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Hydrolysis of cyclosporin A: Identification of 1,11 seco-cyclosporin A and 4,5 secoisoCyclosporin A by FAB-MSMS
w
Peotides. Vol. 16. No. 8. DD. 133551341. 1995 dopyright 0 1995’Ikevier Science Inc. Printed in the USA. All rights reserved Ol96-9781/9<$9.50 + .IJO
1 Pergamon 0196-9781(95)02025-R
Hydrolysis of Cyclosporin A: Identification of 1,ll seco-Cyclosporin A and 4,5 secoisoCyclosporin A by FAB-MS/MS FULVIO MAGNI,*
LOLITA ARNOLDI,*
MARINA
DEL PUPPOt AND MARZIA
GALL1 KJENLEt’
*Scienti$c Institute H. San Raffaele, Via Olgettina 60, 20132 Milan, Italy and iDepartment of Medical Chemistry and Biochemistry, University of Milan, Via Saldini SO, 20133 Milan, Italy Received
4 April 1995
MAGNI, F., L. ARNOLDI, M. DEL PUPPO AND M. GALL1 KIENLE. Hydrolysis ofcyclosporin A: Idenfijcurion of I,11 secocyclosporin A and 4.5 seco-isocyclosporin A by FAB-MS/MS. PEPTIDES 16(S) 1335% 1341,1995.-We have previously reported that treatment of CsA with aqueous HCI gives rise to the formation of a number of water-soluble compounds. Two of these were identified from their FAB-MSNS spectra as open-chain nona- and decapeptides. We describe here the identification of two other main compounds deriving from the same treatment. Identification was rendered possible from the comparison of their FAB-MS/ MS spectra with tho,se of methyl and acetyl derivatives. The two compounds are water-soluble, open-chain undecapeptides corresponding to I.1 1 seco-CsA and of 45 seco-isoCsA, respectively. Cyclosporin A Cyclic peptides
Acidic hydrolysis
Mass spectrometry
A (CsA) is the well-known cyclic undecapeptide with immunosuppressant activity (7). CsA metabolism has been thoroughly investigated in vitro and in vivo (8). Some work has also been carried out to clarify the behavior of CsA in aqueous acidic media (4) to understand whether degradation of the peptide chain occurs at the igastric level and to clarify the kinetics of the acid-catalyzed degradation (10). Only isomerization of CsA has been reported to occur, with formation of the isomer named isocyclosporin A (isoCsA), which was previously shown to be formed by acidic treatment of CsA in organic solvents via N,O-acyl migration (11). In. a previous work on CsA degradation in acidic aqueous medium, in addition to isoCsA we identified from their FAB-MS/MS spectra two open-chain peptides maintaining the amino acid sequences unmodified with respect to that of CsA for the I- 10 and l-9 residues, respectively (9). Two other main compounds were shown to be formed but their structures were not predictable from the FAB-MS/MS spectra. We report identification of the structures of these two compounds from FAB-MS/MS spectra of the parent compounds and of their methyl and acetyl derivatives. They are open-chain undecapeptides deriving from the cleavage of the 1- 11 peptide bond of CsA and of the 4-5 peptidle bond of isoCsA.
Tandem mass spectrometry
(Milan, Italy). Other reagents were of analytical grade. Solvents for HPLC were used after being degassed and filtered using 0.22pm filters.
CYCLOSPORIN
METHOD
Materials
CsA was a generous gift of Dr. G. Corbetta (Sandoz S.p.A., Milan, Italy). 3-Nitrobenzyl alcohol was from Aldrich Chimica
seco-Cyclosporins
HPLC Analysis
A Jasco liquid chromatograph system (Jasco International Co. Ltd., Tokyo, Japan) equipped with a Jasco pump 880 PU connected with a mixing module 880-02 and with an 875-W detector was used. Peptide separation was carried out with an acetonitrile:trifluoroacetic acid (TFA) gradient slightly modified with respect to that previously described (9). A Beckman ODS Ultrasphere column either 150 X 4.6 mm (5 pm) or 150 X 10 mm (5 pm) was used for analytical and semipreparative purposes, respectively. Peptides were eluted with acetonitrile:0.057% TFA (32:68, v/v) (A) and acetonitrile:0.052% TFA (80:20, v/v) (B), performing a linear gradient of B from 0% to 100% in 70 min (9). The flow rate with the analytical column was 0.5 ml/mitt, and the UV detector was set at 210 nm. For the separation of the compounds from each other the semipreparative column was used (four injections of 150 ~1 each) and elution was carried out at 2 ml/n-& flow rate. Fractions at retention time of isoCsA and of the two unknown peptides (A and B in Fig. 1) were collected and concentrated to dryness. Each fraction was then further purified by HPLC for complete separation from impurities. Each collected peak was analyzed again by HPLC to check its purity and by FAB-MS for structure identification.
’ Requests for reprints should be addressed to Marzia Galli Kienle. 1335
1336
MAGNI ET AL.
FAB-MS Analysis
To obtain information on the molecular weight of the unknown peptides, spectra by fast ion bombardment (FAB-MS) were recorded by using a Finnigan MAT95 double-focusing mass spectrometer equipped with a cesium gun, recording positive ions and operating at an ion accelerating voltage of 5 kV. The cesium ions were accelerated through 22 kV. Spectra were recorded from mlz 100 to m/z 1500 at 5- 10 s/decade. Calibration of the instrument was checked daily with a saturated solution of CsI in glycerol. The instrument operated at a resolution of 1300 (10% valley). 3-Nitrobenzyl alcohol was used as matrix as previously reported (9). Dried fractions purified by HPLC were dissolved in-methanol to a final concentration of 1- 10 pg/pl and l~1 aliquots were analyzed. Additional structural information was obtained recording collision-induced dissociation spectra (CID spectra) of the protonated molecular ion [M+H]’ of the peptides and of their derivatives by introducing helium as collision gas into the first field free region. The collision gas pressure in the collision cell was adjusted to attenuate the primary ion beam by 70%. The CID spectra were obtained using the field-controlled B/E linked scan in profile mode at 10 s/decade. Identification of daughter ions was carried out allowing a mass tolerance error of f 1.5 amu around the expected value.
