Substantia. An International Journal of the History of Chemistry 2(2): 125-130, 2018 Firenze University Press www.fupress.com/substantia ISSN 1827-9635 (print) | ISSN 1827-9643 (online) | DOI: 10.13128/substantia-66 Citation: L. Colli, A. Guarna (2018) The dextrorotatory sweet asparagine of Arnaldo Piutti: the original product is conserved in Florence. Substantia 2(2): 125-130. doi: 10.13128/substantia-66 Copyright: © 2018 L. Colli, A. Guarna. This is an open access, peer-reviewed article published by Firenze University Press (http://www.fupress.com/substan- tia) and distribuited under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. Data Availability Statement: All rel- evant data are within the paper and its Supporting Information files. Competing Interests: The Author(s) declare(s) no conflict of interest. Historical Article The dextrorotatory sweet asparagine of Arnaldo Piutti: the original product is conserved in Florence Laura Colli*, Antonio Guarna Dipartimento di Chimica “Ugo Schiff ” dell’Università degli Studi di Firenze, Via della Lastruccia 3-13, Polo Scientifico e Tecnologico, 50019 Sesto Fiorentino – Firenze (Italy) E-mail: laura.colli@unifi.it Abstract. In 1886, Pasteur presented a note on the work of the Italian chemist Arnaldo Piutti concerning the difference between the two physical isomers (enantiomers) of asparagine. The octahedral crystal of asparagine appeared only as “levorotatory hemi- hedralism” but, in principle, should also exist as a dextrorotatory asparagine with a symmetric crystalline form. In 1886 Arnaldo Piutti isolated the dextrorotatory aspara- gines while he was working as an assistant of Ugo Schiff in Florence. He obtained also another unexpected information, of which only Pasteur immediately understood the importance: the dextrorotatory aspargine had a sweet taste. The dextrorotatory sweet asparagine of Arnaldo Piutti is conserved in the Schiff Col- lection of the Department of Chemistry “Ugo Schiff ” at the University of Florence, and is the first compound where a relationship between the optical isomerism of a molecule and a different response of human receptors, in this case the taste, was observed. Keywords. Asparagine, Chemical Heritage, history of enantioselectivity. This research stems from a purely museological question. A few years ago, during the reordering of the products synthesized by Ugo Schiff (1834- 1915) at that time conserved at the Department of Chemistry “Ugo Schiff ” of the University of Florence, and now merged into the Museum of Natural History, a small bottle was found. It carried a “Ugo Schiff ” museum label, written by “our” German chemist, referring to: “Asparagina destrogira dolce” (Dextrorotatory sweet asparagine)1. After reading the work by Joseph Gal2-4 in 2008, Antonio Guarna (the scientific director of the museum project for his- torical chemical finds) immediately had the intuition that the product could contain the famous dextrorotatory sweet asparagine isolated by Arnaldo Piutti. The historical research that was conducted, within the Chemical Her- itage project of the University of Florence, proved him right. In this contri- bution we discuss some insights about the discoverer and how this discovery took place. 126 Laura Colli, Antonio Guarna “PIUTTI’S ASPARAGINE”: WAS IT AN IMPORTANT DISCOVERY? The work of Gal on the discovery of Piutti reports in the conclusions, the following statements4: Piutti was a highly original chemist who carried out nota- ble investigations in a wide variety of research topics. His discovery of a difference in the taste of D- and L-aspara- gine was a milestone first observation of enantioselectiv- ity at a biological (human) receptor. The discovery was also the first observation of stereoselectivity of any kind in taste; the first finding of biological enantioselectivity in an organism higher than microorganisms; the first example of biological enantioselectivity in an effect other than enzyme action; and one of the two earliest reports of the prepara- tion of a D-amino acid. The sample preserved in the Schiff Collection of the University of Florence therefore has a considerable his- torical and scientific relevance. The discovery had a good resonance at the time of Piutti. Louis Pasteur (1822-1895) himself showed a great interest in this research, and in 1886, at the French Academy of Sciences, commented on the work of the Italian chemist with these words5: Why this big difference in taste between these two aspara- gines? One might assume the existence of a very special isomerism, but I think otherwise […] if two dissymmetrical inverse bodies offer in their interaction with inactive bod- ies, physical and chemical properties that are very similar and almost identical, these dissymmetrical inverse bodies will give combinations