Short Communication
`
`[Agr. Biol. Chem., Vol. 33, No. 11, p. 1669~1671, 1969]
`
`Confirmation of the Structure of Surfactin
`by Mass Spectrometry
`
`Sir:
`of peptides. Lederer et al. reported a proce-
`Surfactin is a crystalline peptidelipid sur- dure for the permethylation of the -CONH-
`factant (mp 140°C) isolated from the culture groups in peptides.61 Thomas developed this
`fluids of Bacillus subtilis as a potent clotting
`technique11 and, using the permethylation
`inhibitor in the thrombin-fibrinogen reaction." method described by. Hakomori,81 established
`In the previous reports, the main component a more rapid and complete methylation tcch-
`of the surfactin-composing fatty acids was nique applicable to peptides containing aspartic
`determined to be 3-hydroxy-13-methyl-tetra- or glutamic acid, which gave undesirable
`results under the condition of Lederer's reac-
`decanoic acid (Cu iso /5-OH acid)2'31 and CI3
`and CH /9-OH acids were also detected by gas
`tion. We also have applied the permethylation
`chromatography as minor components.31 The method of Thomas to the alkali-treated de-
`amino acid sequence of the heptapeptide linked
`rivative of surfactin, in which the lactone
`to these fatty acids was determined chemically
`ring present in surfactin has opened, and have
`by the use of Edman's method.4' Thus the obtained an
`interpretable mass spectrum
`total structure of surfactin was established as
`(Fig. 1).
`The permethylated structure of this deriva-
`I.5'5'
`
`CH3 >CH(CH2)9CHCH2CO-Glu-Leu~D-Leu-Val-Asp-D-Leu-Leu
`CH;
`O
`
`I
`
`The present authors describe the confirma­
`tion of the structure of surfactin by mass
`spectrometry.
`Recently mass spectrometry has found prom­
`ising applications in the elucidation of the
`primary structure of oligopeptides. An im­
`portant factor for the success of this technique
`is to prepare satisfactorily volatile derivatives
`
`1) K. Arima, A. Kakinuma and G. Tamura,
`Biochem. Biophys. Res. Commun., 31, 488 (1968).
`2) A. Kakinuma, G. Tamura and K. Arima,
`Experientia, 24, 1120 (1968).
`3) A. Kakinuma, H. Sugino, M. Isono, G. Tamura
`and K. Arima, Agr. Biol. Chem33, 973 (1969).
`4) A. Kakinuma, M. Hori, M. Isono, G. Tamura
`and K. Arima, ibid., 33, 971 (1969).
`5) A. Kakinuma, M. Hori, H. Sugino, I. Yoshida,
`M. Isono, G. Tamura and K. Arima, ibid., 31, 1523
`(1969).
`
`tive is assumed as II.
`In the spectrum of Fig. 1, the molecular
`ion peak (M, mje 1207) is not recognized.
`The fragment peak (M-32, mje 1175) at the
`high mass end is due to the loss of MeOH
`from the fatty acid moiety of II. The next
`peak at mje 1144 is due to the further loss of
`OMe from the C-terminal amino acid, since
`the peak, mje 986 (m/e 1144-MeLeuOMe), is
`not observed. Peaks in the lower mass region
`Each
`form a series of fragment peak groups,
`group shows a characteristic feature consisting
`of five peaks, P* (the most abundant peak,
`
`6) B. C. Das, S. D. Gero and E. Lederer, Biochem.
`Biophys. Res. Commun., 29, 211 (1967).
`7) D. W. Thomas, ibid., 33, 483 (1968).
`8) S. Hakomori, J. Biochem., 55, 205 (1964).
`
`PETITIONERS
`
`EXHIBIT NO. 1022 Page 1 of 3
`
`

