Study of an actinomycin complex by mass spectrometry-mass spectrometry

Talanta, Vol. 35, No. 8, pp. 605-611, 1988 Printed in Great Britain. All rights reserved

0039-9140/88 $3.00+0.00 Copyright © 1988PergamonPress pie

STUDY OF AN ACTINOMYCIN COMPLEX BY MASS SPECTROMETRY-MASS SPECTROMETRY M. BARBER, D. BELL, M. MORRIS, L. TETLER* and M. WOODS Department of Chemistry, UMIST, Manchester, England B. W. BYCROFT Department of Pharmacy, University of Nottingham, England J. J. MONAGHANand W. E. MORDEN ICI C&P Group, Runcorn Heath, Cheshire, England B. N. GREEN VG Analytical, Floats Road, Wytbenshawe, England

(Received 26 January 1988. Accepted 12 April 1988) Summary--The characterization of components within actinomycin complexes may often be complicated by the lack of material and standards of known actinomycins. Mass spectrometry-mass spectrometry can be employed both as a separatory device and as a means of structural analysis. This technique has been applied to an actinomycin complex obtained from a previously unidentified Streptomyces strain. The method involved initial work on a known material, in this case actinomycin D, and application to the unknown material. Three major components within the unknown complex were characterized as actinomycins D, Fs and Fg.

Some years ago, in a study related to the biosynthesis of actinomycin D, actinomycin complexes from two unidentified Streptomyces strains were isolated. Although it was possible to identify actinomycin D as a major component in both mixtures by comparative chromatography, the identification of the other compounds in the mixtures was not possible because of lack of material and of the various standards of the known actinomycins. Comprehensive reviews of the structural variations and methods employed for identification have been published. 13 It is well established that the actinomycins are produced as mixtures of closely related compounds differing only in the amino-acid residues at the sites indicated in Fig. 1. The most useful techniques that have been employed to identify unknown actinomycins by comparison with known samples are papeP and thin-layers chromatography. In addition, amino-acid analysis6 of the hydrolysates of the separated components, as well as gas chromatographic analysis7 of the dioxapiperazines formed on thermolysis, has been employed to identify the various amino-acid residues. Until recently, mass spectrometry had not figured significantly as a tool for the characterization of these molecules, because of their involatility and thermal instability. We have recently utilized fast atom bombardment (FAB) mass spectrometry-mass

spectrometry (MS/MS) to characterize previously unidentified components of the Tyrothricin complex, s We now report our initial findings on the application of this method to one of the actinomycin complexes.

CH(CH3)2 CH(CHa)2 COm CH

O

HaCCH~

CHACO

NMe

NMe

A

X

B

Y

C

Z

CO

CO

CH

CH~CHCH3

NH

NH

CO

CO

O

OI "~H "~O Fig. I. Structural variations in the actinomycin family. A, X = site 4, B, Y = site 3, C, Z = site 2.

