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CHEMISTRY OF PQQ
PROEFSCHRIFT
ter verkrijging van de graad van doctor
aan de Technische Universiteit Delft,
op gezag van de Rector Magnificus,
prof. drs. P.A. Schenck,
in het openbaar te verdedigen
ten overstaan van een commissie aangewezen
door het College van Dekanen . - o ^
1op donderdag 18 mei 1989 te 14.00 uur
door
JAAP JONGEJAN
geboren te Dirksland
Scheikundig ingenieur
Krips Repro Meppel
1989
r
Dit proefschrift is goedgekeurd door de promotor Prof. dr. ir. J.A. Duine
Overige leden: Prof. dr. ir. H. van Bekkum
Prof. dr. J.G. Kuenen
Prof. dr. J. Reedijk
Prof. dr. R.A. Sheldon
Dr. R. Wever
Dr. H. van Koningsveld
This study was carried out at the Department of Microbiology and
Enzymology of the University of Technology Delft, The Netherlands
C o n t e n t s
Abbreviations
Preface, PQQ and Quinoproteins, A Narrative Publications
Chapter
I Introduction and Summary page 1
References 10 Quinoproteins (Table 1) 15
II Synthesis of Pyrroloquinoline Quinone 19
Initial attempts 23 Preparation of PQQ according t o Corey
& Tramontano 26
Synthesis of PQQ analogues 30 Experimental part . 34
References and notes 41 III The Structure of PQQ and Related Compounds 45
Materials and Methods 46
Results 47 3-D structure of PQQ 49
Complexes of copper(II) and PQQ SI
Discussion 61 References 66 IV Reactions of PQQ. 69
Chemical methods for the detection and quantification of PQQ
Formation of 'periodate-product' 74 Detection of enzyme-bound PQQ 77 Reaction of PQQ with hydrazines 80 Reaction of PQQ with amino acids 91
Experimental part 95 Appendix: Preparation of cyclopropanol 99
References and notes 102 V Binding and Activity of PQQ 107
Materials and Methods 108
Results 113 Discussion 118 Conclusions 123 References 124 VI Implications and Prospects 127
References 136
A b b r e v a t l o n s PQQ PQQH PQQH2 P Q Q H4 PQ DNPH PH W u r s t e r ' s b l u e DAO M D H MADH PAO D0H GAO LiP NMR EPR MS FAB-MS SDS E D T A HPLC TLC TMS CAN 2,7,9-tricarboxy-lH-pyrrolo[2,3-/\lquinoline-4,5-dione semiquinone o f PQQ quinol of PQQ 4,S-dihydro-2,7,9-tricarboxy-l 7/-pyrrolo[2,3-/]quinoline-4,S-diol 2,7,9-tricarboxy-l i/-pyrrolo[2,3-/]quinoline 2,4-dinitrophenylhydrazine phenylhydrazine
N,N,N',N'-tetramethyl-p-phenylene-diamine (radical form) diamine oxidase (from porcine kidney)
methanol dehydrogenase (from Hyphomicrobium X) methylamine dehydrogenase (from Thiobacillus versutus) plasma amine oxidase (copper-containing)
dopamine 0-hydoxylase (from adrenal medulla) galactose oxidase (from Dactylium dendroides)
'ligninase', lignin peroxidase (from Phanerochaete chrysosporium) nuclear magnetic resonance spectroscopy
electron paramagnetic resonance spectroscopy mass spectroscopy
fast-atom bombardment mass spectroscopy sodium dodecyl sulphate
ethylenediamine-tetra-acetate
high performance liquid chromatography thin-layer chromatography
tetra-methylsilane eerie ammonium nitrate
PREFACE
PQQ a n d Q u i n o p r o t e i n s , A N a r r a t i v e
Cofactors and coenzymes
Textbooks of biochemistry are not famous for their plots, yet they do have an enormous cast. Of all the characters that play a role in the reactions of the living cell, I have learned, both as a student and as a teacher of biochemistry, t o appreciate those biomolecules t h a t are known as cofactors and coenzymes. I can think of several reasons for this preference.
Within the framework of cellular activities cofactors and coenzymes occupy central positions. In their absence, the proteins with which they are normally associated to form functional enzymes, are reduced to helpless entities unable to sustain the reactions of life. By consequence, in most enzymes it is the cofactor or prosthetic group that determines 'where the action is'. Despite their important role in metabolism, several factors are not always available to the (mammalian) organism by biosynthetic routes. These m u s t be provided by dietary intake. This specific nutritional requirement (as a vitamin), has served as a key to their detection and isolation. Other factors were recognized more or less by chance. After almost a century of cofactor and coenzyme research, some may still be hiding out in ecological niches like archae-bacteria, or evade our attention by mimicking the behaviour of established members of the group.
Exept for DNA and proteins, few biomolecules have become public property to the extent of the vitamins. Notably ascorbate (vitamin C) and the B-vitamins, have become widely known, albeit t h a t their beneficial effects have been associated largely with the relief of the common cold. In the scientific community t h e study of these factors has offered a certain degree of 'scientific shelter' to researchers that developed a long-term addiction to a single theme. This is illustrated by flourishing symposium series on flavins, pteridines, pyridoxal phosphate (and, hopefully, PQQ and quinoproteins).
As a group, cofactors and coenzymes represent a variety of chemical reactivities. Yet, most of these molecules possess simple structures, offering a firm foothold for (biomimetic) chemistry. In biochemistry class, they serve to exemplify the pronounced effects of small molecules on cellular metabolism, especially with regard to vitamin deficiency and chemo-therapy. Historical accounts of the detection and elucidation of their structures and functions have allowed a clear illustration of the methodology of classical biochemistry.
Considering these arguments, one might expect research on cofactors and coenzymes to be a major topic of modern biochemistry. This, however, is no longer the case. With due respect, quite a few biochemists consider this subject t o be a closed chapter, fit to judge a textbook by its style but not by its up-dating. The reasons for this situation most probably stem from the fact that conceivably all of the factors involved in well-established (vitamin) deficiency diseases, appear to have been characterized during the 'great days' of vitamin and cofactor research. This era was founded at the turn of the century with the pioneering work of Eykman*) and Grijns on the detection of vitamin B]. It gained momentum with the detection of nicotinamide by Goldberger in 1926, vitamin A by Stepp and Hopkins in 1929 and riboflavin by Kuhn and Warburg in 1933. It came to an end in 19S5 with the s t r u c t u r e elucidation of vitamin Bj2 by Smith and Crowfoot-Hodgkin. Since that time, novel factors have occasionally been isolated from various sources. However, it appeared that either their existence was restricted to specific bacteria, or they lacked a well-defined vitamin character.
