2304
Anal. Chem. 1988, 6 0 , 2304-2306
Structural and Reaction Assignments for Some Common Polysaccharides Using Laser Desorption Ionization Fourier Transform Ion Cyclotron Resonance Spectroscopy Sir: Commercially available polysaccharides obtained from biological sources have important industrial applications (1). The structural characterization of these polysaccharides is very complex and traditionally has been performed by using a variety of enzymatic and wet chemical methods (2,3). Mass spectrometry has played only a small role in polysaccharide analysis, being limited mostly to the use of gas chromatography/mass spectrometry (GC/MS) for detection and identification of derivatized monosaccharides obtained by chemical degradation of the biopolymers ( 4 ) . The advent of novel desorption/ionization mass spectrometric techniques, such as fast atom bombardment (FAB) and desorption chemical ionization (DCI), combined with derivatizations to improve volatility, has enabled some oligosaccharide structures, obtained from the polysaccharides by enzymatic and wet chemical methods, to be partially delineated (5, 6). Proper sequencing of a carbohydrate polymer or oligomer requires identification of the reducing end of the chain to select which of two possible sequences is correct. FAB spectra of underivatized oligomers generally do not give any information as to which is the reducing end. Therefore, for complete sequencing, including locating the reducing end, an oligosaccharide has to be reduced, and then derivatized prior to mass spectrometric analysis (7). Laser desorption ionization Fourier transform ion cyclotron resonance (LDI-FT-ICR)spectroscopy has shown some success when applied to a variety of underivatized biomolecules (8, 9). Coates and Wilkins first demonstrated the potential of LDI-FT-ICR for the analysis of some common polysaccharides and glycoconjugates (10-12). From the LDI-FT-ICR mass spectra of nine commercially available polysaccharides (12) they observed "extensive fragmentation of the saccharide chains, from both within the sugar rings and between them . ... Although simlarities are seen in the spectra of some compounds, each displays a characteristic fragmentation pattern." The observed ion masses in their fingerprint mass spectra can be described by the empirical formula [(162), X K]+ n = 1, 2, 3, ... (1)
+ +
K is from an added potassium salt, n corresponds to the number of hexose rings in the fragment ion, and the integer X corresponds to the mass of a submonosaccharidefragment. They observed 16 series of fragment ions, with differing X, each of which was assigned an arbitrary label from A to R. The most common ion series were A (X= 01, D (X= 42), F (X= 60), and Q (X= 1441, with the A and F series ions being the most intense. These four series were observed in each of the mass spectra of the polyhexoses reported and thus we feel are likely to be fragmentation patterns charadristic of hexose rings, rather than unique fingerprints of the individual polysaccharides. Some insight into the chemical origin of the ions in the Coates-Wilkins A-R series is provided by writing their empirical formula ( I ) in a somewhat different form. Any polyhexose has a molecular weight given by the generic formula [(162)n + 181 (2) where n is the number of residues. The molecular weight of a CSHI0O6,anhydrohexose residue is 162, and 18 is the molecular weight of water. Successive loss of hexose units in the mass spectrum of this polysaccharide would give rise to fragment ions whose formula is given by
[(162),
+ 181
n = n - 1, n - 2, n - 3,
...
(3)
Loss of water from the ions in the mass spectrum would give rise to ions of the formula [(162),
+ 18 - (18),J,
n = n, IZ - 1, n - 2 ... m = 1, 2, 3,
... (4)
Ring cleavage of ions in the mass spectrum would give rise to ions of the formula [(162),
+ 18 - (18), + yl n = n - 1, n - 2, n - 3,
...
