Ortho-Diacetylbenzene (o-Di) and Some Analogues in Amino Acid Analytics and as Marker in Criminalistics

 o-Di Competitor of Ninhydrin?^*)

 

by Prof Dr Dr Randolph Riemschneider

Institute of Biochemistry, Free University of Berlin (FUB), Germany

and

Central Institute of Chemistry, Universidade Federal de Santa Maria (UFSM), Santa Maria, Rio Grande do Sul, Brazil

 

 

Dedicated to Prof Dr Conrad WEYGAND († 1945), who introduced the author to the subject in 1937.

 

The author developped the chemistry of o-diacetylbenzene from the beginning until Finished-product stage ( sales catalogues, SCHUCHARDT, MERCK) and made possible its application in analytical chemistry as a ninhydrin competitor and its use as a marker in criminalistics. The study of o-Di-analogues resulted into a kind of Polyacyl Chemistry: Table 1,2 and Plate 1.

Isolation and identification of ninhydrin-reductone brought light into the ninhydrin amino acid reaction: Plate 4 and 3.

 The editors

 

The author first prepared o-diacetylbenzene (o-Di) in 1937, using a special oxidation method, namely the permanganate oxidation of o-ethyl acetophenone in buffered solution (1, 11). o-Di is a white powder, from m p 39 - 40^oC, which can be ground to an almost invisibly fine dust and stains the fingers dark-blue/violet on contact. An indelible dye begins to form some 10 minutes after contact. We took considerable interest in this interesting substance and some of its analogues with neighbouring acyl groups on ring systems, primarily aromatic ones; cf Tab 1 and 2, Pl 1 in appendix.

 

 

 

 

 

 

 

 

 

 

 

 

 

 

_______________

*) Bull. IL of series „Acyl derivates of cyclic compounds“. Bull. I in ref. (11), bull. III in ref. (3) et al.

The readily visible, intensive staining by o-Di with amino acids and proteines suggested:

 

1)    to test the compound for analytical applicability and whether it could compete with            the accepted amino acid reagent ninhydrin

 

2)    if so, to find a simple and practicable pathway for synthesizing o-Di

 

3)    parallel to this, to seek among o-Di analogues compounds which, like o-Di and        ninhydrin, are characterized by neighbouring carbonyl functions on aromatic ring          systems

 

 

                                               

                        o-Diacetylbenzol                                           Ninhydrin[1]⁾

 

                 

1)    The analytical chemistry of o-Di

 

Both o-Di and ninhydrin are well suited for determining the concentration on amino acid solutions by colorimeter: Tables 3 and 4 in appendix. Ninhydrin is more sensitive for chromatographic determinations, the dyestuffs form o-Di and amino acids in solution are characterized by greater stability (2,3,4,)[2]⁾ Experience in amino acid analyser present in (5).

 

The continuing investigations into the use of o-diacetylbenzene[3]⁾ as a fluorescent reagent for determining amines, amino acids and proteins showed that o-Di is well-suited in the case of histamine, glutamine, ornithine, lysine, and taurine: Lecture in July 1958 (6a).

Dyes from o-Di and histamine (glutamine, lysine) were isolated and analyzed in collaboration with Dr H Höllriegel, and Dipl-Chem H-J Hein in 1980, working at temperatures below -25°C in organic solutions, in varying pH conditions and using modern physical methods (6b).

Our fluorescence-microscopic investigations with o-diacetyl-benzene into fixed paraffin sections of guinea pig organs and frozen sections of fresh tissue showed that the tissue protein NH₂-groups are linked to the reactions with o-Di: there is no fluorescence reaction if the NH₂-groups are blocked; blocking the COOH-groups increases the fluorescence reaction (7,8).

Despite great efforts[4], we did not succeed in clarifying the mechanism of color reactions of o-Di with amino acids, but were able to make progress with respect to the mechanism of the ninhydrin reaction:  cf. plate 4 in appendix. The familiar ninhydrin was included in our investigations of the color reactions of cyclic di- and tricarbonyl compounds with amino acids, peptides, proteins, and amines. In this way, we came to renewed investigation of color reaction of ninhydrin with amino acids.

 

Modification of ninhydrin reaction mechanism postulated[5]⁾ (10 a,b,c):

Starting from the postulated mechanism of ninhydrin reaction with amino acids, we initially endeavored to recover and identify an expected tautomeric reaction intermediate: Ninhydrin Reductone (II a ⇋ II b) received namely by alkaline saponification of 2-acetoxy-indandione-(1,3) (III) in ultrapure nitrogen: Plate 2 in appendix (10a).

By reason of our investigations into the reductone II b, we have formulated the ninhydrin reaction since 1960 as in Plate 4. This plate is taken from the author’s “Material for biochemical introductory lectures” 1969, 1^st ed, p 43 (10c,e).