*Author to whom correspondence should be addressed. 605

M. BARBERet al.

606

amino-acid residues. The relevant fragmentation can be interpreted as due to initial opening of one of the MS/MS data were acquired with two instruments, namely a VG Analytical ZAB-4F and a VG Analytical ZAB--HSQ. lactone linkages at the bond between the bridging The geometries of these instruments have been described oxygen atom and the threonine side-chain, followed elsewhere?.t° by sequential loss of amino-acid residues by cleavage With both instruments, the parent ion of interest was preselected by using the magnetic and electric field of the at the appropriate peptide bonds. Towards lower masses, fragments derived by first instrument (MS I). Collisional activation (CA) with inert gas atoms to enhance fragmentation was then under- the breakdown of both peptide rings are apparent, taken, the resulting daughter ions being subjected to providing information concerning the amino-acid mass analysis by the second instrument (MS II). The ZAB-4F provides daughter ions arising from high (8 keV) sequence and assisting in the identification of the energy collisions, and the ZAB-HSQ, those from low (up to subsequent amino-acid replacements for the other 500 eV) energy collisions. components of the actinomycin complex. These fragStandard FAB mass spectra were obtained with a VG mentations are listed in Table 1. updated MS902 mass spectrometer. It appears that there is no discrimination as to Samples were prepared by dissolution of 1-2/~g of the solid in 3-5 /~1 of a suitable liquid matrix directly on the which of the two lactone bonds is opened first. probe tip of the sample-introduction system. The liquid Furthermore, no significant fragmentation of the matrices employed were thioglycerol, m-nitrobenzyl alcohol chromophore is observed. and a 2:1 v/v glycerol and thioglycerol mixture containing When it had b,'en established that this method 1% v/v trifluoroacetic acid. afforded not only molecular weight information, but The samples were ionized by FAB with xenon atoms at also data that are structurally significant within a discharge current of 1 mA and a potential of 8 kV. Actinomycin D was isolated as described by Roussos and a defined mass range for the standard compound, Vining,H from cultures of Streptomyces parvullus (ATCC, the technique was applied to a total actinomycin 12434). complex. The two unidentified Streptomyces strains, isolated from The molecular ion region of the actinomycin "SA" soil and provided by the Boots Company, Nottingham, England, were grown on the same medium in rapidly shaken complex is depicted in Fig. 3, and reveals three major submerged culture at 27°. After four days of growth, the components [as (M + H) ÷ ] at m/z 1255, 1229 and mycelium was filtered off and the filtrate repeatedly ex- 1203. Species of lower intensity, interpreted as the tracted with ethyl acetate. Removal of the solvent afforded a red solid which was further purified by dissolution in (M + N a ) ÷ ions from these components, can be ethyl acetate-methanol (9:1 v/v) and filtration through a observed at m/z 1277, 1251 and 1225. Spectra obshort column of silica; evaporation of the effluent gave the tained from specimens in m-nitrobenzyl alcohol and actinomycin complexes. Paper chromatography (Whatman other matrices indicate the presence of minor comNo. 3 paper, ethyl acetate as mobile phase) revealed several ponents of low intensity at m/z 1189, 1215, 1243 and components, including actinomycin D, which was identified 1269. by comparison with an authentic sample. In application of MS/MS to this mixture, the first mass spectrometer allows passage of one ionic species, thus effectively acting as a separatory device. RESULTS AND DISCUSSION This is then followed by collisional activation, and To evaluate the structural information available subsequent analysis of the resultant daughter ions. in the MS/CA/MS spectra, it was decided to use The MS/MS spectrum of (M + H) ÷ at m/z 1255 was actinomycin D as a standard compound I of well identical to that obtained from actinomycin D. Both high and low collision-energy daughter-ion established structure. The FAB mass spectrum of actinomycin D is spectra were obtained for the ions occurring at m/z characterized by an intense line for the protonated 1229 and 1203. These are shown in Figs. 4 and 5. molecular ion (M + H ) ÷, observed at m/z 1255, Again, the MS/MS spectra recorded for high and low together with limited fragment ions leading to in- energy collisions are complementary, with the highcomplete sequence information concerning the mass ions being more pronounced at high collision identity of the peptide rings. energy (8 keV). Interpretations of the spectra are The application of CA, in conjunction with given in Figs. 4 and 5, and Table 2. The spectra reveal MS/MS, leads to enhancement of those peaks which are of importance in characterizing the structure. Table 1 Furthermore, the ratio of these analytical signals to m /z Interpretation the ubiquitous background associated with FAB is considerably enhanced. 956 Loss of (H-Pro-Sar-MeVal--OH) 857 Loss of (H-Val-Pro-Sar-MeVal-OH) The daughter ion spectra obtained from the pro657 Lossof (H-Pro-Sar-MeVal-OH) from both rings tonated molecular ion with the ZAB-4F and ZAB558 Loss of (H-Pro-Sar-MeVal-OH) from one ring HSQ are shown in Fig. 2. These prove to be comand (H-Val-Pro-Sar-MeVaI-OH) from the other plementary in that the high-mass ions are more 459 Loss of (H-Val-Pro-Sar-MeVal-OH) from both pronounced when high-energy collisions are used. rings 399 (H-Val-Pro-Sar-MeVal-OH+ H) ÷ These ions clearly indicate the fragmentation of the 300 (H-Pro-Sar-MeVal-OH + H) + depsipeptide ring, and allow sequencing of the EXPERIMENTAL

