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Leu (3), Arg (1) and Three Unusual Amino Acids

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Leu (3), Arg (1) and Three Unusual Amino Acids VOL. XLI NO. 6 THE JOURNAL OF ANTIBIOTICS 719 THE STRUCTURES OF KATANOSINS A AND B Toshiyuki Kato, Hiroshi Hinoo, Yoshihiro Terui, Jun'ko Kikuchi and Jun'ichi Shoji* Shionogi Research Laboratories, Shionogi & Co., Ltd., Fukushima-ku, Osaka 553, Japan (Received for publication December 8, 1987) 2H and 13C NMRstudies on katanosin A confirmed the presence of eight usual amino acid residues which were previously deduced by amino acid analysis and suggested the presence of /3-hydroxyaspartic acid, /3-hydroxyleucine and /3-phenylserine residues. These amino acids were isolated and confirmed, including their stereochemistries, by comparison with the re- spective authentic specimens. Stereochemistries of the usual aminoacids weredetermined by comparing the L-leucylated amino acids with reference compoundsby HPLC.Lithium borohydride reduction and chromic acid oxidation of katanosin A and alkali-treated katanosin A elucidated a lactone linkage between the C-terminal Ser and phenylserine residues. Edman degradation on alkali-treated katanosin Aclarified the total amino acid sequence. The dif- ference in katanosins A and B was determined to be replacement of Val in A by He in B. Thus, the structures of katanosins Aand B were elucidated. Katanosins A (1) and B (2) are basic peptide antibiotics with molecular formulae of A: C57H95N15O17 and B: C58H97N15O17. Amino acid analysis of the acid hydrolysate suggests that the amino acid constitution of 1 is Asp (1), Thr (1), Ser (1), Gly (1), Val (1), Leu (3), Arg (1) and three unusual amino acids (cited as U-l, U-2 and U-3 in the previous paper) and the difference between 1 and 2 is thought to be replacement ofVal in 1 by He in 21}. The XHand 13C NMRstudies on 1 and 2, in which assignment of proton signals were made by 1H-1Hcorrelation spectroscopy (COSY) spectra and spin decoupling experiments and assignment of carbon signals were made by CSCMand selective decoupling experiments (Table 1) clarified the pres- ence of the above eight usual amino acid residues and furthermore suggested the presence of /3-hy- droxyaspartic acid, /3-hydroxyleucine and /3-phenylserine residues, which were considered to cor- respond to the three unusual amino acids (U-l, U-2 and U-3). The signals of quaternary carbons were assigned using 1H-13C long range coupling relations. These unusual amino acids were isolated from the hydrolysates of a complex of 1 and 2 by pre- parative paper chromatography. The isolated amino acids were comparedwith the authentic speci- mensof L-Z/zrefl-^-hydroxyaspartic acid, L-er^/zro-^-hydroxyaspartic acid, D,L-^re0-/3-hydroxyleucine, D,L-er>tf/zr0-/3-hydroxyleucine, D,L-^r£0-/3-phenylserine and DjL-eryf/zro-^-phenylserine by XHNMR, and then measured for their CDspectra. Consequently, U-l was identified with L-^re<?-/3-hydroxy- aspartic acid (HyAsp), U-2 with L-?/zra?