of different absolute properties when they merge with asymmetric and optically active bodies. The active dissymmetrical bodies that will interact with the nervous system, leading to a sweet taste in one case and almost tasteless in another, won’t be anything else in my opinion than like the nervous matter itself, dissymmetri- cal, just like all the basic substances of life: albumin, fibrin, gelatin, etc. THE SCIENTIFIC CONTEXT OF ARNALDO PIUTTI’S DISCOVERY At the end of the Nineteenth century stereochem- istry did not exist yet. In 1867 the German chemist Friedrich August Kekulé von Stradonitz (1829-1896) proposed for the first time the tetrahedral structure of carbon. Two years later the Italian chemist and politi- cian Emanuele Paternò (1847-1935) applied this hypoth- esis to saturated organic compounds6. On the relation- ship between structure and optical properties of a mol- ecule, in 1874, Jacobus Henricus van’t Hoff (1852-1911), a Dutch chemist awarded with the Nobel Prize in 1901, proposed that compounds with asymmetric carbon may exist in two different forms corresponding to the two optical antipodes, which could be separated into two optically active compounds. Van’t Hoff ’s theory, consid- ered too audacious and abstract, was almost rejected by the academic world. Only when Hermann Emil Fischer (1852- 1919), a German chemist, almost contemporary of Schiff, and Nobel Prize winner in 1902, in 1894 adduced evidence in favor of this thesis by synthesizing the levo- rotatory (not natural) antipode of glucose, the proposi- tions of van‘t Hoff became accepted by the Academics, paving the way to modern stereochemistry7,8. ARNALDO PIUTTI BIOGRAPHY Arnaldo Piutti was born on January 23, 1857 in Udine. He graduated in 1875 at the Technical Institute of Udine, in the Physics-Mathematics section and from there he moved to Turin where he enrolled in the Fac- ulty of Natural Sciences9. In 1879 he brilliantly com- pleted his studies under the guidance of Ugo Schiff (1834-1915), a German chemist that served as professor of chemistry in Florence for fifty years and in his Floren- tine laboratory discovered the bases and the Schiff reac- tion10. Piutti had the opportunity to meet Schiff just in the two years when the German scientist was lecturing in Turin. In fact, Schiff moved from Florence to Turin in 1877, because of the disagreements he had with the man- agement of the Florentine Royal Institute of Practical and Advanced Studies (Regio Istituto di Studi Pratici e di Perfezionamento). He eventually returned to Florence in 1879 after the promise of more funds for his laboratory11. After his graduation, Piutti remained in Turin as an assistant to Angelo Mosso and Icilio Guareschi, two important Italian chemists of the time. In 1881 he joined Ugo Schiff in Florence as assistant at the Laboratory of General Chemistry. In Florence, Piutti helped with the course of General Chemistry and later in 1885 also of Organic Chemistry. In 1886 he received a degree in Pharmacy, a professional pratice that was quite usual at that time. In this way he was able to apply for a position in Pharmaceutical Chemistry and in the same year he obtained a professorship at the University of Sassari in Sardinia9. In 1888 he moved from Sassari to Naples, where in 1890 he was appointed as full professor, with the maxi- mum score6 (Figure 1). He also won the position in Gen- eral Chemistry in Padua, but he preferred to remain in Naples where he could organize the new Institute, fol- lowing the example of Schiff in the reorganization of the Chemistry Institute Florence11. 127The dextrorotatory sweet asparagine of Arnaldo Piutti: the original product is conserved in Florence Piutti was a member and correspondent of numer- ous academies and scientific societies, including the prestigious Accademia dei Lincei. He held the office of Dean of the Faculty of Science and of the School of Pharmacy in Naples for several years, he became Vice- Rector and represented the Minister of Education at the International Congresses of Applied Chemistry in 1896, 1890, 1900, 1903 and 1910. In Naples he was for a long time appreciated Director of the Institute of Pharmaceu- tical Chemistry and Toxicology and he personally fol- lowed with great commitment the construction of the new headquarters in San Marcellino9. Piutti’s scientific contributions are mainly focused on the study of aspartic acid, asparagines and their derivatives and on the optical rotation of organic com- pounds. His research ranged also in other fields of chemistry. He studied the toxicity of the combustion products of locomotives and he traced the presence of helium in a mineral of the Vesuvian area, demonstrating the diffusion of this gas in solids. He patented the Piran- tina solubile Piutti (soluble Pirantina