in vacuo to leave an oil which was dissolved in ethyl acetate and washed successively with 1 M HCl water 1 M NaOH and saturated aq. NaCl. The combined organics were dried over Na2SO4 filtered and concentrated in vacuo to leave an oil which was purified by chromatography on a silica gel column.General procedure B Removal of N-tert-butoxycarbonyl protection with 50% TFA solution in CH2Cl2. A stirred solution of the N-tert-butoxycarbonyl-protected peptide in CH2Cl2 was cooled in an ice-bath and TFA was added. The mixture was stirred at room temperature until TLC analysis indicated complete consumption of the starting material and was then evaporated in vacuo to leave an oil. The crude product was used without purification for the next coupling. General procedure C Removal of N-tert-butoxycarbonyl protection with pure TFA. The N-tert-butoxycarbonyl-protected peptide was treated with TFA (0.5 cm3 for 1 mmol of peptide). The mixture was stirred at room temperature until TLC analysis indicated that the totality of the peptide was deprotected and it was then evaporated in vacuo to give an oil.The crude product was used without purification for the next coupling. Boc-Pro-Leu-OMe. HCl H-Leu-OMe (0.92 g 5.07 mmol) and Boc-Pro-OH (1.20 g 5.58 mmol) were treated with BOP (2.47 g 5.58 mmol) and DIEA (2.7 cm3 15.71 mmol) via procedure A to yield the crude product which was used without purification. Boc-Aib-Pro-Leu-OMe. Boc-Pro-Leu-OMe (1.73 g 5.07 mmol) was deprotected according to procedure C. The crude TFA salt and Boc-Aib-OH (1.12 g 5.50 mmol) were treated with BOP (2.43 g 5.50 mmol) and DIEA (2.7 cm3 15.7 mmol) according to procedure A to yield after purification by chromatography on silica gel (ethyl acetate) 1 g of a powder; TLC Rf (ethyl acetate) 0.40; LSIMS m/z 434 [M 1 Li]1 (100) 378 (5) 334 (27) 332 (2) and 247 (9); HR-LSIMS [M 1 Li]1 434.2822 (Calc.for C21H37LiN3O6 m/z 434.2842); dH(300 MHz; CD3OH) 0.91 (d J 6.0 3 H d Leu) 0.94 (d J 6.0 3 H d Leu) 1.36 (s 3 H b Aib) 1.45 (s 12 H b Aib Boc) 1.81 (m 6 H 2 × b Leu g Leu b9 Pro 2 × g Pro) 2.19 (m 1 H b Pro) 3.59 (m 1 H d9 Pro) 3.68 (s 3 H ester) 3.81 (m 1 H d Pro) 4.37 (m 1 H a Leu) 4.45 (dd J 8.4 and 5.9 1 H a Pro) 7.29 (s 1 H NH Aib) and 8.23 (d J 7.7 1 H NH Leu). Boc-Leu-Aib-Pro-Leu-OMe. Boc-Aib-Pro-Leu-OMe (1.00 g 2.34 mmol) was deprotected according to procedure B. The crude TFA salt and Boc-Leu-OH (0.64 g 2,58 mmol) were treated with BOP (1.14 g 2.58 mmol) and DIEA (1.3 cm3 7.3 mmol) according to general procedure A. The mixture was purified by chromatography on silica gel eluted by CH2Cl2– MeOH (92 8) to yield 1.06 g (85%) of a powder; TLC Rf (CH2Cl2–MeOH 92 8) 0.50; LSIMS m/z 547 [M 1 Li]1 (100) 491 (8) 447 (17) 445 (3) and 247 (8); HR-LSIMS [M 1 Li]1 547.3657 (Calc.for C27H48LiN4O7 m/z 547.3683); [a]D 22 277 (c 0.1 MeOH); dH(300 MHz; CD3OH) 0.93 (m 12 H d Leu1 d Leu4) 1.44 (s 12 H b Aib Boc) 1.46 (s 3 H b Aib) 1.47 (m 2 H 2 × b Leu1) 1.60 (m 1 H b9 Leu4) 1.69 (m 2 H g Leu1 g Leu4) 1.75 (m 1 H b Leu4) 1.88 (m 3 H b9 Pro 2 × g Pro) 2.08 (m 1 H b Pro) 3.62 (m 2 H 2 × d Pro) 3.68 (s 3 H ester) 4.09 (m 1 H a Leu1) 4.34 (m 1 H a Leu4) 4.47 (m 1 H a Pro) 6.68 (d J 8.1 1 H NH Leu1) and 8.21 (br s 2 H NH Aib NH Leu4). Boc-Iva-Leu-Aib-Pro-Leu-OMe. Boc-Leu-Aib-Pro-Leu-OMe (150 mg 0.28 mmol) was deprotected according to procedure B. The crude TFA salt and Boc-Iva-OH (73 mg 0.34 mmol) were treated with BOP (149 mg 0.34 mmol) and DIEA (180 mm3 1 mmol) according to procedure A.The mixture was purified by chromatography on silica gel (CH2Cl2–MeOH 94 6) to yield 152 mg (85%) of the expected product; TLC Rf (CH2Cl2– MeOH 93 7) 0.43; LSIMS m/z 662 [M 1 Na]1 (15) 495 (3) 398 (44) 313 (4) and 243 (100); HR-LSIMS [M 1 Li]1 646.4385 (Calc. for C32H57LiN5O8 m/z 646.4367); dH(300 MHz; CD3OH) 0.84 (m 3 H Meg Iva) 0.95 (m 12 H d Leu2 d Leu5) 1.34 (s 3 H Meb Iva) 1.47 (s 15 H b Aib Boc) 1.62 (m 2 H b9 Leu2 b9 Leu5) 1.70 (m 2 H g Leu2 g Leu5) 1.77 (m 2 H b Leu2 b Leu5) 1.78 (m 1 H b9 Iva) 1.85 (m 1 H b9 Pro) 1.88 (m 2 H g Pro) 1.92 (m 1 H b Iva) 2.16 (m 1 H b Pro) 3.53 (m 1 H d9 Pro) 3.3 (m 1 H d Pro) 3.68 (s 3 H ester) 4.35 (m 2 H a Leu2 a Leu5) 4.48 (m 1 H a Pro) 6.93 (s 1 H NH Iva) 7.94 (d J 7.0 1 H NH Leu2) 8.14 (s 1 H NH Aib) and 8.16 (d J 8.3 1 H NH Leu5).Boc-Pro-Iva-Leu-Aib-Pro-Leu-OMe. Boc-Iva-Leu-Aib-Pro- Leu-OMe (140 mg 0.22 mmol) was deprotected according to procedure B. The crude TFA salt and Boc-Pro-OH (71 mg 0.33 mmol) were treated with BOP (146 mg 0.33 mmol) and DIEA (134 mm3 0.77 mmol) according to procedure A to yield after purification by silica gel chromatography (CH2Cl2–MeOH 95 5) 149 mg (92%) of a powder; TLC Rf (CH2Cl2–MeOH 94 6) 0.44; LSIMS m/z 743 [M 1 Li]1 (100) 643 (14) 641 (5) 473 (3) 388 (9) and 247 (5); HR-LSIMS [M 1 Li]1 743.4897 (Calc. for C37H64LiN6O9 m/z 743.4895); [a]D 22 233 (c 0.1 MeOH); dH(300 MHz; CD3OH) 0.82 (m 3 H Meg Iva) 0.92 (m 12 H d Leu3 d Leu6) 1.36 (s 3 H Meb Iva) 1.49 (s 3 H b Aib) 1.50 (s 12 H b Aib Boc) 1.62 (m 2 H b9 Leu3 b9 Leu6) 1.70 (m 2 H g Leu3 g Leu6) 1.80 (m 2 H b Leu3 b Leu6) 1.82 (m 1 H b9 Iva) 1.83 (m b9 Pro5) 1.86 (m 2 H g Pro5) 1.95 (m 1 H b9 Pro1) 1.98 (m 2 H g Pro1) 2.18 (m 1 H b Pro5) 2.23 (m 1 H b Iva) 4.31 (m 1 H b Pro1) 3.51 (m 2 H d9 Pro1 d9 Pro5) 3.60 (m 1 H d Pro1) 3.69 (s 3 H ester) 3.74 (m 1 H d Pro5) 4.08 (m 1 H a Pro1) 4.36 (m 2 H a Leu3 a Leu6) 4.49 (m 1 H a Pro5) 7.82 (d J 8.5 1 H NH Leu3) 7.87 (s 1 H NH Iva) 8.12 (s 1 H NH Aib) and 8.16 (d J 7.7 1 H NH Leu6).Boc-Ile-Aib-OMe. According to procedure A H-Aib-OMe (1.16 g 7.6 mmol) and Boc-Ile-OH (2.21 g 9.2 mmol) were treated with BOP (4.07 g 9.2 mmol) and DIEA (2.6 cm3 15.2 mmol). The crude product was used without purification for the next coupling. TLC Rf (ethyl acetate–cyclohexane 1 1) 0.61.Boc-Leu-Ile-Aib-OMe. Boc-Ile-Aib-OMe (2.51 g 7.6 mmol) was deprotected according to procedure B. The TFA salt and Boc-Leu-OH (2.27 g 9.1 mmol) were treated with BOP (4.03 g 9.1 mmol) and DIEA (4.2 cm3 24 mmol) according to procedure A. The mixture was purified by silica gel chromatography (ethyl acetate–cyclohexane 5 5) to yield 1.08 g (32% for the two steps) of a solid; TLC Rf (ethyl acetate–cyclohexane, J. Chem. Soc. Perkin Trans. 