m = 0, 1, 2,
--.(5)
where Y is the mass of a carbon-containing fragment. Note that the initial value for n is n in formula 4, but n - 1 in formulas 3 and 5. Also note that the initial value for m is 1 in formula 4 but 0 in formula 5. From our recent high-resolution positive ion LDI-FT-ICR experiments performed on underivatized oligosaccharides obtained by bacteriophage degradation of bacterial surface antigens (13), we are able to propose reactions and corresponding structures for reactants and products for eight of the 16 ion series observed by Coates and Wilkins (12). These eight proposed structures/reactions are for the moat frequently occurring high-intensity peaks. In order to minimize confusion between the classical Kochetkov and Chizhov (14, 15) A to K ion series on permethylated glycosides with the CoatesWilkins series, we proposed to label these new ion series from L onward following Kochetkov's convention. Ionization techniques such as FAB induce fragmentation at the glycosidic bonds of oligosaccharides (5,7). LDI-FT-ICR spectra of carbohydrates also show glycosidic bond fragmentation (13). Following Kochetkov's convention, we label this glycosidic bond cleavage of the saccharide chain the L-type fragmentation. Each ion in the Coates-Wilkins B (X= 18) series fits formula 3 of this work and corresponds to the Lo fragmentation route shown in Figure 1. Note that there are two ways in which the Lo cleavage can take place. One way places the ion charge on the chain fragment containing the nonreducing end of the polymer (Figure 1A); the other places the charge on the fragment containing the reducing end (Figure 1B). For polysaccharides that are polymers of hexopyranose residues, these two fragmentation paths give products that cannot be distinguished from each other. This is the case for all of the Coates-Wilkins polysaccharides (12). For other cases, for example, polymers containing some deoxyhexopyranose residues in the main chain, the two paths can be distinguished (13). High molecular weight oligosaccharide fragment ions can easily lose molecules of water, and the L1, L2, and L3 fragmentations are derived from the Lo fragmentations by successive loss of water molecules. L', L2 and L3fragmentations give rise to Coates-Wilkins A (X= 0), Q (X= 144), and P ( X = 126) series ions, respectively, which fit formula 4 of this work. In our prior (13)oligosaccharide work, we noted that the most prominent ring cleavage, shown in Figure 2, occurs between the ring oxygen and C-l, and between C-2 and C-3,to give a two-carbon unsaturated fragment containing C-1 and C-2. This unsaturated moiety is attached via a glycosidic oxygen atom to a fragment that contains the reducing end of oligomer (13). This type of fragmentation route is designated as the M-type fragmentation in Figures 2 and 3. There are
0003-2700/88/0360-2304$01.50/00 1988 American Chemical Society
ANALYTICAL CHEMISTRY, VOL. 60, NO. 20, OCTOBER 15, 1988
2305
Table I. Proposed Structures and Labels, Following Kochetkov's Conventiona proposed label following Kochetkov's convention
Coates-Wilkins seriesb
mass of submonosaccharide fragment, X
LO L' L2
B
18
A
0 144 126 44 60 42 24
Q
P E F D C
L3
M1 M20 Mz' M22
fragment ion formula,
for fragmentation reactions
this work
and structures see Figure
(3) (4) (m = 1) (4) ( m = 2) (4)( m = 3) (5) ( m = 0) ( 5 ) ( m = 0) (5) ( m = 1) (5) ( m = 2)
1
1 1
1 2 3 3 3
Reference 14. bReference12. HOH2C-
A
HOH,C-
HOH2C-
'r?'\. k?
b?
R2034 HO-
H
-----_
L fragmentation
\
HOH,C
3
0
R2 lRO & 0;&
n
HO
n
OH
HO
OH
L fragmentation
Flgwe 1. L-type glycosidic bond cleavage of underivatized glycosides. R, contains the reducing end of the main chain and R, contains the nonreducing end. Path A gives a product ion containing the nonreducing end of the polymer. Path B gives a product ion containing the reducing end. Lo is the label for the above fragmentation. The L' fragmentation in the text is the label for the same reaction but with the addftionai loss of one water molecule from the product. The L2 (L3) fragmentation in the text is the label for the same reactlon but with the additional loss of two (three) water molecules from the product.
QH:b
HOH,C
HO
no
:.-
*.--
OR,
no
M, fragmentanon
Figure 3. Reducing end ring cleavage (M2 fragmentation) of underivatized glycosides. R, contains the reducing end of the main chain and R, contains the nonreducing end. The unsaturated fra ment formula observed in C2H302,as discussed in the text. The M, fragmentation in the text is the label for the same reaction but with the : (M;) additional loss of one water molecule from the product. The M fragmentation in the text is the label for the same reactlon but with the additional loss of two (three) water molecules from the product.
OH
HO
OH
M I fragmentanon
Figure 2. Reducing end ring cleavage (MI fragmentation) of underivatized 2-ilnked glycosides. R, contains the reducing end of the main chain and R2 contains the nonreducing end. The alkene fragment formula observed is C2H90, as discussed in the text.