The fact that with ninhydrin the individual amino acids yield variously colored products is probably linked, inter alia, to the lability of the reductone formed as an intermediate substance. To what extent dyestuffs of the type X formulated in (10d,e), as obtained by us when we excluded water, requires further investigation.

 

 

2)  Preparation of  o-Di (1, 11,14, 15, 30)

 

We have experimentally tested many possibilities for preparing o-Di over the years and reported on them: Plates 4 and 5 in appendix.

The oxidative degradation of 1,4-dimethyl-naphthalene (C₁₂ > C₁₀)

proved to be the best preparative pathway for recovering o-Di once we had improved synthesis conditions for producing the starting materials (14).

 

 

1-methyl-naphthalene (C₁₀H₇CH₃)

↓

1-chloromethyl-4-methyl-naphthalene (C₁₂H₁₁Cl)

↓

                                   1,4-dimethyl-naphthalene (C₁₂H₁₂)

↓

o-Diacetylbenzene (C₁₀H₁₀O₂)

 

 

 

At the request of Schuchardt Co in Munich, we in 1956 provided the prescriptions (elaborated by us and patented in 1954) for recovering o-Di and its precursor 1,4-dimethyl-naphthalene. Improved instructions were made available to Schuchardt in 1958 [only published in 1973 (14)]. Schuchardt included  o-Di in its sales catalogue from 1958: Leaflet from Schuchardt, Munich, on o-Di, in Appendix. Plate 3,  according to the Riemschneider prescriptions (15,14).

 

3)    o-Di Analogues: „Polyacyl Chemistry“ – a further 50 years of research in the field   of acyl compounds

 

The bibliography in (13) shows that in the course of the following 50 years since 1937 numerous publications appeared – as we casually say today – about “polyacyl compounds”, poly refering in this case to some triacetyl-, one tetraacetyl- and one hexaacetyl compounds. It proved impossible to realize the long striven for pentaacetylbenzene.

Many of the diacyl- thru tetraacyl compounds prepared are set out below in two tables and some plates, namely: in Tab 1 (App) those with at least one ortho position of acyl groups on the aromatic ring system; in Tab 2 most of the acyl compounds synthesized till 1960; in Pl 1 those obtained by diene synthesis from cis- and/or trans-diacetylethylene and unsaturated compounds (29, 28).

Only after some 30 years did it prove possible to obtain a homologue of o-diacetylbenzene crystallized pure, namely to prepare 1-acetyl-2-propionyl-benzene (II) by oxidative degradation of 1-methyl-4-ethyl-naphthalene. See Tab 1: II, mp 49-50°C (16,17,18).

In earlier years we had only obtained II as oil from bp at 13 mm 148-150°C (16) namely on the following synthesis pathway: phthalic anhydride, phthalyl acetate, 2-acetyl-benzoic acid, methyl phthalide, 2-ethyl-benzoic acid + propionic acid (over ThO₂ at 400°C), o-ethylpropiophenone, oxidized with AgMnO₄ to II-crude product (staining hands): Ref (1).

 

 

4)  The   applications  and   incidents  with o-Di over the years were varied and at times strange:

 

Criminalistics, telephone receiver affair, treatment of psoriasis, “cosmetic” applications of o-Diacyl compounds (tattoos).

 

CRIMINALSTIC APPLICATION (from 1947)

On Sundays, a member of staff in the Institute of Physiological Chemistry at the University of Berlin was pilfering the medically pure, drinkable alcohol out of the bottles on the shelves in the lab (1947). To catch the culprit, we dusted them with finest, crystalline, practically colorless o-Di powder.

On Monday, the individual concerned kept his blue-violet fingers for quite a long time and let the bottles well alone from then on.

This was the first criminalistic application. For several years, the detectives in Munich’s police department asked for o-Di from us in order to conduct practical tests (from 1953). It was subsequently recommended for CID use.

 

TELEPHONE RECEIVER AFFAIR

o-Di as “avenger”: As is evident from the numerous bulletins in Acylderivate cyclischer Verbindungen, several students and postgraduates worked in our labs at any one time, especially 1958-62.

Among the students with completely other subjects there was one who was conspicuous by his malevolence and cynicism. For example, he said to Kassahn, who had been working on o-Di for years: “I know a good synthesis, but I’m not going to tell you it.”

This was, of course, not the case but the barb hurt, particularly as the individual concerned was inconsiderate towards his colleagues in other respects as well, eg when solvents were delivered, everything for him before the others even noticed. (note: at that time everything was hard to get - even for money – (Particular circumstances in postwar period of occupied Germany)

He was one unpleasant ‘customer’ among some 20 staff. Wolfgang S., known as a joker and pornographer, thought out a punishment: the receiver of the phone in my office was dusted with o-Di. Then the individual concerned got an outside call and was told to hold the line. So he waited a while, got o-Di on his ear – that was colored violet for weeks.