1 1997 1593 1 1) 0.53; LSIMS m/z 450 [M 1 Li]1 (100) 394 (12) 350 (24) 348 (3) 305 (2) and 192 (8); HR-LSIMS [M 1 Li]1 450.3159 (Calc. for C22H41LiN3O6 m/z 450.3155); dH(300 MHz; CD3OH) 0.93 (m 12 H d Leu Meg Ile Med Ile) 1.16 (m 1 H g9 Ile) 1.42 (s 3 H b Aib) 1.43 (s 9 H Boc) 1.45 (s 3 H b Aib) 1.53 (m 3 H g Ile 2 × b Leu) 1.66 (m 1 H g Leu) 1.77 (m 1 H b Ile) 3.64 (s 3 H ester) 4.07 (m 1 H a Leu) 4.18 (m 1 H a Ile) 6.86 (d J 7.7 1 H NH Leu) 7.71 (d J 8.6 1 H NH Ile) and 8.35 (s 1 H NH Aib).Boc-Asn-Leu-Ile-Aib-OMe. Boc-Leu-Ile-Aib-OMe (1.03 g 2.3 mmol) was deprotected according to procedure C. The crude TFA salt and Boc-Asn-OH (0.65 g 2.8 mmol) were treated with BOP (1.24 g 2.8 mmol) and DIEA (1.3 cm3 7.5 mmol) according to procedure A. After purification by silica gel chromatography (CH2Cl2–MeOH 95 5) it yielded 890 mg (69%) of a powder; TLC Rf (ethyl acetate) 0.17; LSIMS m/z 564 [M 1 Li]1 (100) 508 (4) 464 (31) 462 (3) 419 (4) and 306 (3); HR-LSIMS [M 1 Li]1 564.3583 (Calc. for C26H47LiN5O8 m/z 564.3585); dH(300 MHz; CD3OH) 0.89 (m 3 H Med Ile) 0.92 (m 6 H d Leu) 0.94 (m 3 H Meg Ile) 1.17 (m 1 H g9 Ile) 1.41 (s 3 H b Aib) 1.42 (s 3 H b Aib) 1.43 (s 9 H Boc) 1.43 (m 1 H b9 Leu) 1.55 (m 1 H g Ile) 1.62 (m 1 H b Leu) 1.73 (m 1 H g Leu) 1.90 (m 1 H b Ile) 2.66 (m 2 H 2 × Asn) 3.64 (s 3 H ester) 4.12 (m 1 H a Ile) 4.37 (m 1 H a Leu) 4.41 (m 1 H a Asn) 6.86 (d J 7.7 1 H NH Asn) 6.94 (s 1 H d syn Asn) 7.60 (s 1 H d anti Asn) 7.96 (d J 8.7 1 H NH Ile) 8.03 (s 1 H NH Aib) and 8.17 (d J 7.2 1 H NH Leu).Ac-Aib-Asn-Leu-Ile-Aib-OMe. Boc-Asn-Leu-Ile-Aib-OMe (0.54 g 0.97 mmol) was deprotected according to procedure C. The TFA salt and Ac-Aib-OH (0.27 g 1.84 mmol) were treated with BOP (0.81 g 1.84 mmol) and DIEA (0.5 cm3 3.0 mmol) according to procedure A. After purification by silica gel chromatography (CH2Cl2–MeOH 90 10) it yielded 190 mg of a powder; TLC Rf (CH2Cl2–MeOH 88 12) 0.30; LSIMS m/z 591 [M 1 Li]1 (100) 333 (6) 291 (16) 220 (11) 209 (40) 122 (7) and 106 (15); HR-LSIMS [M 1 Li]1 591.3678 (Calc.for C27H48LiN6O8 m/z 591.3694); dH(300 MHz; CD3OH) 0.90 (m 12 H d Leu Meg Ile Med Ile) 1.21 (m 1 H g9 Ile) 1.43 (m 12 H b Aib1 b Aib5) 1.55 (m 2 H b9 Leu g Ile) 1.69 (m 1 H b Leu) 1.87 (m 2 H g Leu b Ile) 1.99 (s 3 H Ac) 2.71 (ABX system J 7.6 and 15.4 1 H b9 Asn) 2.81 (ABX system J 6.3 and 15.4 1 H b Asn) 3.65 (s 3 H ester) 4.14 (m 1 H a Ile) 4.27 (m 1 H a Leu) 4.41 (m 1 H a Asn) 6.94 (s 1 H d syn Asn) 7.38 (d J 8.9 1 H NH Ile) 7.69 (s 1 H d anti Asn) 7.83 (s 1 H NH Aib5) 8.11 (d J 7.2 1 H NH Leu) 8.46 (d J 6.2 1 H NH Asn) and 8.54 (s 1 H NH Aib1). Ac-Aib-Asn-Leu-Ile-Aib-OH. A stirred solution of Ac-Aib- Asn-Leu-Ile-Aib-OMe (140 mg 0.24 mmol) in MeOH (1.4 cm3) was cooled in an ice-bath and 1.0 cm3 of 1 M NaOH was added.The mixture was stirred at room temperature until TLC analysis indicated complete consumption of the methyl ester. The cooled mixture was then neutralized with 1 M HCl and evaporated in vacuo. The crude product was purified using Sephadex LH-20 with MeOH as eluent to yield 116 mg (85%) of a solid; LSIMS m/z 593 [M 1 Na]1 (100) 468 (7) 355 (14) 242 (23) and 128 (18); HR-LSIMS [M 1 Na]1 593.3293 (Calc. for C26H46N6NaO8 m/z 593.3275); [a]D 22 210 (c 0.1 MeOH); dH(300 MHz; CD3OH) 0.87 (m 6 H Med Ile d Leu) 0.94 (d J 6.8 3 H Meg Ile) 0.95 (d J 6.5 3 H d Leu) 1.21 (m 1 H g9 Ile) 1.42 (s 3 H b Aib) 1.45 (s 3 H b Aib) 1.46 (s 3 H b Aib) 1.47 (s 3 H b Aib) 1.54 (m 1 H g Ile) 1.57 (m 1 H b9 Leu) 1.73 (m 1 H g Leu) 1.81 (m 1 H b Leu) 1.95 (m 1 H b Ile) 1.99 (s 3 H Ac) 2.73 (ABX system J 4.9 and 15.4 1 H b9 Asn) 2.81 (ABX system J 6.3 and 15.4 1 H b Asn) 4.16 (m 1 H a Ile) 4.28 (m 1 H a Leu) 4.45 (m 1 H a Asn) 6.93 (s 1 H d syn Asn) 7.45 (d J 9.1 1 H NH Ile) 7.69 (s 1 H d anti Asn) 7.78 (s 1 H NH Aib5) 8.12 (d J 7.5 1 H NH Leu) 8.45 (d J 6.5 1 H NH Asn) and 8.53 (s 1 H NH Aib1).Ac-Aib-Asn-Leu-Ile-Aib-Pro-Iva-Leu-Aib-Pro-Leu-OMe. Boc-Pro-Iva-Leu-Aib-Pro-Leu-OMe (100 mg 0.13 mmol) was deprotected according to procedure B. The TFA salt and Ac- Aib-Asn-Leu-Ile-Aib-OH (50 mg 0.087 mmol) were treated with BOP (38.5 mg 0.087 mmol) and DIEA (45 mm3 0.26 mmol) according to procedure A to yield after purification by silica gel chromatography (CH2Cl2–MeOH 85 15) 42 mg (41%) of a powder; TLC Rf (CH2Cl2–MeOH 85 15) 0.42; LSIMS m/z 1195 [M 1 Li]1 (100) 798 (6) 641 (27) 628 (6) 531 (13) 446 (16) 333 (9) and 291 (8); HR-LSIMS [M 1 Li]1 1195.7598 (Calc.for C58H100LiN12O14 m/z 1195.7642); dH(300 MHz; CD3OH) 0.83 (m 3 H Meg Iva) 0.87 (m 3 H Med Ile) 0.89 (m 18 H d Leu3 d Leu8 d Leu11) 0.95 (m 3 H Meg Ile) 1.30 (m 1 H g9 Ile) 1.45 (s 3 H Meb Iva) 1.46 (s 6 H b Aib) 1.49 (s 9 H b Aib) 1.50 (s 3 H b Aib) 1.55 (m 1 H g Ile) 1.76 (m 9 H b and g Leu3 b and g Leu8 b and g Leu11) 1.77 (m 1 H b9 Iva) 1.80 (m 1 H b9 Pro6) 1.83 (m 1 H b9 Pro10) 1.86 (m 2 H 2 × g Pro10) 1.92 (m 1 H g9 Pro6) 1.96 (m 1 H b Ile) 2.02 (s 3 H Ac) 2.12 (m 1 H g Pro6) 2.20 (m 1 H b Pro10) 2.32 (m 1 H b Pro6) 2.46 (m 1 H b Iva) 2.76 (d J 5.7 2 H b Asn) 3.42 (m 2 H d9 Pro6 d9 Pro10) 3.69 (s 3 H ester) 3.81 (m 2 H d Pro6 d Pro10) 4.20 (m 3 H a Leu3 a Ile4 a Pro6) 4.34 (m 3 H a Asn2 a Leu8 a Leu11) 4.50 (m 1 H a Pro10) 7.04 (s 1 H d syn Asn) 7.35 (d J 8.8 1 H NH Ile4) 7.49 (s 1 H NH Iva) 7.62 (d J 8.5 1 H NH Leu8) 7.76 (s 2 H NH Aib5 NH Aib9) 7.80 (s 1 H d anti Asn) 8.11 (d J 7.1 1 H NH Leu3) 8.16 (d J 7.8 1 H NH Leu11) 8.61 (d J 5.5 1 H NH Asn) and 8.67 (s 1 H NH Aib1).Ac-Aib-Asn-Leu-Ile-Aib-Pro-Iva-Leu-Aib-Pro-Leuol HB I. A solution of Ac-Aib-Asn-Leu-Ile-Aib-Pro-Iva-Leu-Aib-Pro- Leu-OMe (32 mg 0.027 mmol) in EtOH (1 cm3) was cooled in an ice-bath and NaBH4 (6 mg 0.162 mmol) was added. The mixture was stirred at 50 8C for 8 h and the solvent was evaporated off in vacuo. The residue was dissolved in ethyl acetate and washed with water. The organic layer was dried over Na2SO4 filtered and concentrated in vacuo to yield 25 mg (80%) of HB I; TLC Rf (CH2Cl2–MeOH 85 15) 0.37; LSIMS m/z 1167 [M 1 Li]1 (55) 947 (64) 553 (81) 468 (34) 395 (28) 355 (63) 310 (25) 242 (100) 197 (69) and 128 (70); HR-LSIMS [M 1 Na]1 1183.7333 (Calc.for C57H100N12NaO13 m/z 1183.7430); [a]D 22 17 (c 0.1 MeOH); 1 NMR data were identical with those described for natural HB I. Acknowledgements We are indebted to Dr M. F. Roquebert (Laboratoire de Cryptogamie du Muséum National d’Histoire Naturelle) who provided the T. harzianum strain and to Dr A. Galat (CEA Saclay France) for the dichrograph facility. We thank Dr M. Becchi and the Centre de Spectroscopie du CNRS (Lyon France) for LSIMS measurements and Dr Duclohier for macroscopic current–voltage experiments. The 500 MHz facilities used in this study were funded by the Région Haute- Normandie France.This work was supported in part by a grant from the Centre National de la Recherche Scientifique (GDR 1153). References 1 R. C. Pandey J. C. Cook Jr. and K. L. Rinehart J. Am. Chem. Soc. 1977 99 8469. 2 B. Bodo S. Rebuffat M. El Hajji and D. Davoust J. Am. Chem. Soc. 1985 107 6011. 3 A. Iida S. Uesato T. Shingu M. Okuda Y. Nagaoka Y. Kuroda and T. Fujita J. Chem. Soc. Perkin Trans. 1 1993 367. 4 S. Rebuffat L. Conraux M. Massias C. Auvin-Guette and B. Bodo Int. J. Pept. Protein Res. 1993 41 74. 5 C. Goulard S. Hlimi S. Rebuffat and B. Bodo J. Antibiot. 1995 48 1248. 6 A. Iida M. Sanekata S. Wada T. Fujita H. Tanaka A. Enoki G. Fuse M. Kanai and K. Asami Chem. Pharm. Bull. 1995 43 392. 1594 J. Chem. Soc. Perkin Trans. 