water-soluble compounds are formed (9). Previously reported (9) retention times of the various compounds differ from those reported in Fig. 1 because for the present work the gradient was modified in an attempt to decrease the run time without decrease of the chromatographic resolution. Moreover, different from the previous work, extraction with diethyl ether before HPLC to separate unreacted CsA and part of isoCsA was not carried out. Peaks corresponding to IsoCsA, and to the two peaks at rt 40 and 28 min (Fig. 1, upper panel, compounds A and B) were isolated and purified by HPLC. Preparative HPLC allowed to obtain fractions containing single peaks (Fig. 1, lower panel). Compound A was recovered mainly unaltered (79%) after treatment with 3 M HCl under the conditions described in the Method section. Nevertheless, HPLC showed the presence of peaks at rt 26 and 36 min corresponding to those of nona- (5%) and decapeptides (S%), respectively. FAB-MS of these peaks showed ions at m/z 1108 and 98 1, thus confirming that compound A gives rise to the already identified peptides.
HYDROLYSED CsA. *B
CsA Hydrolysis
Hydrolysis was carried out essentially as described previously (9) with slight modifications. CsA (25 pmol) was mixed with 3 M HCl(5 ml) in 50-ml tubes. Samples were heated (45°C) under vacuum using a Heto centrifuge until dried. Complete evaporation occurred in 4 h. The residue was dissolved with 0.6 ml of methanol:water (l:l, v/v) and aliquots of this solution were analyzed by HPLC.
iso&
Hydrolysis of Compounds A and B and of isoCsA
Hydrolysis was carried out dissolving about 80 nmol(lO0 pg) of each peptide in 1 ml of 3 M HCl. Samples were then dried under vacuum and analyzed by HPLC as described below. Peaks were collected and analyzed by FAB-MS. Peptide Methylation
Aliquots of each fraction purified by HPLC (2-20 nmol calculated on the basis of the molar absorption of CsA) were dried and treated with 50 ,ul of methanolic HCl freshly prepared by addition of acetyl chloride (160 ~1) to anhydrous methanol (1 ml). The samples were vortexed and the mixture was left at room temperature for 2 h. The solvent was then evaporated under vacuum and the dried residue was dissolved in methanol (5).
PURIFIED FRACTIONS.
Peptide Acetylation
Acetylation of the fractions purified by HPLC was carried out by dissolution of the dried samples (2-20 nmol) with 20 ~1 of 50 m&f ammonium acetate at pH 8.5 followed by addition of 50 ~1 of acetic anhydride in methanol (methanolacetic anhydride, 3:l; v:v). After 1 h at room temperature the excess reagent was evaporated and samples were dissolved in methanol (5). RESULTS
HPLC Analysis of Hydrolyzed
CsA
Under the conditions used by us implying evaporation to dryness of a CsA suspension in aqueous HCl, a number of
FIG. 1. Purification of isuCsA and open-chain CsA from hydrolyzed CsA by HPLC. Analyses were carried out using the residue obtained after evaporation to dryness of CsA in 3 M HCI as described in the text. Fractions were collected at the retention time of isoCsA, nonapeptide (*rt 26 min) and decapeptide (*rt 36 min) (9) and of the unknown peaks A and B. HPLC conditions are described in the text. Lower panel shows HPLC chromatograms obtained from purified fractions collected at the retention times of isoCsA and of peaks A and B. #Unknown impurity present also in blank samples.
1337
CsA HYDROLYSIS
gave three intense ions at m/z 1221 corresponding to the unmodified molecule, at m/z 1263 corresponding to a monoacetylated derivative, and at m/z 1305 corresponding to a diacetylated derivative. This suggested that compound B should contain two amino groups.
Compound B was 100% stable under the described treatment with 3 M HCl as showed by the absence of newly formed peaks in the HPLC chromatogram. In contrast, acidic treatment of isoCsA gave rise to the formation of compound B (3.3%) as confirmed by the FAB-MS analysis of the peak at rt 28 min, which gave an intense ion at m/z 1221.
FAB-MS/MS FAB-MS
By FAB-MS both unknown compounds A and B (Fig. 1) showed an intense ion at m/z 1221, allowing two possible interpretations for their formation: either hydration of the double bond of AA’ (MeBmt) or opening of the peptide chain. CsA, isoCsA, and aliquots of the fractions corresponding to the unknown compounds were metbylated and analyzed by FAB. As expected, CsA and isoCsA were unmodified because neither compound presents a free carboxylic group (Table 1). Metbylated nona- and decapeptides showed instead a spectrum with an intense ion at m/z 995 and at m/z 1122 (+ 14 Da with respect to the parent compound), corresponding to the [M+H]’ of the methyl ester derivatives, as could be expected from the previously suggested open-chain structures (9). Compound B (rt 28 min) was completely transformed into its methyl ester, which results from the FAB-MS spectrum showing the [M+H]+ ion shifted from m/z 1221 to 1235. In contrast, analysis of the reaction mixture after methylation of compound A (rt 40 min) suggested the reaction to be not complete; HPLC analysis showed two peaks at the retention time of the substrate and at a longer rt (47 min). FABMS spectrum of the mixture showed two intense ions at m/z 1221 and 1235. The two compounds were separated by HPLC and the fraction corresponding to the umeacted compound with m/z 1221 was methylated again. FAB analysis of the sample after derivatization still showed two main ions at m/z 1221 and 1235, suggesting an equilibrium for the methylation reaction. The CID spectrum of the ion at m/z 1221 was identical to that before methylation, confirming this hypothesis. Attempts to improve the yield of methylation by increasing the molar ratio between the methylating agent and the substrate were not successful. Acetylation of the two unknown compounds was also carried out to determine the presence of N-terminal amino groups. CsA did not react, as expected. On the contrary, the free amino group of isoCsA reacted, giving rise to a derivative with [M+H]’ at ml z 1245 in the FAB-MS spectrum (Table 1). The FAB-MS spectrum of the residue obtained after acetylation of the unknown compound A showed two prominent ions at mfz 122 1 and 1263, indicating that part of the substance did not react and part was monoacetylated. Compound B treated under the same conditions
