sign in sign up
<<
Home , Eduard Buchner, Emil Fischer

REVIEWS

100 Years “Schlussel-Schloss-Prinzip” : What Made Use this Analogy?**

Frieder W. Lichtenthaler*

Emil Fischer’s famous lock-and-key structive reasoning and the thought pat- others--he rather expounded on the analogy (Schliissel-Schloss-Prinzip) for terns underlying the fundamental in- scope of the lock-and-key picture: ‘‘I am the specifity of action has pro- sight. Accordingly, this account at- far from placing this hypothesis side by vided successive generations of scientists tempts to trace how Fischer was led to side to the established theories of our with a mental picture of molecular the lock-and-key analogy, based on the science, and readily admit. that it can recognition processes, and thus has state of knowledge and the views pre- only be thoroughly tested, when we are shaped to a marked degree the develop- vailing at the time. It reveals that Fi- able to isolate the in a pure ment not only of organic , but, scher, who had a pronounced tendency state and thus investigate their configu- through its extension to basic live pro- against any sort of theoretical specula- ration.” Others, most notably P. Ehrlich cesses, that of biology and medicine as tion, refrained from taking this meta- und F. Lillie, by introduction of the con- well. The hundredth anniversary of the phor any further, that is to the obvious cept of stereocomplementarity into first use of this most fertile metaphor extensions of what turns the key, and medicine and biology, induced the lock- provides a welcome opportunity not what kind of doors are then opened. Ex- and-key analogy to become something only for highlighting its paramount im- cept for a small refinement -the differ- of a dogma for explaining principal portance, but for gaining an under- entiation of main key and special keys to processes. standing and appreciation of the cre- account for the fact that some can ative processes involved, of the con- ferment a larger number of than

Id1 halte Lehr-e und Studium der historischm from oblivion, but for gaining an understanding and apprecia- Enttvicklung drr Wissenschafi fur zmenthrhrlich. . . . tion of the creative processes involved, of the thought patterns Unsere Lehrhiicher versageri darin. underlying the fundamental insight, and the constructive rea- Richard Willstitter”’ soning that eventually led to it. A comprehension of these fac- tors appears to be required to get a true measure of the magni- Emil Fischer’s famous lock-and-key analogy for the specifity tude and significance of Fischer’s basic contribution. of enzyme action has provided successive generations of scien- Any attempt-after a 100 years-to trace what led Fischer to tists with their mental picture of molecular recognition pro- the lock-and-key analogy, must go back to the state of knowl- cesses, and. thus has shaped to a marked degree the develop- edge and the views pevailing at the time, that is around 1890, ment not only of organic chemistry, but, by extension to basic and to the scientific school from which Fischer emerged. In life processes, that of biology and medicine as well. 1871, he had entered the , where he attended Fischer’s seminal paper in which he first used the lock-and- lectures by A. Kekule and R. Clausius. yet, in the following year key metaphor appeared in Berichte d~rDeutschen Clirmischen transferred to the University of Strassburg to study with Adolf Gesrllschuft of 1894.[21Thus, a century has passed away since Baeyer, earning his doctorate with him in 1874 at the age of 22. and accordingly, this provides a unique opportunity to com- A year later. while working already independently in Baeyer’s memorate the 100th anniversary of this most fertile hypothe- laboratory, he accidentally discovered phenylhydra~ine‘~] sis-not only for historical purposes or for keeping pivotal facts which was to become the key reagent for his exploration of the . when, ten years later, he finally applied it to the then [*I Prof. Dr. F. W. Lichtenthaler Institut fur Organische Chemie der Technischen Hochschule existing Petersenstrasse 22. D-64287 Darmstadt (FRG) The research school of Adolf Baeyer (1835-1917[51), from Telefax: lnt. code + (61511166674 which Fischer emerged -first in Strassburg, and then for [**I Based on a CommemoraLive Lecture presented at the European Research Conference “: 100 Years Schloss-Schlusscl-Prin- 40 years after 1875 at the University of -was ii major zip’.. Mainz. August 12, 1994. “forge” of talent. A group photograph[61 of 1878 (Fig. 1) 100 Years “Schlussel-Schloss-Prinzip” .REVIEWS

Fig. 1. Photograph of the Baeyer group in early 1878 at the laboratory of the University of Munich (room for combustion analysis). wlth inscriptions from Fischer’s hand [6] attests to that almost literally: the unusually wide hood in the the left Jacob Volhard (1834-1910), who was in charge of the background is certainly more reminiscent of a forge than of a analytical division in Baeyer’s institute, and whose successor laboratory. In the center Adolf Baeyer, wearing a prominent Fischer was to become in Munich a year later (1879), and at the hat; since several others also wear headgear, we may deduce that University of Erlangen in 1882. in the winter of 1878 the heating was deficient in that laboratory. Fischer, at Munich, pursued several classical organic research To the right of Baeyer the 25-year-old Emil Fischer, in a peaked topics : the phenylhydrazones of acetaldehyde, benzaldehyde. cap and strikingly self-confident three years after his Ph.D.; to and furfural were unequivocally characterized and structurally

Frieder W Lichtenthaler, born 1932 in Heidelberg, studied chemistry at the Universitj. of Heidelberg from i952-1956 and received his doctorate there in i959 under E Cramer ,for research on enol phosphates. Thefollowing three years he spent as a postdoctoralfelloiti at the University of California, Berkeley, with Hermann 0. L. Fischer-the only of Emil Fischer’s sons who survived thefirst World War. He subsequently worked as an assistant at the Technische Hochschule Darmstadt, where he acquired his “Habilitation” in i963, was appointed associate professor in 1968, and was promoted to,fullprofessor in 1972. His research activities center on the generation of enantiopure building blocks from sugars, their utilization in the synthesis of’ oligosaccharides and complex non-carbohydrate natural products, the computer simulution of chemical and biological properties of sugars, and studies towardk the utilization of carbohy- drates as organic raw materials.