Pyrroloquinoline quinone, PQQ
These considerations seemed to be applicable also to the low-molecular weight organic factor present in methanol dehydrogenase of methylotrophic bacteria. From the spectroscopie properties of this enzyme it was deduced t h a t the chromophoric group responsible for the absorbance in the visible part of the spectrum could not be attributed to a known flavin or pteridine derivative. In 1979 several lines of evidence suggested it to be a heterocyclic quinone. The structure elucidation of the compound isolated from acetone-treated enzyme showed these ideas to be correct. Subsequently, conclusive evidence for the structure of this novel cofactor a s tri-carboxy-pyrroloquinoline quinone, PQQ, was obtained by Duine and coworkers in 1980. *) And not Eichmann (!), as reported by H.F. DeLuca(1979), Vitamin D Metabolism
At that time, I was working in the same department on a more or less related subject. According to the prevalent research strategy at, what was then called, the Laboratory of Biochemistry and Biophysics, the detection and proper characterization of small biomolecules was considered to be an attractive approach t o biochemistry. This conception had already led t o some important findings, including the detection of bongkrekic acid as (one of) the toxic principle(s) of Pseudomonas cocovenenans, a bacterium responsible for severe poisoning of processed cocos; and the isolation of thymine dimers from UV-irradiated DNA. Another research topic concerned the chemistry of flavins and closely related pteridines. My contribution t o this subject was to study the mechanism of hydroxylation reactions catalyzed by phenylalanine hydroxylase, an enzyme requiring a tetrahydropteridine cofactor for the activation of dioxygen.
The one-time tentative identification of the cofactor of methanol dehydrogenase as a pteridine derivative, stimulated my interest for this novel cofactor. Shortly after the structure of the cofactor was established, I joined Hans Duine and- Hans Frank in their investigations of this factor. As an organic chemist at heart, the chemical synthesis of this novel compound offered a prime challenge. I set o u t t o investigate a synthetic approach suggested by Dr. de Groot from Wageningen University. The initial steps of this route offered no particular difficulties. So, we expected the total synthesis of PQQ to be only a matter of time... This assumption turned out to be correct: two months later an elegant method was published by Corey and Tramontano.
The appearance of two more synthetic schemes within several months offered small comfort to my frustrations. Yet, it forced us to consider our options. Impressed. by the compact prescriptions of the Corey and Tramontano account, we embarked on the synthesis of PQQ by this method. In another month, a first lot of several milligrams of synthetic PQQ was obtained. Stimulated by this success, we decided to a t t e m p t the synthesis of more substantial amounts. Invthe following two years, we managed to produce several grams of PQQ. However, it soon became clear that the published procedure contained some major bottle-necks, impeding a medium-scale production of PQQ with the over-all 10% yield t h a t could be obtained in small-scale experiments. In view of the rapid developments on the quinoprotein scene, we felt pressed for time and decided to prepare the- amounts of PQQ, needed-to 'seed' what we considered needed-to be a fertile field, by sheer force: disregarding low yields we worked our way through several kilograms of starting material and suc ceeded to make PQQ available to the community of potentially interested researchers.
For the preparation of derivatives of PQQ, this policy was inadequate. However, by reinvestigating the Corey and Tramontano method we were able to devise a more efficient procedure, amenable also to the synthesis of derivatives and analogues of PQQ.
PQQ from microbe to man
To determine the distribution of PQQ and quinoproteins (enzymes containing PQQ) among different species, reliable detection methods were needed. It was soon realized t h a t the o-quinone group that is present in PQQ, is particularly vulnerable to attack by a variety of nucleophiles. Moreover, it appeared that the biological t e s t methods t h a t were suitable for the detection of free PQQ did not respond to the products t h a t resulted from these reactions. We decided to investigate whether chemical detection methods might be of help to overcome this problem. The relatively large amounts of (synthetic) PQQ at our disposal opened the possibility to check a number of promising reactions for this purpose. In most cases, however, it turned out t h a t chemical transformations, leading to unique products with suitable properties when conducted on a milligram-scale, were impractical for the detection of the microgram amounts present in biological samples. Considerable experimentation was required before we succeeded to develop a suitable method. With this so-called 'hydrazine-method' we were able t o detect PQQ not only in enzymes from microbial sources, but also in a (still growing) number of enzymes from higher organisms.
Another aspect of PQQ chemistry concerned the mechanism of action of quino-protein enzymes. It appeared t h a t several quinoquino-protein alcohol dehydrogenases could be specifically inhibited by cyclopropanol. The structure elucidation of the cofactor-cyclopropanol reaction product that was formed upon inactivation of the enzyme, was facilitated considerably by the fact that we were able t o prepare both PQQ and cyclopropanol in sufficient quantities. The preparation of cyclopropanol by a combination of chemical and enzymatic s t e p s , provided a first example of a profitable merging of (fundamentally oriented) quinoprotein research and (practically oriented) enzyme technology in our laboratory. According to the trend that was set by Klibanov and coworkers, we investigated the performance of other enzymes under the conditions that are routinely employed in organic chemistry. Following a fundamental study of the enantioselective hydrolysis of glycidyl esters by Upases, we have recently started t o investigate the potential of 'domestic' quinoproteins for similar conversions.
The future of PQQ and quinoprotein research
The first International Symposium on PQQ and Quinoproteins, September 1988, organized by the Delft group, constituted a milestone marking the first decade of PQQ and quinoprotein research. As expected, results and ideas were exchanged in a highly stimulating atmosphere. However, despite all compliments for our achieve ments, few groups appeared to have adopted the methods we developed for the detection and quantification of PQQ. Thus, further sophistication of t h e s e methods will be required. In the meantime, X-ray diffraction studies on methylamine dehydrogenase from Thiobacillus versutus by the Groningen group, have pointed at the existence of a so-called 'pro-PQQ' cofactor. When these claims can be s u b s t a n tiated, some new and challenging questions will call for an answer. I do not doubt that chemistry will participate in this process.
The question, whether or not PQQ is a vitamin, has yet to be settled. As it should be in scientific research, the outcome of further experiments must be awaited before a conclusion regarding the development and direction of PQQ and quinoprotein research can be drawn. However, if we look a t the impressive list of quinoprotein enzymes from both bacteria and eukaryotic organisms, it is clear that 'PQQ is here to stay'.