Study of an actinomycin complex

607

(a) 100

857

(k~-H)+ --Val

~ l

986

Pro--l--hr --I~HMeV,IOH

1285

1240 1212 928 i

I 1

, ,1 . go0

800

1124 1141

,

10()0

m/z

1100

1200

13(~o

100,

(M+H)'*"

(b)

1285

90'

80' 70,

60' 50 ¸

40,

30'

20.

300

887

399 /

888

10'

m/z

Fig. 2. (a) Daughter-ion spectra of m/z 1255 obtained at high energy (8 keV) collision with He; (b) daughter-ion spectra of m/z 1255 obtained at low-energy (59 eV) collision with Ar. the replacement of one or both proline residues by a group with 'molecular weight' 71 (presumably sarcosine, since alanine has not been observed as a replacement at this site). This would identify these components as actinomycins F 9 and Fs, respectively. To date, the minor components have only been characterized by molecular weight. Although, at present, the concentration of these actinomycins is low, proving problematic for MS/MS, chromatographic enrichment is currently under study. However, in view of the known site replacements within

the actinomycins, it is possible to tentatively assign the structures shown in Table 3. CONCLUSIONS

It has been shown that FAB ionization can produce high-quality molecular weight information from microgram quantities of the actinomycins. The fragmentation, however, is sparse, and this combined with the multicomponcnt nature of the sample material makes interpretation impracticable. By use

608

M. BARBER et al. 1229

1203

1251 1225

1255

1269 1277

m/z

Fig. 3. Molecular-ion region of actinomycin 'SA' complex obtained by FAB-MS, with m-nitrobenzyl alcohol as matrix.

of MS/MS techniques, the molecular ions of the individual components of complex mixtures may be selected from the high-mass region of the total spectrum for the complex. These selected ions can then be subjected to collisional activation, enhancing the fragmentation, to give sequence information about the depsipeptide rings. This technique has been successfully applied to one actinomycin complex, resulting in facile characterization of the major com-

m/z

ponents. It has also provided important information concerning the amino-acid composition of the minor components, which needs to be confirmed by additional information from amino-acid analysis and a further combination of MS/MS and degradative studies (to be described later). The potential of this method for monitoring biosynthesis, and the production of semi-synthetic actinomycins by controlled biosynthesis is currently under investigation.

Table 2 Interpretation

(a) Parent ion m/z 1229 956 Loss of (H-Sar-Sar-MeVal-OH) 930 Loss of (H-Pro-Sar-MeVal-OH) 857 Loss of (H-VaI-Sar-Sar-MeVaI-OH) 831 Loss of (H-Val-Pro-Sar-MeVaI-OH) 657 "~ 558 ~- Cleavages from both rings to give same products 459 J as actinomycin D 399 as Table 1 373 (H-Val-Sar-Sar-MeVaI-OH + H) + 300 as Table 1 274 (H-Sar-Sar-MeVaI-OH + H) + (b) Parent ion m/z 1203 930 Loss of (H-Sar-Sar-MeVal-OH) 831 Loss of (H-Val-Sar-Sar-MeVaI-OH) The following ions offer the same interpretation as above: m/z 657, 558, 459, 373 and 274

Study of an actinomycin complex

609

(a) 857

[

Val--[~Sar--I--8at--I--HMeValOH 1214

(M÷H)* 1229

1186 986 (D

I - - -- Pro ~

631--1--Val--

LI

.o

I

1027

I 800

900

t

1098

. _llL,,

10C)0

1100

1200

1300

m/z

(b) 100

1188 831

(M'H)* 1203

v"| ~ l - -

HMeValOH --

S"r--I--S,,r--I

1160

930

!

