TETRAZOLIUM SALTS AS CHEMICAL REAGENTS' NICHOLAS D. CHERONIS and HERMAN STEIN Brooklyn College, Brooklyn, New York T m m z o L I u ~salts are quaternary ammonium compounds and are somewhat soluble in water. Their solutions are colorless or very pale yellow and upon reduction yield highly colored insoluble substances called formazans, as shown in equation (1).
ClTet~rasoliums d t (soluble in water and colorless)
N=N-Ra Formman (insoluble i n water and hiehlv colored)
Owing to this property and their low reduction potentials, they have been introduced as reagents during the past 10 years for the detection and study of reducing enzyme systems and for the detection and estimation of reducing functions of organic compounds. An extensive review on tetrazolium salts bas appeared recently (1). Their discovery and introduction as reagents is of interest. The tetrazole ring was discovered in 1885 by the Swedish chemist Bladin, and about the same time Pinner described a reaction of diazonium salts with benzimino ethyl ether which gave rise to colored compounds later called formazans. In 1892 Von Pechman (2) and Bamberger (3) independently synthesized the colored formazans. A year later Von Pechman noted that oxidants attacked the imide hydrogen of hydrazones to form an N-N linkage, and by analogy he oxidized the formazan to close the ring and form the tetrazolium salts. Pechman and his co-workers prepared a large number of formazans and about 15-20 tetrazolium salts, and studied their properties. After that, interest in this group of compounds waned until they were taken up by Kuhn (4) and his co-workers who were primarily interested in the bactericidal properties of quaternary ammonium compounds. As a result of the study on the properties of the tetrazolium salts, Lakon (6) proposed their use for testing the viability of seeds. For example, corn seeds, if soaked in water for a few hours and then cut into halves and immersed for 30 minutes in one per cent solution of a tetrazolium salt, develop deep colors of the formazans along the areas of the germ if the seeds are viable, and no colors if the seeds are dead.
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Presented before the Division of Chemical Education a t the 128th Meeting of the Amoriaan Chemical Society, Minneapolis, September, 1055.
Application of tetrazolium salts to the detection of viability of seeds was pointed out by Dutcher (6) who interrogated Lakon in 1945 as a member of the American Intelligence Branch in Germany. Subsequently, Porter, Durrell, and Romm (7) ; Mattson, Jensen, and Dutcher (8); Waugh (9); and Cottrell (10) confirmed the observations of Lakon and indicated that tetrazolium salts might be useful as reagents to detect differences in the viability of seeds and other tissues. This outstanding property is due to the fact that tetrazolium salt solutions on reduction by hydrogenases, or generally bx reductants, yield colored formazans as shown in equation % .,
(1).
The application of tetrazolium salts t o the study of carcinomatous tissues n-as made by Straus, Cheronis, and Straus (ii), who showed that tetrazolium salt solutions become reduced in uivo or vitro a t a greater rate than by normal tissues. As a result of this, a prodigious amount of interest has been accumulating in the literature on the properties of these reagents. NATURE, PREPARATION, AND NOMENCLATURE OF TETRAZOLIUM SALTS
There are three types of tetrazolium salts: monotetrazolium, N-N ditetrazolium, and C-C ditetrazolium salts, shown by equation (I), and formulas (2) and (3), respectively:
+
+
CICI S-T Ditetl.azolinm salt
Of these, the first t v o are of greater importance owing to their greater ease of preparation and application. Of the various methods of preparation, the most practical is to react an aldohydrazone with a diazonium hydroxide to give the formazan, and then oxidize the latter to the tetrazolium salt as shown in equation (4) which illustrates the preparation of 2,3,5-triphenyl tetrazolium chloride:
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VOLUME 33, NO. 3, MARCH, 1956
,c
C6H6NHNHz
C,H,-C-Q
H N
carbon and proceeds clockwise including next the other
- CeHrNztOH-, three nitrogen atoms and finally the carbon atom. It' should be noted that the formazan is named after
H\ i
I
H
H
Aldohydrazone
C,H,C=N-C,H,
[ a
]
Cs~,-~=s Rearr.
C,H,-C / h-CsHs R-ON0 \T
I I v=v ..
/ -
Y
..
