Axinastatin 1 or malaysiatin? proof of constitution and 3D structure in solution of a cyclic heptapeptide with cytostatic properties

Abstract

Recently, a cyclic heptapeptide with cytostatic activity was isolated from marine sources by three different groups independently. The sequence of the isolated peptide was ambiguous, since two different isomers have been proposed: cyclo(-Asn'-Pro2-Phe3-Val4-Val5-Pro6-Val7-) (1) also called axinastatin 1 resp. pseudoaxinellin and cyclo(-Asn1"-Pro2"-Pro3"-Phe4*-Val5"-Val6*-Val7"-) (2) called rnalaysiatin. We synthesized both peptides 1 and 2 and compared their optical rotation, FAB-MS, 'H-and 13C-NMR data with those of the native compounds. Our results prove that peptide 1 has been assigned correctly, whereas the data of 2 differ significantly from those of the natural peptide. Peptide 1 adopts two conformations (90: 10 ratio) in DMSO, interconverting slowly on the NMR time scale. According to MD simulations, using NOES and J couplings as experimental restraints, a PVIa-turn with a cis peptide bond between Val5-Pro6 and a PI-turn in the Asn'-Val4 region are the characteristic secondary structural elements of the major conformer. Its backbone conformation is very similar to the X-ray structure of a related peptideDuring the past years many cyclic peptides like hymenistatin[',*], ev~lidine [~I, hymenamide~[~] and pseudostell~rins~~] were isolated from different biological sources. Recently, three cyclic heptapeptides were found independently in marine organisms: axinastatin I in Axinella spongueL6], pseudoxainellin in Pseudoaxinella ma~sa [~], and malaysiatin in Pseudoaxinyssa spongud81. Although similar biological activities and almost identical NMR data were found, two significantly different structures have been proposed: cyclo-(-Asn'-Pro2-Phe3-Val4-Vals-Pro6-Val7-) (1) and cyclo-(-Asn'#-Pro2#-Pro3#-Phe4#-Val5*-VaI6#-Val7#) (2). Both peptides have the same amino acid composition but differ in their sequence.Pettit et al. were the first to describe the isolation of 1 from Axinella spongue. This compound displayed a strong activity in a P388 test of murine lymphoblastic leukemia [6]. The same substance was isolated from Pseudoaxinella massa by Kong et al.L7]. They confirmed the structure and activity of 1. Fernandez et al. also isolated a highly cytotoxic substance (P388 test) from Pseudoaxinyssa spongue [8]. They proposed a different sequence 2 for this compound, although the NMR data, especially the characteristic I3C resonances, were very similar to those of the previously isolated substances. To reveal the correct sequence and structure of this cytotoxic material, both cyclic peptides 1 and 2 were synthesized and their physical data compared with those of the native compounds. Synthesis and Characterizationusing the Fmoc strategy['o,ll] and TBTU { 0- (1H-benzotriazol-l-yl)-N,N,N',N ["] were applied to the synthesis of the linear peptides. These precursors were also cyclized to 1 and 2 by the use of TBTU/HOBt[141. Purification was performed by using RP HPLC and a standard acetonitrile/water gradient system. The products were characterized by FAB-MS, optical rotation analysis and NMR spectroscopy. FAB-MSThe ami...

Cyclic Heptapeptides Axinastatin 2, 3, and 4: Conformational analysis and evaluation of the biological potential

“…A rare conformational class which is found for the synthetic compound cyclo(-G1y'- shows a PI1 turn about Leu7 and Gly' and a PI turn about Pro4 and Leu', whereas yunnanin A (cyclo(-G1y'-Tyr2-G1y3-Gly4-Pro5-Phe6-Pro7)) [33] has a PI1 turn about [34], hymenamide B-F [35], stylopeptide [7], isophakellistatin 3 [36], stylostatin 1 [S] [28], and axinastatin 1 [25], form the cis amide bond at a proline residue. For all these cyclic heptapeptides, the overall backbone conformation is similar to the structures of axinastatins 2 -4, investigated here.…”

“…To our knowledge, phakellistatin 1 (cycle(-Pro '-Ile2-Pro3-Ile4-Phe'-Pro6-Tyr7)) [32] is the only known structure with two cis amide bonds in the p turns about Ile2/Pro3 and Phe'/Pro6. There have been few examples for transltrans configuration in the , 8 turns: pseudostellarin D (cycle(-G1y'-Tyr2-Cly3-Pro4-Leu5-Ile6-Leu7)) [16] shows a PI1 turn about Leu7 and Gly' and a PI turn about Pro4 and Leu', whereas yunnanin A (cyclo(-G1y'-Tyr2-G1y3-Gly4-Pro5-Phe6-Pro7)) [33] [34], hymenamide B-F [35], stylopeptide [7], isophakellistatin 3 [36], stylostatin 1 [S] [28], and axinastatin 1 [25], form the cis amide bond at a proline residue. For all these cyclic heptapeptides, the overall backbone conformation is similar to the structures of axinastatins 2 -4, investigated here.…”

“…According to the experimental data, Pro6 forms a y-endo envelope. This conformation which is indicated by the small 3J(H-C(a),H,,,-,-C(P)) (1 Hz) seems to be characteristic for cis-proline (see Table 2, [25] [26]).…”

“…carteri also seems to be accompanied by highly active by-products, such as the sponge halichondrins and halistatins [13]. Val'-)), the first member of this family of cyclic heptapeptides [lo], a very similar backbone conformation is found too [25]. Axinastatins 1-4 have proline residues in common in position two and six, while the residues at the remaining positions differ.…”

What are the structures of phakellistatin 2 and phakellistatin 4?

“…Some recent examples are the synthetic investigations of dolastatin 3 [10], malaysiatin [11], fenestin A and B [12] and konbamid [13]. Our interest in anticancer compounds [11,14,15] led us to check the proposed structures of phakellistatins 2 and 4.…”