The “punished” guy went to the police, who just laughed at him, particularly as he could not name the miscreant. Public mischief – he did not suspect Wolfgang Schneider who was working on his thesis in a completely different field that was in any case secret^)[6].

The “punished” colleague so refrained from annoying others from then on.

 

“TREATING” PSORIASIS

A colleague named KOKA from India had psoriasis. We tried using o-Di as a “medicament”, the strong reaction with proteins when forming colors having induced us. The local treatment was successful and even coloring blue-violet was not a problem for Mr Koka as he was completely black and you can’t see violet on black.

We pursued this approach in several directions:

Initially with a number of polyacyl compounds that colored less strongly but still reacted with proteins. Parallel to this, we conducted systematic experiments with esters of fumaric acid:

 

 

HC-COOR

   ║

  ROOC-CH               R = allyl, alkyl, aryl, aralkyl, alkynyl, alkenyl

 

This pathway was more promising and soon led to a commercial preparation, in 1958. Between 1958 and 1960 we succeeded in finding highly skin compatible fumaric acid esters that permitted successful treatment of psoriasis. Experience with over 500 patients was gained in collaboration with Japanese and Brazilian dermatologists: 1958-63.

 

TATTOO EXPERIMENTS

o-Diacetylbenzene (I) and 4,5-diacetyl-cyclohexene-(1) (II) were subjected to intensive pharmacological and toxicological testing before we had practical tattoo experiments conducted on mammals and then on humans too (19). The documentation on the experiments, conducted in Brazil, is deposited with Consulting-Development-Engineering of São Paulo. No application for doing tattoos with I and II on humans has been submitted in Germany. In Brazil, tattoos with I and II were only done under medical supervision.

It is of great interest that temporary tattoos are possible (19). The dermatological and toxicological tests done to date, as well as the Ames test, make practical use seem possible.

   

APPENDIX: Tables 1-4, Plates 1-6

 

Table 1:           o-acylated benzene and toluene derivatives and

                        o-diacetylnaphthalene

 

Table   2: Cyclic compounds with 2 to 3 carbonylfunctions            (1937-62)[7]

 

 

Table 3:   Color reactions of o-acyl compounds with amino acids: Limiting concentration and coloration

Key:    1            o-diacetylbenzene, mp. 40°C

2   1-acetyl-2-propionyl-benzene, mp. 49-50°C

3   1,2,3-triacetyl-benzene, mp. 149°C

4   1,2,4-triacetyl-benzene, mp. 75°C

5   1-ethyl-2,3,5-triacetyl-benzene, mp. 105°-106°C

6   1,3-dietyhl-2,4,5-triacetyl-benzene, mp. 102°C

7   1-methyl-3-ethyl-5,6-diacetyl-benzene (nur im Gemisch mit 96% 1-methyl-3-ethyl, 2,5-  diacetyl-benzene, bp 117°C (0,4Torr), n: 1,5350 (20°C)

8   1-methyl-2,3,5-triacetyl-benzene, mp 106,5°C

9   1-methyl-3-etyhl-2,5,6-triacetyl-benzene, mp 149,5°C

10 1-methyl-2,3,5,6-tetraacetyl-benzene, mp 183,6°C

for comparison:

11 ninhydrin, mp 241° - 243°C

 

legend of colours (pH: 8,00):  bl blue; gr grey; or orange; br brown; gn green; r red; d dark, h (hell) light, rs (rosa) pink,                              g (gelb) yellow, o (olive), v violet.

 

Legend to table 3:

Color reactions on spot plates: 14 amino acids were dissolved in concentrations of 10 ^-1 to 10 ^-6 M in Kolthoff buffer pH 8.0 (10.1 g borax/l (a); 13.62 g KH₂PO₄ / litre (b)); buffer solution: 53.5 ml a) and 46.5 ml b). The test reagents were 0.01 molar in 96% ethanol. - 100 μl each of amino acid solution and reagent solution were pipetted into test tubes, where some of the more concentrated amino acid solutions colored after a few minutes. The blends that were still uncolored after being left to stand for 30 mins were heated to boiling over a low flame in order to volatilize the ethanol, resulting in coloration in further mixtures. The limiting concentration of the amino acids was determined by transferring still uncolored or very weakly colored solutions on to spot plates and heating in the drying oven to 80°C till dry (approx 30 mins). Limiting concentrations and colorations are set out in the table.