Acknowledgements
The large number of contributions from the Delft group to t h e Symposium mentioned above, can be taken as an example of the extensive cooperation and coordination that has occurred within this relatively small group. In fact, joining forces has been our major credo. It is my pleasure to acknowledge the contribution of several colleagues to the present account. Of these, Barend Groen, Rob van der Meer, and Hans Frank deserve special mention. Most of all, though, I wish to thank Hans Duine, who not only set the stage for our joint performance, b u t who also was and is our inspired director. His constant guidance and clear decisions kept me off the numerous tempting side-ways, straying away from the main road t h a t should lead t o the unraveling of the mysteries of PQQ and quinoprotein chemistry. I find myself particularly lucky to have been allowed a part in this exiting process.
P U B L I C A T I O N S
Pteridines and related subjects
Jongejan, J.A., Mager, H.I.X. and Berends, W. (1975), Activation and Transfer of Oxygen - X, A new autoxidative rearrangement of Tetrahydropteridines,
Tetrahedron, 31, 533-540
Jongejan, J.A., Mager, H.I.X. and Berends, W. (1979), Autoxidation of 5-alkyl-tetra-hydropteridines, the oxidation product of 5-methyl-THF, in Chemistry and
Biology of Pteridines (R.L. Kisluik and G.M. Brown, eds) Elsevier/North-Holland,
N.Y. pp. 241-246
Jongejan, J.A., Mager, H.I.X. and Berends, W. (1983), Pyrimidine models for the cofactor of Phenylalanine Hydroxylase, in Chemistry and Biology of Pteridines (JA. Blair, ed) Walter de Gruyter & Co, Berlin, N.Y. pp. 357-361
PQQ and Quinoproteins
Duine, J.A., Frank Jzn, J. and Jongejan, J.A. (1983), Detection and determination of pyrroloquinoline quinone, the coenzyme of quinoproteins, Anal. Biochem., 133, 239-243
van der Graaff, W., Duine, J.A., Frank Jzn, J. and Jongejan, J.A. (1984), Does Tryptophan Side Chain Oxidase from Pseudomonas ATCC 29574 contain a quino-protein?, in Progress in Tryptophan and Serotonin Research (H.G. Schlossberger, W. Kochen, B. Linzen and H. Steinhart, eds) Walter de Gruyter & Co., Berlin, N.Y. pp. 761-764
Duine, J.A., Frank Jr, J., Jongejan, J.A. and Dijkstra, M. (1984), Enzymology of the bacterial methanol oxidation step, in Microbial growth on Cj-compounds,
Proceedings of the 4tn International Symposium (Crawford, R.L. and Hanson,
R.S., eds), pp 91-96
Dijkstra, M., Frank Jzn, J., Jongejan, J.A. and Duine, J.A. (1984), Inactivation of quinoprotein alcohol dehydrogenases with cyclopropane derived suicide substrates,
Lobenstein-Verbeek, CL., Jongejan, J.A., Frank Jzn, J. and Duine, J.A. (1984), Bovine serum amine oxidase: a mammalian enzyme having covalently bound PQQ as prosthetic group, FEBS Lett, 170, 305-309
van Koningsveld, H, Jansen, J.C., Jongejan, J.A., Frank Jzn, J. and Duine, J.A. (1985), Structure of the 2,4-dinitrophenylhydrazine adduct of Pyrroloquinoline Quinone (PQQ) Dimethyl Ethyl triester, C2 4H1 8N601 1, Ada Cryst. C41, 89-92
Duine, J.A., Frank, J. and Jongejan, J.A. (1985), The coenzyme PQQ and Quinoproteins, A novel class of oxidoreductase enzymes, in Proceedings of the 16tn FEBS Congress Part A (Y. Ovchinikov, ed) VNU Science Press, Utrecht, pp. 79-88
Duine, J.A., Frank, J. and Jongejan, J.A. (1986), PQQ and quinoprotein enzymes in microbial oxidations, FEMS Microbiol. Rev., 32, 165-178
Jongejan, J.A., van der Meer, R.A. and Duine, J.A. (1986), A vitamin in disguise?,
Trends Biochem. Sci., 11, 511
van der Meer, R.A., Jongejan, J.A., Frank Jzn, J. and Duine, J.A. (1986), Hydrazone formation of 2,4-dinitrophenylhydrazine with pyrroloquinoline quinone in porcine kidney diamine oxidase, FEBS Lett, 206, 111-114
Jongejan, J.A., van der Meer, R.A., van Zuylen, G.A. and Duine, J.A. (1987) S p e c t r o -photometric studies on pyrroloquinoline quinone-copper(II) complexes as possible models for copper-quinoprotein amine oxidases, Reel. Trav. Chim. Pays-Bas,
106, 365
Duine, J.A., Frank, J. and Jongejan, J.A. (1987), Enzymology of Quinoproteins, Adv.
EnzymoL, 42, 169-212
van der Meer, R.A., Jongejan, J.A. and Duine, J.A. (1987), Phenylhydrazine as probe for cofactor identification in amine oxidoreductases, FEBS Lett, 221, 299-304 Duine, J.A., Jongejan, J.A. and van der Meer, R.A. (1987) Copper-containing amine
oxidases (EC 1.4.3.6) have covalently-bound PQQ and not PLP as organic cofactor, in Biochemistry of Vitamin B5 (Korpela, T., and Christen, P., eds.), Birkhauser Verlag, Basel, pp. 243-2S2
van der Meer, R.A., Jongejan, J.A. and Duine, J.A. (1988), Dopamine (3-hydroxylase from bovine adrenal medulla contains covalently-bound pyrroloquinoline quinone,
FEBS Lett., 231, 303-307
Jongejan, J.A., Bezemer, R.P. and Duine, J.A. (1988), Synthesis of 1 3C - and 2H-labelled
Duine J.A. and Jongejan, J.A. (1989), Quinoproteins, enzymes with pyrrolo-quinoline quinone (PQQ) as cofactor, Anna. Rev. Biochetn., 58, 403-426
Duine, J.A. and Jongejan, J.A. (1989), Pyrroloquinoline Quinone (PQQ): A Novel Redox Cofactor, Vitamins and Hormones, 45, in press
Jongejan, J.A., Groen, B.W. and Duine, J.A.(1989), Structural properties of PQQ involved in the activity of quinohaemoprotein alcohol dehydrogenase from
Pseudomonas testosteroni, in PQQ and Quinoproteins, Proceedings of the 1st International Symposium (Jongejan, J.A. and Duine, J.A., eds), Kluwer Academic
Publishers, Dordrecht, pp. 205-216
van Kleef, M.A.G., Jongejan, J.A. and Duine, J.A. (1989), Factors relevant in the reaction of PQQ with amino acids, Analytical and mechanistic implications, in
PQQ and Quinoproteins, Proceedings of the 1st International Symposium
(Jongejan, J.A. and Duine, J.A., eds), Kluwer Academic Publishers, Dordrecht, pp. 217-226
van Koningsveld, H. and Jongejan, J.A. (1989), The three-dimensional structure of PQQ and related compounds, in PQQ and Quinoproteins, Proceedings of the 1st International Symposium (Jongejan, J.A. and Duine, J.A., eds), Kluwer Academic
Publishers, Dordrecht, pp. 243-251
van der Meer, R.A., Jongejan, J.A. and Duine, J.A. (1989), Identification and quanti fication of PQQ, in PQQ and Quinoproteins, Proceedings of the 1st International Symposium (Jongejan, J.A. and Duine, J.A., eds), Kluwer Academic Publishers,