two different fragment structures derived from this M fragmentation. From our previous work (13), we noted that for a 2-linked rhamnose, the unsaturated fragment formula is C2H30. This type of fragmentation is labeled as the M1 reaction in Figure 2. The M1 fragmentation gave the second most intense ion in the spectrum of our pentasaccharide (13). The Coates-Wilkins E (X= 44) series ions could be formed via an M1 fragmentation. These E series ions were of low intensity and were only observed in white dextrin. White dextrin is derived from starch, a 1,4- and 1,g-linked polymer, by acid treatment. It is known that this treatment causes rearrangements, and the possibility of some 2-linked units in white dextrin is likely (16). Further work will be needed to ascertain exactly how definitively the M1 fragmentation characterizes 2-linked residues. For glycosides that are not 2-linked, the unsaturated fragment formula from ring cleavage is C2H3O2 (13). This ring cleavage reaction is termed the M2 reaction in Figure 3. The Mzofragmentation gives rise to the Coates-Wilkins F series (X= 60) ions. Successive loss of water molecules from the
fragmentation products gives the M21 and the M22 products that correspond to Coates-Wilkins D series (X= 42) and C series (X = 24) ions, respectively. All M fragmentations give products that fit formula 5 of this work. Mass spectra of glycoconjugateshave been reported which show this M2-typefragmentation (8, 17). I t should be noted that while a particular ion formula in the Coates-Wilkins scheme allows us to assign its formation to a specific reaction type, it is understood that the last reaction forming the ion does not necessarily have to be of that type. For example, consider an ion in the Coates-Wilkins C series. As argued above, the formation of this ion can be assigned to a reaction of the M2?- type in which the ion is formed by a type M2cleavage with additional loss of two water molecules. The last reaction forming this ion could have been a type M 2 fragmentation, or it could have been a type L1 fragmentation preceded by a type M2' fragmentation. Similarly, an ion in the Coates-Wilkins P series (X= 126) could have been formed by an L3 reaction or three successive L' reactions. Usually, several combinations of reactions could be written to account for the formation of any particular fragment ion. MS-MS or other experiments, which delineate fragmentation pathways, will be needed to ascertain the precise sequence of reactions leading to each of the observed fragment ions. Table I is a summary of the proposed fragmentation labels named after Kochetkov's convention. This table also shows the correspondence between the empirical Coates-Wilkins formulae (formula 1)and the structurallreadion concepts of this work (formulas 2-5). In conclusion, analysis of polysaccharide LDI-FT-ICR mass spectra should allow for the formation of both L and M series ions. The glycosidic bond cleavage fragmentations (L series) indicate possible primary sequences (13), while the reducing end ring cleavage fragmentations (M series) not only provide tentative assignments of the positions of linkage of the monosaccharide units but also serve to distinguish the reducing end of the oligosaccharide, removing the necessity of a prior reduction step (13). We believe that the characteristic nature of the Coates-Wilkins fingerprint spectra (12),together with M20
2306
Anal. Chem. 1988, 60,2306-2309
the structural and reaction concepts described herein, testifies to the potentid of LDI-FT-ICR for the analysis of carbohydrates.
LITERATURE CITED Sandford, P. A.; Balrd, J. I n The Polysaccharides;Aspinall, G. O., Ed.; Academic: New York, 1983; Vol. 2, 411-490. Matheson, N. K.; McCleary, E. V. I n Ths pOiysacchari&s; Aspinall, G . O., Ed.; Academic: New York, 1984; Vol. 3, 1-105. Aspinall, 0.0.I n The Polysaccherides; Aspinall, G. O., Ed.; Academic: New York, 1982, Voi. 1, 35-131. Bjorndal, H.; Hellerqvlst, C. G.; Llndberg, B.; Svensson, S . Angew. Chem., Int. Ed. Engi. 1970, 9 , 810-616. Morris, H. R.; Dell, A.; Panlco, M.; McDowell, R. A. I n Mass Spechometry h the Health and LMe Scknces; Burllngame, A. L., Castagnoll, N., Jr., Eds.; Elsevler: Amsterdam, 1985. pp 363-378. Dutton, 0.G. S.;Elgendorf, 0. K.; Lam, 2.; Llm, A. V. S. Blomed. Envlmn. Mass Spectrom. 1988, 15, 459-460. Dell, A.; Tiller, P. R. Biochem. Biophys. Res. Commun. 1986, 135, 1126-34. McCrery, D. A.; Gross, M. L. Anal. Chlm. Acta 1985, 178, 91-103. Cody, R. E.; Khslnger, J. A.; Ghaderl, S.;Amster, J. L.; McLafferly,F. W.; Brown, C. E. Anal. Chim. Acta 1985, 178, 43-66. Coates, M. L.; Wllkins, C. L. Biomed. Mass Spectrom. 1985, 72, 424-428. Coates, M. L.; Wllkins, C. L. Biomed. Envifon. Mass Spechom. 1986, 73, 199-204.