In part other colorations were observed at pH values other than 8.0

 

Table 4:   Increasing the detection sensitivity of amino acids as compared with the  standard reagents o-diacetylbenzene (I) and ninhydrin (II)

 

 

 

                

 

 

 

 

Key to Tab 4: List of substances more sensitive to a specific amino acid than the standard reagents I and II (key as in Tab 3)

Plate 1: The o-diacetyl compounds I- XIV obtained from DIEN synthesis (28, 29)

 

Plate 2: “Ninhydrin-Reducton” [IIa    tautomer   IIb]  (10a)

Legend to plate 2:

The structure of the reductone II b was proved in 1960 as follows (previously unpublished) Lecture on 4 Sept 1960 in Tokio (10a) and  on  October 1960 in Höchst (10b)

Aqueous solutions of II b are colored reddish-orange. The action of O₂ results in decolorization while forming ninhydrin; the reaction can be followed by spectroscope.

Reductone characteristics:

Tillmann reagent (acetic acid dichloro-indophenol solution) is decolored by II b, AgNO₃ solution immediately reduced.

Dimethyl ether mp 95°C (IV) results from IIb with diazomethane; cf also Bull I in “On knowledge of ninhydrin reactions” (10a).

Quantative determination of II b is possible by indirect iodometric titration.

Comparison of the UV spectra peaks of II b, IV, V and VI in ethanol by strictest exclusion of oxygen showed: Fig 1: (10a,b)

 

II b      1,2-dihydroxy-indenone-(3)  475 nm            4 x 19^-4 mol/l [8]⁾

IV       1,2-dimethoxy-indenone-(3)  430 nm            2 x 10^-4

V         1-methoxy-indenone-(3)                    380 nm            5 x 10^-4

VI       1,3-diketo-indane                               420 nm            7 x 10^-5

 

Both II b and VI are largely enolized and dissociated in alcohol/water, resulting in a strong bathochromic shift up to 40 nm in the mesomerism of the compounds vis-à-vis IV and V. Monoenolates from II b and VI:

 

 

 

fig.1: UV-Spektren von IIb, IV, V und VI

 

IR spectroscopic investigations (10e)

 

KBr pressed disks with 2 mg/g KBr

The author discussed the IR spectra of II b, IV, V and VI in detail in the lectures of  Sept 60 and Oct 1960 (10b, c, e):

The peaks of CO valence vibration for the enol ethers IV and V are at 1701 - 1702 cm^-1. For VI, two band maxima occur, namely at 1704 cm^-1 and 1740 cm^-1; for II b at 1748 and 1720 cm^-1. Wave number of C=C vibrations of V maximum at 1564 cm^-1, corresponding band of IV at 1600 cm^-1. IV and V show split bands at 1630/1941 and 1605/1617 cm^-1, stronger for diether. Deformation vibrations of H atoms of methyl groups of IV and V: in the range of 1460/1468 cm^-1.

Plate 3:

 

Plate 4: Reformulation of ninhydrin reaction (10a-d)

 

 

Plate 5:       Synthesis pathways to o-Diacetylbenzene adopted in own work (13)

 

Plate 6: Synthesis pathways to o-Diacetylbenzene adopted  in own work (13)

 

 
References:

 

 (1)  R Riemschneider

Oxidation of ethylbenzene, o-,m-,p-diethylbenzene and o-ethylacetophenone with potassium permanganate in buffer solution:  o-diacetylbenzene[9]⁾  -  Permanganate oxidation in organic solvents[10]⁾ (Oxydation von Ethylbenzol, von o-,m-,p-Diethyl-benzol und von o-Ethylacetophenon mit Kaliumpermanganat in gepufferter Lösung: o-Diacetylbenzol – Permanganatoxidation in organ. Lösungsmitteln).

Voluntary Year’s Paper in chemistry for graduation at Matthias Claudius High School in Hamburg-Wandsbek, done from June 1937 till December 1938, submitted in Jan 1939: ms 96 pages.

Consultants for Year’s Paper in chemistry were: Dr W Hirsch (chemistry) and R Bach (mathematics and physics). Professor Dr H H Schlubach, Organic Chemistry at University of Hamburg, also gave an expert opinion  -  Summary of some results published 1947 as bulletin I in series “Acylderivate cyclischer Verbindungen”, deposited on 12 Sept. 1940[11]⁾ with editors of GAZZ. CHIM. ITAL., Rome: (11).

 

(2)   R Riemschneider, C Weygand †

Bull III: On the suitability of 1,2-diacyl-benzenes for proving and determining of amino acids (Über die Eignung von 1,2-Diacyl-benzolen zum Nachweis und zur Bestimmung von Aminosäuren)

Mh Chem 86, 201 - 209 (1955)

Prof Weygand was drafted into the Volkssturm (Hitler’s last-ditch levy) and fell in February 1945, shortly before the end of the second world war. He is cited as coauthor in gratitude for introducing me to the subject, and in reverence.

 

(3)   R Riemschneider, J Wierer

Bull XII: o-Diacetylbenzene as amino acid reagent – a comparison with ninhydrin     (o-Diacetylbenzol als Aminosäurereagens – ein Vergleich mit Ninhydrin)

Z analyt Chemie 193, 186-189 (1962)

 

(4)   R Riemschneider, K Hennig, T Wons

Bull XXVII: Color reactions of polyacylated aromatics with amino acids (Farbreaktionen mehrfach acylierter Aromaten mit Aminosäuren)

Mh Chem 118, 831-835 (1987); see table 3 and 4.