Dordrecht, pp. 111-122
Jongejan, J.A. and Duine, J.A. (1989) Multigram synthesis of pyrroloquinoline quinone, PQQ, in preparation
Vellieux, F.M.D., Huitema, F, Groendijk, H., Kalk, K.H., Frank Jzn, J., Jongejan, J.A., Duine, J.A., Drenth, J. and Hol, W.G.J. (1989), Structure of Quinoprotein Methyl-amine Dehydrogenase at 2.25 A1 resolution, in preparation
van der Meer, R.A., Jongejan, J.A. and Duine, J.A. (1989) Pyrroloquinoline quinone (PQQ) as cofactor in galactose oxidase (EC 1.1.3.9), J. Biol. Chem., in press van Kleef, M.A.G., Jongejan, J.A., and Duine, J.A. (1989) Factors relevant in the
reaction of PQQ with amino acids, Eur. J. Biochem., submitted
van der Meer, R.A., Mulder, A.C., Jongejan, J.A., and Duine, J.A. (1989) Analysis of covalently bound Pyrroloquinoline quinone (PQQ) and derivatized PQQ with the hexanol extraction procedure, in preparation
van Koningsveld, H., and Jongejan, J.A. (1989) Three-dimensional structure of PQQ and a PQQ-potassium complex, in preparation
Jongejan, J.A., Groen, B.W., and Duine, J.A. (1989) Binding and Activity of PQQ and PQQ analogues, in preparation
Frank Jzn, J., van Krimpen, S.H., Verwiel, P.E.J., Jongejan, J.A., Mulder, A.C., and Duine, J.A. (1989) Eur. J. Biochem., submitted
Application of enzymes in organic synthesis
Jongejan, J.A. and Duine, J.A. (1987), Enzymatic hydrolysis of cyclopropyl acetate, a facile method for medium- and large-scale preparations of cyclopropanol,
Tetrahedron Lett., 28, 2767-2768
Philippi, M.Chr., Jongejan, J.A. and Duine, J.A. (1987), Enantioselective hydrolysis and transesterification of glycidyl butyrate by lipase preparations from porcine pancreas, in Biocatalysis in Organic Media (C. Laane, J. Tramper and M.D. Lilly, eds) Elsevier, A'dam, pp. 279-284
Philippi, M.Chr., Jongejan, J.A. and Duine, J.A. (1987), Enantioselective hydrolysis of glycidydol esters: analytical and enzymatical aspects, in Proc. 4tn European Congress on Biotechnology 1987, Volume 2 (O.M. Neijssel, R.R. van d e r Meer
and K.Ch.A.M. Luyben, eds) Elsevier Science Publishers B.V., A'dam, pp. 281-284 Jongejan, J.A., and Duine, J.A., (1989) Non-Michaelis-Menten kinetics in the enantio
selective hydrolysis of glycidyl esters by lipase from porcine pancreas, in preparation
CHAPTER I
I n t r o d u c t i o n and Summary
Cofactors, Coenzymes and Prosthetic groups
With few exceptions, enzymes are made up of polypeptides. Enzymatic catalysis, resulting from a decrease of the activation energy of the reaction, can be attained by proper positioning and activation of the substrates in the active site of the enzyme. The mechanism of action of e.g. NAD-dependent lactate dehydrogenase can be taken as an example for this type of catalysis [21. Alternatively, the enzyme may contribute to a lowering of the activation energy by providing a third species as a mediator. Such species may be derived directly from the enzyme polypeptide, e.g. a tyrosine residue [3,4], or more common a serine residue; or indirectly by rearrange ment of the polypeptide, e.g. a pyruvoyl group [5,6]. However, in the majority of enzymes that have been described up till now [1 ], this role is performed by certain metal ions or by organic molecules that are either covalently bound t o the poly peptide or that are more or less firmly associated with the protein.
In this respect, some confusion may arise regarding the use of the t e r m s cofactor, coenzyme and prosthetic group. Historically, these terms were coined t o describe low-molecular weight factors for which the enzyme showed an (absolute) dependence (cofactors) and covalently-bound organic compounds undergoing a (reversible) change during the catalytic process (prosthetic groups). According to these notions, both cofactors and prosthetic groups are required in catalytic a m o u n t s , with stoichiome-tries relating to the enzyme species. Coenzymes (from co-zymase [7], the historical precedent), are compounds (co-substrates) that, in the presence of a suitable enzyme, can react with a substrate to lead t o product(s). In this respect, coenzymes are stoichiometrically related to the substrate and product of the enzyme catalyzed reaction*). Today, these terms are used rather loosely [8-10], while a tendency
) Curiously, the (set of) coenzymes originally detected by Harden and Young [7], functioned as cofactors under the conditions of the test.
exists t o label a factor with its more common function; the coenzyme NAD, the
cofactor PQQ. Approximately one-third of the enzymes that have been described
depend for their activity on one of the four common coenzymes, NAD(P), ATP and coenzyme A [11]. Flavins and haems are frequently present as prosthetic groups, while pyridoxal phosphate functions as a cofactor in a number enzymes.
Since 1980, pyrroloquinoline quinone (PQQ) has been established as a cofactor in several enzymes from bacterial origin [26], while evidence has been presented for its presence as a prosthetic group in enzymes from higher organisms [27].
Cofactors, coenzymes and prosthetic groups have been the subjects of numerous chemical investigations. In the early days, chemical (total) synthesis served to ascertain their identity. Later on, the application of more sophisticated physical methods largely obviated the necessity for total synthesis as the ultimate structural proof. In this respect, the total synthesis of cobyric acid, a vitamin B1 2 degradation
product, by the groups of Woodward [12] and of Eschenmoser [13], appears rather redundant, although it may be argued that a major contribution to (theoretical) organic chemistry was gained in the process (the Woodward-Hoffmann rules [14]). The chemistry t h a t is involved in the mechanism of action of the enzyme with which t h e factor is associated, has been a major topic of chemical research. In addition, chemical modification of factors has been used as an approach to unravel their mechanistic role in t h e enzyme, as well as to obtain specific inhibitors for certain enzymes.
