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《天然药物化学》课程教学资源（文献资料）Structure Determination of Natural Products by Mass Spectrometry

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1.INTRODUCTION In the fall of 1945,I entered the University of Innsbruck(Austria),the city I was born in nineteen years earlier,to study pharmacy,a tradition in my family.By the time I finished with a master's degree in February 1948,I had realized that I did not want to spend my life in an apothecary. I had become interested in organic chemistry,so I continued in that field,which was quite easy,because for both disciplines the same laboratory courses,and lectures taught in the same classroom,were required.So I had to take only a few additional courses and exams.and then continue with ate work the only student in that category,because men my age and older either did not surviv World War II or were still in prisoner-of-war camps,a fate I had avoided at my own great risk.A that time,women rarely studied chemistry;they generally went into pharmacy.When I graduated in February 1951 with a PhD in organic chemistry,I was appointed Instructor and taught a course in the analysis of pharmaceuticals;I was also charged with running the organic chemistry laboratory for the chemis try and pharmacy students. My PhD thesis was carried out under the direction of Professor Hermann Bretschneider,an organic che ustry,first in Hungary and then in Germany.He moved rckerWord War Itohead the organicchemisty department theniversity there. He himself had studied in Vienna under Professor Ernst Spath.His research was in the design and synthesis of organic molecules that could be of therapeutic use.Consequently,my graduate work was in the same field,and,following the hierarchical principles of academia at that time, continued so after my graduation. thesis,I be restless aftera few years By chan e,I noticed one day at the dean's off Cambridge that was offering to host voung scientists and engineers over the summer of 1954. The application process was simple,just a personal letter outlining a prospective research project, publications,if any,and proof of an adequate command of the English language.I already had six papers with H.Bretschneider,and,fortunately,had five years of English in high school.Ad- ditignally,I was at that time in the process per Bretschneider's request of translating an organic chemistry tbook from English in My application s approved and at the end of May 1954 I was on my way,by boat fro Rotterdam (The Netherlands),to Boston and Cambridge,via New York.The program at MIT had been conceived and was run by a group of undergraduate students who had spent the final vears of World War l in the armed forces.They had witnessed the devastation of many cities and damage to universities,and thus wanted to help by providing facilities to carry out work that we could not do at home.I was assigned to the research group of Professor George Buichi,an organie chemist trained at the fidg ssische Technische Hochschule,Zurich(Switzerland)and wh research ntere was the structure and syn thesis o about modern instrumentation,such as ultraviolet (UV)and infrarec spectros opy,and about topics like reaction mechanisms.While still in Innsbruck,I discovered the 1954 Annual Congres of the American pharmaceutical association would be held in Boston the following august.so l took a chance and submitted an abstract about my work on the synthesis of a pyridine analog of the antibiotic chloramphenicol (1).To my surprise.it was accepted and I presented my work on August 26. When the ended rted me as a ral fell w un en whe sa exp ired.On ce home my brief stay in America had not had the blessing of Professor Bretschneider,and he practically

AC08CH01-Biemann ARI 10 June 2015 13:25 1. INTRODUCTION In the fall of 1945, I entered the University of Innsbruck (Austria), the city I was born in nineteen years earlier, to study pharmacy, a tradition in my family. By the time I finished with a master’s degree in February 1948, I had realized that I did not want to spend my life in an apothecary. I had become interested in organic chemistry, so I continued in that field, which was quite easy, because for both disciplines the same laboratory courses, and lectures taught in the same classroom, were required. So I had to take only a few additional courses and exams, and then continue with graduate work. I was the only student in that category, because men my age and older either did not survive World War II or were still in prisoner-of-war camps, a fate I had avoided at my own great risk. At that time, women rarely studied chemistry; they generally went into pharmacy. When I graduated in February 1951 with a PhD in organic chemistry, I was appointed Instructor and taught a course in the analysis of pharmaceuticals; I was also charged with