 

(5)   R Riemschneider

Bull XXXVI: “Application of o-diacetylbenzene and 1-ethyl-2,3,5-triacetyl-benzene as color reagent in amino acid analyser” (Einsatz von o-Diacetylbenzol und 1-Ethyl-2,3,5-triacetyl-benzol als Farbreagens im Aminosäureanalysator)

Ms 1985, 6 p

Part of lecture given in colloquium of Institute of Chemistry, SAITAMA University, Urawa, Japan on 23 August 1985, (chair: Prof Dr J T Shimozawa)

 

(6a)   R Riemschneider (lecturer), M Somplatzki, H G Kassahn, T H Kong

“o-Diacetylbenzene as reagent for fluorescence, above all for histamine, ornithine, lysine, taurine. Selective determination of L-lysine” (o-Diacetylbenzol als Reagenz für die Fluoreszenz, vor allem für Histamin, Ornithin, Lysin, Taurin. Selektive Bestimmung von L-Lysin)

Ms 1958, 14 p

Lecture given in colloquium of Department of Biochemistry at Free University of Berlin

Manuscript replicated (100 copies distributed to students)

Starting point for experimental results presented was early (1948-49) investigations (7,8). Instructions for determining histamine, glutamine, ornithine, lysine and taurine. Ibidem also with regard to o-phthalic aldehyde reaction with amino compounds.

 

(6b)   R Riemschneider, H Höllriegel, H-J Hein

Dyestuff formed from o-diacetilbenzene and histamine, glutamine, lysine.

Isolation and analysis of dyestuffs formed at -25°C, in large scale experiments, from o-diacetilbenzene and histamine (glutamine, lysine) by extraction, lyophilisation, followed by a series of chromatographic steps, by semipreparative high performance liquid chromatograohy, NMR and mass-spectroscopy

Ms 1980, 12 p (microfilm, in possession of Dipl.- Chem. H. J. Hein)

 

(7)   R Riemschneider, A Küchenmeister, M Somplatzki, C Winter

Fluorescent microscopy experiments with o-diacetylbenzene on mounted paraffin sections of guinea pig at pH 9.0 (Fluoreszenzmikroskopische Versuche mit         o-Diacetylbenzol an fixierten Paraffinschnitten von Meerschweinchenorganen bei pH 9,0.)

Lab reports 1948, 10 p plus appendix to Somplatzki’s 1952 diss.

Flourescence evident after approx 1 min. Lasts 4 - 6 mins with 0.15% o-di diluted      1 : 800 (ZEISS luminescence apparatus).

The short-lived light-blue fluorescence is bound to NH₂-groups in tissue, as various blocking tests show: fluorescent compound(s) stable for short period only as a function of pH and then change(s) into familiar colored product(s).

No fluorescent reaction when NH₂-groups blocked by acylation but increased reaction if acid groups blocked by esterification.

 

(8)   R Riemschneider, M Becker, M Somplatzki

Fluorescence microscopy experiments with o-diacetylbenzene on frozen sections of fresh tissue from South African clawed frog Xenopus laevis (Fluoreszenzmikroskopische Versuche mit o-Diacetylbenzol an Gefrierschnitten von frischen Geweben des südafrikanischen Krallenfrosches Xenopus laevis)

Lab reports 1949, 6 p

Strong light-blue fluorescence after 2 mins, stable approx 20 mins, then changes into familiar blue coloration (fluorescence fades).

The experiments to (28, 29) were conducted in the Institutes of Physiology and Physiological Chemistry of University of Berlin from 1947 - beginning 1948. Thanks to Prof Dr E Fischer, Deputy Director of Institute of Physiology, for invaluable advice on how to conduct these experiments.

On the advice of Dr Siebenmarck of the University of Leipzig, the two lab reports (7,8) were sent via Prof Dr E Fischer to Prof Dr H Voss, Director of the Institute of Anatomy, University of Jena, as he had been interested in the color reaction of o-diacetylbenzene during the second world war already (discussions with Weygand, Siebenmarck and Voss). Unfortunately, Wartenberg and Voss made no reference to the experimental data sent to them in their later publications. It must be assumed that Cold War political realities played a part, as E Fischer and the author were working in the West at the Free University of Berlin from 1950.

 

(9)     R Riemschneider, H Koch, Y Morino

o-Phthalaldehyde for detection and determination of amines and amino acids: formation of strongly fluorescent derivatives (o-Phthaldialdehyd zum Nachweis und zur Bestimmung von Aminen und Aminosäuren: Bildung stark fluoreszierender Derivate)

Ms 1947, 16 pages (unpublished)

Most favorable conditions established for producing and isolating dyestuff from o-phthalaldehyde and histamine at -10 – -20°C are described in more detail at (13).