Several factors (e.g. thiamine) have been manufactured by chemical synthesis. Analogues (e.g. methotrexate), serving as (specific) inhibitors, were likewise produced by synthetic methods. Design of chemically modified coenzymes, suitable for efficient regeneration during enzymatic turn-over of enzymes in bio-conversions, constitutes a relatively novel field of cofactor and coenzyme chemistry.
Chemistry of Pyrroloquinoline Quinone (PQQ)
The discovery and structure elucidation [15-20] of a novel factor in methanol dehydrogenase from certain methylotrophic bacteria raised some interesting questions regarding its function, mechanism of action and distribution. To answer these questions, knowledge of the chemical and physical properties of PQQ (Figure 1) was required.
H 0
2C
Figure 1. Pyrroloquinoline Quinone, PQQ
Although some important properties could already be established by studying the limited amounts of factor that were obtained from microbial sources [21 ], we reasoned t h a t future investigations might require more substantial amounts of material. This prompted us to investigate the preparation of PQQ by chemical synthesis. When it became clear that the presence of PQQ was not restricted to methanol dehydrogenase (see [22-28] for reviews), chemical methods for its detection and quantification in other enzymes that contained this factor (so-called quino-proteins) were developed [293. In addition, aspects of the chemical reactivity of PQQ, related to the mechanism of quinoprotein catalysis were investigated [303. To gain insight in the structure-activity relationship of PQQ, its three-dimensional s t r u c t u r e (and that of some related compounds) was established [31,323. Anticipating a possible role of PQQ as a vitamin, synthetic methods for the preparation of isotopically labelled PQQ and of PQQ analogues, suitable for studies on distribution and excretion in higher organisms, were developed [33,343.
Current chemistry of pyrroloquinoline quinone by other research groups has been directed at the chemical synthesis of PQQ and PQQ analogues [35-383, t h e role of PQQ in enzymatic catalysis by quinoproteins [39,403, and the development and application of chemical methods for the detection, isolation and quantification of PQQ [41-463. Biosynthesis of PQQ in certain bacteria has recently been shown to involve tyrosine and possibly glutamic acid as precursors [47-493. Several reports concerning the biomimetic chemistry of PQQ have appeared [50-553.
Synthetic aspects of PQQ chemistry
Shortly after we started our investigations on the chemical synthesis of PQQ, an elegant method for the total synthesis of PQQ was described by Corey and Tramontano [56]. Although it appeared that this method was suitable for small-scale (milligram) preparation of PQQ, preparation of PQQ in the amounts (and with the purity) required for our purposes was obstructed by the presence of several bottle-necks in the described procedure. Eventually, we were able to find a solution for these problems, and devise an efficient procedure, suitable for the synthesis of gram-amounts of PQQ with > 40% over-all yield [34].
In order to investigate the incorporation and excretion of PQQ in biological material, e.g. mammalian tissues, the use of isotopically-labelled PQQ could be of great value. Preliminary investigations proved the (revised) method of Corey and Tramontano to be suitable for the preparation of 2H - and ' 3C-labelled PQQ
[25]. Practical methods for the preparation of PQQ labelled with 3H and
(preferably) 1 4C , in non-exchangeable positions, require introduction of isotopically
substituted precursors in the final step(s) of the synthesis. Incorporation of solvent-derived hydrogens a t C(8) of PQQ appeared possible by a novel method for the synthesis of PQQ. The feasibility of this approach could be demonstrated by preparing 8 -2H - and 8-methyl-PQQ [33]. Preparation of 8 -1 4C - P Q Q by this
method, will be attempted in the near future.
The role of the carboxyl groups of PQQ in binding and activity of quinoprotein (apo-)enzymes has been reported [57-59]. To investigate the effect of less conspicuous substituents, several alkyl-substituted PQQ analogues were prepared by the methods mentioned above. Synthesis of N(l)-methyl-PQQ, as described by Itoh and coworkers [35], proved to be of general applicability for the preparation of N(l)-alkylated PQQ derivatives.
Our contribution to the development of chemical synthetic procedures for the preparation of PQQ and PQQ analogues is discussed in Chapter II. Future developments with regard t o PQQ synthesis will be highly dependent on whether or not PQQ turns out to be a vitamin [60]. Compared to other cofactors, notably folic acid and thiamine, PQQ appears to be an equally likely candidate for large-scale production by chemical synthesis. Judged from the (relatively) non-complicated character of the molecule, possessing no chiral centra, while its chemical reactivity is largely restricted to the ortho-quinone moiety, the development of a short and efficient industrial synthesis would seem to be only a matter of dedicated research.
As far as economy is concerned, existing methods appear t o be inadequate mainly because of the number of steps involved [61]. As we show in Chapter II, the use of a-ketoglutaconic acid ester, representing the single expensive starting compound employed in the synthesis of PQQ according t o the method of Corey and Tramontano, can be circumvented by applying pyruvate in a Pfitzinger quinoline synthesis. Ceric ammonium nitrate (CAN), that is used as an (expensive) oxidant in several methods, may be replaced by a potentially cheaper enzymatic peroxidation step [34]. Large-scale preparation of PQQ by microbial methods has been announced (J- Nagai, personal communication). PQQ obtained by fermentation is already marketed by several firms, albeit at a discouragingly high price.
The structure of PQQ and related compounds
Although the chemical structure of PQQ was already well established in 1981, by the work of Forrest and coworkers [18-20] and of Duine and coworkers [15-17], and the subsequent confirmation by total synthesis [56], the three-dimensional structure of PQQ itself could be determined only recently [32]. In addition, the three-dimensional s t r u c t u r e of a PQQ-potassium complex was established. A comparison of the structural features of these molecules with those of PQQ-acetone adduct [18,19] and PQQ-C(5)-2,4-dinitrophenyhydrazone triester [31], t h a t were investigated earlier, is now possible. Preliminary conclusions with respect to the structural properties of PQQ can be drawn.
The functional presence .of metal-ions in several quinoproteins, notably copper in amine oxidases [29, 63-65], galactose oxidase [66] and dopamine g-hydroxylase [67], as well as iron in lipoxygenase [68] and nitrite hydratase [69], has stimulated ideas about possible ligation of PQQ to these metal centres [27]. We found t h a t free PQQ is an adequate chelator of metal ions [701. Results concerning complex formation of PQQ and reduced PQQ with copper(II), discussed in relation to the structural properties of PQQ and PQQ-potassium complex, are presented in Chapter III.
