Four Decades of Structure Determination by Mass Spectrometry: from Alkaloids to Heparin
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View metadata, citation and similar papers at core.ac.uk brought to you by CORE provided by Elsevier - Publisher Connector Four Decades of Structure Determination by Mass Spectrometry: From Alkaloids to Heparin Klaus Biemann Department of Chemistry, Massachusetts Institute of Technology, Cambridge, Massachusetts, USA The early (1950’s and 1960’s) use of mass spectrometry in natural products chemistry and its evolution to the present significance in biochemistry is recounted. This methodology allowed the facile and speedy determination of the structure of a number of indole alkaloids, such as sarpagine, quebrachamine, and two groups isolated from the roots of Aspidosperma quebracho blanco. At the same time, the first strategy for the sequencing of small peptides by mass spectrometry was demonstrated. It slowly advanced, over a period of two decades, to an important alternative of the ubiquitous automated Edman degradation. Further advances in methodology and instrumentation established mass spectrometry as today’s indispensable tool for the characterization of proteins in biochemistry and biology. A new concept of the ionization of highly acidic compounds as the protonated complexes with basic peptides, which allows the accurate determination of the molecular weights of the former, a highly sensitive method for the sequencing of heparin fragments and related sulfated glycosaminoglycans was developed more recently. (J Am Soc Mass Spectrom 2002, 13, 1254-1272) © 2002 American Society for Mass Spectrometry n the 1950’s the most vigorously pursued research pounds and establishing their identity using elemental efforts in organic chemistry concerned the study of analysis (by combustion) and “mixed melting point” Ithe structure and chemistry of natural products and [the melting point of a mixture of two identical com- the elucidation of the detailed mechanism of chemical pounds is the same as that of each of the components, reactions. Both were fostered by developments before, while it is depressed if two different compounds of during, and after World War II. At that time rational same melting point are mixed], the gold standard of the drug design was still a relatively unknown concept. time. The understanding of reaction mechanisms in Instead, a search for pharmacologically and medically vitro had led to the formulation of biosynthetic path- useful plant products was on all over the world, espe- ways in vivo, which aided in the postulation of struc- cially in the tropics. Many of these were alkaloids, tures that then could be proven by conventional means. because they could be most easily isolated by aqueous Ultraviolet spectra were used routinely for the detection acid from an otherwise complex plant extract. of chromophores, particularly aromatic substitution An early success was an alkaloid isolated in 1932 patterns and infrared spectra came slowly into use. from the root of Rauwolfia serpentina and named reser- pine [1]. Its structure was not determined until 12 years Alkaloids later [2]. Reserpine was one of the first antihypertensive drugs and became a huge financial success for CIBA The circumstances that led me to choose mass spectro- (Basel, Switzerland) in the early 1950’s. Research labo- metry—a field at that time well established in physics, ratories in the pharmaceutical industry and academia the petroleum industry, and then analytical chemistry, raced to duplicate this achievement and shelves began but virtually unknown among organic chemists—as my to fill with alkaloids, which had names and melting area of research have been described earlier [3]. In 1957, points, but unfortunately, mostly lacked useful pharma- when I made the transition from a synthetic organic cological activity. chemist to instructor (in those days the bottom rung of The determination of the structure of these complex the tenure-track at MIT) in analytical chemistry, I had molecules (aronatic-alicyclic, about 20 carbon atoms, been considering the determination of the structure of two nitrogens and a few oxygens) was tedious, time sarpagine, an indole-alkaloid also isolated from R. ser- and material-consuming. At that time (1950’s) it gener- pentina. ally required degradation or conversion to known com- However, before I could design a proper strategy, three research groups independently suggested [to limit the number of references listed in this historical paper, Published online September 17, 2002 Address reprint requests to Dr. K. Biemann, Department of Chemistry, MIT, the interested reader is referred to further details Cambridge, MA 02139-4307, USA. E-mail: [email protected] quoted in those cited here] structure 1. The aromatic © 2002 American Society for Mass Spectrometry. Published by Elsevier Science Inc. Received April 22, 2002 1044-0305/02/$20.00 Revised July 1, 2002 PII S1044-0305(02)00441-5 Accepted July 1, 2002 J Am Soc Mass Spectrom 2002, 13, 1254-1272 FOUR DECADES OF STRUCTURE DETERMINATION 1255 Figure 1. Mass spectra of 3 from sarpagine, 2 from ajmaline, and of ibogaine (4) and ibogamine (5) (reproduced from Reference [6], copyright 1960, with permission of Elsevier Science). 