 

(10a)    R Riemschneider

Bull I: “On ninhydrin and the tautomerism of bis-1,2-hydroxy-indanone-(3) with 2-hydroxy-indandion-(1,3)” (Über Ninhydrin und die Tautomerie des Bis-1,2-hydroxy-indanon-(3) mit dem 2-Hydroxy-indandion-(1,3))

Mh Chem 93, 841-842 (1962)

Presented to Japanese Chemical Society in Tokyo in Sept 1960

 

(10b)   R Riemschneider

Bull II: “On knowledge of ninhydrin reaction. Summarized presentation of ninhydrin reaction with regard to own experiences and investigations” (Zur Kenntnis der Ninhydrin-Reaktion. Zusammenfassende Darstellung über die Ninhydrin-Reaktion unter Berücksichtigung eigener Erfahrungen und Untersuchungen)

Lecture in chemical colloquium of Farbwerke Hoechst in Frankfurt-a-M in October 1960, at instigation and invitation of Head of Department, Dr F Scherer

 

(10c)    R Riemschneider, R Koka, H Kieseler

Bull III: Reaction of triketohydrindene with “labeled” α-amino acids under exclusion of water (Umsetzung von Triketohydrinden mit „markierten“ α-Aminosäuren unter Ausschluß von Wasser)

Mh Chem 94, 1131-1132 (1963)

 

(10d) R Riemschneider

Material for introductory lectures on biochemistry   at Free University of Berlin 1969, p 43: Reformulation of ninhydrin reaction   („Material für biochemische Einführungsvorlesungen“),

Freie Universität Berlin 1969, 1. Auflage, Seite 43: Neuformulierung der Ninhydrin-Reaktion) (based on results of Bull I and IV); cf. PROJ: I and XXVI in (13).

 

(10e) R Riemschneider, H Kieseler

Bull IV: On knowledge of ninhydrin reaction. Further experimental data on results of Bull I – III (Zur Kenntnis der Ninhydrin-Reaktion. Weitere experimentelle Daten zu Ergebnissen der Mitt. I bis III)

Ms 1963, 60 p

 

(10f)  R Riemschneider, J Wierer

Bull V: Comparison of ninhydrin and o-diacetylbenzene as reagent for amino acids and amines (Vergleich von Ninhydrin und o-Diacetylbenzol als Reagens für Aminosäuren und Amine)

Z analyt Chemie 193, 186-189 (1962); see also table 3 and 4.

 

(11)   R Riemschneider

Mitt I: Derivati acetilici di combinazioni isocicliche: o-, m-, e p-diacetil benzolo

Gazz Chim Italiana 77, 607 - 611 (1947), date of receipt 12 September 1940, deposited in agreement with Prof C Weygand, University of Leipzig.

Reason for the late release for publication; cf. also  footnote 11 to (1) and PROJ I 2 in (13)

At his express wish, Prof Weygand is not cited as coauthor as the results pertain to investigations conducted externally [Year Paper of Chemistry, Matthias Claudius High School, Hamburg (1)]

 

(12a) R Riemschneider (lecturer), H-J Hein, H Kahl

            “Permanganate oxidations in organic solvents (acetic anhydride, pyridine (AgMno4), glacial acetic acid), use of permanganates of potassium, sodium, lithium, barium, strontium, calcium, aluminum and silver for permanganate oxidation.” (Permanganatoxydationen in organischen Lösungsmitteln)

Summary of lecture given end of July 1943 in chemical colloquium of works laboratory of RUHRÖL GmbH, Bottrop; ms 44 pages. Given as habilitation lecture at Faculty of Math and Nat Sc of Kaiser Wilhelm University of Berlin on 21 January 1948, incorporating test results from 1938 to 1947 (1): certified copies of habilitation procedures of 09 January 1950; cf also (12b).

Only the permanganates of potassium and silver are suitable as oxidizing agents.

 

(12b) R Riemschneider

Habilitation colloquium at Fac of Math & Nat Sc of Humboldt University of Berlin: “Permanganate oxidations in organic solvents” (Permanganatoxydationen in organischen Lösungsmitteln); on 21 January 1948; cf also (1,12a).

Confirmation of habilitation in writing by Prof Dr K Lohmann on 3 March 1948.

 

(13)   R. Riemschneider

            Re-reading  -  66 years of chemistry (Nachlese – 66 Jahre Chemie) with approx 1,500 citations (own publications, lectures, lab reports, patents) and descriptions of PROJECTS I - XXVI plus vita

            (in preparation). Here relevant PROJ. I.