800

900

1000

m/z

1100

1200

1300

Fig. 4. (a) Daughter-ion spectra of m/z 1229 obtained after high-energy (8 keY) collision with He; (b) daughter-ion spectra of m/z 1203 obtained after high-energy (8 keV) collision with He. Table 3 MR

Trivial name

A

B

C

X

Y

Z

Actinomycin "SA" complex--major components 1254 1228

Actinomycin D, C2 Actinomycin F9 Actinomycin F 8

Sar Sar Sar

Pro Pro Sar

Val Val Val

Sar Sar Sar

Pro Sar Sar

Val Val Val

Actinomycin C 2 and/or Actinomycin iso-C 2 Actinomycin F 2

Sar

Pro

Val

Sat

Pro

aIle

Sar Sar Sar Sar Gly

Pro Pro Pro Sat Sar

aIle Val Val Val Val

Sat Sar Gly Gly Gly

Pro Sar Sat Sar Sar

Val aIle Val Val Val

1202 Actinomycin 'SA' complex--minor components 1268

1242

TAL. 35/~-B

1214

--

1188

--

1174

--

M.B.~d~BER et al.

610 100 (a)

(M,H)*

90

1229

80 70

857

60 50 40 30 831

20

300

459

986

556

930

10

m/z

100 ( b )

lM,m÷

90

1203

80

831

70

60 50 40 930

30 373

2O 10

274

459

657

m/z Fig. 5. (a) Daughter-ion spectra of m/z 1229 obtained after low-energy (41 eV) collision with Ar; (b) daughter-ion spectra of m/z 1203 obtained after low-energy (41 eV) collision with Ar.

Acknowledgements--M.M. and M.W. would like to thank SERC for financial support and D.B. to thank ICI for funding. Thanks are also due to S. Evans for assistance with the data analysis.

REFERENCES

1. A. B. Mauger, The Actinomycins, Topics In Antibiotic Chemistry, Vol. 5, P. Sammes (ed.), pp. 225-257. Horwood, Chichester, 1980.

2. W. A. Remers, Actinomycin, Chemistry of Antitumour Antibodies, Vol. 1, Wiley, New York, 1979. 3. V. Hollstein, Chem. Rev., 1980, 74, 625. 4. K. Zepf, Experimentia, 1958, 14, 207. 5. E. Katz, W. K. Williams, K. T. Mason and A. B. Mauger, Antimicrob. Agents Chemother, 1977, 11, 1056. 6. A. B. Mauger and E. Katz, Antibiotics, Isolation, Separation and Purification, M. J. Weinstein and G. H. Wagman (eds.), Elsevier, New York, 1978. 7. A. B. Mauger, J. Chem. Soc. Perkin Trans. I, 1975, 1320.

Study of an actinomycin complex 8. M. Barber, L. Tetler, D. Bell, J. J. Monaghan, W. E. Morden, B. Bycroft and B. N. Green, Presented at the 35th A S M S Conference on Mass Spectrometry and Allied Topics, Denver, Colorado, 24-29 May 1987. 9. J. R. Hass, B. N. Green, R. H. Bateman and P. A. Bott, Presented at the 32nd Annual Conference on Mass

611

Spectrometry and Allied Topics, San Antonio, Texas, 27 May-I June 1984. I0. A. E. Harrison, R. S. Mercer, E. J. Reiner, A. B. Young R. K. Boyd, R. E. March and C. J. Porter, Int. J. Mass Spectrom. Ion Phys., 1986, 74, 213. 1I. G. G. Roussos and L. C. Vining, J. Chem. Soc., 1956, 2469.