CsA and isoCsA. To obtain additional structural information for the two unknown compounds we recorded the MS/MS spectra. It has been shown for cyclic peptides that unless the peptide contains only one strongly basic site that drives protonation and leads to a dominant series of fragmentation ions, the [M+H]+ ions are actually “mixtures” of different ring-opened acylium ions with the same mass value, each one generating a specific series of daughter ions (2). Therefore, the CID spectra of CsA and isoCsA (Fig. 2) were not unambiguously interpretable because CsA and isoCsA are cyclic peptides with no strong basic amino acid and consequently they can be broken by the collision gas at various peptide bonds. Previously reported interpretation of the daughter ions of CsA obtained with a triple quadrupole by Bowers (1) unclear about structural assignments that, to our knowledge, were never published elsewhere. Compound A. The presence of four residues of N-metbyl-leutine and of three amino acids with mol.wt. 89 (alanine and sarcosine) in the structure of these peptides does not allow to assign
a definitive structure to each daughter ion present in the spectrum (Fig. 3). Based on the fact that the compound should be an openchain peptide because its methyl ester was obtained, two possible structures could be derived from interpretation of daughter ions (Fig. 4), corresponding to l,ll-seco-CsA and 2,3-seco-CsA. The former was confirmed from the CID spectrum of the methyl ester showing an ion at m/z 967, which likely contains the terminal carboxyl group because it is shifted by 14 mass units with respect to that in the CID spectrum of the parent compound (m/z 953). This would be a y9, assuming for compound A the structure of 1,l I-seco-CsA from either CsA or isoCsA by cleavage at the MeVal”-MeBmt’ peptide bond. Final evidence for the amino acid sequence of compound A was obtained by comparison of the CID spectrum of the unmodified peptide with that of its acetate (Fig. 3). In the latter spectrum intense ions of the b series, shifted by 42 Da with respect to those in the CID spectrum of the parent compound, were easily identified. Ion at m/z 1132 corresponds to the loss of MeVal” (b,,); the next b ion at m/z 1005 derives from the loss of MeVal”. The b, and the b7 ions, originating from the loss of D-Ala and Ala, are weaker, as already reported for the CID spec-
TABLE 1 SPECTRA OF THE MAIN COMPOUNDS DERIVING FROM CsA HYDROLYSIS IN AQUEOUS ACIDIC MEDIUM
[M + H]’ IN FAB-MS
m/z (70 Relative Intensity)
CsA IsoCsA
Compound A Comound B Decapeptide Nonapeptide
Parent
Methyl Ester
I203 1203 1221 1221 1108 981
1203 1203 1221 (60)-1235 (100) 1235 1122 995
Acetates
I203 1203 (80)-1245 (100) 1221 (100-1263 (80) 1221 (lo)-1263 (lOO)-1305 (60) 1150 1023
Values represent the more relevant signals over 800 Da. For samples with more than one relevant signal percent relative intensity of each ion is shown in parentheses.
MAGNI
1338
935 694
I
ET AL.
1159 I
426 I
69.5
FIG. 2. CID spectra of CsA and of isoCsA. Analyses were carried out on about 5 mg of CsA and 5 mg of isoCsA under MS/MS conditions described in the text.
tra of the previously identified nona- and decapeptides deriving from the hydrolysis of CsA (9). The b6 ion is still very intense but the following b ions are 10 times weaker. Also some ions of the y series are detectable in the spectrum. From these data the tested compound was identified as a seco-CsA originating from the cleavage of either the peptide bond between MeBmt’ and MeVal” of CsA or, with lower probability, the ester bond of isoCsA. Comported B. Comments similar to those reported above for compound A apply to compound B. The CID spectrum of its methyl derivative (Fig. 4) displays y ions at m/z 740 and 1137 (14 mass units over ions at m/z 1122 and 726 of the parent compound). Both ions suggested ring opening of either CsA or isoCsA between MeLeu4 and Val’. Moreover, as reported above, compound B gave a diacetate, suggesting that two free amino groups were present and that the compound may derive from the
hydrolytic degradation of a peptide bond of isoCsA. As a matter of fact, the CID spectrum of the parent compound (Fig. 4) could be mainly interpreted (y series ions at mlz 1122,995,726; b series at m/z 227, 298, 369, 497, 624, 737, 1005, 1076) assuming an amino acid sequence deriving from isoCsA by cleavage of the bond between MeLeu4 and Val’. DISCUSSION
Acidic degradation of CsA as described here gives rise to open-chain peptides as reported previously by us for slightly different conditions (9). Products on which we have been focusing our attention are isoCsA, a nonapeptide, and a decapeptide reported previously (9), and the two undecapeptides are identified by FABNS-MS as seco-CsA cleaved between MeBmt’ and MeVal” and seco-isoC!sA cleaved between MeLeu4 and Va15.
1339
CsA HYDROLYSIS
A
However, neither cleavage between amino acids 1 and 11 nor hydrolysis of an amide bond of isoCsA was reported. Results reported in the experimental section also clearly show that isoCsA is hydrolyzed to seco-isoCsA but is not transformed by the acid into seco-CsA. Besides, seco-CsA is hydrolyzed to the deca- and nonapeptide whereas isoCsA is not. Therefore, acidic attack of CsA can be depicted as in Scheme 1. It is difficult to establish at present whether deca- and nonapeptide are obligatory intermediates of the complete degradation of the peptide
1”
5 "
1
27,l
FlG. 3. CID spectra of compwnd A and its derivatives. Purification of the parent compound, preparation of derivatives, and analytical conditions are described in the texi. Identification from CID spectra of the target compounds and of their methyl esters and aceltates is unambiguous for the seco-CsA. For seco-isoCsA, evidences presented are in good agreement with the assigned structure, but the possibility of the presence in the purified fraction of seco-isoCsA of an isomer cleaved at a different position cannot he excluded completely. To the best of our knowledge, the seco-CsA described here has not been described before. When drafting of this manuscript was in progress, a paper was published dealing with the synthesis of various secoCsAs with the peptide chain cleaved at different positions (3).
