Concealed

THE CONCEALED SIDE OF THE HISTORY OF COMBINATORIAL CHEMISTRY

Árpád FURKA*, István HARGITTAI**

*Department of Organic Chemistry, Eötvös Loránd University
H-1518 Budapest 112, PO Box 32
**Institute of General and Analytical Chemistry, Budapest University of Technology and Economics and Structural Chemistry Research Group of the Hungarian Academy of Sciences at Eötvös University, H-1521 Budapest, PO Box 91, Hungary

Received: May 14. 2004

Abstract

Combinatorial chemistry today is an accepted new branch of science that comprises methods widely applied in drug research and other research fields withinchemistry and even outside of it. Its beginnings were slow and painfulin the 1980s. The present account describes a story that has not beenheretofore revealed in the history of its discovery. The format isa conversation in which Árpád Furka, the original discoverer,provides the answers to István Hargittai’s questions.

Keywords: combinatorial chemistry, split-mix method, peptide synthesis, science history

How and when did you come to the idea of the combinatorial method?

Around 1980, I began to think about the possibility of preparing all membersof families of peptides: dipeptides, tripeptides, tetrapeptides, or pentapeptides.It was clear from the beginning, that this could be accomplished only bya new method since a very simple calculation showed that using the availabletechniques, thousands of years would be needed to synthesize, for example,all the pentapeptides. Considering the possible solutions, it was also clearthat multi-component mixtures of peptides could easily be prepared by usingmixtures of amino acids - instead of the usually applied single ones - intheir solid phase synthesis.
On the other hand, this did not seem to be an acceptable solution becauseof the differences in the reactivity of the activated amino acids that wouldlead to the formation of peptides in significantly different concentrations, thus causing problems in screening. The goal was to eliminate the problem stemming from reactivity differences. I solved the problem in 1982, working in Budapest.
My idea was to make the couplings with single amino acids on equal samplesof the resin, then mix the samples after coupling. This made it possible to force the coupling reactions to completion with each amino acid and thus assuring the equal molar formation of peptides. This is the simple and well-known split-mix combinatorial synthetic method that reduced the time needed toprepare the peptide libraries from thousands of years to a few days.

Did you have any idea of how to utilize the peptide mixtures?

Finding the bioactive peptides in a mixture of millions of components seemedlike finding a needle in a haystack. So a strategy was needed to solve thisproblem and this strategy (later known as the iteration method) was also ready in 1982.

Did you try to find applications for the idea?

I discussed the problem of making a patent with a patent attorney Dr. ÉvaSomfai with whom we worked together in filing other patents for a Budapestpharmaceutical company. It turned out that patenting would cost more thanI could afford. The pharmaceutical companies were not interested in patentingour method. As a safeguard, she suggested to describe the method ina document and notarize it. This could help in the future in possible prioritydisputes. I did so and the document was notarized on June 15, 1982.The document can be seen in both the original Hungarian and its English versionin my home page [1] and it was also published in Drug Discovery today [2].

When did you first publish the method?

When I discussed the new synthetic method with colleagues I experienced astrong disbelief. This was the reason why I thought for a long time about finding the best way of publishing. Finally we published the synthetic method on posters at two international congresses in 1988: The 14th International Congress of Biochemistry, Prague [3] and The 10th International Symposium of Medicinal Chemistry, Budapest [4]. Although no particular interest orresponse was forthcoming, in hindsight it was most fortunate that at leastthese Abstracts about the initiation of combinatorial chemistry had goneon record. However, the lack of responses by chemists and my failureto find cooperating biologists quite disappointed me. It took a whilebefore I decided to submit a manuscript for a full paper.

What was your choice then for your publication?

It was clear to me that it would be impossible to publish the method in ageneral journal like Nature. So I chose a more specialized periodical, theInternational Journal of Peptide and Protein Research in which we had publishedbefore although we knew that the chief editor of the journal had changedsince that time.
In our experiments we focused on proving that the expected peptides do formin the synthetic procedure. For this reason only small libraries could beused. We developed a computer-aided paper electrophoretic procedure [5] thatwas successfully used in identifying the components of the synthetic mixtures. The manuscript, entitled "General method for rapid synthesis of multi-component peptide mixtures" was sent to Professor Hruby, University of Arizona, Tucson, and was received on February 12, 1990. The manuscript was reviewed by three reviewers. One of them, although accepted the paper with minor revisions, wrote in his comments: "Having spent years endeavoring to prepare peptides in the pure state, manuscripts like this ‘compromising with mixtures’ (p.3) cause me some anxiety." In his letter of May 15, 1990, Professor Hruby wrote that the paper may be acceptable only after major revision. After doing further peptide separations using HPLC, the revised version of the paper was accepted on November 21, 1990, and appeared in print in June 1991 [6].