 

(14)   R Riemschneider, K Nolde, K Hennig

Bull XXII: Notice on preparation of diacetylbenzene (Notiz zur Darstellung von        o-Diacetylbenzol)

Mh Chem 104, 987-989 (1973). Schuchard Co (Munich) produced the o-di offered in its sales catalog according to this prescription since 1960: plate 3, later adopted by Merck (Darmstadt)

 

(15)   R Riemschneider

Bull IV: o-Diacetylbenzene derivatives (o-Diacetylbenzol-Derivate)

Patent registration R 21633 IV b/12b of 05 August 1957,

 

           

 

(16)   R Riemschneider, D Y Kwok

1-Acetyl-2-propionyl-benzene from [phthalic anhydride, 2-acetyl-benzoic acid, methylphthalide, 2-ethyl-benzoic acid + propionic acid (via ThO₂ at 400°C)], o-ethyl-propionophenone, oxidized to acetyl-2-propionyl-benzene bp  148 - 150°C (13 mm).

Mitt Physiolog-chem Institut Berlin, 1948, 11 pages.

 

(17)  R Riemschneider, T Wons

Bull XXIX b: 1-Acetyl-2-proprionyl-benzene, m.p. 49°-50°C

Presented to J Am Chem Soc (received in Columbus, Ohio on 28 May 1985): Table1.

 

(18)  R Riemschneider (lecturer), T Wons

Bull XXIX a: “Homologues of o-diacetylbenzene: 1,2-diproprionyl-benzene and 1-acetyl-2-proprionyl-benzene” (Homologe des o-Diacetylbenzols: 1,2-Dipropionyl-benzol und 1-Acetyl-2-propionyl-benzol)

Ms 1985, 6 p; cf lecture (31)

 

(19a) F R Pesserl, R Riemschneider, H Fereira

Bull XLVI: Continuation of investigations from Bull XLIII:

1985-90/1999-2003 long-term experiments to test for carcinogenic effect of o-diacetyl-benzene and 4,5-diacetyl-cyclohexene  (skin of rats, pigs), simultaneous continuation of practical tattooing tests (temporary tattoos), Ames tests (Langzeittestversuche zur Prüfung auf kanzerogene Wirkung von o-Di und 4,5-Diacetylcyclohexen: 1985–1990, 1999-2003 (Rattenhaut, Schweinehaut), gleichzeitig auch Fortsetzung der praktischen Tätowierungsversuche (zeitlich begrenzte Tätowierungen), AMES-Teste)

Ms 1991 (unpublished)

Tests conducted in labs of Consulting-Development-Engineering in Sao Paulo, Brazil

Results: o-diacetylbenzene shows no mutagenic effect; it is suitable for tattooing.

 

(19b) R Riemschneider, M Salvioni

Bull XXXIV: Application of diacetylbenzene (I) and some analogues, for instance 4,5-diacetyl-cyclohexene isomers (II) as  dye stuffs in cosmetics: long-term colorings of skin (tattooing) toxicol. expriments of I and II. Experiences from 1985.  [Anwendung von o-Diacetylbenzol (I) und einiger Analogen, z.B. 4,5-Diacetyl-cyclohexen-Isomere (II) als Farbstoffe in der Kosmetik: Langzeitfärbungen der Haut (Tätowierung) – Toxikolog. Untersuchungen mit I und II . Erfahrungen ab 1985]

Ms 12 p (secreted).

 

(20)   R Riemschneider, B Dietrich

Bull XVII: 1-Ethyl-2,3-diacetyl-benzene and 1,2,3-triacetyl-benzene (1-Ethyl-2,3-diacetyl-benzol und 1,2,3-Triacetyl-benzol)

Liebigs Ann Chemie 646, 18-23 (1961).

 

(21)  R Riemschneider, T Wons

Bull XXIV: 1,2,4-Triacetyl-benzene from 6-acetyl-1,4-dimethyl-naphthalene (1,2,4-Triacetyl-benzol aus 6-Acetyl-1,4-dimethyl-naphthalin)

Mh Chem 114, 1267-1268 (1983).

 

(22)  R Riemschneider, K Hennig, T Wons

Bull XXVII: 1-Ethyl-2,3,5-triacetyl-benzene, an amino acid reagent (1-Ethyl-2,3,5-triacetyl-benzol, ein Aminosäurereagens)

Z analyt Chemie 325, 561-562 (1986)

 

(23)  R Riemschneider, K Hennig, T Wons

Bull XXVII: Color reactions of polyacylated aromatics with amino acids (Farbreaktionen mehrfach acylierter Aromaten mit Aminosäuren)

Mh Chem 118, 831-835 (1987)

 

(24)  R Riemschneider, K Hennig

Bull XXVI: 2,3,5-Triacetyl-toluene (2,3,5-Triacetyl-toluol)

MhChem116, 873-876 (1985)

 