Aii,qJii Clitwi Irir Ed Engl 1994, 33. 2364-2374 2365 REVIEWS F. W Lichtenthaler secured;"] over a number of years (1876-1880) hedid extensive "During the winter semester of 1876/1877 I again was in investigations on rosaniline dyes with his cousin Otto Fischer Strassburg, and there, through Dr. Albert Fitz, a wealthy (Fig. 1. far left. sitting).['] and in 1881 he started work on puri- winegrower from the Palatinate, was introduced to the book nes. investigating the structure of ,lgl research that even- of Pasteur "Etudes sur la biere". that had just appeared. tually led to his classification of the . In 1882 at the age Therein, this ingenious researcher had laid down his experi- of 30, he moved from Munich to Erlangen, accepting the chair ences on the contamination of beer- by other microor- of chemistry at that university, and there he was intensely occu- canisms and their harmful effect on the quality of the beer. pied with the conversion of into N-heterocy- When I reported on this to my father, he urged me to study cles,""] which led to the Fischer synthesis." 'I It was in this subject very thoroughly, which I gladly did since it inter- Erlangen in 1884, that is after having left Baeyer's sphere of ested me scientifically. A fine microscope was immediately influence for over two years, that he began his studies on sugars, acquired, and with the help of Dr. Fitz and the botanist Prof. by reaction of those that were known at the time (. fruc- de Bary I made studies on moulds. sprouts, and yeasts. from tose, galactose, maltose. sucrose, and lactose) with phenylhy- which I later profited immensely in my investigations of the dra~ine.'~.21 The and osazones obtained thereby sugars. For the time being, however. I had to make practical have not only rendered invaluable service for the identification use of this new knowledge. and isolation of the then existing sugars, but also have been Accordingly, I moved with my microscope to Dortmund for instrumental in the preparation of new ones. In 1888 Fischer several weeks, to train the workers of the brewery in the new had moved to the University of Wiirzburg by then -he discov- identification procedures. Presumably. I was the first ered a new in this way:"3] gentle oxidation of inannitol in who attempted this, and have to admit, that I with nitric acid gave a mixture which could not be characterized was met with substantial distrust by the men. They made as such, but on exposure to phenylhydrazine afforded a crys- every effort to lead me astray with false statements on the talline phenylhydrazone, isomeric with the one generated from origin and the quality of the yeast under examination. They glucose (Fig. 2). By the became more serious-minded though after I could find out, acid hydrolysis of this with the help of the microscope, those yeast types that were mannitol product, an as yet un- spoiled. Yet. I did not succeed in instructing any of the men known hexose was ob- in the correct use of the microscope." IINO* 1 tained. which he named mannose. Through these activities. Fischer obviously had developed a mannose glucose It is in this stage of. keen interest in the subject, because he remarks: "The chemistry II+ T 1PhNIINIi~ j, I.hNHNH2 Fischer's purely chemi- of yeasts interested me so highly, that 1 certainly would have cal-synthetic studies of done own research in this field had I stayed longer in Strass- phenylhydrazonc phenylhydrarone sugars, in the first burg."["] m.p. I RX 'C m.p. 144-14s~ of four papers with Seen in this context. it was a quite obvious move for Fischer Hirschberger on man- (Fig. 3) to test whether the newly prepared hexose, of which the \ PhEIIINH, nose.[13- 151 that we find. rather unpre- phenylglucosazone paredly, the lapidary m.p. 204 'C sentence:Ii3] "Mannose Fig. 2. Synthesis of mannose from niannitol is avidly ferlnented by in 1888 [13] iooii thercafter to he discmered in nature [16]. beer yeast at room tem- perature even in strong- ly diluted aqueous solu- tion." For Fischer, however. it was not a peculiar. remote thing to incorporate yeast into his investigations. since he had devel- oped a curiosity in yeast as a youth already - sparked by the entrepreneurship of his father. Laurens Fischer was a successful businessman. and in 1870-Emil was 18 by then-he invested a large amount of money in the foundation of a beer brewery in Dortmund. an enterprise that was later turned FiS. 3. Emil Fischer (1852- 1919) in into a stock company, the "Dortmunder Aktienbrauerei" of 1x89 [18]. today; Laurens Fischer was chairman of the board for several decades. In the winter of 187611 877, Emil Fischer spent three months set of reactions summarized in Figure 2 had shown it to be the at the University of Strassburg on Baeyer's suggestion obvi- 2-epimer of glucose, would also be fermented by yeast. Similar- ously, since he held the position of an assistant at his Munich ly. when racemic sugars became available by his investigations institute to acquire more expertise in quantitative analysis of the formose reaction, it became standard practice to expose in the laboratory of Prof. Rose. A delightful passage of them to "ordinary beer yeast" for evaluation of their fer- Fischer's autobiography elaborates on his encounter with yeast mentability. Thus, besides proving that D-mannose indeed there :I ' 'I formed ethanol on yeast fermentation (Fig. 4).[15] it was estab-

2366 100 Years “Schlussel-Schloss-Prinzip” REVIEWS

I) - mannose C02 + ethanol [13,15] clarity to the field. To Fischer it was the incentive for now D, L-fNctose L-fNctose [19,201 venturing into topics of much higher complexity, that is into mannose nose P L-mannose POI biological phenomena :rZ91 D, L-glucose - [.-glucose [211 I), L-galactose P L-galactose PI “After the classification of the monosaccharides has essential- 1)-gulose -X- ~31 ly been concluded by the establishment of their configura- i.-gulose -X- ~41 tional formulae, it is now obvious to utilize the experiences, u-munnu-heptose -X- ~251 which have led to this goal, for the purposes of biological u-glrrco- heptose -X&+ 1261 research.” Fig. 4. Fischer’\ early observations (1888- 1892) on the fermentation of sugars with beer yemt [27] Following the early observations on the fermentability of sug- ars (Fig. 4), which had more the character of orientative tests than carefully planned experiments, Fischer apparently realized lished that in the case of racemic glucose, mannose, galactose, that the ordinary brewer’s yeast (“gewohnliche Brauereihefe”) and , only the D component was devored, allowing the he had been using was not pure, and that therefore the results isolation and characterization (as hydrazones and osazones) of could be misleading. So he made, together with Hans Thier- the corresponding L-sugars. felder,[301 a comparative study of natural and synthetic The study of the fermentation of these sugars was a by- monosaccharides with respect to their behavior towards various product of his synthetic work, until, at the end of 1891, he had families of yeast. This resulted in a landmark paper in the proceeded so far as to have reached the goal: the relative config- “Berichte” of 1894.[311Fischer, thereby, was in the fortunate urations of the sugars had been unravelled. This proof not only position, that his studies had left him with a rich stock of put carbohydrate chemistry on a rational basis but-more im- rare sugars--nowhere else in chemistry was such a fine invento- portantly for that time-provided unequivocal proof for the ry of isomers available-yet some of these were only accessible validity of the Le Bel-van’t Hoff theory of stereoisomerism.[281 in small amounts.[3‘1 It became the basis for the sugar family tree (Fig. 5) as it is-I 00 years later-in our textbooks today. “Since the preparation of the artifical sugars is in part quite The completion of this most remarkable, classic piece of laborious and the experiments had to be varied frequently we work, accomplished by ingeniously planned organic syntheses have used a small fermentation tube of the form shown below and brilliant mathematical reasoning had brought order and to save material” (Fig. 6).