I have heard rumors that there were some disappointing events connectedwith your publication. Could you tell us what these events were?

Our paper appeared 16 months after submitting the manuscript. During thisperiod, patent applications were submitted by four different groups all basedon our split-mix method: Huebner and Santi (Chiron group) [7] on May 15,1990, Lam et al. (Salmon-Hruby group) [8] on July 2, 1990, Houghten et al.(Richard Houghten's group) [9] on November 21, 1990 and DiMarchi et al. (EliLilly group) [10] on June 18, 1991. It was particularly painful that ProfessorHruby, the Editor-in-Chief of the journal where our manuscript was submitted,participated in patenting and his group also submitted a paper to Nature[11] and presented a lecture to the 12th American Peptide Symposium [12],claiming in both papers that they invented the split-mix method. Shortly after the appearance of our paper, Dr. Richard Houghten and his group (Torrey Pines Institute for Molecular Studies, San Diego) made also a presentation at the Symposium on the Innovation & Perspectives in Solid Phase Synthesis and Related Technologies [13] and submitted a paper to Nature [14] in which our split-mix synthesis was described. I would also like to call your attention to a remarkable coincidence. The number of the filed patents was exactlythe same as the number of those that had had access to our manuscript: theeditor of the journal and three reviewers. Do you think that such a radicallynew idea like that of the split-mix synthesis may suddenly and independentlyoccur in four different heads not earlier and not later but exactly at thesame time when our manuscript is being reviewed for publication, andone of those heads is that of the chief editor of the journal (or of oneof his associates) to which our manuscript had been submitted?

Possible, but unlikely. Let me ask you this: Did you have personalcontacts with Professor Hruby at this time?

I did. Our contacts began in the following way. In January 1991, Igot a letter from Dr. Kaubisch, Vice President of Selectide Corp. and heoffered to visit us to discuss the possibilities of cooperation. He explainedthat Selectide was founded at the end of 1990 (well after our paper had beensubmitted) by Professor Hruby and others at the University of Arizona. Dr. Kaubisch also mentioned in the letter that they saw the abstract of our presentation to the 10th International Symposium of Medicinal Chemistry (Budapest, 1988). Dr. Kaubisch came to Budapest in February 1991 and heexplained that one of them found out that in the split-mix synthesis onepeptide forms on each bead and they developed a screening method based onthat. He invited me to visit Selectide to give a seminar and discuss thepossibilities of cooperation. I accepted the invitation and my visit tookplace in early April 1991. I gave a seminar on April 2 at the CancerCenter of University of Arizona, Tucson, speaking about the split synthesisand the stepwise screening strategy I described in the 1982 document. Dr.Lam (first author), Professors Hruby and Salmon (director of the ArizonaCancer Center and Dr. Lam’s boss) as well as other authors of theirlater papers and the earlier patent application were among the audience.After they asked me to sign a confidentiality document, with which I complied,they offered me a consultancy (which was never realized), and showed me theirscreening method. As a possibility of cooperation, however, they suggestedto me to do analytical experiments with non-natural amino acids they usedin peptide synthesis. I found this offer inappropriate and told them thatour cooperation should be connected with our synthetic method. Finally weagreed to continue the discussions by mail but the cooperation was never realized because of later events.

When did you sense for the first time that something went wrong?

A few months after my visit to Tucson, one of my former students attendedthe 12th American Peptide Symposium in Boston in June 1991. She told me thatDr. Lam had an oral presentation reporting about the split-mix synthesis and their screening method [12] but made no reference to our work. Lateron, one of my colleagues took part in the Symposium on the Innovation &Perspectives in Solid Phase Synthesis and Related Technologies in August1991, where Richard Houghten presented our synthetic method as his own [13].Yet later, I found in Nature the papers of Lam et al. [11] and Houghten etal. [14] that appeared in September 1991 and in which they described thesplit-mix synthesis as their own invention and without any reference to ourpapers.

How did you feel about this and what could you do in this situation?

I have to tell you that this situation has caused me much bitterness throughthe years. As my first reaction, I wrote a letter to both parties askingfor a correction in Nature and citation of our papers.