FIG. 4. CID spectra of compound B and its derivatives. Purification of the parent compound, preparation of derivatives, and analytical conditions are described in the text.
MAGNI
1340
I
D-d-
I Ad-
f&L*"6 _
I8
va16
Q-Ala
Ala’-
-
H20
MeLeus-
I
’ 8 D-Ah
I:
-4
Ala’-
MaLeu6
-
Val 5
$_
c
x
H;CH’fil/0 :+t3
t&Leleu9
-
‘5
Val
H+
et-b :1> 0 rnL,‘“-
ET AL
B .AblL3&U3 I M.3L.3U4
I D-Ala6 -
11 - MeVal -4
Ala’-
h,bLeU6 -
val 5
Mele~MeLe~~D-Ala8-Ala~~Le~-Va~-Mele~~~A~MeBmtl
.,,:d
DECAPEPTIDE NONAPEPTIDE Scheme
1. Hydrolysis of CsA in aqueous acidic medium.
chain to amino acids observed previously by degrading CsA with 6 M HCl(6). Previous work on CsA hydrolysis in aqueous acidic media has been carried out for two kinds of purposes: to clarify the effect of structural modifications on CsA to isoCsA isomerization and thus ascertain the mechanism of the N,O-acyl migration, on which the isomerization is based (lo), and to verify whether variability of oral bioavailability of CsA may be related to its intragastric degradation (4). Acidic conditions and CsA concentrations used to investigate these aspects were much milder than those giving rise to the degradation products described here, particularly because chain opening by the acid in our experiments occurs during concentration under vacuum of the CsA solution, as we will describe elsewhere in detail. Both the above-reported groups of authors found isoCsA to be the predominant degradation product of CsA. Loss of CsA at pH < 2 (4) was accompanied by the formation of isoCsA in more than 80% yield but no peaks other than isoCsA appeared in the HPLC chromatogram. At pH 2-4, less isoCsA was formed and the authors concluded that the nature of the main degradation reaction remains unknown, which appears true if one considers that the rate constant for CsA degradation in the l-4 pH range was pH independent. We have tried to repeat their conditions, but unfortunately, even at pH 1 for 5 h, which was described to degrade CsA efficiently at 5 x 10e6 M, we recovered more than 90% CsA by diethyl ether extraction. Besides, direct injection of the reaction solution under our HPLC conditions did not allow detection of any peak, due to the too low concentration. The aqueous fraction of this extraction was concentrated to dryness
to analyze it by HPLC. Peaks corresponding to the open-chain peptides were detected in this fraction. However, we cannot exclude their formation during evaporation of the acidic solution despite the fact that CsA had been almost completely eliminated from the aqueous solution by the ether extraction before evaporation to dryness. Our inability to reproduce conditions described by these two groups of authors (4,lO) may depend on difficulties in interpreting their experimental procedures. Nevertheless, it is important to note that both groups have proposed intermediary formation of an open-chain CsA during CsA isomerization to isoCsA. We have presented here the direct evidence that open-chain undecapeptides can be formed during acidic treatment of CsA. As expected, open-chain peptides obtained by us are all water soluble. As a matter of fact, when the residue obtained after CsA hydrolysis in the conditions described is suspended in water and extracted with diethyl ether, residual CsA and a great part of isoCsA are recovered in the organic fraction whereas the openchain peptides are almost completely found in the aqueous fraction (data not shown). Solubility in water could represent an important property of the compounds described here in relation to a possible use as immunosuppressants. Nevertheless, their activity is still unknown and for its evaluation larger amounts of material will need to be available. ACKNOWLEDGEMENT This
work was supported by the CNR Target Project “Biotechnology
and Bioinstmmentation.”
REFERENCES 1. Bowers, L. D.; Norman, D. D.; Yan, X.; Scheeler, D.; Carison, K. L. Isolation and structural identification of ghydroxy-‘desmethylcyclosporine. Clin. Chem. 36: 1875- 1879; 1990.
2. Cemey, R. L.; Gross, M. L. Tandem mass spectrometry for determining the amino acid sequence of cyclic peptides and for assessing interactions of peptides and metal ions. In: Desiderio,
CsA HYDROLYSIS
3.
4.
5.
6.
D. M., ed. Mass spectrometry of peptides. Boston: CRC Press; 1990:289-314. Eberle, M. K.; Jutzi-Eme. A. M.: Nuninger, F. Cyclosporin A: Regioselective ring opening and fragmentation reactions via thioamides. Route to semisynthetic cyclosporins. J. Org. Chem. 59:72497258; 1994. Friis, G. J.; Bundgaard, Hi. Kinetics of degradation of cyclospotin A in acidic aqueous solution and its implication in its oral absorption. Int. J. Pharmacol. 82:79-83; 1992. Griffin, P. R.; Kumar, S.; Shabanowitz, J.; et al. The amino acid sequence of the sex steroid-binding protein of rabbit serum. J. Biol. Chem. 264: 19066- 1907tl; 1989. Hartman, N. R.; Jardine, I. Mass spectrometric analysis of cyclosporine metabolites. Biomed. Mass Spectrom. 13:361-372; 1986.
1341
7. Kahan, B. D. Cyclosporine: The agent and its actions. Trasplant. Proc. XVII(4):5-17; 1985. 8. Kahan, B. D.; Shaw, L. M.; Holt, D.; Grevel, J.; Johnston, A. Consensus document: Hawk’s Cay meeting on therapeutic drug monitoring of cyclosporine. Clin. Chem. 36/S: l510- I5 16; 1990. 9. Magni, F.; Arcelloni, C.; Paroni, R.; et al. Open-chain pcptides obtained by acidic hydrolytic cleavage of cyclosporin A. Biol. Mass Spectrom. 23:514-518; 1994. IO. Oliyai, R.; Safadi, M.; Meier, P. G.; Hu, M.; Rich, D. H.; Stella, V. J. Kinetics of acid-catalyzed degradation of cyclosporin A and its analogs in aqueous solution. Int. J. Pept. Protein Res. 43:239-247; 1994. I I. Rtiegger, A.; Kuhn, M.; Lichti, H.; et al. Cyclosporin A, ein immunsuppressiv wirksamer Peptidmetabolit aus Trichoderma polysporum (Link ex Pers.) Rifai. Helv. Chim. Acta 59: 1075-1092; 1976.