Piutti), an antipy- retic and analgesic9. Despite having personally contributed to the con- struction of the new laboratories in San Marcellino, he finally abandoned those in the place called “Il Salvatore” only when they were declared unfit. During that period, not being able to carry out experimental work, he devot- ed himself to theoretical studies on the spatial represen- tation of chemical elements, arranging them in an alter- native way to the Mendeleev table9. He died in Conegli- ano, near Treviso, on October 19, 1928. ISOLATION OF ASPARAGINE Asparagine is an α-amino acid (Figure 2) that was identified for the first time by the French chemists Louis Nicolas Vauquelin (1763-1829) and Pierre Jean Robiquet (1780-1840) in 1815, in the asparagus sprouts. Short- ly afterwards Joseph Bienaimé Caventou (1795-1877), another French chemist who pioneered the research in amino acids and plant alkaloids, and Heinrich Hlasivetz isolated asparagine from Glycyrrhiza glabra (liquorice) and Robinia pseudoacacia. Later Asparagine was found in many other plant species: in the roots of althea, in potatoes, in hop, in legumes sprouted in darkness and in sweet almonds12. To isolate the asparagine molecule, the juice squeezed from the plant was boiled until it formed an abundant coagulation of albumin. At this stage it was filtered, purified and crystallized, obtaining crystals that looked like “sugar candy”12,13. The isolated asparagine was an optically active molecule that caused a left-hand- ed rotation of the polarization plane. In 1835 William Hallowes Miller (1801-1880) a British mineralogist and physicist, determined the crystallographic constants and measured its refractive indices. In 1848 Pasteur identified a relationship between the crystalline form of the asparagine and the rotation of the polarization plane, arguing that in principle, a dextrorota- tory asparagine with symmetrical crystalline form should exists, albeit the occurrence of the octahedral crystal of the asparagine only as “levorotatory hemihedralism”14. Even Karl Friedrich Rammelsburg, (1813-1899), a German mineralogist and chemist, in 1855 advanced the possibil- ity of crystallization in the form of a left or right-handed tetrahedron15, but the isolation of the dextrorotatory asparagine was carried out only in 1886. Figure 1. A portrait of Arnaldo Piutti in his laboratory at Naples9. Figure 2. The two forms of asparagine. 128 Laura Colli, Antonio Guarna THE DISCOVERY OF DEXTROROTATORY “SWEET” ASPARAGINE Piutti guessed that the failure in identifying the dex- trorotatory asparagine was presumably due to its low abundance in Nature. In the factory of Mr. Galgano Par- enti, near Siena, Piutti prepared a large quantity of the product from sprouted vetch, a legume15. Through frac- tional crystallization he observed the precipitation of two species of crystals: ordinary levorotatory asparagine, described at that time as “almost tasteless”, and dextroro- tatory, which turned out to be “sweet”. Thus the dextro- rotatory asparagine could be detected and separated from the levorotatory form on the basis of its taste (Figure 3). The two types of crystals were analyzed and found to be chemically identical, with the same refractive indices and same plane of optical axes. In the laboratory of Phys- ics headed by Prof. Roiti at the Royal Institute of Practi- cal and Advanced Studies in Florence, Piutti determined the optical rotation of the two species with a Laurent polarimeter, measuring the following values: [α]D = -5.43 for the ordinary or levorotatory asparagine and [α]D = +5.41 for the dextrorotatory sweet asparagine15 . The dextrorotatory asparagine, whose existence was theoretically proposed by Pasteur, was finally discovered. THE SYNTHESIS OF SWEET ASPARAGINE After separating the dextrorotatory asparagine in 1886, Piutti succeeded in setting up the synthesis in hislaboratory in 188716. The synthesis was carried out through the reduction of the oxime of oxaloacetate ether. Piutti separated two monoethyl esters, written with the formulae: COOC2H5-CH-CHNH2-COOH and COOC2H5-CHNH2-CH2-COOH (according to the origi- nal notation). From the former derivative he obtained the amidate, and finally the two rotatory asparagines, identical to the natural products and called by Piutti as a whole “β-asparagine”, with the formula: CONH2-CH2- CHNH2-COOH (Figure 4). This is of course a historical nomenclature, which does not comply with the com- mon rules currently in use. From the second amidate he obtained the inactive “α-asparagine”, an isomeric form of the β-asparagines. Thus, he came to the conclusion that he had obtained three asparagines. He did not know whether the inactive form, called “α-asparagine”, could be separated into two optically active asparagines. He successfully separated