Mass Spectrometric Peptide Sequencing: Cyclochlorotine Robert J. Anderegg and K. Biemann Department of Chemistry, Massachusetts Institute of Technology, Cambridge, Massachusetts 02139, USA

Awinash Manmade and Anil C. Ghosh SISA, Inc. and Sheehan Institute for Research, Inc., Cambridge, Massachusetts 02138, USA

A potent toxin isolated from Peniciliium islandieum Sopp was found to have the composition Cz4H31N507CI~. Mass spectrometric investigation of a partial acid hydrolyzateshowed that it has the structure of cyclochlorotine. The mass spectral characteristics of polyamino alcohol-related derivatives of peptides containing P-phenylalanine, a-amino butyric acid and dichloroproline were determined in the course of this work.

INTRODUCTION Over the past decade or so, mass spectrometry has been used in a number of laboratories for the determination of the amino acid sequence of small peptides.' These studies, which have involved the entire range from simple model peptides of known structure to complex mixtures derived from proteins of unknown sequence, have been quite successful. A special class are cyclic peptides which differ from those derived from proteins by a number of characteristics. They are usually of limited size, but have no defined N-terminus or C-terminus, which makes them unsuitable for any sequential method that begins on either end (and thus specifically eliminates the Edman technique). This also greatly complicates the mass spectrometric sequencing approaches because transannular bond formation either prior to or after ionization of the molecule, followed by cleavage after electron impact may lead to fragments representing two amino acids which in the original structure were not attached to each other. The mass spectrum of a cyclic peptide is therefore very complex and its interpretation is fraught with potential pitfalls. The logical way of solving this problem is the conversion of the cyclic peptide to a linear one by the cleavage of one of the peptide bonds. If this could be accomplished in a specific manner, a linear oligopeptide would result which then is susceptible to either Edman degradation or derivatization followed by mass spectrometric sequencing. Unfortunately, most cyclic peptides lack unique bonds which can be cleaved specifically by chemical means; and enzymatic reactions are often hampered by the frequent occurrence of Damino acids which are not attacked by natural proteolytic enzymes. Nonspecific cleavage by partial acid hydrolysis may overcome these problems because it is irrelevant which peptide bond is cleaved first: while the intermediate mixture consists of different oligopeptides, they all retain the original sequence information. Further hydrolysis of some of the peptide bonds in each one of

these linear molecules leads to a mixture of small peptides from which the original structure can be reassembled in the same way as is the case with the linear oligopeptides encountered in protein sequencing, except that it is not necessary to identify a specific N- or C-terminus. Recently we demonstrated this principle in the course of the determination of the structure of malformin C , a cyclic pentapeptide consisting of the common amino acids cysteine, valine and leucine.2 The present paper illustrates the use of this technique for a cyclic peptide which contains a number of uncommon amino acids including a chlorine-containing proline. The common storage mold Penicillium islandicum Sopp has been found to contaminate a wide variety of foodstuffs including rice, wheat, soybean, peanuts, flour and ~ o r n . A ~ large - ~ number of toxic as well as nontoxic compounds are produced by this mold. One of the potent toxins is cyclochlorotine C24H31N507C12 (l),a water soluble cyclic pentapeptide (LD50,0.475 mg kg-', subcutaneous injection in mice) isolated from P. islandicum in 1955.8'9 When injected into mice, the toxin produces violent symptoms such as necrosis, vacuolation of liver cells and development of blood lakes.33'" OH