CHO CHO CHO CHO CHO CHO CHO CHO I I I I I I ! I HCOH HOCH HCOH HOCH HCOH HOCH HCOH HOCH I I I I I I I I HCOH HCOH HOCH HOCH HCOH HCOH HOCH HOCH I I I I I I I I HCOH HCOH HCOH HCOH HOCH HOCH HOCH HOCH I I I I I I I I HCOH HCOH HCOH HCOH HCOH HCOH HCOH HCOH I I I I I I I 1 CHzOH CHzOH CHzOH CHiOH CHzOH CHzOH CHiOH CHzOH o-dose D-altr ose o-glucose o-mannose o-gulose o-idose 0-g 01 act 0 se 0-1 OlD Se t b CHO CHO CHO CHO I I I HCOH HOCH HCOH HOCH I I I HCOH HCOH HOCH HOCH I I I HCOH HCOH HCOH HCOH I I , I CHzOH CHzOH CHzOH CHIOH

o- o-arabinose o-xylose o-lyxose * t 4 t CHO CHO I I HCOH HOCH - I I HCOH HCOH I I CH20H CHzOH o-erythrose o-threose CHO t I 1 HCOH I 1 CHzOH o-glycerinaldehyde Fig. 5. The sugar family tree of ~-aldoses. h,qe’h‘. Chnti. Itif. Ed. Eng/. 1994. 33. 2364-2374 2367 REVIEWS F. W. Lichtenthaler

tose, and, to a lesser extent d-galactose resemble d-glucose, as- does sucrose (“Rohrzucker”) and maltose, whilst one of the yeasts (“Milchzuckerhefe”) fermented sucrose and lactose (“Milchzucker”), yet left maltose untouched. All of the yeasts were indifferent towards a variety of synthetic sugars. Fig. 0. Semimicro scale assay for the fermentation Fischer seemed to be particularly intrigued by the fact, that ~f sugars by yeaTts [311 in original size. a = d-talose, the 2-epimer of galactose, was not fermented (Fig. 7). Ferment;ition flask. b = S-trap for CO, generated, c = aqueous Ba(OH),. Example: 70 mg sugar in since he notes:1311 0.35 mL H20, 0.35 mL aqueous yeast abstract; stcriliiation, addition of 13 mg of a pure yeast spe- “d-Talose relates configurationally to rl-galactose as does cies. 3 lOd iit 24-2X’C. d-mannose to d-glucose. As d-galactose already ferments less readily than the two others, any further small change in geometry eliminates fermentability altogether.” This microscale fermentation assay was quite elaborate for thc time, allowing one to work with 70 mg of sugar; the bulb holding the sample has a volume of about 1 mL only. It is COH COH COH COH interesting to perceive today Fischer’s keen sense for meticulous H-~.OHHO.~.H H.~.OH ~0.L.i~ observations:‘.’ I I HO-~-HHO.C.H ~0.6.~IIO L.H ”In all cases. even when the sugar is not fermented, a small H.~.OH II-L-OH rto.6 H riu.C: 11 amount of evolves, which covers the surface of H.k-OH H.6 OH H.kOtI H k.OlC I I I the barium hydroxide with a thin layer of carbonate. Since CH, . OH CH, OH dH, OH CI1,OH this phenomenon occurs even when no sugar has been added to the solution, it is obviously caused by the small amount of d-glucose d-mannose d-galactose d-talose carbohydrate present in the yeast itself or the extract. +++ +++ + - The situation is quite different, when the material is readily Fig. 7. Fermentability of hexoses by yeast [31]. fermentable: the barium hydroxide is not only becoming strongly turbid, but is neutralized. Interincdiate c:iscs are these, where material has to be brought Considerations such as these led to the cautious rationaliza- into ;I fcrmentablc state first, as with the glucosides; fermen- tion, “that the yeast cells with their asymmetrically formed tation proceeds slowly . . . ., yet here too, the amount of agent are capable of attacking only those sugars of which the carbon dioxide developed is always large enough, that one geometrical form does not differ too widely from that of d-glu- cannot be in doubt about the real occurrence of fermenta- co~e.’”~‘1 Ii o ti .” On extending this inquiry to natural and artificial glucosides, Fischer found that these materials arrange themselves into dis- Obscrvations of this sort led to the data collected in Scheme 1, tinct groups with respect to their behavior towards air-dried ;I rcproduction from the first’”’of four papers to appear on the yeast extract and the aqueous extract of bitter almonds (“invert- sub.iect in the second half of 1894:‘’~ 29. 31. 321 d-mannose. d-fruc- in” and “emulsin”, respectively). Although both were later shown to be crude mixtures of enzymes, the former only cleaved - .- a-glucosidic linkages, whereas the other, just as specifically. only hydrolyzed fl-glucosides (Table 1). ti Y The second of these four 1894 papers on yeast fermentation J carries the unassuming title “influence of the configuration on -6: -: s. Pastorianus I . . ttt ttt ttt ttt ttt s. Paltorianua I1 . ttt ttt tt ttt ttt S. Pastoriaow 1x1 . ttt ttt ttt ttt ttt Table 1. Fermentability of glycosides [2, 321. S. cerevisiae I . . . ttt ttt ttt ttt ttt S. elllpsoidcus I . . ttt ttt tt ttt ttt Glycoside Yeast eniyme Ernulsin [bl S. elbpsoideus XI . . ttt ttt t ttt ttt (invertin) (a] S. Memianus . . , ttt ttt ttt ttt ttt S. mcmbransefsclca~ __- - - Brsucreihefe . . . . ttt ttt ttt ttt ttt methyl-a-o-glucoside Brennereihefe . . . ttt tti t ttt ttt ethyl-a-u-glucoside S. productivull. . . ttt ttt - t ttt saccharose MUchzuckerhefc . - . tt ttt t ttt maltose methyl-a-L-glucoside bedniirt kcinr ReduWor drr Prlilingrhru Lorvng nach 8 TLgen. dX, rohtundigc VcrgiWng. methyl-a-D-mannoside tf ,, eioc pnz rbr.che Reduktloo nsch 8 Tagen. methyl-rr-u-galactoside dx, 1-1 v~mdi*evcrgilung. ethyl-a-o-galactoside t ,, dauthrbe Rcauktlen ~.CL 8 lagen. .kr un- rrdclhafte Gi-. methyl-8-u-glucoside - ,, hint Gimoy. phenyl-p-D-glucoside Sclicnic I. 1kIi;ivior cil.\ug;irs towards pure yeasts (from ref. [31]). ttt denotes no methyl-8-D-galactoside icduclioti III’ the l’rhling’s wlutioii ;ilier X days, thua complete fermentation. tt lactose dciiolch vcry ucah icduction ;tfter X days, thus almost complete fermentation. t dciiokh sipnilicmit reduction ;iflcr X day.;. but undoubted fermentation. -denotes [a] Aqueous extract of air-dried beer yeast (Succhrrromyw.s wrcwsirrc, type Froh- no lcrnlclll~itioil. berg). [b] Aqueous extract of bitter almonds.