running the organic chemistry laboratory for the chemistry and pharmacy students. My PhD thesis was carried out under the direction of Professor Hermann Bretschneider, an organic chemist in the pharmaceutical industry, first in Hungary and then in Germany. He moved to Innsbruck after World War II to head the organic chemistry department at the university there. He himself had studied in Vienna under Professor Ernst Spath. His research was in the design ¨ and synthesis of organic molecules that could be of therapeutic use. Consequently, my graduate work was in the same field, and, following the hierarchical principles of academia at that time, continued so after my graduation. Although this work was interesting and productive, and I learned a great deal of organic synthesis, I became restless after a few years. By chance, I noticed one day at the dean’s office an announcement for a summer program at the Massachusetts Institute of Technology (MIT) in Cambridge that was offering to host young scientists and engineers over the summer of 1954. The application process was simple, just a personal letter outlining a prospective research project, publications, if any, and proof of an adequate command of the English language. I already had six papers with H. Bretschneider, and, fortunately, had five years of English in high school. Additionally, I was at that time in the process, per Bretschneider’s request, of translating an organic chemistry textbook from English into German. My application was approved and at the end of May 1954 I was on my way, by boat from Rotterdam (The Netherlands), to Boston and Cambridge, via New York. The program at MIT had been conceived and was run by a group of undergraduate students who had spent the final years of World War II in the armed forces. They had witnessed the devastation of many cities and damage to universities, and thus wanted to help by providing facilities to carry out work that we could not do at home. I was assigned to the research group of Professor George Buchi, an organic ¨ chemist trained at the Eidgenossische Technische Hochschule, Zurich (Switzerland) and whose ¨ research interest was the structure and synthesis of natural products. During this time, I learned about modern instrumentation, such as ultraviolet (UV) and infrared spectroscopy, and about topics like reaction mechanisms. While still in Innsbruck, I discovered the 1954 Annual Congress of the American Pharmaceutical Association would be held in Boston the following August. So I took a chance and submitted an abstract about my work on the synthesis of a pyridine analog of the antibiotic chloramphenicol (1). To my surprise, it was accepted and I presented my work on August 26. When the MIT program officially ended September 15th, Professor Buchi supported me as a ¨ postdoctoral fellow until the end of November, when my visa expired. Once home, I realized that my brief stay in America had not had the blessing of Professor Bretschneider, and he practically 2 Biemann Annual Rev. Anal. Chem. 2015.8:1-19. Downloaded from www.annualreviews.org Access provided by 45.58.110.168 on 08/27/16. For personal use only

ignored me after my return.I was never asked what I did or learned;I had to continue to put my myacademic career would be what was referred to as the Habl process through which one has to demonstrate independent research.After a while,Bretschneider would permit me to publish some work under my own name,to fulfil these requirements.During my briefstay at MIT,I had learned about the American way of academic life,so different from the autocratic system prevailing at that time in Austria(and Germany).It took me only a few months to decide that I would be better off in the former than in the latter. When Geo orge Buchi heard about my desi e to continue to tudy in the Us he offered me a new postdoctoral position,which Iaccepted an started October1,1955,in the by then familia environment at MIT.The major project I worked on was the synthesis of muscopyridine for the purpose of proving a structure that George had proposed on biogenetic grounds.This compound had been isolated previously from the perfume gland of the musk deer.Such a proof of structure must begin with a compound of known structure and must involve reactions of well-established mechanisms.The svnthesis george had designed involved eleven steps.of which six had been ried out by a graduate ntil he ut of material it wa ugh to earn him a PhD.It was now my job repe. at his work on a larg cale and carry ou the ning five ns.At each step,the product of the reaction has to be isolated,purified,and fully characterized by taking melting points and infrared and UV spectra and burning a few precious milligrams for elemental analysis.The product of the final step was indeed identical to the natural product,thus proving the proposed structure(2). In the fall of 1956,while I was still working on this synthesis,the head of the Department of Chemistry at MIT,Professor Arthur C.Cop an eminent o addan nic chemist analytical divi analysis of pl fnr器t the U with my work in Buchi's lab.He offered me a position as an instructor-at the time the lowest rung of the academic ladder-starting July 1,1957.Although it meant a rather dramatic change from the synthetic chemistry I was trained in,I accepted.I now had to think of an area of research that could benefit from that training but involved analytical chemistry. 