Peotides. Vol. 16. No. 8. DD. 133551341. 1995 dopyright 0 1995’Ikevier Science Inc. Printed in the USA. All rights reserved Ol96-9781/9<$9.50 + .IJO
1 Pergamon 0196-9781(95)02025-R
Hydrolysis of Cyclosporin A: Identification of 1,ll seco-Cyclosporin A and 4,5 secoisoCyclosporin A by FAB-MS/MS FULVIO MAGNI,*
LOLITA ARNOLDI,*
MARINA
DEL PUPPOt AND MARZIA
GALL1 KJENLEt’
*Scienti$c Institute H. San Raffaele, Via Olgettina 60, 20132 Milan, Italy and iDepartment of Medical Chemistry and Biochemistry, University of Milan, Via Saldini SO, 20133 Milan, Italy Received
4 April 1995
MAGNI, F., L. ARNOLDI, M. DEL PUPPO AND M. GALL1 KIENLE. Hydrolysis ofcyclosporin A: Idenfijcurion of I,11 secocyclosporin A and 4.5 seco-isocyclosporin A by FAB-MS/MS. PEPTIDES 16(S) 1335% 1341,1995.-We have previously reported that treatment of CsA with aqueous HCI gives rise to the formation of a number of water-soluble compounds. Two of these were identified from their FAB-MSNS spectra as open-chain nona- and decapeptides. We describe here the identification of two other main compounds deriving from the same treatment. Identification was rendered possible from the comparison of their FAB-MS/ MS spectra with tho,se of methyl and acetyl derivatives. The two compounds are water-soluble, open-chain undecapeptides corresponding to I.1 1 seco-CsA and of 45 seco-isoCsA, respectively. Cyclosporin A Cyclic peptides
Acidic hydrolysis
Mass spectrometry
A (CsA) is the well-known cyclic undecapeptide with immunosuppressant activity (7). CsA metabolism has been thoroughly investigated in vitro and in vivo (8). Some work has also been carried out to clarify the behavior of CsA in aqueous acidic media (4) to understand whether degradation of the peptide chain occurs at the igastric level and to clarify the kinetics of the acid-catalyzed degradation (10). Only isomerization of CsA has been reported to occur, with formation of the isomer named isocyclosporin A (isoCsA), which was previously shown to be formed by acidic treatment of CsA in organic solvents via N,O-acyl migration (11). In. a previous work on CsA degradation in acidic aqueous medium, in addition to isoCsA we identified from their FAB-MS/MS spectra two open-chain peptides maintaining the amino acid sequences unmodified with respect to that of CsA for the I- 10 and l-9 residues, respectively (9). Two other main compounds were shown to be formed but their structures were not predictable from the FAB-MS/MS spectra. We report identification of the structures of these two compounds from FAB-MS/MS spectra of the parent compounds and of their methyl and acetyl derivatives. They are open-chain undecapeptides deriving from the cleavage of the 1- 11 peptide bond of CsA and of the 4-5 peptidle bond of isoCsA.
Tandem mass spectrometry
(Milan, Italy). Other reagents were of analytical grade. Solvents for HPLC were used after being degassed and filtered using 0.22pm filters.
CYCLOSPORIN
METHOD
Materials
CsA was a generous gift of Dr. G. Corbetta (Sandoz S.p.A., Milan, Italy). 3-Nitrobenzyl alcohol was from Aldrich Chimica
seco-Cyclosporins
HPLC Analysis
A Jasco liquid chromatograph system (Jasco International Co. Ltd., Tokyo, Japan) equipped with a Jasco pump 880 PU connected with a mixing module 880-02 and with an 875-W detector was used. Peptide separation was carried out with an acetonitrile:trifluoroacetic acid (TFA) gradient slightly modified with respect to that previously described (9). A Beckman ODS Ultrasphere column either 150 X 4.6 mm (5 pm) or 150 X 10 mm (5 pm) was used for analytical and semipreparative purposes, respectively. Peptides were eluted with acetonitrile:0.057% TFA (32:68, v/v) (A) and acetonitrile:0.052% TFA (80:20, v/v) (B), performing a linear gradient of B from 0% to 100% in 70 min (9). The flow rate with the analytical column was 0.5 ml/mitt, and the UV detector was set at 210 nm. For the separation of the compounds from each other the semipreparative column was used (four injections of 150 ~1 each) and elution was carried out at 2 ml/n-& flow rate. Fractions at retention time of isoCsA and of the two unknown peptides (A and B in Fig. 1) were collected and concentrated to dryness. Each fraction was then further purified by HPLC for complete separation from impurities. Each collected peak was analyzed again by HPLC to check its purity and by FAB-MS for structure identification.
’ Requests for reprints should be addressed to Marzia Galli Kienle. 1335
1336
MAGNI ET AL.
FAB-MS Analysis
To obtain information on the molecular weight of the unknown peptides, spectra by fast ion bombardment (FAB-MS) were recorded by using a Finnigan MAT95 double-focusing mass spectrometer equipped with a cesium gun, recording positive ions and operating at an ion accelerating voltage of 5 kV. The cesium ions were accelerated through 22 kV. Spectra were recorded from mlz 100 to m/z 1500 at 5- 10 s/decade. Calibration of the instrument was checked daily with a saturated solution of CsI in glycerol. The instrument operated at a resolution of 1300 (10% valley). 3-Nitrobenzyl alcohol was used as matrix as previously reported (9). Dried fractions purified by HPLC were dissolved in-methanol to a final concentration of 1- 10 pg/pl and l~1 aliquots were analyzed. Additional structural information was obtained recording collision-induced dissociation spectra (CID spectra) of the protonated molecular ion [M+H]’ of the peptides and of their derivatives by introducing helium as collision gas into the first field free region. The collision gas pressure in the collision cell was adjusted to attenuate the primary ion beam by 70%. The CID spectra were obtained using the field-controlled B/E linked scan in profile mode at 10 s/decade. Identification of daughter ions was carried out allowing a mass tolerance error of f 1.5 amu around the expected value.