1 1997 7 S. Rebuffat C. Goulard and B.Bodo J. Chem. Soc. Perkin Trans. 1 1995 1849. 8 C. Auvin-Guette S. Rebuffat Y. Prigent and B. Bodo J. Am. Chem. Soc. 1992 114 2170. 9 T. Fujita S. Wada A. Iida T. Nishimura M. Kanai and N. Toyama Chem. Pharm. Bull. 1994 42 489. 10 M. S. P. Sansom Prog. Biophys. Mol. Biol. 1991 55 139. 11 S. Rebuffat H. Duclohier C. Auvin-Guette G. Molle G. Spach and B. Bodo FEMS Microbiol. Immunol. 1992 105 151. 12 T. Le Doan M. El Hajji S. Rebuffat M. R. Rajesvari and B. Bodo Biochim. Biophys. Acta 1986 858 1. 13 M. El Hajji S. Rebuffat T. Le Doan G. Klein M. Satre and B. Bodo Biochim. Biophys. Acta 1989 978 97. 14 S. Hlimi S. Rebuffat C. Goulard S. Duchamp and B. Bodo J. Antibiot. 1995 48 1254. 15 A. R. Artalejo C. Montiel P. Sanchez-Garcia G. Uceda J. M. Guantes and A. G. Garcia Biochem.Biophys. Res. Commun. 1990 1204. 16 A. Iida M. Okuda S. Uesato Y. Takaishi T. Shingu M. Morita and T. Fujita J. Chem. Soc. Perkin Trans. 1 1990 3249. 17 S. Rebuffat P. Drognat-Landré C. Goulard I. Augeven-Bour C. Auvin and B. Bodo Peptides 1992 Proceedings of the 22nd European Peptide Symposium ed. C. H. Schneider and A. N. Eberle Escom Science Publishers Leiden 1993 pp. 427–428. 18 P. Roepstorff P. Höjrup and J. Möller Biomed. Mass Spectrom. 1985 12 181. 19 S. Wada A. Iida N. Akimoto M. Kanai N. Toyama and T. Fujita Chem. Pharm. Bull. 1995 43 910. 20 G. Esposito J. A. Carver J. Boyd and I. D. Campbell Biochemistry 1987 26 1043. 21 S. Rebuffat Y. Prigent C. Auvin-Guette and B. Bodo Eur. J. Biochem. 1991 201 661. 22 Y. Nagaoka A. Iida and T. Fujita Chem. Pharm. Bull. 1994 42 1258.23 C. Toniolo M. Crisma F. Formaggio C. Peggion V. Monaco C. Goulard S. Rebuffat and B. Bodo J. Am. Chem. Soc. 1996 118 4952. 24 C. Griesinger G. Otting K. Wuthrich and R. R. Ernst J. Am. Chem. Soc. 1988 110 7870. 25 A. Bax and D. G. Davis J. Magn. Reson. 1985 63 207. 26 D. J. States R. A. Haberkorn and D. J. Ruben J. Magn. Reson. 1982 48 286. 27 M. Piotto U. Savdek and V. Sklenar J. Biomol. NMR 1992 2 661. 28 J. N. Weinstein S. Yoshikami P. Henkari R. Blumenthal and W. A. Hagins Science 1977 195 489. 29 R. A. Boissonnas S. Guttmann P. A. Jaquenoud and J. P. Waller Helv. Chim. Acta 1955 38 1491. 30 M. Bodanszky and A. Bodanszky The Practice of Peptide Synthesis Springer-Verlag Berlin Heidelberg New York and Tokyo 1984 p. 20. Paper 6/05629F Received 12th August 1996 Accepted 11th February 1997 © Copyright 1997 by the Royal Society of Chemistry J.Chem. Soc. Perkin Trans. 1 1997 1587 Harzianin HB I an 11-residue peptaibol from Trichoderma harzianum isolation sequence solution synthesis and membrane activity Isabelle Augeven-Bour,a Sylvie Rebuffat,a Catherine Auvin,a Christophe Goulard,a Yann Prigent b and Bernard Bodo *,a a Laboratoire de Chimie URA 401 CNRS GDR 1153 CNRS Muséum National d’Histoire Naturelle 63 rue Buffon 75231 Paris Cedex 05 France b Laboratoire de RMN URA 464 CNRS Institut Fédératif de Recherche Multidisciplinaire sur les Peptides No 23 INSERM Université de Rouen 76821 Mont-Saint-Aignan Cedex France Harzianin HB I is a minor component of the peptaibol mixture biosynthesized by a Trichoderma harzianum strain. It is isolated by a multi-step chromatography procedure including HPLC; its sequence has been elucidated by LSIMS and two-dimensional 1H NMR experiments.This 11-residue peptaibol is an analogue of the 14-residue peptaibol harzianin HC IX resulting from the deletion of the tripeptide Aib- Pro-Ala. The CD and NMR data (NOE data and amide proton temperature coefficients) are similar to those of HC IX suggesting a right-handed helix-type conformation for the two peptides. Harzianin HB I was synthesized by the solution-phase method using BOP as coupling reagent. The membrane properties examined on liposomes are compared with those of other known peptaibols. Introduction Peptaibols biosynthesized by Trichoderma soil fungi are amphipathic linear peptides with a high proportion of a,adialkylated amino acids such as a-aminoisobutyric acid (Aib U) an N-terminal acetylated residue and a C-terminal amino alcohol.They can be classified into the long-sequence group containing 18 to 20 residues,1–5 the short-sequence group with 11 to 16 residues 6,7 and the lipopeptaibols with 7 to 11 residues and an N-terminal amino acid acylated by a lipid chain.8,9 The main interest in such peptides stems from their ability to form voltage-gated ion channels in planar lipid bilayer membranes 10,11 and therefore they can be viewed as a prototypic pore. In the absence of a voltage peptaibols induce permeabilization of liposomes;12,13 long-sequence ones being more effi- cient than short-sequence ones. Their membrane activity is modulated by both the length and hydrophobicity of the sequence. The known biological properties of these peptides are their antibiotic 14 and catecholamine secretion-inducing abilities 15,16 which have been suggested to be related to their membrane activity.Recently we isolated peptaibols organised in helical structures which still have significant membrane activity in spite of their short sequences such as the 11-residue lipopeptaibol GA IV and the proline-rich 14 residue peptaibols harzianins HC.7 From a Uruguayan strain of Trichoderma harzianum we previously isolated two groups of peptaibols trichorzins HA and harzianins HC containing 18 and 14 residues respectively. From the particularly complex mixture of the 14-residue peptides a minor component of the same polarity harzianin HB I was isolated. Its structure was determined from liquid secondary ion mass spectrometry (LSIMS) and NMR spectroscopy.It was synthesized in order to test its antibiotic and membrane properties. Harzianin HB I was shown to modify slightly the permeability of liposomes and induce voltage-gated macroscopic conductance in planar bilayers. Sequence of harzianin HB I Results and discussion Isolation and characterization of HB I 17 The culture broth extract of the Uruguayan M-903603 T. harzianum strain was fractionated by exclusion chromatography; the peptide fraction was further chromatographed over silica gel to yield two peptaibol groups of different polarity. The first group was composed of 18-residue peptaibols the trichorzins HA,5,14 and the second one mostly of the 14-residue harzianins HC.7 The HPLC chromatogram of the crude HC mixture was very complex showing more than 16 peaks one of them being assigned to a minor component HB I.Its isolation was undertaken by repetitive semi-preparative reversed-phase high-performance liquid chromatography (RP-HPLC) and its purity was checked by analytical HPLC (Fig. 1). Further LSIMS and 1H NMR experiments confirmed HB I to be homogeneous. It reacted neither with diazomethane nor with ninhydrin indicating the presence of neither free carboxy nor amino groups. The