3.368 Angrw. Chrm. In!. Ed. Et1:NRI. 1994. .?.?, 23h4 2374 100 Years “Schlussel-Schloss-Prinzip” REVIEWS

545. Emil Fischer: Einfluss der Configuration Emil Fischer left many contributions of great brilliance in the auf die Wirkung der Enzyme. annals of science: [ Aus dem I. Berliner UnivenitLts-Laboratorium.1 (Vorgetragen in der Sitzuog vom Verfasser.) the unravelment of the sugar configurations as the classical piece of exact mathematical reasoning in any experimental Das verschiedene Verhalten der stereoisomeren Hexosen gegen science“ 2l Hefe hat Thierfelder iind micb zu derHypothese gefuhrt, dass die activen chemiscben Agentien der Hefezelle nur in diejenigeo Zucker the classification of purines[331and the synthesis of the first eingreifeLl kijnnen, mit denen sie eine rerwandte Configuration he nucle~sides[~~~ sitzen 1). the laying of the chemical and biological foundation of Diese stereochemisehe Auffassung des Gibrprocesses 113~4880 Wabrscbeiuliehkeit gewinnen , wenn es mBglicb war, ihnliche Ver- chemistry by his extensive work on amino acids, pep- schiedeiibeiten aucb bei den vom Organismus abtrennbaren Fermenteo, tides, and ,[351 den sogenauuteri Eutymen, feetzustellen. the first unifying concept on the structures of the complex nae ist mir inin in unzweideutiger Weise zuniicbst fiir zwei glu- cosidspaltende Enzyme, das lurertin und Emulsin, gelungen. Die natural products depsides and Mittel dazu boten die kiinstlichen Glucoside, welche nach den1 roii mir a~fgefundeiieti Verfahren aus den verscbiedenen Zuckern und den But here, an analogy, a metaphor, almost casually thrown in Alkoboleii in grosser Zahl bereitet werden kBnnen I). Zum Vergleich the end of a paper, develops a life of its own. to become one wurden aber auch niehrere natiirlicbe Producte der aromatischen Reibe und ebenso einige Polyssccharide, welclie ich als die Gluco- of the most frequently invoked concepts of the past 100 years. side der Zucker selbst hetrachte, in den Rreis der Untersuchung Apparently, the lock-and-key analogy met a conceptual need of gezogen. Das Ergebnivs derselben liisst sich iu deu Satz zusammen- the time, for within a very short period it formed an interpace fas8er1, dass die Wirkuug der heiden Enzyme in auffallender Weise von der Configuration des ~lucosidmolekulsabbingig ist. between chemistry, biology, and medicine very much to the surprise of Fischer himself, since he did not expound on it. Versucbe mit Invertin. Particularly, he refrained from going any further-at least in Das Enzym lisst sich bekanntlich nus der Rierhefe mit Wasser auslaugen und sol1 nus der LKsung diirch Alkohol unverindert ge- print-although I am sure, in his thoughts, he must have taken fillt werdeo. Aua den spiitrr sngefiihrten Griinden hahe ich auf die this picture to the obvious questions, what turns the key, and Isolirun~desselben verzichtet. Die nachfolgenden Versuche siad viel- what kind of doors are then opened. The only extension to be mehr direct mit einer klar filtrirten Liisung angestellt, welche durch 15 atuodige Digestion vnn 1 Theil lufttrockener Hierhefe (Saccharo- found in print, in an extensive 43-page review on his investiga- mS-cee cerevisiae, Typus Frohberg, Reincoltur) mit 15 Theilen Wasser tions on sugars of 1894, is a small refinement:[291 bei 30-35O bereitet war. “The action of enzymes involves a far-reaching chemical pro- 3 Diese Berichte 27, 20.36. cess which takes place readily or not at all. Here, apparently, Fig. 8. Title page of the second [2] of Fischer’s four landmark papers in 1894 on the the geometrical structure exerts such a profound influence on influence of the configuration on the action of enzymes. the playing of the chemical affinities, that it appeared legiti- mate to me to compare the interacting molecules with key and lock. the action of enzymes” (Fig. 8), reporting some of these results If one wants to do justice to the fact, that some yeasts can in a very sober, purely scientific diction.12JTowards the end-as ferment a larger number of hexoses than others the picture usually found in the majority of Fischer’s publications-he may be completed by the differentiation of main key and gives clear indications on what he is to do next: incorporation special keys.” of other enzymes into the study, such as glucase, ptyalin, myros- in. and the ferments of pancreas, and their extension to the rare It was obvious to apply the concept of lock-and-key comple- oligosaccharides, as for example isomaltose, turanose, melibiose mentarity to the question of asymmetric synthesis in plants, and melitriose (Fig. 9). Then, very much towards the end of this most notably to the process of assimilation. Along the way of paper, in the coda quasi, in musical terms, Fischer tries to sum the gradually unfolding interrelationships between the sugars, up and rationalize the observations available. The resulting sec- Fischer, in 1889, had made another key discovery that was to tion contains the crucial metaphor: have major bearing on biological questions. He uncovered the phenomenon of asymmetric synthesis:[371the cyanohydrin ex- “The restricted action of the enzymes on glucosides may tension of natural L-arabinose does not only give L-mannonic therefore be explained by the assumption that only in the case acid on hydrolysis, as Kiliani had previously shown,[381but a of similar geometrical structure can the molecules so closely second product, the 2-epimeric L-gluconic acid, as evidenced by approach each other as to initiate a chemical action. To use a their distinctly different, well-crystallizing phenylhydrazides : picture I would like to say that enzyme and glucoside have to fit together like lock and key in order to exert a chemical effect on each other. The finding that the activity of enzymes is COOH COOH limited by molecular geometry to so marked a degree, should CHO H-LOH HO-LH be of some use in physiological research. Still more important H-b-OH LHCK HA-OH H-LOH though appears to me the proof, that the previously assumed HO-LH2,H+ HOLH’ HO-~-H difference between the chemical activity of a cell and the HO-LH H~-H HO-C!-H mode of action of chemical reagents is, factually, non-exis- &H,OH iH,OH ~H,OH tent .” L21 L-arabinose L-mannonic acid L-gluconic acid