2.PEPTIDES,PROTEINS,AND MASS SPECTROMETRY It was just three years since Fred Sanger at the University of Cambridge(United Kingdom)had determined the first structure of a protein,insulin(3).This was accomplished by separating the two disulfide-linked chains,cleaving them by partial acid hydrolysis into a mixture of small peptides. and separating them by paper chromatography.Sanger had developed a method for marking the N-terminal amino group of peptides by reaction with 2,4-dinitro-fuorobenzene.Upon complete acid hydrolvsis,follo red by acids can be ified.For paper chrom A D A or A-C dipep que fora tripeptide,there are can De d for overlaps. Clearly,marking also the C-terminal amino acid would greatly facilitate protein sequencing and simultaneously combine svnthesis with analvsis.It so happened that my final work in Innsbruck involved the synthesis of 3-amino-1,2,4-triazoles from carboxylic acid hydrazides(4).Carrying out this reaction on a peptide hydrazide,produced by partial hydrazinolysis of a protein,would label the C-terminal amino acid with a ver stable marker.Combined with Sange ent,a tripe tide,'A-B-C reas for a te the proposal ww.annualrecieesorg.Strucure Determination of Natural Prodncts 3

AC08CH01-Biemann ARI 10 June 2015 13:25 ignored me after my return. I was never asked what I did or learned; I had to continue to put my nose to the grindstone and just keep quiet. The next step in my academic career would be what was referred to as the Habilitation, a process through which one has to demonstrate independent research. After a while, Bretschneider would permit me to publish some work under my own name, to fulfil these requirements. During my brief stay at MIT, I had learned about the American way of academic life, so different from the autocratic system prevailing at that time in Austria (and Germany). It took me only a few months to decide that I would be better off in the former than in the latter. When George Buchi heard about my desire to continue to study in the US, he offered me a ¨ new postdoctoral position, which I accepted and started October 1, 1955, in the by then familiar environment at MIT. The major project I worked on was the synthesis of muscopyridine for the purpose of proving a structure that George had proposed on biogenetic grounds. This compound had been isolated previously from the perfume gland of the musk deer. Such a proof of structure must begin with a compound of known structure and must involve reactions of well-established mechanisms. The synthesis George had designed involved eleven steps, of which six had been carried out by a graduate student, until he ran out of material. It was enough to earn him a PhD. It was now my job to repeat his work on a larger scale and carry out the remaining five reactions. At each step, the product of the reaction has to be isolated, purified, and fully characterized by taking melting points and infrared and UV spectra and burning a few precious milligrams for elemental analysis. The product of the final step was indeed identical to the natural product, thus proving the proposed structure (2). In the fall of 1956, while I was still working on this synthesis, the head of the Department of Chemistry at MIT, Professor Arthur C. Cope, an eminent organic chemist of his day, decided to add an organic chemist to the analytical division. He knew that I had taught a course in qualitative analysis of pharmaceuticals at the University of Innsbruck, and apparently was quite impressed with my work in Buchi’s lab. He offered me a position as an instructor—at the time the lowest ¨ rung of the academic ladder—starting July 1, 1957. Although it meant a rather dramatic change from the synthetic chemistry I was trained in, I accepted. I now had to think of an area of research that could benefit from that training but involved analytical chemistry. 