(12) Coates, M. L.; Wllkins, C. L. Anal. Chem. 1987, 5 9 , 197-200. (13) Lam, 2.; COmimrOW, M. 6.; Dutton, G. G. S.;Weil, D. A.; Bjarnason, A. RaDid Commun. Mass Soectrom, 1987. 1 . 83-87. (14) Chizhov, 0. S.; Kochetkov.'N. K. Adv. Carbohydr. Chem. Biochem. 1966, 21, 39-93. (15) Lonngren, J.; Svensson, S. Adv. Carbohydr. Chem. Blochem. 1974, 29, 41-106. (16) Greenwood, C. T. I n The Carbohydrates. Pigman, W., Horton, D., Eds.; Academic: New York, 1970; Vol. 28. Chapter 38, p 504. (17) Takayama, K.; Qureshl, N.; Hyver, K.; Honovich, J.; Cotter, R. J.; Mascagni, P.; Schneider, H. J . Biol. Chem. 1988, 267, 10824-10631.
Zamas Lam Melvin B. Comisarow* Guy G. S. Dutton Department of Chemistry University of British Columbia Vancouver, British Columbia Canada V6T 1Y6
RECEIVED for review October 19,1987. Resubmitted June 27, 1988. Accepted July 19,1988. This research was supported by the Natural Sciences and Engineering Research Council of Canada.
Quantitative Determination of Impurities in Polyene Antibiotics: Fourier Transform Raman Spectra of Nystatin, Amphotericin A, and Amphotericin B Sir: Macrolide polyenes such as nystatin, amphotericin A, and amphotericin B represent a class of natural products exhibiting potent antifungal activity. Nystatin, the first of these antibiotics to be isolated, is produced naturally by the bacterial strain Streptomyces noursei (1). Amphotericin A and B are also derived naturally from the related organism Streptomyces nodosus (1);however, the total synthesis of amphotericin B has recently been reported (2). Although a number of chemical studies, as well as NMR, X-ray, and mass spectral data, have been utilized in elucidating the structures of nystatin and amphotericin B (3-51, the structure of amphotericin A has only been c o n f i e d recently (6,7). As shown in Figure 1,the structures of these three antibiotics are similar, with the main differences related to the placement of hydroxyl groups and to the scheme of conjugated double-bond linkages. There is evidence that nystatin A, also exists in the hemiketal form ( 4 ) ) as shown in Figure 1 for amphotericin A and B. On the basis of the amphipathic structure of the polyene antibiotics, it has been suggested that they perform a very important physiological function as channel formers in the membrane matrix (see, for example, ref 8). In order to examine these interesting membrane interactions and to understand generally both the chemical and physicochemical properties of these systems, it is extremely important to know the purities, specifically in terms of the conjugated doublebond groupings, of the antibiotics used. For example, amphotericin B is commercially available in two different preparations that are distinguished only by their relative amounts of impurities (9). The most convenient method for measuring the impurity levels in the macrolide polyenes has been high-performance liquid chromatography (HPLC) (9-11), although microbiological assays are available (IO). Quantitative estimates of purity can be obtained from HPLC provided that the relative detector responses are known for the different compounds. Because polyenes of different lengths of conjugation have UV-vis T - X * electronic absorption maxima at different
wavelengths, single-wavelength detection in HPLC has obvious limitations. For example, tetraenes analogous to nystatin and amphotericin A absorb between 250 and 350 nm, while heptaenes, similar to amphotericin B, absorb between 350 and 450 nm (12). The length of the conjugated system in a polyene governs the vibrational frequencies (13) and intensities of the C=C stretching modes. Since these double-bond structures are highly polarizable moieties, Raman spectroscopy is extraordinarily useful in characterizing these units. The intensities in spontaneous Raman spectra vary linearly with the concentrations of scattering species, and under certain conditions, quantitative measurements can be made (14). Nystatin, amphotericin A, and amphotericin B, however, are highly colored compounds (yellow/orange) and exhibit selective resonance or preresonance enhancement and fluorescent effects when visible or UV laser radiation is used to excite the Raman spectra (15, 16). Since the newly developed technique of Fourier transform (FT) Raman spectroscopy employing near-infrared excitation (1064 nm) offers a method of recording vibrational Raman spectra in the absence of resonance and fluorescence effects (17,18)) the technique offers a spectroscopic approach for characterizing complex mixtures of these conjugated double-bonded species. In this report we apply FT-Raman methods to perform rapidly and easily quantitative measurements of the double-bond impurities in these biologically important antifungal and antitumor compounds.
EXPERIMENTAL SECTION Instrumentation. FT-Raman spectra were collected with a
Bomem, Inc., DA3.02 Fourier transform interferometer operating at 4-cm-I resolution. An external sample compartment was positioned adjacent to the Bomem instrument in order to focus the Raman scattered radiation into the emission port of the interferometer. A 90° scattering geometry was used, and the instrument response function was measured by directing a broad-band source through the Raman collection optics. The
This article not subject to US. Copyright. Published 1988 by the American Chemical Society