Angeii Clwii. Inl. Ed. Enxl. 1994. 33, 2364-2374 2369 REVIEWS F. W. Lichtenthaler

As it turned out. this is the first example of an asymmetric CHIOH CH@H CHZOH synthesis recorded in the literature, on which Fischer comment- LO HO-~H H-t-OH ed in the following way:'""] HO-~-H NrHg HO-~-H HO--C-H H-i-oH H-~-OH HA-OH '-The simultaneous formation of the two stereoisomeric prod- ___. H-&--OH H-C-OH H-t-0~ ucts on the addition of hydrogen cyanide to , which ~H~OH tH@H CH~OH was observed here for the first time. is quite remarkable in theory a5 well 21s in practice." This first example of an asymmetric synthesis was soon to be followed by a second case. since sodium amalgam reduction of Four years later. in one of these 1894 papers.['"] the biological D-fructose gave rise to two stereoisomeric products. namely D- significance of these sober chemical findings had been fully real- mannitol and u-sorbitol.[""] Fischer clearly realized the basic ized and applied to assimilation by invoking the lock-and-key importance of this result: concept: "The reduction of fructose is the second reaction in the sugar "It seems to me that this concept offers a simple solution for group. which gcneraks two stereoisomeric products due to the enigma of natural asymmetric synthesis. According to the the formation of an asymmetric carbon atom. The same phe- plant physiologists, carbohydrate formation takes place in nomenon will undoubtedly be observed much more frequent- the chlorophyll granule, which itself consists entirely of opti- ly in thc future. and most probably will be generally found cally active substances. I can imagine that the formation of with all compounds that are asymmetric a priori." carbohydrates is preceeded by the generation of a compound

2370 100 Years “Schlussel-Schloss-Prinzip” REVIEWS

of carbonic acid or formaldehyde with those substances; and This had led to the hypothesis, that there must be ii similarity that then. due to the already existing asymmetry of the entire in the molecular configuration between the enzymes and their molecule. the condensation to the sugars takes place in an object of attack. if reaction is to take place. To make this asymmetric f ash’ ion too. thought more perspicuous, I have used the picture of lock and Their asymmetry can thus be readily explained by the nature key. of the material from which they were produced. Of course. I am far from placing this hypothesis side by side to the they also provide the material for new chlorophyll granules established theories of our science, and readily admit, that it which. in turn, produce active sugar. In this manner, the op- can only be thoroughly tested. when we are able to isolate the tical activity propagates from molecule to molecule. as life enzymes in a pure state and thus investigate their confiyra- itself does from cell to cell. Hence, it is not necessary to at- tion,,-[4fi1 tribute thc formation of optically active substances in the Paul Ehrlich, for example. from 1897 on. introduced the lock- plant to asymmetric forces outside the organism. as Pasteur and-key complementarity into the then young disciplinc of im- had supposed. The origin rather lies in the structure of the munology through his so-called “side chain theory of immuni- chlorophyll granule that generates the sugar, and with this ty”, as illustrated in a publication from 1900[4‘1(Fig. 10): each conccption the difference between natural and artificial syn- cell possesses a number of side chains. which bind toxins in ;t thesis is completely eliminated.” lock-and-key type manner. The binding of such toxins c;i uses In a lecture of 1894 on “The chemistry of carbohydrates and the overproliferation of that particular side chain somc of which their iinportance for physiology” he again advocated this view are set free from the cells as antibodies. In the case of diseases in morc gcncral that leave immunity there are so many frec “side chains” (anti- bodies) in the blood that appreciable fixations at thc ccll cannot ”Whoever wants to conclusively elucidate the process of as- occur. similation, will have to tackle the more special question. why the plant exclusively generates optically active sugar whilst chemical synthesis leads to the inactive products. This con- trast appeared so fundamental to Pasteur, who created the precept of molecular asymmetry, that he considered the gen- eration of active substances to be a privilege of the organism. The progress of science has deprived the highly respected lifeforcc of even this last hiding-place: for we are now in a position to artificially prepare such active substances without any assistance from a living organism.” With these words, Fischer had clearly repudiated the accepted view of‘ the time-asserted by Pasteur-that fermentation is inextricably tied to living cells, wherein a “vis vitalis” was sup- posedly operating. is usually credited to have demonstrated in 1897[421that fermentation can occur outside living cells, thus unequivocally refuting Pasteur’s view. The abovc passage of Fischer in 1894 proves, that he had arrived at this conclusion already three years earlier. In 1894, when Fischer first used the lock-and-key analogy to