CH 3

I y*

I

CH,

I

NH-CH-CO-NH-CH I

I I

C,tF: co

NH CHI C,H

N

I

CO-CH-NH-CO-CH,

I

I

CH 2

I

OH

LLAbu-L-Ser-L-P-Phe-L-Ser-L-DCPJ Cyclochlorotine

1

CCC-0306-042X/79/0006-0129$03.00 @ Heyden & Son Ltd, 1979

BIOMEDICAL MASS SPECTROMETRY, VOL. 6, NO. 3, 1979 129

R. J. ANDEREGG, K. BIEMANN, A. MANMADE AND A. C. GHOSH CH,

OH

CH,

CH2

I

I

I

NH-CH-CO-NH-CH

I

I

I

co

co

CHI

NH

I

I

C,H,-CH

I CH-CH,-OH I

I

I

NH-CO-CH-N-CO C

I

c1V

Islanditoxin 2

In an independent series of studies, Marumo isolated islanditoxin ( 2 ) from P. islandicum and assigned an isomeric structure to this peptide.' '-13 However, chemical and toxicological investigations of these two toxins have been seriously hampered due to the scarcity of the material available. Although the physical and chemical properties of these two peptides are almost identical, it appears that no direct comparison has ever been made. Recently we have undertaken a systematic study of the microbial production and isolation of cyclochlorotine which indeed resulted in the isolation of a chlorine-containing peptide of melting point 254255 "C (decomp.), whereas the melting points (decomp.) of cyclochlorotine and islanditoxin are reported to be 251 "C and 250-251 "C r e s p e ~ t i v e l y . ~Mass ~ ~ ' spectrometry seemed to be the best method to determine whether this material was 1,2, or a new compound.

EXPERIMENTAL The details of the isolation of cyclochlorotine from Penicillium islandicum are to be published elsewhere. l 4 The peptide diacetate (about 1 mg) was prepared in acetic anhydride + pyridine (1: 1) at room temperature (20"-25 "C) for 1 h. Partial acid hydrolysis on 800 p g of cyclochlorotine was carried out in evacuated, sealed tubes using 6 M HCl (Ultrex) at 105 "C for 20 min. After lyophilization, the hydrolyzate was methylated in 3 M methanolic HCl at 25 "C for 30 min. The excess reagents were removed in vacuo. The residue was trifluoroacetylated by dissolving it in methyl tryhoroacetate+methanol (1 : 1). The p H was adjusted to > 7.5 with triethylamine. After standing overnight at 25°C and removal of the reagents, the TFA-peptide-methyl esters were dissolved in 0.25 M LiAlD4 in glyme at 0 "C. After stirring for several hours in ice, the mixture was slowly brought to 9 0 "C where it was allowed to react for another 12 h. The excess LiAlD4 was quenched by the careful addition of a few drops of methanol; the aluminate salts were precipitated with water and filtered off. The filtrate was evaporated to dryness, extracted with chloroform, and refiltered. The chloroform was evaporated, and the polyamino alcohols were silylated in 100 p l each of pyridine and trimethylsilyl-diethylamine (TMS-DEA). Portions of the 130 BIOMEDICAL MASS SPECTROMETRY, VOL. 6, NO. 3, 1979

silylation mixture were injected directly on the gas chromatographic column. The GCMS system has been thoroughly described e 1 ~ e w h e r e .A l ~Perkin-Elmer Model 990 gas chromatograph with a flame ionization detector has been coupled via a fritted glass Watson-Biemann separator to a Hitachi-Perkin-Elmer RMU-6L mass spectrometer. Ionization is by electron impact at 7 0 e V . The instrument is magnetically scanned every 4.7 s from m / z 28 to m / z 767 under control of an IBM 1800 computer. The data are processed and a microfilm is generated showing the set of successive mass spectral scans and a set of associated 'mass chromatograms'.'6 Retention indices are computer calculated using internal hydrocarbon standards." Samples were chromatographed on a 6 f t glass column (Q in i.d.) packed with 3% OV-17 on Gas-Chrom Q (100/120 mesh). The temperature w;s linearly programmed from 80-330 "C a t 12 "C min- . Helium was the carrier gas at a flow rate of 30 ml min-'. High resolution mass spectra were determined on a Dupont GEC-110B mass spectrometer using an ion source temperature of 250 "C, an ionizing potential of 70 eV, and an ionizing current of 200 p A. Samples were introduced through a vacuum lock directly into the ion source. A photographic plate was used to record the spectra. Field desorption mass spectra were obtained with a Varian MAT 731 mass spectrometer. The emitter was dipped into a solution of the sample and the solvent carefully evaporated; the sample was desorbed using an emitter current of 20-25 p A .