water-soluble compounds are formed (9). Previously reported (9) retention times of the various compounds differ from those reported in Fig. 1 because for the present work the gradient was modified in an attempt to decrease the run time without decrease of the chromatographic resolution. Moreover, different from the previous work, extraction with diethyl ether before HPLC to separate unreacted CsA and part of isoCsA was not carried out. Peaks corresponding to IsoCsA, and to the two peaks at rt 40 and 28 min (Fig. 1, upper panel, compounds A and B) were isolated and purified by HPLC. Preparative HPLC allowed to obtain fractions containing single peaks (Fig. 1, lower panel). Compound A was recovered mainly unaltered (79%) after treatment with 3 M HCl under the conditions described in the Method section. Nevertheless, HPLC showed the presence of peaks at rt 26 and 36 min corresponding to those of nona- (5%) and decapeptides (S%), respectively. FAB-MS of these peaks showed ions at m/z 1108 and 98 1, thus confirming that compound A gives rise to the already identified peptides.
HYDROLYSED CsA. *B
CsA Hydrolysis
Hydrolysis was carried out essentially as described previously (9) with slight modifications. CsA (25 pmol) was mixed with 3 M HCl(5 ml) in 50-ml tubes. Samples were heated (45°C) under vacuum using a Heto centrifuge until dried. Complete evaporation occurred in 4 h. The residue was dissolved with 0.6 ml of methanol:water (l:l, v/v) and aliquots of this solution were analyzed by HPLC.
iso&
Hydrolysis of Compounds A and B and of isoCsA
Hydrolysis was carried out dissolving about 80 nmol(lO0 pg) of each peptide in 1 ml of 3 M HCl. Samples were then dried under vacuum and analyzed by HPLC as described below. Peaks were collected and analyzed by FAB-MS. Peptide Methylation
Aliquots of each fraction purified by HPLC (2-20 nmol calculated on the basis of the molar absorption of CsA) were dried and treated with 50 ,ul of methanolic HCl freshly prepared by addition of acetyl chloride (160 ~1) to anhydrous methanol (1 ml). The samples were vortexed and the mixture was left at room temperature for 2 h. The solvent was then evaporated under vacuum and the dried residue was dissolved in methanol (5).
PURIFIED FRACTIONS.
Peptide Acetylation
Acetylation of the fractions purified by HPLC was carried out by dissolution of the dried samples (2-20 nmol) with 20 ~1 of 50 m&f ammonium acetate at pH 8.5 followed by addition of 50 ~1 of acetic anhydride in methanol (methanolacetic anhydride, 3:l; v:v). After 1 h at room temperature the excess reagent was evaporated and samples were dissolved in methanol (5). RESULTS
HPLC Analysis of Hydrolyzed
CsA
Under the conditions used by us implying evaporation to dryness of a CsA suspension in aqueous HCl, a number of
FIG. 1. Purification of isuCsA and open-chain CsA from hydrolyzed CsA by HPLC. Analyses were carried out using the residue obtained after evaporation to dryness of CsA in 3 M HCI as described in the text. Fractions were collected at the retention time of isoCsA, nonapeptide (*rt 26 min) and decapeptide (*rt 36 min) (9) and of the unknown peaks A and B. HPLC conditions are described in the text. Lower panel shows HPLC chromatograms obtained from purified fractions collected at the retention times of isoCsA and of peaks A and B. #Unknown impurity present also in blank samples.
1337
CsA HYDROLYSIS
gave three intense ions at m/z 1221 corresponding to the unmodified molecule, at m/z 1263 corresponding to a monoacetylated derivative, and at m/z 1305 corresponding to a diacetylated derivative. This suggested that compound B should contain two amino groups.
Compound B was 100% stable under the described treatment with 3 M HCl as showed by the absence of newly formed peaks in the HPLC chromatogram. In contrast, acidic treatment of isoCsA gave rise to the formation of compound B (3.3%) as confirmed by the FAB-MS analysis of the peak at rt 28 min, which gave an intense ion at m/z 1221.
FAB-MS/MS FAB-MS
By FAB-MS both unknown compounds A and B (Fig. 1) showed an intense ion at m/z 1221, allowing two possible interpretations for their formation: either hydration of the double bond of AA’ (MeBmt) or opening of the peptide chain. CsA, isoCsA, and aliquots of the fractions corresponding to the unknown compounds were metbylated and analyzed by FAB. As expected, CsA and isoCsA were unmodified because neither compound presents a free carboxylic group (Table 1). Metbylated nona- and decapeptides showed instead a spectrum with an intense ion at m/z 995 and at m/z 1122 (+ 14 Da with respect to the parent compound), corresponding to the [M+H]’ of the methyl ester derivatives, as could be expected from the previously suggested open-chain structures (9). Compound B (rt 28 min) was completely transformed into its methyl ester, which results from the FAB-MS spectrum showing the [M+H]+ ion shifted from m/z 1221 to 1235. In contrast, analysis of the reaction mixture after methylation of compound A (rt 40 min) suggested the reaction to be not complete; HPLC analysis showed two peaks at the retention time of the substrate and at a longer rt (47 min). FABMS spectrum of the mixture showed two intense ions at m/z 1221 and 1235. The two compounds were separated by HPLC and the fraction corresponding to the umeacted compound with m/z 1221 was methylated again. FAB analysis of the sample after derivatization still showed two main ions at m/z 1221 and 1235, suggesting an equilibrium for the methylation reaction. The CID spectrum of the ion at m/z 1221 was identical to that before methylation, confirming this hypothesis. Attempts to improve the yield of methylation by increasing the molar ratio between the methylating agent and the substrate were not successful. Acetylation of the two unknown compounds was also carried out to determine the presence of N-terminal amino groups. CsA did not react, as expected. On the contrary, the free amino group of isoCsA reacted, giving rise to a derivative with [M+H]’ at ml z 1245 in the FAB-MS spectrum (Table 1). The FAB-MS spectrum of the residue obtained after acetylation of the unknown compound A showed two prominent ions at mfz 122 1 and 1263, indicating that part of the substance did not react and part was monoacetylated. Compound B treated under the same conditions