illustrate enzyme specifity, he was 42. He lived for another FIB.10. Ehrlich’s side chain theory of itntnunit) as illustr:itt.d in 1900 I371 25 years, during which time he published the imposing number of over 400 further However, he referred to the lock- and-key concept only in another three: in his Nobel lecture in The lock-and-key complementarity also gained headway in 1902, rather incidentally,[441in his Faraday lecture at the Uni- embryology. particularly from 1914 on, when Lillic. at the Uni- versity of London in 1907.[451also quite cursorily. and, at the versity of Chicago, invoked it to describe recognition between end of an extensive, 28-page review of 1898, with the momen- sperm and He crystallized his idcas on the interaction tous title “Significance of Stereochemistry for Physiology.” of the components involved into an explicit lock-and-key dia- Therein,[“’] Fischer apparently felt that he had to state the scope gram (Fig. 11). a dangerously elaborate concept in view of the of the analogy he had proposed, because others were taking it few secure experimental data available then. too far: Accordingly, Ehrlich had brought stereoconiplenientarit) “The reasons for these phenomena are in all probability to be from the realm of chemical reactions in solution to reactions on found in the asymmetric structure of the enzyme molecule. the cell surface, whilst Lillie and othersL4y1extended it to cell-- Although one does not know these substances in a pure state, cell interactions. So, the first two decades following Fischer’s their similarity with proteins is so close and their generation use of the lock-and-key analogy saw a rather free. uncontrolled from these so probable, that they have undoubtedly to be proliferation of the concept from chemistry into medicine and considered as optically active, and, hence, asymmetric molec- biology-and, along the way. its use became more and morc ular forms. speculative.

’371 REVIEWS F. W Lichtenthaler

over, so many limited heads have now jumped on this ques- Symbols tion, that the delight thereon is spoiled completely.” The second example refers to the question still open around 1914 on the ring sizes of the fructose and glucose portions of sperm sucrose (formulations see Fig. 12). and how these sugars are d receptor foreign sperm combining group ‘CH 0’ CHsOH

spermophile 7group fertilizin a ovophile /CH CHaO H group 0./’ CH;;;;‘O-C 6 anti-fertilizin \:;OH /CHOH E. Fischer, 1893 O CHOH 4 egg cHOH ‘GH receptor CH,OH CH, OH

8 blO?d inhibitor

Fig. 1 I. Lillie‘s theory of fertilization as diagrammed in 1914 [48]: segment 1 shows B. Tollens, 1914 the siruation before fertilization; in segment 2. the sperm receptor binds to the apermophile group of “fertilizin”, activating the ovophile group to bind to the egg receptor. Molecules of antifertilizin combine competltively with this site on other fertilizin molecules to forbid the binding of other sperm. The other segments refer to experiments not relevant to the discussion here. Fig. 12. Structural representations of sucrose by Tollens [53, 551 and Fischer [54]. The passage of time corrects many a distortion of perspective. Both theories survived only insofar as they allowed recognition linked. Fischer clearly states his position,[5z1which may right- of the complex relationship in very general terms, and hence, fully be extended to the lock-and-key picture : had a favorable influence on the concretization of research ac- tivities. Wherever they were used for too detailed analyses, they “We know nothing definite on the mode, how the fructose failed, obviously because the ground of scientifically established residue is linked in cane sugar, thus leaving huge room for facts was left too far behind in the quest to explain phenomena speculation. I, however, gladly renounce to use it.” much too complex as to yield to rationalization or comprehen- sion at the time. Unlike these theories from Ehrlich, Lillie. and others along similar veins,[491Fischer’s lock-and-key analogy still stands in the annals of science-a 100 years later-as a most fertile con- cept. Maybe, because it was unspecified in its details, thus leav- ing ample room for the imagination of , biologists, and medical researchers alike. Fischer had an unfailing intuitive perception for identifying important areas of research in organic chemistry and brought unsurpassed creativity to the conception of experiments and their skilful execution. The most striking feature of Fischer’s scientific personality may be found in his pronounced tendency against any sort of theoretical speculation. Two instances may document this attitude further. one concerning the Walden in- version, to which Fischer had contributed[501and which was controversially discussed around 19t2. In a letter to T. W. Richard~,”~]Fischer writes: “I do not derive much pleasure from theoretical things. The occupation with the Walden inversion was rather a digression and recuperation from the extensive work on proteins. More- Fig. 13. Emil Fischer around the turn of the century.

2312 Angar. Clwm. In(. Ed. Eiigt. 1994. 33. 2364-2374 100 Years "Schliissel-Schloss-Prinzip" REVIEWS

In concluding this centennial tribute to one of the really great figures of our science (Figs. 13, 14) and to the lock-and-key concept with which he had a major influence on interrelating chemistry. biology, and medicine, I would like to cite a passage from his 1907 Faraday lecture at the University of London, entitled "Synthetic Chemistry in Relation to Biology", in which he clearly states his conviction to give in one's theoretical rea- sonings expression only to observed f;dcts:[451

"The separation of organic chemistry from biology was nec- essary during the past century while experimental methods were being elaborated; now, that our science is provided with a powerful armory of analytical and synthetical weapons, chemists can once more renew the alliance both to its own honor and to the advantage of biology. The prospect of ob- taining a clearer insight into the wondrous series of processes which constitute animal and vegetable life may well lead or- ganic chemistry and biology to work with definite purpose to a common end. In order, as far as possible, to avoid mistakes in this difficult task and to shield ourselves from the disappointment which is the inevitable consequence of exaggerated hopes, we cannot do better than strive to imitate the great example of Faraday, who always. with rare acumen, directed his attention to actual phenomena without allowing himself to be influenced by pre- conceived opinion, and who in his theoretical conceptions gave expression only to observed facts." Fig. 14. Emil Fischer around 1910 in his laboratory at the Univer~ity of (61. This attitude with respect to the interpretation of experimen- tal results applies to our science today as much as it did a measure of how far we should go with our interpretations today. 100 years ago. Especially in the field of molecular recognition and what we should leave for the next 100 years

which is in a very active phase of its development, we should Recci\ed- August 25. 1994 [A X0 IE] comply with it most rigorously, as it gives us an unfailing German version: Aii,yeii . C/ieiii. 1994. 106. 2456