RESULTS Both cyclochlorotine (1) and islanditoxin ( 2 ) contain p-phenylalanine (p-Phe), a-aminobutyric acid (Abu) and dichloroproline (DCP), as well as two moles of serine. If cyclic, the isomers would have a composition C24H31N507C12.The ion of highest mass in the high resolution mass spectrum of the intact peptide was found at m / z 499.1616 corresponding to C24H26N505C1 (calc. 499.1623) which would be compatible with the composition of 1 or 2 if this ion was formed by the loss of 2H20-tHC1 from the molecular ion. This was further corroborated by an ion due to loss of an additional molecule of HC1 (found m / z 463.1867; calc. 463.1856). A low resolution mass spectrum using field desorption to ionize the peptide gave similar results. An ion was observed for [M- HCI]+, but again no molecular ion was present and very little fragmentation occurred. However, when the peptide was acetylated, a protonated molecular ion at m / z 656 corresponding to the peptide diacetate was observed with isotope peaks at m / z 658 and 660 in the expected ratio of 9 : 6 : 1 for two chlorine atoms. Peak matching on m / r 656 was hampered by interference from a marker compound ion; but peak matching on m/z 658 gave an accurate mass measurement of 658.1908 (calc. for :658.1860). C28H3609N~35C137Cl These findings were compatible with either 1 or 2, but in order to distinguish between the two, the sequence of the amino acids had to be determined. Since the structures differ essentially by the position of the p-Phe @ Heyden & Son Ltd, 1979

PEPTIDE SEQUENCING: CYCLOCHLOROTINE

Ser-B-Phe, P-Phe-Se:

1

1

1

'''

Ser-B-Phe-Ser

Table 1. Oligopeptide fragments identified (see Fig. 1) and reassembled sequence of cyclochlorotine Scan

Ser-P-Phe Ser-p-Phe-Ser p-Phe-Ser Ser-DCP-Abu DCP-AbU Ser-P-Phe-Ser-DCP-Abu

65 111 65 59 47

-

survive and must be identified in separate enzymatic hydrolyzates. "-" Peptides which would be characteristic of islanditoxin (2),for example Ser-Ser or p-Phe-Abu, were searched for but none could be found. The mass chromatogram for m/z 252 (base peak in the spectrum of Ser-Ser) is shown in Fig. 3. While peaks are observed for p-Phe-Ser and Ser-/3-Phe-Ser, no corresponding peak appears with the retention index of Ser-Ser. None of the mass spectra exhibited the typical chlorine multiplets; the dichloroproline apparently dehalogenated during the lithium aluminium deuteride reduction, giving rise to two derivatives: (1)a dideuteroproline (3) and (2) a pyrrole analogue of proline (4). Which of these two derivatives was formed may depend on the position of the cleavage of the original peptide during the acid hydrolysis, and the reasons for this difference are outlined in the discussion.

Retention index t t + i i + + + t + + - 4

20

40

1

60

i

i

i

~

i

100

80

++

:

120

++-++++++-ttcci

140

160

180

200

Spectrum index number

Figure 1. Mass resolved gas chromatogram of the O-trimethylsilyl polyarnino alcohols obtained by derivatization of an acid hydrolyzate of cyclochlorotine. The hydrocarbons CZ2and Cs2were co-injected as internal retention index standards.

residue, it would suffice to identify its neighbors, but with our gas chromatographic mass spectrometric technique,'*-*' information about the entire sequence was obtained in a single experiment. The cyclic peptide was hydrolyzed in 6 N aqueous HCI for 20 min at 105 "'C. The resulting hydrolyzate was methylated, trifluoroacetylated, reduced, and silylated as described previously.'' When this mixture of 0TMS-polyamino alcohols was subjected to GCMS analysis, the mass resolved gas chromatogram" shown in Fig. 1 resulted. Five peptide derivatives were identified. The mass spectrum of one of these, Ser-pPhe-Ser, is shown in Fig. 2. This tripeptide could only arise from cyclochlorotine (l),and in fact all of the peptides identified in this partial hydrolyzate could be uniquely reassembled to structure (l), as shown in Table 1. In the case of this small cyclic pentapeptide enough peptide bonds were retained in one or more degradation peptides to reassemble the entire sequence. This is in contrast to partial acid hydrolyzates of the much larger polypeptides encountered in protein sequencing where particularly acid labile peptide bonds often d o not

DHC-CHD I

H,C,

\

N

,cH-cD,-$

HC-CH N

\\

HC\~/CH-CD~--

1

I

CD,

I

CHR

I

_N

3

The data summarized in Table 1 demonstrate that the sample isolated in the course of this work was in fact cyclochlorotine (1) and not islanditoxin (2).