presence of a sharp singlet at dH 2.02 in the 1D NMR spectrum suggested an acetylated N-terminal residue as usually found in peptaibols. The amino acid composition and absolute configuration of the residues resulted from GLC analysis of the total acid hydrolysate derivatives on a Chirasil L-Val capillary column Aib (3) L-Asn (1) L-Ile (1) D-Iva (1) L-Leu (2) L-Pro (2) L-Leuol (1).Sequence determination of HB I Positive liquid secondary-ion mass spectrometry [(1)-LSIMS]. The amino acid sequence of HB I was examined by positive LSIMS. A relative molecular mass of 1160 was assigned to HB I from the sodium ion adduct [M 1 Na]1 observed at m/z 1183 in agreement with the postulated molecular mass arising from the amino acid and amino alcohol composition. The LSIMS spectrum exhibited several abundant b-type acylium ions 18 at m/z 947 553 468 355 242 and 128 and two lower-abundance ion regions between m/z 553 and 947 and between m/z 947 and 1183. This fragmentation pattern suggested the presence of two labile Aib–Pro bonds in HB I. The series of b-type ions ranging from m/z 128 to 553 gave the N-terminal pentapeptide sequence.The mass difference of 394 amu between m/z 947 and 553 was in agreement with the formation of the tetrapeptide ion observed at m/z 395 and 1588 J. Chem. Soc. Perkin Trans. 1 1997 Fig. 1 HPLC chromatogram of purified natural harzianin HB I (Spherisorb ODS2 5mm) 4.6 × 250 mm MeOH–water (83 17) flow rate 1 cm3 min21; absorption monitored at 220 nm obtained via cleavage of the Aib5–Pro6 amide bond.6,19 The Cterminal dipeptide resulting from cleavage of the Aib9–Pro10 amide bond was observed as a y2 ion at m/z 215. Furthermore a series of weak and unusual but significant [xn 1 Na]1 ions at m/z 1083 969 856 743 462 and 167 was observed and allowed the confirmation of the HB I sequence.18 Nevertheless the respective locations of the isomeric residues Leu/Ile remained to be determined.1H NMR spectroscopy. The complete amino acid sequence of HB I was determined by two-dimensional 1H NMR spectroscopy. Assignments of 1H chemical shifts to specific protons of individual residues (Table 1) were obtained by 2D homonuclear (COSY) and phase-sensitive 2D total (TOCSY) chemical-shift correlation experiments showing complete spin systems of one Asn two Pro and one Leuol (Fig. 2) as well as those of one Ile and two Leu residues in agreement with 13C resonances of one Ile Ca at dC 64.1 and two Leu Ca at dC 53.2 and 54.1 (data not shown). The sequence-specific assignments of the backbone NH proton signals arose from the rotating-frame nuclear Overhauser effect (ROESY) spectrum and were completely carried out by using inter-residue connectivities dNN(i,i 1 1) and daN(i,i 1 1) [Fig.3(a)]. The lowest-field singlet NH proton showing a cross-peak with the acetyl CbH3 protons was assigned to Aib1. Then dNN(i,i 1 1) connectivities extending from Asn2 to Aib5 and from Iva7 to Aib9 confirmed the location of Ile at position 4. Finally sequential assignment of Pro6 and Pro10 arose from dNd(i,i 1 1) connectivities between their d protons and the amide protons of Aib5 and Aib9 [Fig. 3(b)]. H3C C O NH C C O NH CH C NH2 O C O NH CH C O NH CH C O NH C C O N CH C O NH C C O NH CH C O NH C C O N CH C O NH CH CH2OH (x10 + Na)+ 1083 (6) (x9 + Na)+ 969 (1) (x8 + Na)+ 856 (2) (x7 + Na)+ 743 (2) (x4 + Na)+ 462 (2) (x1 + Na)+ 167 (2) (b) Mass fragmentation pattern of harzianin HB I (positive-ion LSIMS) exhibiting the bn ions (a) and the xn 1 Na ions (b) (relative intensities in parentheses) J.Chem. Soc. Perkin Trans. 1 1997 1589 Table 1 1H sequential and stereospecific assignments of harzianin HB I (500.13 MHz; CD3OH; 296 K). Chemical shifts (ppm) are given to the nearest three or two decimal places when obtained from 1D or 2D spectra respectively; 3JNH-CaH coupling constants (Hz) arise from the 1D spectrum and are given in parentheses Residue NH a-H b-H/b-Me Other groups Ac Me 2.027 s Aib1 8.679 s pro-R 1.46*/pro-S 1.450 Asn2 8.610 d (5.6) 4.38 2.76 e syn 7.04/e anti 7.77 Leu3 8.111 d (7.1) 4.25 1.93/1.56 g 1.74/d1 0.89/d2 0.96 Ile4 7.356 d (9.1) 4.22 1.95 g 1.55/g9 1.32/g-Me 0.95/d-Me 0.86 Aib5 7.806 s pro-R 1.498*/pro-S 1.501 Pro6 4.21 pro-R 1.78/pro-S 2.35 pro-R g 1.94/pro-S g 2.10/pro-R d 3.85/pro-S d 3.41 Iva7 7.484 s 2.45/1.77/b-Me 1.46 g 0.82 Leu8 7.627 d (8.5) 4.39 1.75/1.75 g 1.75/d1 0.85/d2 0.96 Aib9 7.836 s pro-R 1.450*/pro-S 1.501 Pro10 4.42 pro-R 1.78/pro-S 2.29 pro-R g 1.87/pro-S g 1.93/pro-R d 3.88/pro-S d 3.44 Leuol11 7.534 d (8.9) 3.96 1.60/1.35/3.53 g 1.64/d1 0.90/d2 0.95 * May be exchanged.Conformation of HB I The conformation of HB I in methanol solution was examined by CD and NMR spectroscopy using inter-residue NOE connectivities 3JNH-CaH coupling constants and amide temperature coefficients. The CD spectrum of HB I showed two transitions at 192 (1) and 205 (2) nm characteristic of a right-handed helix. Fig. 2 Expansion of the TOCSY spectrum of HB I in CD3OH (spin lock period 120 ms) (a) w2 = 0.6–4.5 ppm w1 = 7.3–8.7 ppm; (b) w2 = 4.1–4.6 ppm w1 = 1.7–4.6 ppm; spin-systems are labelled with the sequential residue positions A stretch of strong sequential dNN(i,i 1 1) accompanied by daN(i,i 1 1) and by a series of medium and strong daN(i,i 1 3) all along the sequence was observed in the ROESY spectrum (Fig.3) in agreement with a helical structure. The presence of daN(i,i 1 2) NOEs all along the sequence and the complete absence of daN(i,i 1 4) connectivities suggested a succession of turns stabilized by 4Æ1 intramolecular hydrogen bonds as observed for the 310-helix. This was in agreement with the amide protons’ thermal coefficients (Dd/DTNH) (Fig. 4). Little information was obtained from the 3JNH-CaH coupling constants as only half of the residues could give such data (Fig. 4). The Asn2 and Leu3 residues showed values <7 Hz whereas Ile4 Leu8 and Leuol11 had higher values around 9 Hz apparently inconsistent Fig.3 Parts of the ROESY spectrum of HB I in CD3OH (mixing time 250 ms) (a) w2 = 7.3–8.8 ppm w1 = 7.1–8.7 ppm; (b) w2 = 7.3–8.0 w1 = 3.2–4.8 ppm 1590 J. Chem. Soc. Perkin Trans. 