-/3-hydroxyleucine (HyLeu) and U-3 with L-threo-P-phenyl- serine (PhSer), respectively. W/*n?o/3-Hydroxyaspartic acid has already been isolated as an antibiotic substance and "L-threo- /3-hydroxyleucine has been isolated from a hydrolysate of an antibiotic2~4). The stereochemistries of the usual amino acid residues were examined by the following method. The amino acid mixture by acid hydrolysis of the antibiotic was L-leucylated and compared by HPLC with respective reference compounds. As a result, D-a/fo-threonine (aThr), L-serine, L-valine, l- 720 THE JOURNAL OF ANTIBIOTICS JUNE 1988 Table 1. ^ and 13C NMRdata for katanosins A and B hydrochloridea. Assignment Hb LUb Leu1 CO 171.2 (s) 171.4 (s) NH3+ 8.32 br 8.28 br a-CH 4.13 brm 51.3(d) 4.ll brm 51.3 (d) /3-CH2 -1.58 m 40.0(t) -1.57 m 40.1 (t) -1.53 m -1.53 m r-CH -1.62m 23.9(d) -1.61 m 24.0 (d) CH3 0.95 d(6.0) 22.0 (q) 0.94 d(6.0) 22.0 (q) CH3 0.91 d(6.0) 22.3 (q) 0.89 d(6.0) 22.3 (q) Leu2 CO 175.2 (s) 175.3 (s) NH 9.60 br 9.57 br 3.78 br a-CH 1.85m(« 55.2 (d) 3.80 br 55.3 (d) /3- CH2 12.8, -3.0) 39.3(t) 1.86 m(-13.2, -3.3) 39.4 (t) 1.33 m(-2.3, -ll.4, 1.33 m(-2.3, -ll.4, -13.8) -13.6) r-CH -1.92 m 23.5(d) 1.95 m 23.6 (d) CH3 0.92 d (6.4) 23.1 (q) 0.93 d(6.4) 23.1 (q) CH3 0.76 d (6.4) 19.5(q) 0.78 d(6.4) 19.6 (q) PhSer CO 171.6 (s) 171.8 (s) NH 8.91 d(8.8) 60.4 (d) 8.93 d(8.5) 5.56 dd(8.5, 10.0) a-CH 5.57 dd(8.8, 9.7) 60.4 (d) /3-CH 6.84 d (9.7) 74.1 (d) 6.80 d(10.0) 74.2 (d) Ph-z 136.2 (s) 136.2 (s) Fh-o 7.41 d-like 127.3 (d) 7.40 d-like 127.3 (d) Ph-m 7.23 t-like 128.0 (d) 7.23 t-like 128.0 (d) Ph-p 7. 19 t-like 128.2 (d) 7.18 t-like 128.3 (d) HyLeu CO 171.8 (s) 171.9 (s) NH 9.05 br 9.05 br a-CH -3.62 m 59.5(d) -3.61 m 59.5 (d) /3-CH 3.43 m 74.6(d) 3.43 m 74.7 (d) /3-OH 5.35 br 5.38 br T-CH 2.35 m 29.9(d) 2.37 m 30.0 (d) CH3 0.98 d (6.4) 19.3 (q) 0.99 d(6.4) 19.3 (q) CH3 0.80 d(6.4) 19.1 (q) 0.81 d(6.4) 19.1 (q) Leu5 CO 172.2 (s) 172.8 (s) NH 7.12 d(6.4) 7.04 d(6.6) a-CH 4.06 m 52.0(d) 4.09 m 51.9 (d) i3-CH2 -1.62 m 40.9(t) -1.59 m 40.5 (t) 1.46 m(-3.3, -10.5, 1.46 m(-2.5, -10.8, -13.8) -12.3) r-CH -1.92 m 23.5 (d) 1.92 m 23.6 (d) CH3 0.79 d (6.4) 20.9 (q) 0.80 d (6.4) 20.7 (q) CH3 0.75 d(6.4) 23.1 (q) 0.75 d(6.4) 23.4 (q) Arg CO 171.9 (s) 171.9 (s) NH 6.89 d(5.6) 6.93 d(5.5) a-CH 3.76 brm 54.6(d) 3.72 brm 54.8 (d) i3-CH2 -1.50 m 27.8 (t) -1.49 m 27.6 (t) -1.60m -1.59m r-CH2 -1.50 m 26.0(t) -1.49 m 26.0 (t) -1.23 m -1.24m 2.83 brm 40.1 (t) 2.83 brm 40.1 (t) 7.27 brt -7.26 brt VOL. XLI NO. 6 THE JOURNAL OF ANTIBIOTICS 721 Table 1. (Continued) Assignment lHb 1Hb 13Ce 13CC NH 6.60 brs 156.7 (s) 6.60 brs 156.7 (s) II c NH3+ Val CO 170.7 (s) NH 7.50 d(9.0) a-CH 3.72 dd(9.0, 10.3) 60.9 (d) /3-CH 2.01 m 29.2 (d) CH3 0.92 d(6.4) 19.0 (q) CH3 0.83 d(6.4) 19.6 (q) He CO 171.0 (s) NH 7.51 d(ll.0) a-CH 3.75 dd(ll.0,8.8) 59.4 (d) /3-CH -1.80m 35.0 (d) £-CH3 0.89 d(6.4) 15.4 (q) r-CH2 -1.05m 25.7 (t) -1.51 m CH3 0.75 t(6.5) 10.1 (q) aThr CO 172.2 (s) 172.3 (s) NH 6.98 d(9.8) 7.01 d(9.7) a-CH 4.39 t(9.8) 57.0 (d) 4.38 t(9.7) 57.1 (d) /3-CH 3.56m 69.6 (d) -3.95 m 69.5 (d) /3-OH 5.23 d(5.6) 5.20 d(5.7) CH3 1.19 d(6.4) 20.6 (q) 1.20 d(6.4) 20.6 (q) Gly CO 168.3 (s) 168.4 (s) NH 9.07 t(5.7) 9.06 t(6.0) a-CH2 4.02 dd(5.7, 16.8) 43.2 (t) 4.00 dd(6.0, 17.0) 43.3 (t) 3.54 dd(5.7, 16.8) 3.55 dd(6.0, 17.0) HyAsn CO 168.9 (s) 169.0 (s) NH 7.54 d(10.0) 7.49 d(10.0) a-CH 4.76 dd(10.0,2.0) 55.4 (d) 4.77 dd(10.0, 1.8) 55.4 (d) /3- CH 4.35 dd(2.0, 5.6) 71.0 (d) 4.36 dd(1.8,5.7) 70.9 (d) ^-OH 5.50 d(5.6) 5.49 d(5.7) £-CONH2 7.14 br 173.0 (s) 7.14 br 173.2 (s) Ser CO 167.4 (s) 167.4 (s) NH 7.01 d(9.8) 6.99 d(-9) a-CH 4.51 dt(9.8,6.3) 55.4 (d) 4.51 dt(9.3,6.5) 55.3 (d) /3- CH2 '3.62m 61.7 (t) -3.61 m 61.7 (t) /3-oh 5.00 t(6.2) 5.00 t(6.4) a The data measured in DMSO-^6at 60°C were listed.
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