OTMS I Cf,-

.CD2

100

-

. OTMS

200

300

400

500

600

m/z

Figure 2. Mass spectrum corresponding to the derivative of Ser-p-Phe-Ser from cyclochlorotine.

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BIOMEDICAL MASS SPECTROMETRY, VOL. 6, NO. 3, 1979

131

R. J. ANDEREGG, K. BIEMANN, A. MANMADE AND A. C. GHOSH

I\ 9

I P

+ 50

100

150

200

Figure 3. Mass chromatograin of m/z 252 from the GCMS experiment depicted in Fig. 2-1.

DISCUSSION The field desorption mass spectra were useful for the determination of the molecular formula and demonstrated the presence of two chlorine atoms, but gave no sequence information. The electron ionization mass spectra were also of little help because in general the spectra of cyclic peptides are difficult to interpret unambiguously. In the ion source of the mass spectrometer, the cyclic peptide can open into a linear peptide by cleaving one of several bonds. All of the possible linear sequences formed can fragment independently leading to a series of superimposed spectra. In addition, rearrangements are possible where a fragment of the cyclic structure is ejected without opening the ring.23The results of most sequencing attempts on single mass spectra of cyclic peptides have been far from convincing. In at least one case, the same mass spectral data was used to support two different struct u r e ~ . Only ~ ~ , by ~ ~careful comparison with synthetic samples can conclusive evidence be obtained.26 Our studies on cyclochlorotine are an example of the use of mass spectrometry not only as a primary sequencing tool, but also as a fast and effective method for distinguishing sequence-isomeric peptide structures. Furthermore, they show that the presence of unusual amino acids is no problem, and in fact provides for an interesting extension of the rules for sequence ion determination which have been developed previously. According to the conventions proposed earlier,19 sequence ions originating from the amino-terminus of the peptide are designated A l , Az, A 3 , .. . ; and those originating from the carboxyl-terminus are labeled z 1 , z,, .... p-Phenylalanine, the presence of which places an additional methylene unit in the backbone of the peptide, gives rise to a doublet separated by 14 amu in the mass spectra while a-Phe would result in a single sequence ion. For example, in Fig. 2 the AZ ion for Ser-p-Phe-Ser appears as a doublet at m/z 337 and m / z 351. In contrast, the spectrum of Ser-a-Phe-Ser would show only a single peak at m / z 351. a-Aminobutyric acid presents no particular difficulty and behaves as a regular amino acid with a sidechain mass of 29 amu. The dichloroproline (DCP) data are especially interesting. If one assumes that the chlorine atoms remain on 132 BIOMEDICAL MASS SPECTROMETRY, VOL. 6, NO. 3, 1979

the proline throughout the acid hydrolysis, the multiplicity of derivatives must be produced at a later step, presumably during the reduction. If the peptide bond involving the amino portion of DCP was cleaved in the acid hydrolysis to produce a peptide DCP-X, the pyrrolidine nitrogen is then trifluoroacetylated and, during the reduction, eliminates two molecules of HCl to form the pyrrole analogue of Pro (4). However, if the acid hydrolyzed the original cyclochlorotine to give a peptide of the type X-DCP-Y, the chlorine atoms are replaced (via a nucleophilic substitution) during the reduction, rather than eliminated, to give a dideutero-Prc (3).The difference appears to be caused by the electron-withdrawing trifluoroacetyl group which makes the hydrogens on the proline ring nearest the nitrogen more acidic. The hydrogen at C-2 is now sufficiently acidic to be abstracted by deuteride ion, followed by loss of chloride ion. The resulting 2,3-double bond makes C-4 an allylic carbon, thus facilitating the loss of a chloride ion from that position. Loss of a proton from C-5 is then favored, producing the stable pyrrole moiety. On the other hand, trifluoroacetylation of X-DCP-Y leads to a derivative in which the TFA group is too far away to affect the acidity of the pyrrolidine hydrogens, and therefore substitution of the chlorine atoms predominates. Scheme i summarizes the proposed

co I

CHR I

CHR I

Scheme 1. Proposed reactions of dichloroproline-containing peptides during LAD reduction.