Elucidation of the three-dimensional structure of PQQ-C(S)-2,4-dinitrophenyl-hydrazone triester [31 ] served to establish the identity of the product of PQQ and 2,4-dinitrophenylhydrazine that could be detached from hydrazine-inhibited plasma amine oxidase [40]. As a result of this investigation, plasma amine oxidase was recognized as the first quinoprotein from mammalian sources. In the meantime, the use of hydrazines as protective agents in the isolation and characterization of PQQ from enzymes in which it is tightly bound, has been extended [42].
Application of 2,4-dinitro-phenylhydrazine and phenylhydrazine in the so-called 'hydrazine method' for detection and quantification of PQQ, resulted in the formation of other types of PQQ-hydrazine adducts with different properties. In the past, the two types of adduct, that were obtained by treatment of selected quinoproteins with 2,4-dinitrophenylhydrazine, have been tentatively identified as 'azo' and 'hydrazone' tautomers [63]. In view of the structural features of PQQ-C(5)-2,4-dinitrophenylhydrazone triester, these assignments are reconsidered in Chapter IV.
Chemical methods for the detection and quantification of PQQ
The development of biological and chemical methods for the detection and quantification of PQQ has been a major part of our work. Reconstitution of quinoprotein apoenzymes was found to be a very sensitive method for the detection of free PQQ [41]. However, to determine the presence of PQQ in enzymes in which it is so tightly bound that the polypeptide must be hydrolysed first, a different strategy was needed. To design suitable methods, further knowledge of the chemical reactivity of PQQ was required.
As far as the chemical reactivity of PQQ is concerned, nucleophilic addition at the C(S)-carbonyl group of PQQ appears t o be of prime importance. Reversible adduct formation has been reported for HzO , alcohols, ammonia, urea and
cyanide [21]. Aldol-type adducts are formed with suitable aldehydes and ketones [41]. Formation of C(5)-adducts has been implied as a primary step in the reaction of PQQ with several other nucleophiles, including amines [SI ,54], amino acids [55, 71 -73] and thiols [52]. Reactions with amino acids were found t o be of particular importance, as these reactions gave rise to the formation of stable adducts that escape detection by (most) biological methods.
On the other hand, we found that adducts, suitable for detection and quantification of PQQ, may be obtained by reacting the quinoprotein with carbonyl group reagents, notably (dinitro-)phenylhydrazines. Extraction of quinoproteins with (acidic) hexanol according to the so-called 'hexanol-method' [42,62], constitutes a novel method for the detection and quantification of PQQ. Other transformations, including reaction of PQQ with hydrogenperoxide or persulfate to give ring-opened products; and treatment of PQQ with strong alkali to give ring-contracted products [41,26] were investigated. The chemistry that is involved in these reactions as well as their relevancy for the detection and quantification of PQQ is discussed in Chapter IV.
Binding and activity of PQQ
The involvement of PQQ in the mechanism of action of several quinoprotein enzymes from microbial sources has been properly established [26]. As far as enzymes from mammalian sources are concerned, the function of PQQ in amine oxidases seems evident [27,74]. Less information is available with regard t o the role of PQQ in other quinoproteins, although appealing mechanisms have been proposed for a number of these enzymes [27]. Of the four redox forms of PQQ that are known to exist (Figure 2), only PQQ itself and its one- and two-electron reduced forms appear t o occur in individual enzymes. The role of the semiquinone in catalysis in vivo, is still not completely clear.
Figure 2. PQQ, PQQH', PQQH2 and PQQH4, the four redox forms of PQQ.
From a physiological perspective, PQQ appears to be a multi-purpose cofactor. Examples of quinoprotein catalysis have been found for the dehydrogenation of alcohols, aldoses and aldehydes, the hydrogenation and oxidation of amines, hydroxylatibn of dopamine, oxygenation of unsaturated fatty acids, decarboxylation of dopa and possibly hydration of nitriles. A listing of established and alledged quinoproteins is given in Table 1.
A role for PQQ in quinoprotein-catalyzed redox reactions involving the a-carbon of amines and amino acids can be readily visualized [71,72]. Dehydrogenation of alcohols by a similar mechanism seems less straigthforward. A working hypothesis for the mechanism of action of quinoprotein alcohol dehydrogenation, based on the results of reconstitution studies with quinohaemoprotein alcohol dehydrogenase apo-enzyme from Pseudomonas testosteroni and several analogues and derivatives of PQQ, is developed in Chapter V.
Implications and Prospects
In the past ten years, the scope of PQQ and quinoprotein enzymology has evolved at a tremendous pace. While the initial detection of PQQ as the cofactor of methanol dehydrogenase from Hyphomicrobium X, could still be considered to be an interesting finding with limited impact on enzymology in general, the finding of PQQ in other bacterial enzymes already pointed at a more important role of this novel cofactor. By establishing the presence of PQQ in amine oxidases, its occurrence in both p r o - and eukaryotic species became apparant. Recent finding of PQQ in other classes of enzymes from higher organisms will no doubt stimulate further interest in this topic.
Now that quinoprotein enzymology appears to be firmly established, several additional questions deserve our attention. The question whether or not PQQ (or a derivative) will serve as a vitamin has been adressed earlier [60]. In the meantime, several reports on the beneficial effects of admistering PQQ t o rats and mice [75,76], plants [77], and microorganisms [78,79] have appeared. Although the r e s u l t s appear to be in favor of a role for PQQ as a vitamin, conclusive evidence has not yet been presented. Indeed, several lines of evidence seem to support the opposite view (discussed in Chapter VI).
Directly related to this question are the recent insights in the biosynthesis of PQQ by microorganisms. Both Unkefer and Houck [48,49] and van Kleef and Duine [47] have reported tyrosine as a precursor for the indole-ring of PQQ in methylotrophes. The former authors have provided additional evidence for glutamate or a closely related compound as a precursor for the pyridine-ring of PQQ. We have proposed a tentative biosynthetic scheme [47], t h a t takes into account the fact t h a t we have been unable to detect (any) biosynthetic intermediates [83]. Genes involved in the biosynthesis of PQQ in Acinetobacter calcoaceticus have been cloned [80]. Expression of these genes in Escherichia coli was found to provide them with PQQ. From the small number of genes that are needed to induce the biosynthesis of PQQ in E. coli, a limited sequence of reactions may be assumed, although other explanations cannot be excluded.