(25)  R Riemschneider, K Hennig

Bull XXV: 2,3,6-Triacetyl-5-ethyl-toluene and 2,3,5,6-tetraacetyl-tuluene (2,3,6-Triacetyl-5-ethyl-toluol und 2,3,5,6-Tetraacetyl-toluol)

Z Naturforschg 39 b, 835-838 (1984)

 

(26)  R Riemschneider

Bull XLI: 1,2,4,5-Tetraacetyl-benzene (I) thru oxidation of 1,4-dimethyl-6-ethyl-7-acetyl-naphthalene and/or thru oxidation of 1-ethyl-2,4,5-triacetyl-benzene (II) [ I ← II ] (1,2,4,5-Tetraecetyl-benzol (I) durch Oxydation von 1,4-Dimethyl-6-ethyl-7-acetyl-naphthalin bzw. durch Oxydation von 1-Ethyl-2,4,5-triacetyl-benzol (II)      [ I ← II ].

Ms December 1985, 9 p

 

 

 

Proof of structure for I and II thru MS, IR, NMR

Experiments conducted in Central Institute of Chemistry at the Federal University of Santa Maria (UFSM), Santa Maria, Rio Grande do Sul, Brazil

 

(27)   R Riemschneider (lecturer), H-J Hein

Bull XVIII: “o-Diacetylnaphthalene through a) degradation of 1,4-dimethyl-anthracene b) synthesis, starting from naphthalene-dialdehyde-(2,3)” [o-Diacetylnaphthalin,     a) durch Abbau von 1,4-Dimethyl-anthracen, b) durch Synthese, ausgehend von Naphthalin-dialdehyd-(2,3)]

Lecture of 12 November 1962: “o-diacetylnaphthalene (I)

[2,3-diacetyl-naphthalene] mp 118°C” in chemical colloquium of University of Santa Maria (USM), S Maria, RS, Brazil

Ms January 1961, 12 p, 100 hectographed copies distributed (in English) as manuscripts.

Extract from lecture manuscript: The constitution of 2,3-diacetyl-naphtalene (I) m.p. 118-119^° C, was proven by IR-spectroscopy (fig.) and by colour reactions of I with amino acids (tabl). Colours observed: dark violet with Gly; violet: Ala, Phe; red/violet: Ser, Lys, β-Ala, Leu, Thr, Pro; blue: His; yellow-green: Try. The formation of oximes and hydrazones was disturbed; the high activity of CO-groups results in polymerization and aldol condensation: the IR-spectrum shows OH-bands.

The starting material, the diol, oxydized to I (via b), is melting at 131^°C, its dibenzoat at 153^°C.

 

(28)   R Riemschneider, K Preuß

Bull XI: Color reactions of amino acids with 4,5-diacetyl-cyclohexene-(1), [Farbreaktionen von Aminosäuren mit 4,5-Diacetyl-cyclohexen-(1)]

Mh Chem 90, 924-928 (1959): plate 1

 

(29)   R Riemschneider (lecturer), P Claus

Bull XXI a: “Color reactions with cyclic di- and triacetyl compounds, received by DIEN-synthesis” (Farbreaktionen mit cycl. Di- und Triacetyl-Verbindungen): plate 1

Mh Chem 93, 844-850 (1962); cf also lecture of 04 September 1960 to Japanese Chemical Society

 

(30)   R Riemschneider, S Foerster

Bull XX: o-Diacetylbenzene from 1,4-dimethyl-naphthalene (o-Diacetylbenzol aus 1,4-Dimethyl-naphthalin)

Mh Chem 93, 616-617 (1962) For patent reasons, no refererence is made to earlier bulletins [for instance (15)]  about the synthesis of o-diacetylbenzene and substitution products from 1,4-dimethyl-naphthalenes, ie through degradation reactions i.e.  C₁₂ →C₁₀ (14).

 

(31)  R Riemschneider (lecturer), K Hennig, T Wons, H-J Hein, G Buchlow, M Salvioni

Lecture „Recent results in the field of the chemistry of acyl derivatives of cyclic compounds and some side reactions” (Neuere Ergebnisse auf dem Gebiet der Chemie der Acylderivate cyclischer Verbindungen und einiger Nebenreaktionen), given on 10 August 1984 in colloquium of Central Institute of Chemistry at the Federal University of Santa Maria (UFSM), Santa Maria, Rio Grande do Sol, Brazil and based on the results of bulletins XXVI - XXXI and XXXV (13)

Lecture published in Portuguese as special edition of UFSM 1985, 21 p

 

(32)  R Riemschneider, K Hennig

Bull XXV: 2,3,6-triacetyl-5-ethyl-toluene and 2,3,5,6-tetraacetyl-tuluene (2,3,6-Triacetyl-5-ethyl-toluol und 2,3,5,6-Tetraacetyl-toluol)

Z Naturforschg 39 b, 835-838 (1984)