the β-asparagines and found that the compounds, like the natural products, differed in the rotation of the plane of polarization as well as in taste, in solubility and in density. Piutti wrote: The synthetic asparagines, thus obtained, differ from each other, just like the natural ones. Besides the different hemi- hedralism and the opposite rotatory power, even the taste, which in the levorotatory is insipid, while the dextrorota- tory is “sweet”. Furthermore “…as the rotatory asparagines have the same chemical composition, they are to be consid- ered as physical isomers. Piutti continued: This result […] also shows how the dextrorotatory aspara- gine, discovered by me in the vetch and now obtained by synthesis, is the physical isomer of the ordinary asparagine15. According to Piutti “the second [amidate ] supplied the inactive species, chemically isomeric, and until today unknown” (inactive α-asparagine) that he specified with Figure 3. The words of Piutti about the first discover of sweet asparagine: “From the mother liquor from which the asparagine was crystallized, some crystals formed whose pronounced sweet taste I immediately caught”15. Figure 4. Nomenclature given by Piutti to the “asparagines” he found: on the left the inactive asparagines that he called α-asparagine and the “mixture” of the two optically active asparagi- nes, that he called β-asparagine (α-asparagine and β-asparagine are two racemic mixtures; Piutti reported this observation, but was not able to confirm this assumption). On the right the products obtained by separation of the mixture of β-asparagine: the asparagine levoro- tatory, tasteless, and the dextrorotatory, with a sweet taste. 129The dextrorotatory sweet asparagine of Arnaldo Piutti: the original product is conserved in Florence the formula: CONH2-CHNH2-CH2-COOH. He conclud- ed by saying: I intend to complete the study of this asparagine [inactive α-asparagine] and to determine whether or not it is separa- ble from the two rotatory asparagines of the same composi- tion (such as the presence of an asymmetric C would sug- gest) when I will be able to prepare it in greater quantities. In 1890 Piutti modified the preparation method 17. This time the asparagines were obtained from the silver salt of “acido γ-ossimmidosuccinico” in ether via solvent evaporation and filtration of the iodide excess. The result is an oil: “nitrilosuccinato dietilico”, a nitrogen derivative of diethyl succinate. The oil was treated with bromine in acetic acid solution yielding the compound C6H7N2O3Br which is optically inactive. This compound was reduced in acetic acid solution with sodium amalgam. The moth- er liquor was left resting for a long time together with copper acetate; finally: with a fine sieve the [inactive] α-asparagine, reduced to powder by the loss of its crystallization water, is separated from the crystals of the rotatory β-asparagines, in their turn recognizable and separable by their taste or by their different hemihedralism17. In this way Piutti understood that the formation of the rotatory asparagines is independent on the syn- thesis procedure. This observation also emphasizes the instability of the inactive asparagine for what he ref- ereed to as “reasons of physical order”. However, his goal changed: he was prompted to isolate the inactive α-asparagine, and to separate it into the two rotatory forms. He wrote: The resolution of the inactive α-asparagine into two cor- responding rotatory asparagines acquires therefore more interest now and I am confident of having the means to experiment later17. PRESENCE IN NATURE OF THE SWEET FORM In 1915 Piutti made another important discovery: the two rotatory β-asparagines “coexist in the products of germinated lupins” 18. According to Piutti, as the dex- trorotatory sweet asparagine is mainly used by the plant itself, much more than its optical antipode, only a small quantity can be isolated. Finally the dextrorotatory asparagine disappears with the process of germination18. The presence of dextrorotatory sweet asparagine was not due to racemization during the extraction from the plant, but according to Piutti’s hypothesis, it was already present in the plant itself. The dextrorotatory sweet asparagine is the D-aminoacid: Piutti demonstrated its presence in nature. During the years in which the theories of van ‘t Hoff on optically active compounds were not yet easily accepted, Piutti successfully identified the two enanti- omers of asparagines. He demonstrated the correspond- ence between the crystallographic form and the optical properties of a chiral molecule and discovered a correla- tion between the different optical rotation of the mole- cule and the different response of the human receptors17. Finally, a few years later, he observed the enantio-specif- ic