1 1997 with a helical structure. However such values have frequently been observed for the two amino acids flanking Aib-Pro segments in a-helical peptaibols.4,5,20–22 The studied residues in HB I have such a location in the sequence. Comparison of the thermal coefficients and coupling constants of HB I with those of the longer analogue HC IX which contains an additional Aib-Pro-Ala tripeptide after Leu3 showed extensive similarity of the data.17 The results thus suggested a structure stabilized by 4Æ1-type intramolecular hydrogen bonds forming a ribbon of b-turns.Synthesis of HB I In order to make available a sufficient amount of harzianin HB I for bioassays it was synthesized by a solution-phase method in the presence of the (benzotriazol-1-yloxy)tris(dimethylamino) phosphonium hexafluorophosphate (BOP) coupling reagent in CH2Cl2 at room temperature according to the synthetic route shown in Scheme 1. The penta- and hexa-peptide fragments were built up in a stepwise manner using Boc/OMe Fig. 4 Amino acid sequence of harzianin HB I (the one-letter code of amino acid residues is used with U = Aib J = Iva Lol = Leuol) and a survey of the NOE connectivities involving NH and CaH (dad connectivities observed for prolines are indicated by white boxes) of the 3JNH-CaH coupling constants and of the temperature coefficients of the amide protons.The observed NOEs are classified as strong medium and weak (based on counting the cross-peak contour levels) and shown by thick medium and thin lines respectively. strategy. They were designed so that Aib was placed at the Cterminal position in order to avoid racemization during the deprotection and activation steps. The synthesis of HB I was achieved by reduction of the C-terminal methyl ester group into an alcohol function by NaBH4–EtOH. Synthetic HB I was finally purified by semi-preparative HPLC. The analytical HPLC retention time (tR) and the 1H NMR spectrum of synthetic HB I were identical to those of natural HB I. Antibacterial activity The antibacterial activity of HB I examined against S. aureus and E. coli showed it to be inactive against E. coli in agreement with previous observations on other peptaibols.4,5,7 However no antibacterial activity against S.aureus was detected even at 200 mg pit21 whereas growth inhibition induced by short-sequence peptaibols harzianins HC and trichogin GA IV could be detected up to 50 and 1.5 mg pit21 respectively.7,8 This result was in agreement with the absence of antibacterial activity noticed for C2-GA IV,23 the analogue of GA IV with an acetyl group instead of the lipid chain. The absence of antibacterial activity for this 11-residue peptaibol confirms the role of the Nterminal lipid chain in the lipopeptaibol activity. Membrane-modifying properties of HB I Long-sequence peptaibols have been previously shown to exhibit membrane-modifying properties by increasing the permeability of liposomes.5,11 Optimal membrane activity was observed for a hydrophobic neutral a-helix of 18–19 residues while the liposome permeabilization decreased for shortersequence peptaibols.5 The membrane-modifying activity of HB I was studied by fluorescence spectroscopy following the leakage of a carboxyfluorescein (CF) fluorescent probe previously entrapped at self-quenched concentration in small unilamellar vesicles.The results presented as a percentage of escaped CF at 20 min as a function of Ri 21 = [peptide]/[lipid] were compared with those of other short peptides such as the 14- residue HC IX and the 11-residue lipopeptaibol GA IV (Fig. 5). Comparison of Ri 21 values characteristic of 50% release of the entrapped material showed HB I (Ri 21 = 83 × 1023) to be less efficient than HC IX (Ri 21 = 12 × 1023) and GA IV Scheme 1 Scheme for the total synthesis of HB I J.Chem. Soc. Perkin Trans. 1 1997 1591 (Ri 21 = 4 × 1023). This result points to the major role of the sequence length the presence of the N-terminal lipidic chain also favouring the liposome permeabilization. The voltage-dependent channel-forming properties of harzianin HB I were also examined by macroscopic current– voltage experiments (G. Molle H. Duclohier unpublished results). In such conditions HB I exhibited channel-forming activity for concentrations ranging between 1026 and 1025 M in the same way as the 11-residue peptaibol trichorozin TZ-IV,6 or the 14-residue harzianins HC.7 Experimental Isolation of harzianin HB I The T. harzianum strain (M-903603) collected in Uruguay was obtained from the ‘Collection de souches fongiques du Muséum National d’Histoire Naturelle’ (Paris); the strain was maintained and cultivated as previously described.7 The culture was incubated for 11 days at 27 8C.The filtered fermentation broth was extracted three times with butan-1-ol to give after removal of the solvent under reduced pressure 1.2 g of crude extract. The residue was submitted to gel filtration on Pharmacia Sephadex LH 20 with methanol as eluent. The crude peptide mixture (468 mg) was then chromatographed over silica gel (Kieselgel 60 H Merck Darmstadt) with CH2Cl2–MeOH (9 1 to 5 5) as eluent. The HC/HB mixture (130 mg) was eluted with CH2Cl2–MeOH (80 20). HPLC separation This was carried out with a Waters liquid chromatograph (6000 A and M45 pumps a 680 automated solvent programmer a WISP 712 automatic injector and a 481 UV–VIS detector) on a semipreparative C18 column (Spherisorb ODS2; 5 mm; 7.5 × 300 mm; AIT France); eluent methanol–water (83 17); flow rate 2 cm3 min21.The purity of HB I (3 mg) was confirmed on an analytical column (3.5 × 250 mm); eluent methanol–water (83 17); flow rate 1 cm3 min21; tR 16 min. Amino acid analysis Total hydrolysis of HB I was carried out according to the usual procedure for peptides (6 M HCl at 110 8C in sealed tubes for 24 h). Identification of the amino acids was accomplished by gas chromatography after derivatization.4 Retention times of the Ntrifluoroacetyl isopropyl ester derivatives were compared with those of standard samples. The GLC analyses were performed with a Girdel 3000 chromatograph on a Chirasil-L-Val (Npropionyl- L-valine tert-butylamide polysiloxane) quartz capillary column (Chrompack 25 m length 0.2 mm i.d.) with He (0.7 × 105 Pa) as carrier gas and a temperature programme 50–130 8C 3 8C min21; 130–190 8C 10 8C min21; tR (min) Aib Fig.5 Peptide-induced CF at t = 20 min for different [peptide]/[lipid] ratios (Ri 21) from egg PC/cholesterol 70/30 vesicles (a) HB I (b) HC IX (Ac-Aib-Asn-Leu-Aib-Pro-Ala-Ile-Aib-Pro-Iva-Leu-Aib-Pro-Leuol) and (c) GA IV (Oc-Aib-Gly-Leu-Aib-Gly-Gly-Leu-Aib-Gly-Ile-Leuol) (10.4) L-Asp (29.5) L-Ile (20.4) D-Iva (11.2) L-Leu (24.2) LLeuol (22.2). A special temperature programme was used for the separation of proline enantiomers 50–110 8C 3 8C min21; plateau at 110 8C for 10 min; 100–190 8C 10 8C min21; tR L-Pro (25.1). Secondary ion mass spectroscopy Positive LSIMS was recorded on a ZAB2-SEQ (VG Analytical Manchester UK) mass spectrometer equipped with a standard FAB source and a caesium ion-gun operating at 35 kV.Peptide methanolic solution was mixed with a-monothioglycerol as matrix. The resolution was 1000. Positive HR-LSIMS were recorded on a ZAB-HF spectrometer. The MS spectra were registered with either Li1 or Na1 added to the matrix. NMR Spectroscopy A 0.4 cm3 aliquot of 7 mM methanolic (CD3OH) solution of HB I peptide in a 5 mm tube (Wilmad) was used for all the NMR experiments. Proton NMR spectroscopy experiments were conducted at 296 K on a Bruker AC 300 equipped with