2. PEPTIDES, PROTEINS, AND MASS SPECTROMETRY It was just three years since Fred Sanger at the University of Cambridge (United Kingdom) had determined the first structure of a protein, insulin (3). This was accomplished by separating the two disulfide-linked chains, cleaving them by partial acid hydrolysis into a mixture of small peptides, and separating them by paper chromatography. Sanger had developed a method for marking the N-terminal amino group of peptides by reaction with 2,4-dinitro-fluorobenzene. Upon complete acid hydrolysis, followed by paper chromatography, the marked and unmarked amino acids can be identified. For a dipeptide, the sequence ∗A-B is unique; for a tripeptide, there are two possibilities, ∗A-B-C or ∗A-C-B, which still can be used for overlaps. Clearly, marking also the C-terminal amino acid would greatly facilitate protein sequencing and simultaneously combine synthesis with analysis. It so happened that my final work in Innsbruck involved the synthesis of 3-amino-1,2,4-triazoles from carboxylic acid hydrazides (4). Carrying out this reaction on a peptide hydrazide, produced by partial hydrazinolysis of a protein, would label the C-terminal amino acid with a very stable marker. Combined with Sanger’s reagent, a tripeptide, ∗A-B-C∗∗, would be uniquely identified, whereas for a tetrapeptide two possible sequences would remain. I prepared an application for a research grant from the NIH on the basis of this proposal. www.annualreviews.org • Structure Determination of Natural Products 3 Annual Rev. Anal. Chem. 2015.8:1-19. Downloaded from www.annualreviews.org Access provided by 45.58.110.168 on 08/27/16. For personal use only

Then something happened that would dramatically change my future career.Firmenich&Cie a preeminent Swiss firm active in flavor and fragrances that also funded my position in Biichi' group,asked me to attend a conference on food flavors to be held in Chicago in late spring 1957 and to provide a report.Although I was not interested in that topic,attending provided me with the opportunity to fly to Chicago.Of the papers presented,one in particular caught my attention:that by William H.Stahl of the research labo of the Us Ar y ouarterma ter corns in natick MA,not far fro as acetone,ethyl butyrate,butylacetate,fruit mass spectrometry (MS).It was done by comparing the mass spectra of the compounds isolated with the spectra of authentic compounds. Once I had sent off my report to Firmenich,I looked up what was known about the mass spectra of organic molecules.Most of it was about hydrocarbon analysis,because the method was widely used in the s on aliphatic alcohols (5). aldehydes(⑥，ker s (7),me ster(⑧，and amines(9,to more imp They all dealt with orrelation be twee nown mass spectra to establish fragmentation rules.It soon became clear to me that mass spectrometry is particularly informative about the structure of linear molecules containing heteroatoms-and peptides are such molecules!But I also had learned that to obtain a mass spectrum,the compound has to be vaporized into the ion source,usually held at 250C at low pressure.However,peptides decompose rather than vaporize upon heating due to their zwitterionic character,which is caused by the pre ence of an acidic and a basic p in the well as nds between the and my training me in handy.The carboxyl group can b e converte the amino group can be acylated,and the carbonyl groups can be reduced to CH2 by lithium aluminum hydride,producing a polyamino alcohol that retains the backbone and position of the side chains of the original peptide(Figure 1). 1.MeOH/HCI 2.Ac20/py R. R LiAID./glyme CHCDNH-CH-CONH-CH-CO3-NH-CH-CD-OH Figure 1 sidechains ofa from Reference 46

AC08CH01-Biemann ARI 10 June 2015 13:25 Then something happened that would dramatically change my future career. Firmenich & Cie., a preeminent Swiss firm active in flavor and fragrances that also funded my position in Buchi’s ¨ group, asked me to attend a conference on food flavors to be held in Chicago in late spring 1957 and to provide a report. Although I was not interested in that topic, attending provided me with the opportunity to fly to Chicago. Of the papers presented, one in particular caught my attention: that by William H. Stahl of the research laboratory of the US Army Quartermaster Corps in Natick, MA, not far from Cambridge. He reported on the identification of simple, small molecules, such as acetone, ethyl butyrate, butyl acetate, etc., in fruit extracts using a method I had never heard of: mass spectrometry (MS). It was done by comparing the mass spectra of the compounds isolated with the spectra of authentic compounds. Once I had sent off my report to Firmenich, I looked up what was known about the mass spectra of organic molecules. Most of it was about hydrocarbon analysis, because the method was widely used in the petroleum industry. However, there were also papers on aliphatic alcohols (5), aldehydes (6), ketones (7), methyl esters (8), and amines (9), to name the more important ones. They all dealt with the correlation between the known structure of reference compounds and their mass spectra to establish fragmentation rules. It soon became clear to me that mass spectrometry is particularly informative about the