What was their reaction?

Let me speak first about the Hruby group. Both Professor Hruby and Dr. Lamwrote me an apologizing letter stating that in the first version of theirNature papers, there was a reference to our work but they had to shortenthe manuscript and the reference was deleted. They also promised to publisha correction in Nature. First of all I have to note that if you read thepaper, you don't get the impression that anything was left out. They described the synthetic method as their own invention and in later papers they called it the Selectide process. After almost one year of delay, their correction, with an ambiguous content, appeared in Nature [15]. My name, however, was misprinted: Fukura instead of Furka. After repeated correspondence, a correction of the correction appeared in Nature in December 1992 [16].
I questioned the explanation of omitting the references to our papers byHruby and Lam from the beginning. Later on, I found evidence that convincedme that omitting us from the list of references vas intentional. They havepublished other papers and documents from which citation of our work is alsomissing. Let me mention only two examples.
(i) Around the time of publishing the Nature paper Professor Hruby and someof his co-workers wrote a chapter in a book [17] in which the split-mix synthesisis also mentioned without reference to our contribution.
(ii) Professor Sydney E. Salmon of the University of Arizona (one of theauthors of the Nature paper [11]) had grant applications in cooperation withHruby and others based on the split-mix synthesis. The synthesis was indicatedin their proposals as their own invention. One of the successful proposalsto the National Institutes of Health had the title "Discovery of PeptideAnticancer Drugs," grant No. CA57723. This grant lasted from 1992 to 1995and brought more than 3 million dollars to the applicants. A copy of thegrant application was in my hands and saw no reference in it to our work.
Let's turn now to Dr. Houghten. I also asked him to correct his Nature paper[14]. He promised to do that, called me by phone, and also offered cooperationand financial support. He sent me the copy of the correction letter supposedlymailed by him to Nature. He wrote in the letter "As we were unaware of anyof Dr. Furka's work we did not include it as a reference in our manuscript. We regret this omission ...". The correction, however, was never published. It deserves to look closer what Dr. Houghten states in his letter: "we were unaware of any of Dr. Furka's work." I later found Dr. Houghten's patent application [8] in which he cited the abstract of our 1988 presentation in Prague [3]. The patent was filed on November 21, 1990, and the Nature paper was submitted on July 31, 1991. Let me ask you: Could anybody believe that what he knew in November 1990 was unknown to him eight months later?

Did you try to make other actions in addition to those outlined above?

Yes, but all of them were unsuccessful. I tried to publish myself a letterin Nature, but this was declined by Professor Maddox, who was the Editorat that time. I also contacted the Office of Research Integrity ofthe Department of Health & Human Services of the U.S. I had long correspondence with Dr. Alan Price of that office and sent him all the evidences. Finally, he concluded that in his opinion no plagiarism occurred and advised me to turn to the University of Arizona. I also contacted the Publisher of the International Journal of Peptide and Protein Research, again withoutany success.

What was the effect of Drs. Hruby's and Houghten's actions on you?

In addition to the bitterness that lasted for years, for a long time onlytheir papers were cited in the literature. Later on, and still nowadays, their work is often cited as independent from that of ours. I had difficulties for years in publishing papers and had no possibility to give oral presentations at peptide symposia. Many years later, a friend of mine who was chairmanof a combinatorial session of a peptide symposium told me that he was instructed by the organizers not to give me possibility for questions or comments.
Despite all this, the situation has gradually changed. I remember that shortlyafter the appearance of Lam's and Houghten's Nature papers, I got a letterfrom Professor W. C. Still of Columbia University, asking me to clarify thepriority question by sending him earlier publications. I sent him copiesthe two 1988 abstracts [3, 4] and the 1982 document [1, 2]. I also made availablecopies of the 1982 document as a supplement to my posters presented at symposia.As a consequence, people have realized that the real birthplace of combinatorialchemistry was our laboratory in Budapest, and have begun to be called the“pioneer” or the “father of combinatorial chemistry.” I was elected Honorary President of the European Society of CombinatorialSciences when this organization held its first symposium in Budapest in 2001.

Some years ago, I already made an interview with you [18] and you alsopublished a paper about the history of combinatorial chemistry [19], butyou did not speak about these problems. Why?