reactions and Fig. 4 shows examples of the mass spectra of the two derivatives. In the pyrrole analog [Figure 4(a)], the cleavage which normally gives rise to the A l ion is suppressed because it requires scission of a bond attached to an aromatic system. Instead, cleavage occurs at the bond which is allylic to the pyrrole ring, resulting in an A l + 16 ion. For the same reason, the Z1ion is of very low abundance. In the course of our work with these peptide derivatives, it has been found that their Kovats retention indices are predictable and can be used as an aid to @ Heyden & Son Ltd, 1979

PEPTIDE SEQUENCING: CYCLOCHLOROTINE

Table 2. Sequence ions and retention increments for the unusual amino acids from cyclochlorotine

I

I

A1t16

t

A2

Amino acid IM-151'

200

300

,.

-,-"-

z1

I149+Rlb

AN

+

Z,

I 2

A1

I1 13 + Rlb

Abu 29 142 178 375 p-Phe 240 910 77 190,204 DCP-3 43 156 192 495 -a DCP-4 37 186 935 'A, is of low abundance. A, 16 at m/z 166 is characteristic.

i

100

R

I

2.

bl 13= CF$ZDZ--NH-CH-

u)

al

I

t

149 = -CHCDzO--Si(CH,),

-:

I

0 a!

A,: the sequence ion which appears if the residue occurs at the N-terminus of the peptide. Z,:the sequence ion which appears if the residue occurs at the C-terminus of the peptide. ARI: determined empirically.

i

100

300

xx)

500

400

m/z

Figure 4. Mass spectra corresponding to the derivatives of (a) DCP-Abu and (b) Ser-DCP-Abu.

peptide identification. 19,20 Each derivatized amino acid residue has a characteristic retention index increment (which has been determined experimentally for all the common amino acids). The retention index of a peptide derivative then can be calculated by summing the increments of each of the constituent amino acids (regardless of order) and adding a contribution from the oligopeptide backbone. For example, the peptide Ala-Asp-Pro has a calculated retention index of RI = ABackbone + AAla + AAsp APro = 6 3 0 + 3 3 5 + 6 4 0 + 4 9 5 = 2100. The observed retention index is 2080. Although there were only a few peptides produced upon partial hydrolysis of cyclochlorotine, approximate retention index data were calculated for the unusual amino acids or new derivatives. The dipeptide Ser-pPhe had a retention index of 2100. Subtracting 630 and 540 (the contributions of the backbone and of Ser, respectively) leaves a retention index increment of 930 for P-Phe. A similar calculation for the peptide Ser-pPhe-Ser gives: 2600 - (630 + 540 + 540) = 890 for the p-Phe in this case. Averaging these two values results in an approximate ARI of 910 for P-Phe compared with 960 for cu-Phe.20 The dideutero-Pro derivative was assumed to have a ARI identical (within experimental error) to normal Pro, or 495. Using this value and the retention index of the derivative resulting from the tripeptide Ser-DCP-Abu

+

(i.e. the derivative corresponding to Ser-2H2-Pro-Abu) one calculates: 2040 - (630 + 540 + 49.5) = 375 for the experimental ARI of Abu. This is about what one would predict (380) for the homolog falling between Ala (ARI 335) and Val (ARI 425), and thus demonstrates that ARI values of new amino acids can be estimated from known related ones. Finally, using the calculated value of 375 for Abu and the retention index of DCP-Abu (pyrrole derivative of DCP), one obtains: 1940 - (630 + 375) = 935 for DCP. It is realized that all of these values are based o n a very small sample, but the approximate retention increments may be useful should these amino acids appear in future work. Table 2 summarizes the sequence ions and retention index data for those amino acids. In conclusion, these results demonstrate convincingly that the compound isolated in this study was 1 rather than 2.The consistent assignment of the cyclochlorotine structure to cyclic chlorine-containing \pentapeptides from Penicillium islandicum by several different approaches (e.g. conventional peptide sequencing, Xray diffraction and now GCMS) raises some doubt about the original structure assignment of islanditoxin (2).It seems that further direct comparison of the two compounds or a redetermination of the sequence of 2 would have to be undertaken to settle the question whether islanditoxin is a unique compound or is perhaps identical with cyclochlorotine.

Acknowledgments The work at the Massachusetts Institute of Technology was supported bytheNationa1 Instituteof Health(ResearchGrant No.GM 05472and RR 00317). The work at SISA, Incorporated and Sheehan institute for Research, Incorporated was supported by FDA Contract No. 223-742209.

