CID-MS/MS studies of Geninthiocin (130) and the new Val-Geninthiocin (131) (131)

the molecular formula as C50H49N15O14S with one oxygen atom less than 130.

The compound showed an NMR pattern similar to that of 130, except that the oxy-carbon at δ 71.0 (βC of hydroxy-valine) in 130 was replaced by a non oxygen-ated one at δ 29.5 in 131. Alternatively, the gem-dimethyls of the hydroxyvaline residue in geninthiocin (130) were changed into a 6H doublet, corresponding to an isopropyl system (δ 0.97, J= 6.6 Hz) in 131.

Therefore, compound 131 was identified as deoxy-derivative of geninthiocin (130), and named Val-geninthiocin.

N

4.7.3 CID-MS/MS studies of Geninthiocin (130) and the new Val-Geninthiocin (131)

As part of our ongoing structure elucidation of cyclopeptides by MS methods, it was of interest to investigate the fragmentation behaviour of both metabolites in par-allel. For this purpose, MS² and MS³ experiments using a quadrupol ion trap were performed (Table 14), and additionally, high-resolution CID-MS/MS measurements

were carried out on a Fourier transform ion cyclotron resonance (FT-ICR) mass spectrometer to determine the elemental composition of key fragments.

Tandem mass spectrometry is a very powerful method for sequence analysis of peptides and proteins. The fragmentation of linear peptides have been described comprehensively and detailed knowledge of the fragmentation mechanism have been obtained^[196-197]. However, the structure analysis of cyclic peptides, which represent an important class of bioactive natural products, by mass spectrometry, remains a challenging task. Complex fragmentation patterns by two-bond cleavage at different ring positions, ring-opening reactions and uncommon rearrangement reactions com-plicate the interpretation of CID-MS/MS spectra^[198-199]. Frequently, higher order MSⁿ investigations are required. Furthermore, the cyclic peptides produced as secon-dary metabolites from bacteria often contain uncommon amino acid residues leading to different fragmentation reactions.

In general, for sequencing linear peptides, fragmentation of the [M+2H]²⁺ ions is used successfully, but in the case of geninthiocin (130) and his minor congener, fragmentation spectra of those ions were dominated by doubly charged fragment ions without significant sequence information. Furthermore, [M+2H]²⁺ ions were ob-served with very low intensity under the conditions described. Also the MS² spectra of [M+Na]⁺ and [M-H]^- provided little sequence information. Therefore, CID-MS/MS studies were concentrated on the more intense [M+H]⁺ species (Figure 57).

400 500 600 700 800 900 1000 1100 1200 m/z

0 5 10 15 20 25 30 35 40 45 50 55 60 65 70 75 80 85 90 95 100

Relative Abundance

897

1098

949

1088 1099 798

1070

1017 978 1030 852 879

631 780 931

579 746

508 613 660 850

370 465 492 545 729 1116

Figure 57: CID-MS/MS spectrum of Val-Geninthiocin (131,); fragmentation of [M+H]⁺ measured on a quadrupol ion trap instrument.

Beside an unspecific loss of H2O, CO, and NH3, the following main fragmenta-tion pathways were observed (Latin numbers correspond to the fragments indicated in Figure 58):

I Cleavage of a single amino acid residue unit from the peptide ring system is found basically for Hyval (-115 Dalton) and Val (-99 Dalton) in 130 and 131, re-spectively (cleavage of two CO-NH peptide bonds). Therefore, the structural dif-ference between both compounds could be allocated at this amino acid at a very early stage.

II Cleavage of a single amino acid residue unit from the peptide ring system by fragmentation of one CO-NH peptide bond and one CH₂=C-NH bond (H-Oxa-NH2, H-Hyval/Val-NH2, H-Deala-NH2) was detected. Obviously, the -NH-C=CH₂- bond between Oxa(1)-Deala(1) and Oxa(3)-Deala(2) as well as between Deala(2)-Oxa(2) and Hyval/Val-Oxa(3) could be broken favourably leading to a second preferred fragmentation pathway beside peptide bond (-CO-NH-) cleav-age.

III Cleavage of Oxa-Deala dipeptide units with high preference resulted in the prominent fragment ion peaks at m/z 913 (130) and 897 (131), respectively. Fur-thermore, fragmentations of Deala-Hyval/Val occured with lower intensity.

IV Cleavage of the tripeptide unit Hyval/Val-Oxa-Deala gave the key fragment at m/z 798. After cleavage of the tripeptide unit Hyval/Val-Oxa-Deala, a further dipeptide Oxa-Deala loss resulted in the fragment ion m/z 579 (IV+III).