CsA and isoCsA. To obtain additional structural information for the two unknown compounds we recorded the MS/MS spectra. It has been shown for cyclic peptides that unless the peptide contains only one strongly basic site that drives protonation and leads to a dominant series of fragmentation ions, the [M+H]+ ions are actually “mixtures” of different ring-opened acylium ions with the same mass value, each one generating a specific series of daughter ions (2). Therefore, the CID spectra of CsA and isoCsA (Fig. 2) were not unambiguously interpretable because CsA and isoCsA are cyclic peptides with no strong basic amino acid and consequently they can be broken by the collision gas at various peptide bonds. Previously reported interpretation of the daughter ions of CsA obtained with a triple quadrupole by Bowers (1) unclear about structural assignments that, to our knowledge, were never published elsewhere. Compound A. The presence of four residues of N-metbyl-leutine and of three amino acids with mol.wt. 89 (alanine and sarcosine) in the structure of these peptides does not allow to assign
a definitive structure to each daughter ion present in the spectrum (Fig. 3). Based on the fact that the compound should be an openchain peptide because its methyl ester was obtained, two possible structures could be derived from interpretation of daughter ions (Fig. 4), corresponding to l,ll-seco-CsA and 2,3-seco-CsA. The former was confirmed from the CID spectrum of the methyl ester showing an ion at m/z 967, which likely contains the terminal carboxyl group because it is shifted by 14 mass units with respect to that in the CID spectrum of the parent compound (m/z 953). This would be a y9, assuming for compound A the structure of 1,l I-seco-CsA from either CsA or isoCsA by cleavage at the MeVal”-MeBmt’ peptide bond. Final evidence for the amino acid sequence of compound A was obtained by comparison of the CID spectrum of the unmodified peptide with that of its acetate (Fig. 3). In the latter spectrum intense ions of the b series, shifted by 42 Da with respect to those in the CID spectrum of the parent compound, were easily identified. Ion at m/z 1132 corresponds to the loss of MeVal” (b,,); the next b ion at m/z 1005 derives from the loss of MeVal”. The b, and the b7 ions, originating from the loss of D-Ala and Ala, are weaker, as already reported for the CID spec-
TABLE 1 SPECTRA OF THE MAIN COMPOUNDS DERIVING FROM CsA HYDROLYSIS IN AQUEOUS ACIDIC MEDIUM
[M + H]’ IN FAB-MS
m/z (70 Relative Intensity)
CsA IsoCsA
Compound A Comound B Decapeptide Nonapeptide
Parent
Methyl Ester
I203 1203 1221 1221 1108 981
1203 1203 1221 (60)-1235 (100) 1235 1122 995
Acetates
I203 1203 (80)-1245 (100) 1221 (100-1263 (80) 1221 (lo)-1263 (lOO)-1305 (60) 1150 1023
Values represent the more relevant signals over 800 Da. For samples with more than one relevant signal percent relative intensity of each ion is shown in parentheses.
MAGNI
1338
935 694
I
ET AL.
1159 I
426 I
69.5
FIG. 2. CID spectra of CsA and of isoCsA. Analyses were carried out on about 5 mg of CsA and 5 mg of isoCsA under MS/MS conditions described in the text.
tra of the previously identified nona- and decapeptides deriving from the hydrolysis of CsA (9). The b6 ion is still very intense but the following b ions are 10 times weaker. Also some ions of the y series are detectable in the spectrum. From these data the tested compound was identified as a seco-CsA originating from the cleavage of either the peptide bond between MeBmt’ and MeVal” of CsA or, with lower probability, the ester bond of isoCsA. Comported B. Comments similar to those reported above for compound A apply to compound B. The CID spectrum of its methyl derivative (Fig. 4) displays y ions at m/z 740 and 1137 (14 mass units over ions at m/z 1122 and 726 of the parent compound). Both ions suggested ring opening of either CsA or isoCsA between MeLeu4 and Val’. Moreover, as reported above, compound B gave a diacetate, suggesting that two free amino groups were present and that the compound may derive from the
hydrolytic degradation of a peptide bond of isoCsA. As a matter of fact, the CID spectrum of the parent compound (Fig. 4) could be mainly interpreted (y series ions at mlz 1122,995,726; b series at m/z 227, 298, 369, 497, 624, 737, 1005, 1076) assuming an amino acid sequence deriving from isoCsA by cleavage of the bond between MeLeu4 and Val’. DISCUSSION
Acidic degradation of CsA as described here gives rise to open-chain peptides as reported previously by us for slightly different conditions (9). Products on which we have been focusing our attention are isoCsA, a nonapeptide, and a decapeptide reported previously (9), and the two undecapeptides are identified by FABNS-MS as seco-CsA cleaved between MeBmt’ and MeVal” and seco-isoC!sA cleaved between MeLeu4 and Va15.
1339
CsA HYDROLYSIS
A
However, neither cleavage between amino acids 1 and 11 nor hydrolysis of an amide bond of isoCsA was reported. Results reported in the experimental section also clearly show that isoCsA is hydrolyzed to seco-isoCsA but is not transformed by the acid into seco-CsA. Besides, seco-CsA is hydrolyzed to the deca- and nonapeptide whereas isoCsA is not. Therefore, acidic attack of CsA can be depicted as in Scheme 1. It is difficult to establish at present whether deca- and nonapeptide are obligatory intermediates of the complete degradation of the peptide
1”
5 "
1
27,l
FlG. 3. CID spectra of compwnd A and its derivatives. Purification of the parent compound, preparation of derivatives, and analytical conditions are described in the texi. Identification from CID spectra of the target compounds and of their methyl esters and aceltates is unambiguous for the seco-CsA. For seco-isoCsA, evidences presented are in good agreement with the assigned structure, but the possibility of the presence in the purified fraction of seco-isoCsA of an isomer cleaved at a different position cannot he excluded completely. To the best of our knowledge, the seco-CsA described here has not been described before. When drafting of this manuscript was in progress, a paper was published dealing with the synthesis of various secoCsAs with the peptide chain cleaved at different positions (3).