`

`670 A. KAKINOMA, A. OUCHIDA, T. SHIMA, H. SUGINO, M. ISONO, G. TAMUKA and K. ARIMA
`
`100 r
`
`50 -
`
`5 6
`c
`.z.
`
`• -
`
`cs
`
`CH3
`>CH(CH2))CH=CHCO
`CHj'
`MeGluOMe
`
`•»-|< MeLeu
`
`•»!« MeLeu
`
`—MeVal—
`
`507*
`
`X10 r
`
`I 1 11 .
`200
`
`1 n
`
`300
`
`100
`
`380*
`
`352 QCC AltflL
`
`400
`
`493
`479
`1
`
`521
`1
`
`539
`
`500
`
`• 6066?0! 648 666
`
`634*
`
`1^,1 >, I.,
`600
`
`700
`
`MeAspOMe
`
`>]<
`
`MeLeu
`
`MeLeu
`
`OMe
`K—H
`
`100 r
`
`I-
`
`• 100
`/~~m*
`
`50 -
`
`761 779
`
`733
`719
`i
`
`890*
`
`904
`
`876
`862
`
`922
`
`1017*
`1003
`. 'I311049
`989
`i* t i *
`1
`1000
`
`1144 1175
`
`1100
`
`1200
`
`700
`
`800
`
`900
`
`m/e
`FIG. 1. Mass Spectrum of the Permethylated Derivative of Alkali-Treated Surfactin.
`The spectrum was determined with a Hitachi RMS-4 mass spectrometer using <• direct inlet
`system. The accelerating voltage was reduced so that the spectrum could be measured up to m/e
`1250.
`Ionizing energy, 70eV; ion source temperature, 200oC; evaporation temperature, 260oC.
`Alkali-treated surfactin was prepared as described previously.51
`CH3 )CH(CH2)9CHCH2CO-MeGlu-MeLeu-MeLeu-MeVal-MeAsp-MeLeu-MeLeuOMe
`CH3
`OMe
`OMe
`OMe
`
`11
`
`narked with an asterisk in Figure), P* + 32) described above may be produced by the
`** + 14, P* —14, and P*—28. The mass dif- sequential cleavage of peptide bonds without
`erence of 127 between m/e 1144 and the
`the elimination of MeOH from II. P"-14
`dghest P* (m/e 1017) corresponds to the loss and p*-28 peaks, also accompanied by weak
`>f an N-methylated leucine (MeLeu) indicat- peaks 32 m.u. higher would mainly be due to
`ng that the C-terminal amino acid in surfactin
`the fragmentation of surfactin homologues
`s leucine. The P peaks at mle 890, 747, 634, having
`and Cn fatty acids, respectively.
`07 and 380 can be regarded to have derived Although peaks (P* + 14) may suggest
`the
`>y the further successive elimination of MeLeu, possibility of the existence of a surfactin
`/TeAspOMe, MeVal, MeLeu and MeLeu homologue with Cn fatty acid, they are in-
`rom m/e 1017, respectively. The remaining
`terpreted as presumably produced from the
`mino acid, Glu, is therefore directly linked dehydrated fragment of II," since the peaks
`0 the fatty acid moiety. Thus, the amino 32 m.u. higher are not apparently observed.
`,cid sequence in surfactin determined chemi-
`ally by Edman's method was confirmed mass
`pectrometrically. A series of peaks (P* + 32)
`
`9) E. Bricas, J. van Heijenoort, M. Barber, W. A.
`Wolstenholme, B. C. Das and E. Lederer, Biochemistry,
`4, 2254 (1965).
`
`PETITIONERS
`
`EXHIBIT NO. 1022 Page 2 of 3
`
`

`

`Confirmation of the Structure of Surfactin by Mass Spectrometry
`
`1671
`
`In the course of this work
`Thomas and
`Ito101 reported the result of a mass spectrometric
`reinvestigation on an antibiotic esperin (mp
`238°C)11, and proposed for it a revised structure,
`which is similar to that of our surfactin.
`
`The authors wish to thank Dr. S. Tatsuoka for his
`interest and Dr. M. Nishikawa for his helpful discus­
`sion.
`
`10) D. W. Thomas and T. Ito, Tetrahedron, 25,
`1985 (1969).
`11) T. Ito and H. Ogawa, Bull. Agr. Chem. Soc.
`Japan, 23, 536 (1959).
`
`Atsushi KAKINUMA
`Akira OUCHIDA
`Takashi SHIM A
`Hiromu SUGINO
`Masao ISONO
`Gakuzo T AMUR A"
`Kei ARIMA*
`Research and Development Division,
`Takeda Chemical Industries, Ltd.,
`Osaka, Japan
`*Department of Agricultural Chemistry,
`The University of Tokyo, Tokyo, Japan
`Received September 16, 1969
`
`PETITIONERS
`
`EXHIBIT NO. 1022 Page 3 of 3
`
`