V Fragmentation of the linear peptide unit predominantly occurred by cleavage of the NH-CO bond between Deala(4) and Deala(3) forming a b-fragment ion (V b10). Furthermore, formation of a c-type fragment (V c9) by cleavage of CH2 =C-NH bond between Deala(3) and Pyr induced by the dehydroalanine structure is observed.

Deala (3) Deala (4)

Deala (1) Geninthiocin (130) and Val-Geninthiocin (131) obtained on a quadru-pole ion trap mass spectrometer; Table 14.

The fragmentation pathways derived from collision activated dissociation using a quadrupole ion trap (Table 14) were confirmed by high resolution MS/MS in an FT-ICR mass spectrometer providing the elemental composition of key fragments. By comparison of MS² spectra, the position of valine in 131 was determined

unambigu-ously. The information obtained during such fragmentation pathways could be ap-plied to further investigations of this class of substances in order to identify low amounts of derivatives.

Table 14: MS² and MS³ product ions of the [M+H]⁺ precursor ions of Geninthiocin (130) and Val-Geninthiocin (131) obtained on a quadrupole ion trap mass spectrometer

Geninthiocin (130) Val-Geninthiocin (131) MS² fragment ion

896 880 -H-Oxa-Deala-NH2

895 878; 877; 851 879 861; 851 -Oxa-Deala-H2O

889 -C10H17N3O4

-(H-Oxa-NH2)-C3H6O-H2O

850 852 850 -H-Hyval/Val-Oxa-NH2

798 781; 780; 770;

754; 712; 660;

631; 579

798 781; 780; 770; 754;

712; 660; 631; 579 -Hyval/Val-Oxa-Deala

780 780 -Hyval/Val-Oxa-Deala-H2O

746 729; 728; 660;

608; 579 746 729; 728; 660;

(608; 579) -Deala-Oxa-Hyval/Val-COCCH2+2H

Val-Geninthiocin (131) is a new thiopeptide^[194], having a very close similarity with geninthiocin (130)^[195]. Both compounds are thiopeptide antibiotics^[200,201], char-acterized by several common structural features, such as oxazole and thiazole units

and unusual amino acids; especially dehydroamino acids are typical^[202,203]. Among this group are thioxamycin^[204], berninamycin A^[205], sulfomycin I^[206-208] and A10255^[209], possessing the thiazole-pyridine-oxazole substructure. Moreover, thio-cillin I ^[210,211], micrococcin P ^[212] and GE2270 A ^[213] are thiopeptides characterized by a thiazole-pyridine-thiazole moiety. These peptides are used as antibacterial agents against Gram-positive bacteria and anaerobes, including pathogens resistant to antibiotics currently in use^[214,215]. They have also potential activity as growth inhibi-tors of the human malaria parasite^[216]. Recently, thiopeptides have been proved as effective growth promoters for domestic animals^[201,217]. Most of the thiopeptide anti-biotics inhibit protein synthesis in bacteria, and share a common mode of action^[218]. Thiostrepton, whose antibiotic activity is best understood, acts by binding tightly to the prokaryotic ribosome, and thus inhibits translation^[218,219]. Geninthiocin (130) is known as an activating agent for transcription of the tip A promoter in Streptomy-ces^[195].

4.7.4 Chalcomycin A

Compound 132 was isolated as a white solid with a UV absorbing zone, which turned dark blue with anisaldehyde/sulphuric acid. According to ESI MS, the mo-lecular weight was established as 700 Dalton, corresponding to the momo-lecular for-mula C₃₅H₅₆O₁₄. The ¹H/¹³C NMR spectra revealed the presence 35 carbon atoms, classified into a ketone and an ester carbonyl, four sp² methines, representing two double bonds conjugated with carbonyls, two acetals and sixteen oxygenated sp³ carbons of methines and methyls. Finally, three non-hetrobound methines, two me-thylenes and six methyls were observed.

Based on these data, a search in AntiBase deduced the structure as chalcomycin A (132), a 16-membered macrolide, which previously reported from Streptomyces bikiniensis^[220~221].Compound 28, a structural analogue, was recently reported by our research group together with 132 from the culture broth of the marine Streptomycete isolate B7064^[52]. Chalcomycin A (132) has a strong antibacterial activity against Staphylococcus aureus, Escherichia coli and Bacillus subtilis, which is higher than that of erythromycin A (4). Chalcomycin A (132) was investigated here for the first time by ESI MS/MS, and the details are given in Figure 59.

O

Table 15: ¹³C NMR (CDCl₃,125 MHz) spectroscopic data of Chalcomycin A (132).

No. δδδδC No. δδδδC No. δδδδC No. δδδδC

m/z = 549.3 [M-sugar(A)+Na]⁺ m/z = 405.2 [M-sugar(A)-sugar(B)+Na]⁺ Chalcomycin A

A

B

Figure 59: ESI MS/MS of Chalcomycin A (132).