an Aspect 3000 computer or on a Bruker Avance DMX 500 spectrometer equipped with a Bruker Station 1 computer and an indirect quadruple-resonance 1H–31P–13C–15N gradient probehead.Spectra were processed using UXNMR and AURELIA software (Bruker Inc). Chemical shifts were referenced to the central component of the quintet due to the CD2H resonance of methanol at dH 3.313 downfield from SiMe4. J Values are given in Hz. TOCSY24 experiments were run with the MLEV 17 sequence for spin locking and a mixing time of 120 ms (9 kHz). The ROESY25 experiment was carried out with a mixing time of 250 ms and a spin lock field of 2 kHz to reduce Hartmann–Hahn transfers. Two-dimensional spectra were obtained with quadrature detection in both dimensions using the hypercomplex method in the F1 dimension.26 The solvent signal was suppressed using the WATERGATE scheme27 included in the standard and ROESY pulse sequences. A total of 2048 data points were acquired in the F2 dimension and 512 complex points in the F1 dimension.For each complex data point in the F2 4 free induction decays were accumulated with a relaxation delay of 2 s. All spectra were apodized with p/2-shifted sine-bell functions in both dimensions. CD spectrum The spectrum of HB I was recorded with a Jobin-Yvon CD6 dichrograph with a 0.1 mm path cell at 22 8C (1 mmol cm23; CH3OH); l (nm) and [q]M (deg cm2 dmol21) 192 (56 000) and 205 (2110 000). Antimicrobial activity The antibacterial activity of HB I was examined against Staphylococcus aureus (strain 209P) and Escherichia coli (strain RL 65) by the agar diffusion test using the Mueller Hinton culture medium and 6 mm diameter pits. The peptide sample was dissolved in dimethyl sulfoxide (DMSO) such as to give a 4 mg cm23 solution.Eight other concentrations were obtained by successive dilutions and 50 mm3 of each solution was deposited into the pits (1.5 to 200 mg). Inhibition zones were measured after 24 h of incubation at 37 8C. Liposome permeabilization Egg phosphatidylcholine (egg PC) type V E and cholesterol were purchased from Sigma; egg PC was used without further purification and cholesterol was recrystallized from methanol. CF from Eastman Kodak was separated from hydrophobic contaminants and recrystallized from ethanol as previously described.11 Fluorescence spectra were measured at 20 8C on an Aminco SPF 500 spectrofluorometer. The peptide-induced release of intravesicular content was monitored by the method introduced by Weinstein,28 that uses the property of quenching relief upon dilution of an encapsulated fluorescent probe CF.1592 J. Chem. Soc. Perkin Trans. 1 1997 CF-entrapped small unilamellar vesicles (SUV) were prepared as previously described,11 by sonication of an egg PC– cholesterol (7 3) mixture ([lip] = 0.6 mM). The SUV obtained by sonication were separated from unencapsulated CF by gel filtration (Sephadex G 75). Leakage kinetics were obtained for different peptide lipid molar ratios obtained by adding aliquots of methanolic solutions of peptides (methanol concentration kept below 0.5% by volume). Synthesis of HB I Diisopropylethylamine (DIEA) trifluoroacetic acid (TFA) ditert- butyl dicarbonate (Boc2O) and L-leucine were purchased from Sigma-Aldrich Chimie and D-isovaline [(R)-(2)-2-amino- 2-methylbutanoic acid] from Acros (France). All N-tertbutoxycarbonyl- protected L-amino acids and BOP were purchased from Propeptide (France) and used without subsequent purification.H-Leu-OMe was prepared according to Boissonnas et al.29 Boc-Iva-OH was prepared according to Bodanszky and Bodanszky.30 Column chromatography was carried out with 230–400 mesh Merck grade 60 silica gel. Analytical TLC was performed on aluminium sheets covered with Merck grade 60 silica gel. Gel filtration was carried out with Pharmacia Sephadex LH 20. General procedure A BOP-mediated peptide coupling. The Nprotected amino acid and BOP reagent were added to a solution of the TFA salt of the C-protected amino acid or peptide in CH2Cl2. The stirred solution was cooled in an ice-bath and DIEA was added. The mixture was stirred at room temperature until TLC analysis indicated that consumption of the amino component was no longer proceeding.It was then concentrated in vacuo to leave an oil which was dissolved in ethyl acetate and washed successively with 1 M HCl water 1 M NaOH and saturated aq. NaCl. The combined organics were dried over Na2SO4 filtered and concentrated in vacuo to leave an oil which was purified by chromatography on a silica gel column. General procedure B Removal of N-tert-butoxycarbonyl protection with 50% TFA solution in CH2Cl2. A stirred solution of the N-tert-butoxycarbonyl-protected peptide in CH2Cl2 was cooled in an ice-bath and TFA was added. The mixture was stirred at room temperature until TLC analysis indicated complete consumption of the starting material and was then evaporated in vacuo to leave an oil. The crude product was used without purification for the next coupling.General procedure C Removal of N-tert-butoxycarbonyl protection with pure TFA. The N-tert-butoxycarbonyl-protected peptide was treated with TFA (0.5 cm3 for 1 mmol of peptide). The mixture was stirred at room temperature until TLC analysis indicated that the totality of the peptide was deprotected and it was then evaporated in vacuo to give an oil. The crude product was used without purification for the next coupling. Boc-Pro-Leu-OMe. HCl H-Leu-OMe (0.92 g 5.07 mmol) and Boc-Pro-OH (1.20 g 5.58 mmol) were treated with BOP (2.47 g 5.58 mmol) and DIEA (2.7 cm3 15.71 mmol) via procedure A to yield the crude product which was used without purification. Boc-Aib-Pro-Leu-OMe. Boc-Pro-Leu-OMe (1.73 g 5.07 mmol) was deprotected according to procedure C.The crude TFA salt and Boc-Aib-OH (1.12 g 5.50 mmol) were treated with BOP (2.43 g 5.50 mmol) and DIEA (2.7 cm3 15.7 mmol) according to procedure A to yield after purification by chromatography on silica gel (ethyl acetate) 1 g of a powder; TLC Rf (ethyl acetate) 0.40; LSIMS m/z 434 [M 1 Li]1 (100) 378 (5) 334 (27) 332 (2) and 247 (9); HR-LSIMS [M 1 Li]1 434.2822 (Calc. for C21H37LiN3O6 m/z 434.2842); dH(300 MHz; CD3OH) 0.91 (d J 6.0 3 H d Leu) 0.94 (d J 6.0 3 H d Leu) 1.36 (s 3 H b Aib) 1.45 (s 12 H b Aib Boc) 1.81 (m 6 H 2 × b Leu g Leu b9 Pro 2 × g Pro) 2.19 (m 1 H b Pro) 3.59 (m 1 H d9 Pro) 3.68 (s 3 H ester) 3.81 (m 1 H d Pro) 4.37 (m 1 H a Leu) 4.45 (dd J 8.4 and 5.9 1 H a Pro) 7.29 (s 1 H NH Aib) and 8.23 (d J 7.7 1 H NH Leu). Boc-Leu-Aib-Pro-Leu-OMe.Boc-Aib-Pro-Leu-OMe (1.00 g 2.34 mmol) was deprotected according to procedure B. The crude TFA salt and Boc-Leu-OH (0.64 g 2,58 mmol) were treated with BOP (1.14 g 2.58 mmol) and DIEA (1.3 cm3 7.3 mmol) according to general procedure A. The mixture was purified by chromatography on silica gel eluted by