Considering the attachment of the cofactor to the quinoprotein polypeptide, either covalent or non-covalent binding may take place. In practice, these modes can be distinguished to a certain extent by considering whether or n o t the application of agents capable of disrupting covalent bonds is required to detach the cofactor from the protein. In this respect, two situations have been met: PQQ can be detached from several enzymes under denaturing conditions, notably
by the action of detergents or by applying high salt concentrations [41]. Alternatively, the (derivatized) cofactor is released only after hydrolytic cleavage of the poly-peptide [29]. Isolation of an oligo-poly-peptide that contains the derivatized cofactor, from (partial) tryptic digests of diamine oxidase (from pig kidney), has been reported [81]. Covalent attachment of the cofactor appears evident in this case. Recent structure elucidation of methylamine dehydrogenase from Thiobacillus
versutus at 2.2S X resolution may shed light on this matter. Preliminary r e s u l t s
[82] suggest the presence of a 7-(Y-glutamyl)-indole structure (tentatively designated as 'pro-PQQ', Figure 3).
— C Y S - 2 9 — ►
I
S — S -—CYS-61 — S E R - 6 0 G L Y - 5 9 — V A L - S 8 — | X - S 7 J — S E R - 5 6 — - — P H E - 1 0 9 C Y S - 1 0 8 — I X - 1 0 7 I — I L E - 1 0 6 — I L E - I O S —I
S — SI
C Y S - 7 8 ►Figure 3. Tentative structure for the covalently-bound cofactor of methylamine dehydrogenase (MADH) from Thiobacillus versutus
It will be clear, that further substantiation of this finding can have important consequences for the direction of future research on the enzymology of quino-proteins. The chemistry of PQQ t h a t may be relevant to t h e s e issues, is addressed in Chapter VI.
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Table 1 Microbial quinoproteins Enzyme DEHYDROGENASES Methanol dehydrogenase Methylamine dehydrogenase Alcohol dehydrogenase Amine dehydrogenases Quinohaemoprotein alcohol dehydrogenase Quinate dehydrogenase (Form)aldehyde dehydrogenases Glucose dehydrogenase Glucose dehydrogenase (membrane bound) Glucose dehydrogenase (soluble; EC 1.1.99.17) Lactate dehydrogenase Glycerol dehydrogenase
Polyethylene glycol dehydrogenases Polyvinylalcohol dehydrogenase Organism Gram-negative methylotrophes Clostridium thermoautotrophicum * Nocardia spec. 2 3 9 * Gram-negative methylotrophes Ps. aeruginosa, Ps. putida
Rhodops. acidophila, Acetobacter spec. Ps. spec.
Ps. testosteroni Gram-negative bacteria
Acetob. spec, Hyphomicrobium spec* Rhodops. acidophila Zymomonas mobilis* Gram-negative bacteria Acinetobacter calcoaceticus LMD 79.41 Propionibacterium pentosaceum* Gluconobacter industrius Flavobacterium spec. * Ps. spec. references 1 2 3 4 5,6 7,8 9-11 12 13 7,1 14 15 16,17 18,19 1 1 1 20 OXIDASES Methylamine oxidase Galactose oxidase Amine oxidase Nitroalkane oxidase Lysyl oxidase Arthrobacter P1 DaetyHum dendroides Aspergillus spec, yeasts Fusarium oxysporum Pichia pastor is 21 22 23,24 25 26 DECARBOXYLASES
Glutamate decarboxylase E. coli 27
MISCELLANEOUS * Tryptophan side chain oxidase Nitrile hydratase
Atypical pyridoxoprotein amino acid decarboxylases
Ps. spec. Brevibacterium spec. Microbia 1 2 8 12
Table 1 (continued) Plant and mammalian quinoproteins Enzyme
OXIDASES
Plasma amine oxidase (EC 1.4.3.6) Diamine oxidase (EC 1.4.3.6) Lysyl oxidase (EC 1.4.3.13) Amine oxidases MONO-OXYGENASES Dopamine (3-hydroxylase (EC 1.14.17.1)
Peptidyl glycine mono-oxygen;
Organism Bovine serum Pig kidney
Human placenta and arteria chicken cartillage
Plants and mammals
Bovine adrenal medulla ase Mammals * * references 30 31 32,33 36,37 34 38 DIOXYGENASES Lipoxygenase-1 (EC 1.13.11.12) Soybean 35 DECARBOXYLASES Dopa decarboxylase (EC 4.1.1.28) Atypical pyridoxoprotein amino acid decarboxylases
Pig kidney
Plants and mammals
29
29 Quinoprotein status based solely on the similarity with Dopamine p-hydroxylase
References Tabel 1. Quinoproteins
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CHAPTER II
S y n t h e s i s o f P y r r o l o q u i n o l l n e Qulnone, PQQ
Within a year after the structure of the novel cofactor PQQ, present in methanol dehydrogenase from methylotrophic bacteria, was deduced from the combined evidence presented by the groups of Forrest [1-3] and Duine [4-6], Corey and Tramontano [7] confirmed the proposed structure by t o t a l synthesis (Scheme 1). A second method (Scheme 2) was published only a few months later by Gainor and Weinreb [8,9].
Scheme 1. Synthesis of PQQ by the method of Corey and Tramontano
Both methods started with compounds containing the central (B) ring of PQQ, t o which the pyridine ring (A) and pyrrole ring (C) were subsequently attached (B - ~ BC —» ABC and B —► AB — ABC, respectively).
Scheme 2. Synthesis of PQQ by the method of Gainor and Weinreb
J.A. Gainor ans S.M. Weinreb (1981) J. Org. Chem., 46, 4317-4319
Scheme 3. Synthesis of PQQ by the method of Hendricksen and de Vries J.B. Hendricksen and J.G. de Vries (1982) J. Org. Chem., 47, 1148-1 ISO
A convergent synthesis, using a conceptually simpler approach (A + C —► ABC, Scheme 3) was described by Hendrickson and de Vries [10,11]. Buechi and coworkers [12] devised a synthetic scheme based on the alledged biosynthetic precursors of PQQ, tyrosine and glutamic acid [13-15] (Scheme 4).