~

[I] R. Willstiitter. .-1m meinem Leben, Verlag Cheniie, Weinheim. 1973. p. 324 "I [9] E. Fischer. Bcr. Drscii Chm. Gcs. 1881. 14. 637-644. 1905 1915: ihrd 1882. considcr the teaching and stud) of the historical development of science as 1.7. 29-33. 453 -456; Jir.stir.s Lkhig.~,411n C/IN~. 1882. 213. 1; 320. essrnti,il. Our textbooks fail in this respect." 1101 E Fischer. F. Jourdan, Brr. Dr,sc/i. Chcwi. Gr.s 1883, 16. 2241 ~2215:L [2] E. Fischer, Ber. Dtsrh. Clieni. Ge.s. 1894, 27, 2985-2993. Fischer. 0. Hcss. ;hid 1884. 17. 559- 568: E. Fischer. .Jii.\rirs I.rdyqc ,firii. [3] E. Fischer. Ber. D/,di. Clieiii. Ges. 1875, 8. 589-599, G. 9. Kauffman, R. P Chrm. 1886. 236, 116--126 Ciul,~.J Cliem Ed~ic1977, 54. 295. [l I] P. A. Roussel, J Cheiir. Ediic. 1953. 30. 122 125; 9. Robinson. ('/Iw. Kci [4] E. Fischer, Bev Dts

1986. 25. 297-311 F. W. Lichtenthaler, Aiiqw. C/lcm. 1992. 104. 1577 ~ 1593: d,~gr~.Chwi liri

161 a) Thc original and numerous other photographs arecontained in the Collec- Ed EirXI. 1992. 3/, 154 I - 1556. tion (11' Eniil Fischer Papers, housed in the Bdncroft Library at the University [13] E. Fischer. J. Hirschhergcr, Bo.. D(\clr. C/imi. &\. 1888, 2/. 1x05 IXO') olC ahrornia, Berkeley. This xientitic bequest consists of34 boxes of material: 1141 E. Fischer, J Hirschhci-gel-. Brr. Dt,sdi. Chi?.Grs. 1889. 73. 765 376. I155 lab tiotes. manuscripts. drafts of his autobiography "Aus mcinem Leben", and 1156. about 5000 lettei-s. that reached Fischer during his 40-year scientific career; foi- [lj] E. Fischer. J. Hirschberger, Bw. Dtscli C/icvn. Gcs. 1889. 72. 3218 3214 example. IS7 letters during 1889- 1915 from his teacher A von Baeyer, others [th] R. Gans, W E Stone, B. Tollens. Ber. Dtscli. (%em Grs. 1888.71. 214X 2152. from F Beilstein (11). A. Hantsch (56). F. Haber (35). I. H. van't Hoff (31). R. Gatis. B Tollens. Jcr.ws Lid~ig.5Anif. (%eiii. 1888, 249. 245 257; R. K~N. A. Lk Hofmann (5). A. von Kekuli (6). H. Kiliani (16). V Meyer (46). W. Ber. Dt&cli. Clwni. Gr& 1889. 22. 609-613. Nernst (67). 9. Tollens (20). P. Walden (13). 0. W;illach (46). and R. Will- [17] E. Fiseher. Ar4.r manmi Lrhen. Springei-. Berlin. 1987. pp 18 70 and 70 stittcr (33). Flscher's intense interaction with industry IS documented by (Reprint of the original 1922 YCI-sion.edited by M. Bergmatin. \+,ii11 an English 181 lettersfrom C. Duisherg(frorn 1895-1919),from Bayer(Z71). BASF(54). commentary by B. Witkop). Boehringer Mannhem (( 16). Hoechst (50).and Merck (33) The papers even- [I81 Picture from the congratulatory album of pliotogmphs pre\ented 10 August tii.iI1) reached Berkeley through Fischer's son. Hermann Otto Laurens Fischer von Kekule on the occasion of hi5 60th birthday (September 6. 1x89). The [6h] who carried thiscollection through all the stations of his unusually cosnio- original document is in thc collection of Kckt112 pxpers. iii ihc lii~t~t~itLbr politsn carcer: Privatdozent in Berlin, Professor of Pharmaceutical Chemistt-y Organische Chcmie. Technischc Hochschule Darmstadt !n Basel. Dit-ector of the Banting Institute. Toronto, and Professor of Biochem- [I91 E. Fischer. J. Wfd, B

1878. IW. 1x4- 188. 1231 E. Fischer. R. Stahel, Bw Dr.sc/i. C%P~GPS. 1891. 34. 52X ~ 539. E 0. Ber. Disdi C'hem. Ge\. 1876. 9,891 -900. Jlrjtrr~Liehigr [Sl Fischer. Fischer, 1241 E Fischer. 0. Piloty. Bei. Dtsclr. Climii. Gr.5. 1891. 24. 571 ~ 537. hi?.Chriii 1878, 194. 242-303; BPI..Dtsch. Climi. Ges. 1880, 13. 2204-2207. 1251 E. Fischer. F. Passmore. Bet.. Df.dt. C/XWI, Gas. 1890, 2.1. 212h--239.

Ailgtaii.. Chein. 111i. Ed Gig/. 1994, 33, 2364-2374 3373 REVIEWS F. W. Lichtenthaler

[26] E. Fischer. Justu.\ Licd~igsAnn. Cheni. 1892, 270, 64-107. [39] E. Fischer, Ber. D15ch. Clicwi. GES.1890. 23, 2134-2143; quotation from [27] a) In the Figures 4 and 5 the affiliation of the individual sugars to the D- or p. 2334. r-series has already been made. It should be noted though. that Fischer only (401 E. Fischer, Ber. Dtsdi. Chwn GPJ.1890. 23. 368443687, introduced the symbols d and I as late as 1890 [27 b] (cf. Scheme 1 and Fig. 7). [41] E. Fischer, Lecture given on August 2. 1894 in Berlin. on the occasion of the deriving their meaning from the sign of rotation. Rosanoff in 1906 [27c], and foundation day of the School of Military Physicians. A. Hirschwald Verlag,