CH2Cl2– MeOH (92 8) to yield 1.06 g (85%) of a powder; TLC Rf (CH2Cl2–MeOH 92 8) 0.50; LSIMS m/z 547 [M 1 Li]1 (100) 491 (8) 447 (17) 445 (3) and 247 (8); HR-LSIMS [M 1 Li]1 547.3657 (Calc. for C27H48LiN4O7 m/z 547.3683); [a]D 22 277 (c 0.1 MeOH); dH(300 MHz; CD3OH) 0.93 (m 12 H d Leu1 d Leu4) 1.44 (s 12 H b Aib Boc) 1.46 (s 3 H b Aib) 1.47 (m 2 H 2 × b Leu1) 1.60 (m 1 H b9 Leu4) 1.69 (m 2 H g Leu1 g Leu4) 1.75 (m 1 H b Leu4) 1.88 (m 3 H b9 Pro 2 × g Pro) 2.08 (m 1 H b Pro) 3.62 (m 2 H 2 × d Pro) 3.68 (s 3 H ester) 4.09 (m 1 H a Leu1) 4.34 (m 1 H a Leu4) 4.47 (m 1 H a Pro) 6.68 (d J 8.1 1 H NH Leu1) and 8.21 (br s 2 H NH Aib NH Leu4).Boc-Iva-Leu-Aib-Pro-Leu-OMe. Boc-Leu-Aib-Pro-Leu-OMe (150 mg 0.28 mmol) was deprotected according to procedure B. The crude TFA salt and Boc-Iva-OH (73 mg 0.34 mmol) were treated with BOP (149 mg 0.34 mmol) and DIEA (180 mm3 1 mmol) according to procedure A. The mixture was purified by chromatography on silica gel (CH2Cl2–MeOH 94 6) to yield 152 mg (85%) of the expected product; TLC Rf (CH2Cl2– MeOH 93 7) 0.43; LSIMS m/z 662 [M 1 Na]1 (15) 495 (3) 398 (44) 313 (4) and 243 (100); HR-LSIMS [M 1 Li]1 646.4385 (Calc. for C32H57LiN5O8 m/z 646.4367); dH(300 MHz; CD3OH) 0.84 (m 3 H Meg Iva) 0.95 (m 12 H d Leu2 d Leu5) 1.34 (s 3 H Meb Iva) 1.47 (s 15 H b Aib Boc) 1.62 (m 2 H b9 Leu2 b9 Leu5) 1.70 (m 2 H g Leu2 g Leu5) 1.77 (m 2 H b Leu2 b Leu5) 1.78 (m 1 H b9 Iva) 1.85 (m 1 H b9 Pro) 1.88 (m 2 H g Pro) 1.92 (m 1 H b Iva) 2.16 (m 1 H b Pro) 3.53 (m 1 H d9 Pro) 3.3 (m 1 H d Pro) 3.68 (s 3 H ester) 4.35 (m 2 H a Leu2 a Leu5) 4.48 (m 1 H a Pro) 6.93 (s 1 H NH Iva) 7.94 (d J 7.0 1 H NH Leu2) 8.14 (s 1 H NH Aib) and 8.16 (d J 8.3 1 H NH Leu5).Boc-Pro-Iva-Leu-Aib-Pro-Leu-OMe. Boc-Iva-Leu-Aib-Pro- Leu-OMe (140 mg 0.22 mmol) was deprotected according to procedure B. The crude TFA salt and Boc-Pro-OH (71 mg 0.33 mmol) were treated with BOP (146 mg 0.33 mmol) and DIEA (134 mm3 0.77 mmol) according to procedure A to yield after purification by silica gel chromatography (CH2Cl2–MeOH 95 5) 149 mg (92%) of a powder; TLC Rf (CH2Cl2–MeOH 94 6) 0.44; LSIMS m/z 743 [M 1 Li]1 (100) 643 (14) 641 (5) 473 (3) 388 (9) and 247 (5); HR-LSIMS [M 1 Li]1 743.4897 (Calc.for C37H64LiN6O9 m/z 743.4895); [a]D 22 233 (c 0.1 MeOH); dH(300 MHz; CD3OH) 0.82 (m 3 H Meg Iva) 0.92 (m 12 H d Leu3 d Leu6) 1.36 (s 3 H Meb Iva) 1.49 (s 3 H b Aib) 1.50 (s 12 H b Aib Boc) 1.62 (m 2 H b9 Leu3 b9 Leu6) 1.70 (m 2 H g Leu3 g Leu6) 1.80 (m 2 H b Leu3 b Leu6) 1.82 (m 1 H b9 Iva) 1.83 (m b9 Pro5) 1.86 (m 2 H g Pro5) 1.95 (m 1 H b9 Pro1) 1.98 (m 2 H g Pro1) 2.18 (m 1 H b Pro5) 2.23 (m 1 H b Iva) 4.31 (m 1 H b Pro1) 3.51 (m 2 H d9 Pro1 d9 Pro5) 3.60 (m 1 H d Pro1) 3.69 (s 3 H ester) 3.74 (m 1 H d Pro5) 4.08 (m 1 H a Pro1) 4.36 (m 2 H a Leu3 a Leu6) 4.49 (m 1 H a Pro5) 7.82 (d J 8.5 1 H NH Leu3) 7.87 (s 1 H NH Iva) 8.12 (s 1 H NH Aib) and 8.16 (d J 7.7 1 H NH Leu6).Boc-Ile-Aib-OMe. According to procedure A H-Aib-OMe (1.16 g 7.6 mmol) and Boc-Ile-OH (2.21 g 9.2 mmol) were treated with BOP (4.07 g 9.2 mmol) and DIEA (2.6 cm3 15.2 mmol). The crude product was used without purification for the next coupling. TLC Rf (ethyl acetate–cyclohexane 1 1) 0.61. Boc-Leu-Ile-Aib-OMe. Boc-Ile-Aib-OMe (2.51 g 7.6 mmol) was deprotected according to procedure B. The TFA salt and Boc-Leu-OH (2.27 g 9.1 mmol) were treated with BOP (4.03 g 9.1 mmol) and DIEA (4.2 cm3 24 mmol) according to procedure A. The mixture was purified by silica gel chromatography (ethyl acetate–cyclohexane 5 5) to yield 1.08 g (32% for the two steps) of a solid; TLC Rf (ethyl acetate–cyclohexane, J. Chem. Soc. Perkin Trans. 1 1997 1593 1 1) 0.53; LSIMS m/z 450 [M 1 Li]1 (100) 394 (12) 350 (24) 348 (3) 305 (2) and 192 (8); HR-LSIMS [M 1 Li]1 450.3159 (Calc.for C22H41LiN3O6 m/z 450.3155); dH(300 MHz; CD3OH) 0.93 (m 12 H d Leu Meg Ile Med Ile) 1.16 (m 1 H g9 Ile) 1.42 (s 3 H b Aib) 1.43 (s 9 H Boc) 1.45 (s 3 H b Aib) 1.53 (m 3 H g Ile 2 × b Leu) 1.66 (m 1 H g Leu) 1.77 (m 1 H b Ile) 3.64 (s 3 H ester) 4.07 (m 1 H a Leu) 4.18 (m 1 H a Ile) 6.86 (d J 7.7 1 H NH Leu) 7.71 (d J 8.6 1 H NH Ile) and 8.35 (s 1 H NH Aib). Boc-Asn-Leu-Ile-Aib-OMe. Boc-Leu-Ile-Aib-OMe (1.03 g 2.3 mmol) was deprotected according to procedure C. The crude TFA salt and Boc-Asn-OH (0.65 g 2.8 mmol) were treated with BOP (1.24 g 2.8 mmol) and DIEA (1.3 cm3 7.5 mmol) according to procedure A. After purification by silica gel chromatography (CH2Cl2–MeOH 95 5) it yielded 890 mg (69%) of a powder; TLC Rf (ethyl acetate) 0.17; LSIMS m/z 564 [M 1 Li]1 (100) 508 (4) 464 (31) 462 (3) 419 (4) and 306 (3); HR-LSIMS [M 1 Li]1 564.3583 (Calc.for C26H47LiN5O8 m/z 564.3585); dH(300 MHz; CD3OH) 0.89 (m 3 H Med Ile) 0.92 (m 6 H d Leu) 0.94 (m 3 H Meg Ile) 1.17 (m 1 H g9 Ile) 1.41 (s 3 H b Aib) 1.42 (s 3 H b Aib) 1.43 (s 9 H Boc) 1.43 (m 1 H b9 Leu) 1.55 (m 1 H g Ile) 1.62 (m 1 H b Leu) 1.73 (m 1 H g Leu) 1.90 (m 1 H b Ile) 2.66 (m 2 H 2 × Asn) 3.64 (s 3 H ester) 4.12 (m 1 H a Ile) 4.37 (m 1 H a Leu) 4.41 (m 1 H a Asn) 6.86 (d J 7.7 1 H NH Asn) 6.94 (s 1 H d syn Asn) 7.60 (s 1 H d anti Asn) 7.96 (d J 8.7 1 H NH Ile) 8.03 (s 1 H NH Aib) and 8.17 (d J 7.2 1 H NH Leu). Ac-Aib-Asn-Leu-Ile-Aib-OMe. Boc-Asn-Leu-Ile-Aib-OMe (0.54 g 0.97 mmol) was deprotected according to procedure C.The TFA salt and Ac-Aib-OH (0.27 g 1.84 mmol) were treated with BOP (0.81 g 1.84 mmol) and DIEA (0.5 cm3 3.0 mmol) according to procedure A. After purification by silica gel chromatography (CH2Cl2–MeOH 90 10) it yielded 190 mg of a powder; TLC Rf (CH2Cl2–MeOH 88 12) 0.30; LSIMS m/z 591 [M 1 Li]1 (100) 333 (6) 291 (16) 220 (11) 209 (40) 122 (7) and 106 (15); HR-LSIMS [M 1 Li]1 591.3678 (Calc. for C27H48LiN6O8 m/z 591.3694); dH(300 MHz; CD3OH) 0.90 (m 12 H d Leu Meg Ile Med Ile) 1.21 (m 1 H g9 Ile) 1.43 (m 12 H b Aib1 b Aib5) 1.55 (m 2 H b9 Leu g Ile) 1.69 (m 1 H b Leu) 1.87 (m 2 H g Leu b Ile) 1.99 (s 3 H Ac) 2.71 (ABX system J 7.6 and 15.4 1 H b9 Asn) 2.81 (ABX system J 6.3 and 15.4 1 H b Asn) 3.65 (s 3 H ester) 4.14 (m 1 H a Ile) 4.27 (m 1 H a Leu) 4.41 (m 1 H a Asn) 6.94 (s 1 H d syn Asn) 7.38 (d J 8.9 1 H NH Ile) 7.69 (s 1 H d anti Asn) 7.83 (s 1 H NH Aib5) 8.11 (d J 7.2 1 H NH Leu) 8.46 (d J 6.2 1 H NH Asn) and 8.54 (s 1 H NH Aib1).Ac-Aib-Asn-Leu-Ile-Aib-OH. A stirred solution of Ac-Aib- Asn-Leu-Ile-Aib-OMe (140 mg 0.24 mmol) in MeOH (1.4 cm3) was cooled in an ice-bath and 1.0 cm3 of 1 M NaOH was added. The mixture was stirred at room temperature until TLC analysis indicated complete consumption of the methyl ester. The cooled mixture was then neutralized with 1 M HCl and evaporated in vacuo. The crude product was purified using Sephadex LH-20 with MeOH as eluent to yield 116 mg (85%) of a solid; LSIMS m/z 593 [M 1 Na]1 (100) 468 (7) 355 (14) 242 (23) and 128 (18); HR-LSIMS [M 1 Na]1 593.3293 (Calc. for C26H46N6NaO8 m/z 