Scheme 4. Synthesis of PQQ by the method of Buechi and coworkers
G. Buechi, J.H. Botkin, G.C.M. Lee and K. Yakushijin (198S) J. Am. Chem. S o c , 107, SSS5-5SS6
Scheme 5. Synthesis of PQQ and alkyl-substituted PQQ by the method of Jongejan Bezemer and Duine [161
By a modification of the method of Corey and Tramontano, Jongejan, Bezemer and Duine [16,172 prepared PQQ and alkyl-substituted PQQ (Scheme S). Several analogues and derivatives of PQQ were prepared by Ohshiro and coworkers [18] and Bruice and coworkers [19-21]. Using a slightly different strategy, MacKenzie, Moody and Rees [22,23] reported the preparation of PQQ in high yield (Scheme 6). Preliminary evaluations of the basic strategies have appeared [24,25]. Prominent features of individual methods are presented in Schemes 1-6.
co,H N3s, ^C02CH3 C OBz
17
HOjC' ^ N " Y V° H3CO,C O CO,CH3 C03CH3 ,CO,H12
20
19
Scheme 6. Synthesis of PQQ by the method of MacKenzie, Moody and Rees A.R. MacKenzie, C.J. Moody and C.W. Rees (1983) J. Chem. S o c , Chem. Commun., 1372-1373
Production of PQQ
With the method that has been developed by Rees and coworkers as a possible exeption, all strategies have been designed primarily t o achieve 'qualitative' goals. The methods by Corey and Tramontano, and by Gainor and Weinreb were aimed at the confirmation of the proposed structure of PQQ. Hendrickson and de Vries emphasized the synthetic 'logic' of a convergent synthesis. Buechi and coworkers adopted a 'bio-mimetic' approach. None of these methods, however, appears to be fully adequate for the preparation of gram-amounts of PQQ. Meanwhile, multigram preparation of PQQ by the method of Corey and Tramontano has been shown to require the introduction of considerable modifications [17].
Microbial production of PQQ has been described in the past [24]. Recent claims, concerning the use of over-producing strains under iron-limitation have been made [26]. Although it can be expected that future large-scale production of PQQ by microbial methods will compete favorably with organic synthesis a far as economy is concerned [27], preparation of PQQ derivatives and analogues may continue to require organic synthetic methods. We developed a method for the chemical synthesis of 2H and 1 3C labelled PQQ [16]. This methods appears equally suited
for the preparation of radio-labelled compounds of high isotopic purity. On the other hand, PQQ that is either uniformly or specifically labelled with 1 3C , has
been obtained from microbial sources by Unkefer and Houck [14,15] and by van Kleef and Duine [13], as part of a research program on the biosynthesis of PQQ. Microbial production of 1 4C-labelled PQQ has been announced [28].
Synthesis of PQQ, Initial attempts
Considering the methods that have been devised for the synthesis of PQQ, it is clear that a 'logical' approach, as adopted by Hendrickson and de Vries, is not necessarily the most successful.
We selected two types of approach from the vast array of possibilities emerging from a (limited) literature study. In our view, the prominent features of the PQQ molecule might be created by merging the structures of indole-2-carboxylic acid and 2,4-dicarboxy-quinoline (Scheme 7).
,C02H ,|
'foil,
COzH
PQQ
''
v^°
Synthesis of ethyl indole-2-carboxylate has been described by Brehm [30]. Reduction of ethyl o-nitrophenylpyruvate prepared by the method of Wisliscenus and Thoma [31] gave the indole. 2,4-Dicarboxy-quinoline can be readily obtained by Pfitzinger quinoline synthesis [32] (see [33] for a review), starting with the corresponding isatin. We prepared both model compounds, while we started a search for a precursor molecule t h a t was suitably substituted to act as a template for the consecutive annulations. We intended t o introduce the o-quinone group in a final step using nitrosodisulfonate in the Teuber reaction [34] (see [35] for a review).
Scheme 8. Strategy for the synthesis of PQQ
/'ƒ. glycol/H+; HI. benzaldehyde/HOAc; iv. ozonolysis; (alternatively,
Hi/iv/v. SeOz or K M n 04) ; vi. thioglycol/H+; vil. H+/ HzO ; viii.
esterification; ix. p-bromopyruvic ester/base; x. NH3; xi. H+/ HzO / H g ,
then nitrosodisulphonate, then hydrolysis
Meanwhile, we were happy to t e s t an alternative approach (Scheme 8), suggested to us by Prof, de Groot and Dr. de Bie (LU Wageningen). By creating a carbonyl function at C(5) of the tetrahydroquinoline ring we expected to obtain suitable activation of the C(6)-methylene group for subsequent annulation of the pyrrole ring. The carbonyl function at C(8), as a precursor for the C(5)-phenolic hydroxyl group in the pyrroloquinoline, was judged to be essential for t h e introduction
of the o-quinone moiety, an intuition that proved t o be justified in retrospect by the findings of Hendrickson and de Vries [10,11] (considerable experimentation was required to introduce the o-quinone function in the precursor lacking an oxygen substituent at either C(4) or C(S) of the pyrroloquinoline ring system). It was reasoned that differential protection of the two carbonyl groups might well allow the preparation of the monoketal-ketone required for the pyrrole-annulation step. Alkylation of 7,8-dihydro-S(6W)quinolone at C(6) has been described [29].
Synthesis of the tetrahydroquinoline ring by condensation of 4 a m i n o p e n t 3 -en-2-one, prepared by reacting acetylacetone with ammonia according to Combes and Combes [36,37], and dimedone a s described by Curran [38], could be performed in high yield (Scheme 8. R = C H3) . Similar condensations with dihydroresorcinol
( R = H ) , prepared by hydrogenation of resorcinol over Rhodium catalyst (CRL Baker & Co, 'Catalyst 310') according to Smith and Stump [39], were also successful. An elegant method for the oxidation of the C(2)- and C(4)-methyl groups appeared to be the condensation of the 2,4-dimethyl-5,6,7,8-tetrahydroquinoline with benzaldehyde according to Shaw and Wagstaff [40], followed by ozonolysis of the di-styryl derivative according to Kaslow and Stayner [41]. By performing this sequence on the model compound 2,4-dimethyl-S,6,7,8-tetrahydroquinoline, obtained by catalytic reduction of dimethylquinoline over platinum oxide [42], it was found that the 2,4-distyryl-8-benzylidene derivative could be prepared in low yield. Initial a t t e m p t s at small-scale ozonolysis t o give 2,4-dicarboxyl-5H-6,7-dihydro-quinolin-8-one showed promising results. Similar treatment of 2,4rdimethyl-6//-6,7-dihydroquinolin-S-one, however, did not lead t o the desired product.
A compound, tentatively assigned as 2,4-dicarboxy-5H-6,7-dihydroquinolin-8-one was obtained by treatment of the corresponding 2,4-dicarboxy-tetrahydroquinoline with selenium oxide. To probe the feasibility of the pyrrole annulation step, we prepared a-tetralone as a model compound.
Both studies were discontinued as soon as the Corey and Tramontano method came to our attention. This method (Scheme 1) comprises two consecutive annu lation reactions. The indole moiety is obtained by Fischer indolization of the hydrazone resulting from Japp-Klingemann reaction, a Doebner-Von Miller type reaction is used to prepare the quinoline ring. Pfitzinger quinoline synthesis [33] appears to be a rather poor substitute for the latter 'annulation, although we established its usefulness for the synthesis of PQQ analogues substituted, at C(8) [16].