REFERENCES 1. For a review see: K. Biemann, in Biochemical Applications of Mass Spectrometry, ed. by G. R. Waller, p. 405, Wiley-lnterscience, New York (1972) and supplement in press.

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2. R. J. Anderegg, K. Biemann, G. Buchi and M. Cushman, J. Am. Chem. SOC.98,3365 (1976). 3. M. Saito, M. Enomoto and T. Tatsuno, in Microbial Toxins, Vol. VI, p. 299. Academic Press, New York (1971).

BIOMEDICAL MASS SPECTROMETRY, VOL. 6, NO. 3, 1979 133

R. J. ANDEREGG, K. BIEMANN, A. MANMADE AND A. C. GHOSH 4. A. C. Ghosh, A. Manmade and A. L. Demain, in Mycotoxin, Pathotox Publishers, Inc., Park Forest South, Illinois (in press). 5. A. C. Ghosh, A. Manmade, A. Bousquet, J. M. Townsend and A. L. Demain, Paper Presented at 173rd American Chemical Society National Meeting, New Orleans (1977), Abstract ANAL-061. 6. A. C. Ghosh, A. Manmade, B. Kobbe, J. M. Townsend and A. L. Demain, Appl. Environ. Microbiol. 35, 563 (1978). 7. A. E. Pohland and P. Mislivic, in Mycotoxinsand OtherFungal Related food Problems, p. 10. Advances in Chemistry Series, American Chemical Society, Washington, DC (1976). 8. T. Tatsuno, M. Tsukioka, Y. Suzuki and Y . Asami, Chem. Pharm. Bull. 3, 476 (1955). 9. H. K. Yoshioka, M. Nakatsu, M. Sat0 and T. Tatsuno, Chem. Lett 1319 (1973). 10. K. Uraguchi, M. Saito, Y. Noguchi, K. Takahashi, M. Enomoto and T. Tatsuno, Fd. Cosmet. Toxicol. 10, 193 (1972). 11. S. Marumo, Bull. Agric. Chem. SOC.Jpn 19,258 (1955). 12. S. Marumo, Bull. Agric. Chem. SOC.Jpn 23,428 (1959). 13. S. Marumo, K. Miyao, A. Matsuyama and Y. Sumiki, J. Agric. Chern. SOC.Jpn 23,305 (1955). 14. A. C. Ghosh,A. Manmade, J. M.Townsend,A. Bousquet, J. F. Howes and A. L. Demain, Appl. Environ. Microbiol. 35, 1074 (1978).

134 BIOMEDICAL MASS SPECTROMETRY, VOL. 6, NO. 3, 1979

15. 16. 17. 18. 19. 20. 21. 22. 23. 24. 25. 26.

R. A. Hites and K. Biemann, Anal. Chem. 40, 1217 (1968). R. A. Hites and K. Biemann, Anal. Chem. 42,855 (1970). H. Nau and K. Biemann, Anal. Chern. 46,426 (1974). J. A. Kelley, H. Nau, H.-J. Forster and K. Biemann, Biomed. Mass Spectrom. 2,313 (1975). H. Nau, H.-J. Forster, J. A. Kelley and K. Biemann, Biomed. Mass Spectrom. 2,326 (1975). H. Nau and K. Biemann, Anal. Biochern. 73, 139 (1976). H. Nau and K. Biemann, Anal. Biochem. 73, 154 (1976). J. E. Biller and K. Biemann, Anal. Lett. 7,515 (1974). B. J. Millard, Tetrahedron Lett 34,3041 (1965). M. Koncewicz, P. Mathiaparanam,T. F. Uchytil, L. Sparapano, J. Tam, D. H. Rich and R. D. Durbin, Biochem. Biophys. Res. Comrnun. 53, 653 (1973). W. L. Meyer, L. F. Kuyper, R. B. Lewis, G. E. Templeton and S. H. Woodhead, Biochem. Biophys. Res. Commun. 56, 234 (1974). M.Bodanszky, J. B. Henes, S. Natarajan, G. L. Stahl and R. L. Foltz, J. Antibiot. 29, 549 (1976).

Received 1 December 1978

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1979

@ Heyden & Son Ltd, 1979