use of amino acids by plants 18. The existence of the two rotatory asparagines in lupins and their selective use in the plant and the obser- vation that only one of the two enantiomers interacts with our receptors, giving the sweet taste, represented two major discoveries in biological chemistry. These results were of fundamental importance that, perhaps Piutti (unlike Pasteur) did not fully understand. THE ASPARAGINE OF PIUTTI IS CONSERVED IN THE MUSEUM OF NATURAL HISTORY IN FLORENCE The original sample of dextorotary sweet asparagine obtained by Piutti in 1886 is conserved in the Schiff Col- lection of the University of Florence. Recently, the sample of dextrorotatory sweet aspara- gine has been included as a cultural asset to the scien- tific-technological heritage (“PST”, Patrimonio Scienti- fico e Tecnologico), according to the Italian Ministry for Cultural Heritage and Activities (Ministero per i Beni e le Attività Culturali). The information concerning this sample are available online through the SigecWeb sys- tem of the Central Cataloging and Documentation Insti- tute (ICCD)19. The scientific and private correspond- ence of Ugo Schiff is also conserved at the “Ugo Schiff ” Department of the University of Florence, including the correspondence between Schiff and Piutti of those years. A study of these letters will elucidate how and why the sample of the asparagine of Piutti remained in Florence in his master’s laboratory. Furthermore, it can shed light on Piutti’s arguments related to his experimental work and whether he really understood or not the importance of those discoveries. ACKNOWLEDGMENTS The authors gratefully acknowledge the current Head of the Department of Chemistry “Ugo Schiff ”, 130 Laura Colli, Antonio Guarna Prof. Andrea Goti, the past Directors and all the staff of the Department, for the support to their research on the historical roots of the chemistry works carried out in Florence. We thank Dott.ssa Mariagrazia Costa for her constant and timely attention. We are also grate- ful to the Museum of Natural History for the aid to the Chemical Heritage project, in particular we thank Prof. Marco Benvenuti, Dott.ssa Angela Di Ciommo and Dott. ssa Maria Giulia Maraviglia, Prof. Guido Chelazzi and Prof. Giovanni Pratesi. We acknowledge also Fondazione Cassa di Risparmio di Firenze for funding the Chemical Heritage project. REFERENCES 1. A. Guarna, L. Colli, M. Costa, in Rendiconti della Accademia Nazionale delle Scienze detta dei XL, serie V, vol. XXXIII, parte II, tomo II, 391-400, 2009. 2. J. Gal, Chirality, 2008, 20 (1), 5-19. 3. J. Gal, Chirality, 2008, 20 (10), 1072-84. 4. J. Gal, Chirality, 2012, 24 (12), 959–976. 5. L. Pasteur, Académie des sciences, 1886, Comptes ren- dus hebdomadaires des séances de l’Académie des sciences / publiés... par MM. les secrétaires perpé- tuels 6. G. Montaudo, Bollettino Accademia Gioenia di Scien- ze Naturali, 2000, Catania, Vol. 42 N. 371, 41-50 7. J. H. van’t Hoff, La chimie dans l’espace, Rotterdam, 1875. 8. J. I. Solov’ev   Jurij Ivanovič,   L’  evoluzione del pensiero chimico dal ‘600 ai giorni nostri,  Edizioni Scientifiche e Tecniche Mondadori, Milano 1976. 9. L. Pescitelli, Arnaldo Piutti, Napoli, Tip. Cimmaruta, Della R. Università, 1914 10. T.T. Tidwell, Angewandte Chemie, 2008, Volume 47, Issue 6, 1016-1020 11. L. Colli, L. Dei, A. Guarna, M. Costa, in La Palazzina dei Servi a Firenze. Da residenza vescovile a sede uni- versitari, a cura di Cristina De Benedictis, Roberta Roani, Giuseppina Carla Romby, Edifir Edizioni Fire- nze, 2014 12. L. Cantoni Appunti chimici sull’asparagina, Milano, Stab. Della Antica Casa Edit. Dott. Francesco Vallar- di, 1889 13. R. Piria Cimento, 1846, Gennaio-Febbraio 14. L. Pasteur, Annales de Chimie et de Physique, 1848, XXIV, 442. 15. A. Piutti, Gazzetta Chimica Italiana, 1886, XVI. 16. A. Piutti, Ricerche fatte nel laboratorio di Chimica far- maceutica della R. Università di Sassari, 1887-88 17. A. Piutti Rend. della R. Accademia delle Scienze Fisi- che e Matematiche di Napoli, 1890, Fasc. 3° 18. A. Piutti Estratti dalla R. Accademia delle Scienze Fisiche e Matematiche di Napoli, 1915, Allegato alla seduta del 4 Dicembre, Fasc. 11° e 12°. 19. http://www.iccd.beniculturali.it/index.php?it/118/ sistema-informativo-generale-del-catalogo-sigec. Accessed on 13/07/2018. Figure 5. The dextrorotatory sweet asparagine of Piutti: on the left the compound in the original bottle with the original label handwritten by Ugo Schiff; on the right, a particular of the crystals of original asparagines contained in the bottle. This item is conserved in the Schiff Collection of “Ugo Schiff ” Chemistry Department at University of Florence (class: Collezione Schiff, DCO20). Courtesy of the Museum of Natural History of the University of Florence.