As mentioned before, my earlier efforts to clarify the priority problemswere unsuccessful. What happened was a scandal in science and most people,including myself, do not like scandals. Also, it is not easy to speakabout such events in science, particularly if you are the victim. My friendsalso advised me to be silent since those who speak out about such things,the whistle blowers, are often considered as bad guys. Lately, however, colleagues,whose opinion I respect, told me that it's time to bring out the truth tolight.

References

[1]    http://szerves.chem.elte.hu/furka
[2]    FURKA, Á., Combinatorial chemistry: 20 years on...., Drug Discovery today 7 (2002), pp. 1-7.
[3]    FURKA, Á. – SEBESTYÉN, F. – ASGEDOM, M. – DIBÓ, G., Cornucopia of peptides by synthesis, Abstr. 14th Int. Congr. Biochem., Prague, Czechoslovakia, 1988, Vol. 5., p. 47.
[4]    FURKA, Á. – SEBESTYÉN, F. – ASGEDOM, M. – DIBÓ, G., More peptides by less labour, Abstr. 10th Int. Symp. Med. Chem., Budapest, Hungary, 1988, p. 288.
[5]    FURKA, A. – SEBESTYEN, F. – GULYAS, J., Computer made electrophoretic peptide maps, Program of the 2nd International Conference on Biochemical Separations, Keszthely, Hungary, Full Papers & Abstracts, Edited by J. Pick, J. Vajda, pp. 35-42 (1988).
[6]    FURKA, Á. – SEBESTYÉN, F. – ASGEDOM, M. – DIBÓ, G., General method for rapid synthesis of multicomponent peptide mixtures, Int. J. Peptide Protein Res. 37 (1991), pp. 487-493.
[7]    HUEBNER, VERENA D. – SANTI, DANIEL V., Controlledsynthesis of peptide mixtures using mixed resins. US Patent 5,182,366 (FiledMay 15, 1990).
[8]    HOUGHTEN, R. A. – CUERVO, J. H., – PINILLA,C. – APPEL Jr., J. R. – BLONDELLE, S., Synthesis of equimolecular multiple oligomer mixtures, especially of oligopeptide mixtures, PCT patent application, WO 92/09300 ( Filed November 21, 1990).
[9]    LAM, K. S. –SALMON, S. E. – HRUBY, V. J. – HERSH, E. M. – AL-OBEIDI, F., Random bio-oligomer library, a method of synthesis thereof, and a method of use thereof, PCT patent application, WO 92/00091 (Filed July 2, 1990).
[10]    DIMARCHI, R. D. – GESELLSCHEN, P. D. – OWENS, R. A.: Rapid synthesis and screening of peptide mimetics. Patent application, filed June 18, 1991.
[11]    LAM, K. S. – SALMON, S. E. – HERSH, E. M. - HRUBY, V. J. – KAZMIERSKI, W. M. –KNAPP, R. J., A new type of synthetic peptide library for identifying ligand-binding activity, Nature 82 (1991) p. 354.
[12]    LAM, K. S. – SALMON, S. E., HERSH, E. M. –AL-OBEIDI, F. – HRUBY, V. J., Rapid selection and structure determinationof acceptor binding ligands from a large synthetic peptide library, Abstr.12th American Peptide Symposium, Boston, USA, LW3.
[13]    HOUGHTEN, R. A., New approaches for peptide and peptide-mimetic biomedical drug discovery, Innovation & Perspectives in Solid Phase Synthesis & Related Technologies, Second International Symposium, Canterbury, August 27-31, 1991, 10c.1.
[14]    HOUGHTEN, R. A., PINILLA, C. – BLONDELLE, S. – APPEL, J. R. – CUERVO, J. H., Generation and use of synthetic peptide combinatorial libraries for basic research and drug discovery, Nature 84 (1991) p.354.
[15]    Nature 258, 434 (1992).
[16]    Nature 360, 768 (1992).
[17]    HRUBY, V. J. – SHARMA, S. D. – COLLINS, N.– MATSUNAGA, T. O. – RUSSEL, K. C., Application of SyntheticPeptides, In: Synthetic Peptides. A User's Guide, G. A. Grant (Editor), W.H.Freeman and Company, New York, 1992, pp. 270-272.
[18]    I. Hargittai, The Chemical Intelligencer 6(2), 37-40 (2000); I. Hargittai, Candid Science III: Conversations with Famous Chemists. Imperial College Press, London, 2003, pp. 220-229.
[19]    Á. Furka, History of Combinatorial Chemistry, Drug Development Research 36, 1-12 (1995).