structure of linear molecules containing heteroatoms—and peptides are such molecules! But I also had learned that to obtain a mass spectrum, the compound has to be vaporized into the ion source, usually held at 250◦C at low pressure. However, peptides decompose rather than vaporize upon heating due to their zwitterionic character, which is caused by the presence of an acidic carboxyl group and a basic amino group in the same molecule, as well as hydrogen bonds between the carbonyl groups and amide hydrogens. Here my training in synthetic organic chemistry came in handy. The carboxyl group can be converted to an ester, the amino group can be acylated, and the carbonyl groups can be reduced to CH2 by lithium aluminum hydride, producing a polyamino alcohol that retains the backbone and position of the side chains of the original peptide (Figure 1). Figure 1 Reaction scheme for the reduction of peptides to polyamino alcohols. LiAlD4 was used to prevent the sidechains of aspartic acid and serine, and of glutamic acid and threonine, from becoming isobaric. Reprinted from Reference 46. 4 Biemann Annual Rev. Anal. Chem. 2015.8:1-19. Downloaded from www.annualreviews.org Access provided by 45.58.110.168 on 08/27/16. For personal use only

A more formidable obstacle was the fact that MIT did not have a mass spectrometer(MS),nor ates at that time.When ,he replied,"It takesa full-time electric I enginee to keep it running."I remarked that I could do this myself and explained my plans.He listene carefully and responded,"Ifyou promise the instrument will not collect dust,I promise to provide the money."We both kept our word.At that time,federal agencies did not easily fund expensive instruments like mass spectrometers,which cost more than S50.000.What I did not know was that Cope had recently negotiated with MI'Ts president James R.Killiana fund of $250,000 to be r the next t ade the chemistry depar tment.In a letter dated July 22,1964. to the then president of MITJulius A.Stratton,professor Cope stated that the purchase of my mass spectrometer was one of the best uses of the funds:"Subsequently he has become recognized as the foremost person in the world in the application of mass spectrometry to the determination ofstructures of organic compounds..."(10).In addition to the $50,000 for the mass spectrometer Firmenich Cie.provided $10,000 with the understanding that I would provide mass spectra of some of its research products,including their interpretation. IorderedaCEC21-103Cma er.the utinely usedin the from Co orporation CE C)in Pasa a,CA,which deliv early May 1958.In the meantime,the NIH grant I had applied for was approved.It allowed changing the approach to a more promising one,so I was permitted to use the funds for the mass spectrometric sequencing of peptides.It also provided funds for a postdoctoral associate,a position I offered to Josef("Sepp")Seibl,who had recently obtained his PhD in Bretschneider's group and whom Iknew well.He accepted and arrived one day before CEC'sengineer Hank DeQuasie began installing the instrum ent.It took six eks,includine g two weeks of instruction for operation nd intena e test.Thi ole irbons. As I was not interested in hydrocarbons,or ir quantitative analyses,I asked that it be a mixture of the corresponding alcohols.CECmanagement at first refused my request,but Hank persuaded them.The experiment worked satisfactorily and we took official possession of the mass spectrometer. A National Science Foundation (NSF)grant I had been approved for also provided a post- up,Fritz Ga who ed a few nths later at which the three of us started our rk o oject.As Ihac the pept d pre cted,the po yaminoalcohol ery go ss spectra and odue to the preferential clevage at the NHCH(R-CH,N This feature was very important,because we could not use the common method of identifying the unknown by comparison with the mass spectrum of a standard reference compound.There are 20 different amino acids in mammalian proteins;as such,there are 400 different dipeptides,8,000 peptides,et Therefore,it was practically impossible to compilea brary of authent di-,tri-,ete nino alcohols for c and we had to in terpret the cratch.A rt c was the first pa er reporting the in peptide (and prot n)ch try.These were suf ently vo to gas chromatography(GC)(12),thus allowing the separation of the complex mixtures expecte from partial hydrolyses of proteins.Over time.the chemical procedure was improved with the use of trifluoroacetylation(13)and silylation(14),which allowed its extension to penta-or even hexapeptides.With the interfacing of the GC to the MS(15)and to an IBM 1800 computer (16), eof心en ss spect also sed for osteocalcin fron chicken b one,a fifty ar o acid】 ong protein. cont s three y-car oxy-glutami which were converted to y-dideutero-glutamic acid before acid or enzymatic hydrolysis(18). ww.Structure Determination of Natural Prodnc