593.3275); [a]D 22 210 (c 0.1 MeOH); dH(300 MHz; CD3OH) 0.87 (m 6 H Med Ile d Leu) 0.94 (d J 6.8 3 H Meg Ile) 0.95 (d J 6.5 3 H d Leu) 1.21 (m 1 H g9 Ile) 1.42 (s 3 H b Aib) 1.45 (s 3 H b Aib) 1.46 (s 3 H b Aib) 1.47 (s 3 H b Aib) 1.54 (m 1 H g Ile) 1.57 (m 1 H b9 Leu) 1.73 (m 1 H g Leu) 1.81 (m 1 H b Leu) 1.95 (m 1 H b Ile) 1.99 (s 3 H Ac) 2.73 (ABX system J 4.9 and 15.4 1 H b9 Asn) 2.81 (ABX system J 6.3 and 15.4 1 H b Asn) 4.16 (m 1 H a Ile) 4.28 (m 1 H a Leu) 4.45 (m 1 H a Asn) 6.93 (s 1 H d syn Asn) 7.45 (d J 9.1 1 H NH Ile) 7.69 (s 1 H d anti Asn) 7.78 (s 1 H NH Aib5) 8.12 (d J 7.5 1 H NH Leu) 8.45 (d J 6.5 1 H NH Asn) and 8.53 (s 1 H NH Aib1).Ac-Aib-Asn-Leu-Ile-Aib-Pro-Iva-Leu-Aib-Pro-Leu-OMe. Boc-Pro-Iva-Leu-Aib-Pro-Leu-OMe (100 mg 0.13 mmol) was deprotected according to procedure B.The TFA salt and Ac- Aib-Asn-Leu-Ile-Aib-OH (50 mg 0.087 mmol) were treated with BOP (38.5 mg 0.087 mmol) and DIEA (45 mm3 0.26 mmol) according to procedure A to yield after purification by silica gel chromatography (CH2Cl2–MeOH 85 15) 42 mg (41%) of a powder; TLC Rf (CH2Cl2–MeOH 85 15) 0.42; LSIMS m/z 1195 [M 1 Li]1 (100) 798 (6) 641 (27) 628 (6) 531 (13) 446 (16) 333 (9) and 291 (8); HR-LSIMS [M 1 Li]1 1195.7598 (Calc. for C58H100LiN12O14 m/z 1195.7642); dH(300 MHz; CD3OH) 0.83 (m 3 H Meg Iva) 0.87 (m 3 H Med Ile) 0.89 (m 18 H d Leu3 d Leu8 d Leu11) 0.95 (m 3 H Meg Ile) 1.30 (m 1 H g9 Ile) 1.45 (s 3 H Meb Iva) 1.46 (s 6 H b Aib) 1.49 (s 9 H b Aib) 1.50 (s 3 H b Aib) 1.55 (m 1 H g Ile) 1.76 (m 9 H b and g Leu3 b and g Leu8 b and g Leu11) 1.77 (m 1 H b9 Iva) 1.80 (m 1 H b9 Pro6) 1.83 (m 1 H b9 Pro10) 1.86 (m 2 H 2 × g Pro10) 1.92 (m 1 H g9 Pro6) 1.96 (m 1 H b Ile) 2.02 (s 3 H Ac) 2.12 (m 1 H g Pro6) 2.20 (m 1 H b Pro10) 2.32 (m 1 H b Pro6) 2.46 (m 1 H b Iva) 2.76 (d J 5.7 2 H b Asn) 3.42 (m 2 H d9 Pro6 d9 Pro10) 3.69 (s 3 H ester) 3.81 (m 2 H d Pro6 d Pro10) 4.20 (m 3 H a Leu3 a Ile4 a Pro6) 4.34 (m 3 H a Asn2 a Leu8 a Leu11) 4.50 (m 1 H a Pro10) 7.04 (s 1 H d syn Asn) 7.35 (d J 8.8 1 H NH Ile4) 7.49 (s 1 H NH Iva) 7.62 (d J 8.5 1 H NH Leu8) 7.76 (s 2 H NH Aib5 NH Aib9) 7.80 (s 1 H d anti Asn) 8.11 (d J 7.1 1 H NH Leu3) 8.16 (d J 7.8 1 H NH Leu11) 8.61 (d J 5.5 1 H NH Asn) and 8.67 (s 1 H NH Aib1).Ac-Aib-Asn-Leu-Ile-Aib-Pro-Iva-Leu-Aib-Pro-Leuol HB I. A solution of Ac-Aib-Asn-Leu-Ile-Aib-Pro-Iva-Leu-Aib-Pro- Leu-OMe (32 mg 0.027 mmol) in EtOH (1 cm3) was cooled in an ice-bath and NaBH4 (6 mg 0.162 mmol) was added.The mixture was stirred at 50 8C for 8 h and the solvent was evaporated off in vacuo. The residue was dissolved in ethyl acetate and washed with water. The organic layer was dried over Na2SO4 filtered and concentrated in vacuo to yield 25 mg (80%) of HB I; TLC Rf (CH2Cl2–MeOH 85 15) 0.37; LSIMS m/z 1167 [M 1 Li]1 (55) 947 (64) 553 (81) 468 (34) 395 (28) 355 (63) 310 (25) 242 (100) 197 (69) and 128 (70); HR-LSIMS [M 1 Na]1 1183.7333 (Calc. for C57H100N12NaO13 m/z 1183.7430); [a]D 22 17 (c 0.1 MeOH); 1 NMR data were identical with those described for natural HB I. Acknowledgements We are indebted to Dr M. F. Roquebert (Laboratoire de Cryptogamie du Muséum National d’Histoire Naturelle) who provided the T. harzianum strain and to Dr A.Galat (CEA Saclay France) for the dichrograph facility. We thank Dr M. Becchi and the Centre de Spectroscopie du CNRS (Lyon France) for LSIMS measurements and Dr Duclohier for macroscopic current–voltage experiments. The 500 MHz facilities used in this study were funded by the Région Haute- Normandie France. This work was supported in part by a grant from the Centre National de la Recherche Scientifique (GDR 1153). References 1 R. C. Pandey J. C. Cook Jr. and K. L. Rinehart J. Am. Chem. Soc. 1977 99 8469. 2 B. Bodo S. Rebuffat M. El Hajji and D. Davoust J. Am. Chem. Soc. 1985 107 6011. 3 A. Iida S. Uesato T. Shingu M. Okuda Y. Nagaoka Y. Kuroda and T. Fujita J. Chem. Soc. Perkin Trans. 1 1993 367. 4 S. Rebuffat L. Conraux M. Massias C. Auvin-Guette and B. Bodo Int. J. Pept.Protein Res. 1993 41 74. 5 C. Goulard S. Hlimi S. Rebuffat and B. Bodo J. Antibiot. 1995 48 1248. 6 A. Iida M. Sanekata S. Wada T. Fujita H. Tanaka A. Enoki G. Fuse M. Kanai and K. Asami Chem. Pharm. Bull. 1995 43 392. 1594 J. Chem. Soc. Perkin Trans. 1 1997 7 S. Rebuffat C. Goulard and B. Bodo J. Chem. Soc. Perkin Trans. 1 1995 1849. 8 C. Auvin-Guette S. Rebuffat Y. Prigent and B. Bodo J. Am. Chem. Soc. 1992 114 2170. 9 T. Fujita S. Wada A. Iida T. Nishimura M. Kanai and N. Toyama Chem. Pharm. Bull. 1994 42 489. 10 M. S. P. Sansom Prog. Biophys. Mol. Biol. 1991 55 139. 11 S. Rebuffat H. Duclohier C. Auvin-Guette G. Molle G. Spach and B. Bodo FEMS Microbiol. Immunol. 1992 105 151. 12 T. Le Doan M. El Hajji S. Rebuffat M. R. Rajesvari and B. Bodo Biochim. Biophys. Acta 1986 858 1.13 M. El Hajji S. Rebuffat T. Le Doan G. Klein M. Satre and B. Bodo Biochim. Biophys. Acta 1989 978 97. 14 S. Hlimi S. Rebuffat C. Goulard S. Duchamp and B. Bodo J. Antibiot. 1995 48 1254. 15 A. R. Artalejo C. Montiel P. Sanchez-Garcia G. Uceda J. M. Guantes and A. G. Garcia Biochem. Biophys. Res. Commun. 1990 1204. 16 A. Iida M. Okuda S. Uesato Y. Takaishi T. Shingu M. Morita and T. Fujita J. Chem. Soc. Perkin Trans. 1 1990 3249. 17 S. Rebuffat P. Drognat-Landré C. Goulard I. Augeven-Bour C. Auvin and B. Bodo Peptides 1992 Proceedings of the 22nd European Peptide Symposium ed. C. H. Schneider and A. N. Eberle Escom Science Publishers Leiden 1993 pp. 427–428. 18 P. Roepstorff P. Höjrup and J. Möller Biomed. Mass Spectrom. 1985 12 181. 19 S. Wada A. Iida N. Akimoto M. Kanai N.Toyama and T. Fujita Chem. Pharm. Bull. 1995 43 910. 20 G. Esposito J. A. Carver J. Boyd and I. D. Campbell Biochemistry 1987 26 1043. 21 S. Rebuffat Y. Prigent C. Auvin-Guette and B. Bodo Eur. J. Biochem. 1991 201 661. 22 Y. Nagaoka A. Iida and T. Fujita Chem. Pharm. Bull. 1994 42 1258. 23 C. Toniolo M. Crisma F. Formaggio C. Peggion V. Monaco C. Goulard S. Rebuffat and B. Bodo J. Am. Chem. Soc. 1996 118 4952. 24 C. Griesinger G. Otting K. Wuthrich and R. R. Ernst J. Am. Chem. Soc. 1988 110 7870. 25 A. Bax and D. G. Davis J. Magn. Reson. 1985 63 207. 26 D. J. States R. A. Haberkorn and D. J. Ruben J. Magn. Reson. 1982 48 286. 27 M. Piotto U. Savdek and V. Sklenar J. Biomol. NMR 1992 2 661. 28 J. N. Weinstein S. Yoshikami P. Henkari R. Blumenthal and W. A. Hagins Science 1977 195 489.29 R. A. Boissonnas S. Guttmann P. A. Jaquenoud and J. P. Waller Helv. Chim. Acta 1955 38 1491. 30 M. Bodanszky and A. Bodanszky The Practice of Peptide Synthesis Springer-Verlag Berlin Heidelberg New York and Tokyo 1984 p. 20. Paper 6/05629F Received 12th August 1996 Accepted 11th February 1997 © Copyright 1997 by the Royal Society of Chemistry
ISSN:1472-7781
DOI:10.1039/a605629f
出版商:RSC
年代:1997
数据来源: RSC
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