1256 BIEMANN J Am Soc Mass Spectrom 2002, 13, 1254-1272 portion was supported by UV-spectroscopy, but the two alkaloids (Figure 1) indeed showed an identical alicyclic system was chiefly based upon biogenetic pattern (with a shift of 30 u, CH3O vesus H), which is, considerations. One of the papers [4] promised to however, very different from that exhibited by 2 and 3. provide final proof of the structure by conversion of Having thus demonstrated the validity of the new sarpagine to 2, which can be made from another alka- concept, which later became known as the “mass spec- loid, ajmaline, of known structure [5]. No such report trometric shift technique”, it was enticing to look for appeared, probably because the conversion of 1 to 2 other examples, preferably indole alkaloids, for which would be quite tedious, requiring the removal of an structures had been proposed but final proof was aromatic and an aliphatic hydroxyl, introduction of a difficult by conventional methods. One such case was methyl group at the indole-nitrogen, and reduction of a quebrachamine isolated long ago [8] from Aspidosperma double bond. The product would have to be purified to quebracho blanco. It was known to contain an indole- constant melting point, followed by the proof of iden- system unsubstituted on its benzene ring and nitrogen, tity with 2 by mixed melting point. and a tertiary amino group. However, there was no It occurred to me that this correlation could be functionality which could provide a site suitable for accomplished much easier and requiring much less selective degradation. The more drastic “zinc dust dis- material by comparison of the mass spectra of two tillation”, another procedure in the tool chest of the analogous, but not necessarily identical compounds. classical organic chemist, led to a complex mixture of Rather than removing the aromatic hydroxyl group, alkyl indoles and alkyl pyridines [9]. They were te- which is very difficult, it was simply methylated; the diously purified by crystallization as picrates and iden- aliphatic hydroxyl group was removed by tosylation tified by elemental analysis, melting points, and mixed and reduction, and the double bond was catalytically melting points when authentic samples were available. hydrogenated to yield 3. Fortunately, the comparison As one of the earliest examples of a proton-NMR Compound 2 was easier to come by: It required only a spectrum used in this field, the presence of an unsub- quick trip up the river to Professor Robert B. Wood- stituted indole was corroborated, but a previously sug- ward’s laboratory at Harvard where it was sitting on gested N-methyl group could be ruled out [10]. The lack the shelf, left over from earlier work [5]. of resolution and extensive chemical shift data at that The mass spectra of 2 and 3 (Figure 1) exhibited a time did not allow the unraveling of the alicyclic remarkably similar pattern, shifted of course, in the m/z system. On the basis of all these rather meager data, values of the peaks due to the different substituents at the aromatic system (methoxyl versus methyl ϭ 16). Witkop et al. suggested structures 6a or b for que- Thus, what a sizeable Swiss research group could not brachamine [11]. A complete qualitative and quantita- do in a couple of years, was accomplished [6] in a few tive analysis of the pyridine mixture was required to weeks by the use of mass spectrometry and a few differentiate these two possibilities and would have milligrams of sarpagine kindly provided by Dr. A. needed a much larger amount of the alkaloid than was Hofman. available. It should be noted that these spectra were obtained Therefore, we repeated the Zn-dust distillation of by electron ionization. In contrast to today’s widely quebrachamine on a very small scale (13 mg). The mass used “soft” ionization (CI, FAB, ESI, and MALDI) this spectra of the pyridine fraction, after separation on a “hard” ionization generates molecular radical cations of packed GC column and collection of each peak, re- high excess energy which fragment extensively and vealed that the most abundant product by far was very reproducibly. 3-ethylpyridine, while 3-methyl-5-ethylpyridine, which Because this proof of structure was novel and un- had supported Structure 6b was a very minor component. precedented, it had to be shown that another isomer of It could well have arisen by thermal rearrangement from different carbon skeleton exhibits a different mass spec- 6a during the harsh conditions of the experiment. This trum which is, however, specific for that type. Fortu- structure is closely related to aspidospermine, the major nately, Dr. William I. Taylor (CIBA, Summit, NJ) had alkaloid of Aspidosperma quebracho blanco. Its structure such a pair at hand: Ibogaine (4) and ibogamine (5), the (7a) had just been determined by x-ray crystallography structures of which he had determined shortly before [12].