FIG. 4. CID spectra of compound B and its derivatives. Purification of the parent compound, preparation of derivatives, and analytical conditions are described in the text.
MAGNI
1340
I
D-d-
I Ad-
f&L*"6 _
I8
va16
Q-Ala
Ala’-
-
H20
MeLeus-
I
’ 8 D-Ah
I:
-4
Ala’-
MaLeu6
-
Val 5
$_
c
x
H;CH’fil/0 :+t3
t&Leleu9
-
‘5
Val
H+
et-b :1> 0 rnL,‘“-
ET AL
B .AblL3&U3 I M.3L.3U4
I D-Ala6 -
11 - MeVal -4
Ala’-
h,bLeU6 -
val 5
Mele~MeLe~~D-Ala8-Ala~~Le~-Va~-Mele~~~A~MeBmtl
.,,:d
DECAPEPTIDE NONAPEPTIDE Scheme
1. Hydrolysis of CsA in aqueous acidic medium.
chain to amino acids observed previously by degrading CsA with 6 M HCl(6). Previous work on CsA hydrolysis in aqueous acidic media has been carried out for two kinds of purposes: to clarify the effect of structural modifications on CsA to isoCsA isomerization and thus ascertain the mechanism of the N,O-acyl migration, on which the isomerization is based (lo), and to verify whether variability of oral bioavailability of CsA may be related to its intragastric degradation (4). Acidic conditions and CsA concentrations used to investigate these aspects were much milder than those giving rise to the degradation products described here, particularly because chain opening by the acid in our experiments occurs during concentration under vacuum of the CsA solution, as we will describe elsewhere in detail. Both the above-reported groups of authors found isoCsA to be the predominant degradation product of CsA. Loss of CsA at pH < 2 (4) was accompanied by the formation of isoCsA in more than 80% yield but no peaks other than isoCsA appeared in the HPLC chromatogram. At pH 2-4, less isoCsA was formed and the authors concluded that the nature of the main degradation reaction remains unknown, which appears true if one considers that the rate constant for CsA degradation in the l-4 pH range was pH independent. We have tried to repeat their conditions, but unfortunately, even at pH 1 for 5 h, which was described to degrade CsA efficiently at 5 x 10e6 M, we recovered more than 90% CsA by diethyl ether extraction. Besides, direct injection of the reaction solution under our HPLC conditions did not allow detection of any peak, due to the too low concentration. The aqueous fraction of this extraction was concentrated to dryness
to analyze it by HPLC. Peaks corresponding to the open-chain peptides were detected in this fraction. However, we cannot exclude their formation during evaporation of the acidic solution despite the fact that CsA had been almost completely eliminated from the aqueous solution by the ether extraction before evaporation to dryness. Our inability to reproduce conditions described by these two groups of authors (4,lO) may depend on difficulties in interpreting their experimental procedures. Nevertheless, it is important to note that both groups have proposed intermediary formation of an open-chain CsA during CsA isomerization to isoCsA. We have presented here the direct evidence that open-chain undecapeptides can be formed during acidic treatment of CsA. As expected, open-chain peptides obtained by us are all water soluble. As a matter of fact, when the residue obtained after CsA hydrolysis in the conditions described is suspended in water and extracted with diethyl ether, residual CsA and a great part of isoCsA are recovered in the organic fraction whereas the openchain peptides are almost completely found in the aqueous fraction (data not shown). Solubility in water could represent an important property of the compounds described here in relation to a possible use as immunosuppressants. Nevertheless, their activity is still unknown and for its evaluation larger amounts of material will need to be available. ACKNOWLEDGEMENT This
work was supported by the CNR Target Project “Biotechnology
and Bioinstmmentation.”
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2. Cemey, R. L.; Gross, M. L. Tandem mass spectrometry for determining the amino acid sequence of cyclic peptides and for assessing interactions of peptides and metal ions. In: Desiderio,
CsA HYDROLYSIS
3.
4.
5.
6.
D. M., ed. Mass spectrometry of peptides. Boston: CRC Press; 1990:289-314. Eberle, M. K.; Jutzi-Eme. A. M.: Nuninger, F. Cyclosporin A: Regioselective ring opening and fragmentation reactions via thioamides. Route to semisynthetic cyclosporins. J. Org. Chem. 59:72497258; 1994. Friis, G. J.; Bundgaard, Hi. Kinetics of degradation of cyclospotin A in acidic aqueous solution and its implication in its oral absorption. Int. J. Pharmacol. 82:79-83; 1992. Griffin, P. R.; Kumar, S.; Shabanowitz, J.; et al. The amino acid sequence of the sex steroid-binding protein of rabbit serum. J. Biol. Chem. 264: 19066- 1907tl; 1989. Hartman, N. R.; Jardine, I. Mass spectrometric analysis of cyclosporine metabolites. Biomed. Mass Spectrom. 13:361-372; 1986.
1341
7. Kahan, B. D. Cyclosporine: The agent and its actions. Trasplant. Proc. XVII(4):5-17; 1985. 8. Kahan, B. D.; Shaw, L. M.; Holt, D.; Grevel, J.; Johnston, A. Consensus document: Hawk’s Cay meeting on therapeutic drug monitoring of cyclosporine. Clin. Chem. 36/S: l510- I5 16; 1990. 9. Magni, F.; Arcelloni, C.; Paroni, R.; et al. Open-chain pcptides obtained by acidic hydrolytic cleavage of cyclosporin A. Biol. Mass Spectrom. 23:514-518; 1994. IO. Oliyai, R.; Safadi, M.; Meier, P. G.; Hu, M.; Rich, D. H.; Stella, V. J. Kinetics of acid-catalyzed degradation of cyclosporin A and its analogs in aqueous solution. Int. J. Pept. Protein Res. 43:239-247; 1994. I I. Rtiegger, A.; Kuhn, M.; Lichti, H.; et al. Cyclosporin A, ein immunsuppressiv wirksamer Peptidmetabolit aus Trichoderma polysporum (Link ex Pers.) Rifai. Helv. Chim. Acta 59: 1075-1092; 1976.