AC08CH01-Biemann ARI 10 June 2015 13:25 A more formidable obstacle was the fact that MIT did not have a mass spectrometer (MS), nor did any academic chemistry department of a university in the United States at that time. When I asked Professor Cope why we did not have one, he replied, “It takes a full-time electrical engineer to keep it running.” I remarked that I could do this myself and explained my plans. He listened carefully and responded, “If you promise the instrument will not collect dust, I promise to provide the money.” We both kept our word. At that time, federal agencies did not easily fund expensive instruments like mass spectrometers, which cost more than $50,000. What I did not know was that Cope had recently negotiated with MIT’s president James R. Killian a fund of $250,000 to be spent over the next ten years to upgrade the chemistry department. In a letter dated July 22, 1964, to the then president of MIT Julius A. Stratton, professor Cope stated that the purchase of my mass spectrometer was one of the best uses of the funds: “Subsequently he has become recognized as the foremost person in the world in the application of mass spectrometry to the determination of structures of organic compounds...” (10). In addition to the $50,000 for the mass spectrometer, Firmenich & Cie. provided $10,000 with the understanding that I would provide mass spectra of some of its research products, including their interpretation. I ordered a CEC 21-103C mass spectrometer, the type routinely used in the petroleum industry, from Consolidated Electrodynamics Corporation (CEC) in Pasadena, CA, which was delivered early May 1958. In the meantime, the NIH grant I had applied for was approved. It allowed changing the approach to a more promising one, so I was permitted to use the funds for the mass spectrometric sequencing of peptides. It also provided funds for a postdoctoral associate, a position I offered to Josef (“Sepp”) Seibl, who had recently obtained his PhD in Bretschneider’s group and whom I knew well. He accepted and arrived one day before CEC’s engineer Hank DeQuasie began installing the instrument. It took six weeks, including two weeks of instruction for operation and maintenance and for running an acceptance test. This was to be the quantitative analysis of a mixture of low–molecular weight hydrocarbons. As I was not interested in hydrocarbons, or in quantitative analyses, I asked that it be a mixture of the corresponding alcohols. CEC management at first refused my request, but Hank persuaded them. The experiment worked satisfactorily and we took official possession of the mass spectrometer. A National Science Foundation (NSF) grant I had been approved for also provided a postdoctoral position. I offered it to another recent graduate from Bretschneider’s group, Fritz Gapp, who arrived a few months later, at which point the three of us started our work on the peptide project. As I had predicted, the polyamino alcohols produced very good mass spectra and sequence information due to the preferential cleavage at the NHCH(R)-CH2NH bonds (Figure 2). This feature was very important, because we could not use the common method of identifying the unknown by comparison with the mass spectrum of a standard reference compound. There are 20 different amino acids in mammalian proteins; as such, there are 400 different dipeptides, 8,000 different tripeptides, etc. Therefore, it was practically impossible to compile a library of authentic di-, tri-, etc., amino alcohols for comparison, and we had to interpret these spectra from scratch. A subsequent short communication (11) was the first paper reporting the use of mass spectrometry in peptide (and protein) chemistry. These derivatives also were sufficiently volatile to be amenable to gas chromatography (GC) (12), thus allowing the separation of the complex mixtures expected from partial hydrolyses of proteins. Over time, the chemical procedure was improved with the use of trifluoroacetylation (13) and silylation (14), which allowed its extension to penta- or even hexapeptides. With the interfacing of the GC to the MS (15) and to an IBM 1800 computer (16), a powerful system was created for determining, finally, the first primary structure of a protein, subunit I of monellin, entirely by mass spectrometry (17). It was then also used for osteocalcin from chicken bone, a fifty amino acid long protein, which contains three γ-carboxy-glutamic acids which were converted to γ-dideutero-glutamic acid before acid or enzymatic hydrolysis (18). www.annualreviews.org • Structure Determination of Natural Products 5 Annual Rev. Anal. Chem. 2015.8:1-19. Downloaded from www.annualreviews.org Access provided by 45.58.110.168 on 08/27/16. For personal use only

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