Wohl and Freudenberg in 1923 [27d] brought the use of the dand I symbols on Berlin 1894, 1 - 36; quotation from p. 30. il logical. genetic basis by selecting the enantiomeric glyceraldehydes as points [42] E. Buchner. R. Rapp. Ber. Dtsch. Chrnl. Gcr. 1897, 30. 2668-2678. of reference. such that any sugar belongs to the d-series, if it can be derived 14.11 a) Fischer. from about 1896 on. shifted his main research efforts to the from ri-glyceraldehyde by successive Kiliani - Fischer syntheses. The present systematic exploitation of peptldes [35), purines [33].and tannins [36]. Yet he we of the D- and r-notation [27e] started around 1940: b) E. Fischer. Ber. retained a keen interest in sugars [43 b] and their action uith enzymes; his last Dfsch. Chem. G~A.1890. 23. 370-394; c) M. A. Rosanoff. J. Am. Chem. Sue. two papers [43c], received by the editorial office of Hoppe-Sqh-’.s Zritschrifi 1906,ZR. 114- 121: d) A. Wohl. K. Freudenberg. Ber. Dlsch. Chern. Gas. 1923. fur phnio/og/.sc/ie Chemra on July 14. 1919. the day of his death. dealt with 56. 309-313; e) Rules of Carbohydrate Nomenclature. No. 4 and 5 in The sugars, the former being entitled “Einfluss der Struktur der P-Glukoside aufdie Cirrhn/?~drut~~.x,Vol. llB (Eds.: W. Pigman. D. Horton). Academic Press. New Wirkung des Emulsins.” b) E. Fischer. C’izrer.wc/~zingmirhrr Kohlenhjdrure und

York. 1970, p. 809ff. Fwmrntc 11 (1908 ~ 1919). Springer. Berlin. 1922. 543 pages; c) E. Fischer. [2X] Aside of ref. [12]. there are several pertinent accounts of Fischer’s conquest of Huppr-Sc$er’.i Z. Phv~iol.Chrm 1919, 107, 176-202; ihid. 1919. 108, 3-8. the sugar configurations, for example C. S. Hudson, “Emil Fischer’s Discovery [44] Nobel Lccturm Inchirling Prrsrntutwn Spwchrs mid Luurrutrs B~ogruphies, of the Configuration of Glucose”. J. Chem. Educ. 1941. 18, 353-357; “Emil Chrmisfrj, 1901-1921 (Ed.: Nobel Foundation). Elsevier. Amsterdam. 1966, Fischer and His Contribution to Carbohydrate Chemistry”. K. Freudenberg, pp. 15- 39. Adi’. Curhohdr. Chem. 1966. 21. 1-38. [45] E. Fischer, Faraday Lecture on ”Synthetical Chemistry In its Relation to Biol- [2Y] E. Fischer. Brr. DISC^. Cheni. Ges. 1894, 27. 3189-3232; quotation from ogy”. J Chrm. So<. (London) 1907, 91. 1747-1765. p. 3228. 1461 “Bedeutung der Stereochemie fur die Physiologie”: E. Fischer, Hoppe-S 1301 Hans Thierfelder (1858-1930) earned hisdoctorate (Dr. med.) at the Universi- Z. PhJ,sio/.Chrm. 1898, 26. 60-87; quotation from p. 82.’83. ty of Rostock (1884). then joined Hoppe-Seyler’s group at the University of 1471 ”On Immunit>. with Special Reference to Cell Life”: P Ehrlich. Proc. R. Sue. StrdSSbUrg. where he habilitated in 1887 with work on the chemistry and London. 1900, 66. 424-448: quotation from p. 433. of glucuronic acid. With Hoppe-Seyler he coedited the 6th edition [48] “The Mechanism of Fertilization in Arbacia”: F. R. Lillie. J. Exp. Zoo/. 1914.

of the “Hundhuch der ph~~sio/ogi.~ch-und purho/ogiseh-r/ien?ischeu-Anu/~s~~” 16. 523 ~ 590. (1x93). In 1891. he accepted a position at the hygienic institute of the Univer- [49] “Intellectual Traditions in the Life Sciences: Stereocomplementarity”: S. F. sity of Berlin. where he came into contact with Emil Fischer. that eventually led Gilbert. J. P Greenberg, Pcr.~p.Bid Med. 1984, 28, 18-34. to the joint work on the yeasts [31]. From 1909 on. Thierfelder was professor [SO] E. Fischer. Justus Liehigs Ann. Chi 1911, 381, 123-141; &id. 1911. 386. of physiological chemistry at the University of Tubingen. For a biographical 374- 386; hid. 1912. 350-362. memoir. see: E. Klenk, Z. Physiol. Chem. 1931. 203, 1-9. [51] Letter dated November 11.1912. toTheodore William Richards (1868-1928). [31] E. Fischer. H. Thierfelder. Ber. Dlsch. Chem. Gr5. 1894, 27, 2031 -2037. professor at Harvard University. Richards did postdoctoral work with W. [32] E. Fischer. Ber. Dtsch. Chem. Grs. 1894. 27, 3479-3483. Ostwald in Leipzig. and in 1907 had an exchange professorship at Fischer’s 1331 E Fischer. Cintru.xtidwngenin der Puringruppe (1882 - 1906). Springer. Berlin. institute at the University of Berlin. In the Emil Fischer papers at the Bancroft

I 907. Library, Berkeley. CA, there are 39 letters by Rlchards dated 1905 ~ 1915 (orig- [34] E. Fischer. B. Helferich. Bw. Dtsch. Chcm. Crs. 1914. 47, 210 -235. inals) and the responses by Fischer (copies). [35] E. Fisher, C~iiteu.siic/iim~~~~fiihcr Aniinosiiurrn, Po/Jpeptide und Proteine I [52] E. Fischer. Brr. DwA. Cheni. Grs. 1914. 47, 1980- 1989; quotation from (18Y9-19015). Springer. Berlin, 1906; I1 (lYU7- 1919) (Ed.: M. Bergmann). p. 19x4. Springer. Berlin. 1923. [53] B. Tollena. Beu. Dtuh. Chrm. Ges. 1883, 16. 921-924; quotation from [36] E. Fischer. L’ntersuchungen iihcr DepAide und Gerhtoffr (1908- 1919). p. 923. Springer, Berlin. 1921. 524 pages. [54] E. Fischer. Ber. DI.\ch. Chrm. GPS.1893. 26. 2400-2412; quotation from [37] E. Fischer. F. Passmore, Ber. Dt~h.Chem. GK. 1889, 22. 2728-2736; E. p. 2405. Fischer. hid. 1890. 23. 261 1-2624. [55] B. Tollens, Kurze.5 Honrlbuch der Kohlenhdrufe. J. A. Barth Verlag, Leipzlg, 13x1 H Kiliani. Bur. Dfsch. Ch(vn. Crs. 1885. 18, 3068-3072: 1886. 19. 211 -227. 1914. p. 363.

2374 Angebi. Chrni Inf. Ed. Engl. 1994. 33, 2364- 2374