Sudan Black B Chemical Structure & Histochemistry of Blue Main Components

Histochernistry 54, 237-250 (1977) Histochemistry �9 by Springer-Verlag 1977

Sudan Black B: Chemical Structure and Histochemistry of the Blue Main Components*

U. Pf/.iller, H. Franz, and A. Prei13

State Institute for Immune prcparates and Nutrition Media, Klement-Gottwald-Allee 317-321, DDR-112 Berlin, German Democratic Republic, and VEB Chemiekombinat Bitterfeld~ Bitterfeld, German Democratic Republic

Summary. Sudan Black B contains two blue main components, SSB-I and SSB-II. Their chemical structures were determinated by the aid of two- dimensional thin-layer chromatography, column chromatography, absorp- tion, IR, mass, H1-NMR, and CI3-NMR spectroscopy and were proved by alternate synthesis. SSB-I has been found to be 2,3-dihydro-2,2-dimethyl- 4-[(4-phenylazo- 1-naphthalenyl)-azo]- 1H-perimidine. For SSB-II was confirmed the known structure 2,3-dihydro-2,2-dimethyI-6-[(4-phenyIazo-I- naphthalenyl)-azo]-lH-perimidine. Relations of chemical structure of SSB-I and SSB-II to their staining properties are discussed.

Introduction

An enormous amount of literature has accumulated in the field of lipid staining with Sudan Black B (SSB). Much has been written relating to the properties and stabilities of SSB solutions and also to the fractionation of SSB into different colored fractions. Until now, however, only limited studies have been made about the chemical structure of these dyes. The synthesis of the common lipid stain SSB was described first in patent literature (BASF, 1913) some 60 years ago. This dye for staining lipids was introduced in 1934 by Lison. The chemical structure of SSB was derived from its synthesis. The formula for SSB was cited in 1952 in the histochemical literature by Berman (1952).

SSB (Fig. 1) is formed by the coupling of diazotized 4-phenylazonaphthalen- amine-1 (3) with 2,3-dihydro-2,2-dimethyl-lH-perimidine (1). The resulting dye contained many colored and colorless by-products.

Therefore, SSB would have the structure 2,3-dihydro-2,2-dimethyl-6-[(4- phenylazo- 1-naphthalenyl)-azo]- 1H-perimidine (4).

SSB composition was the subject of many studies and in all cases one blue fraction and other dye fractions were found (Bermes and McDonald, 1957;

ProFessor Dr. G. Hollo on the occasion on his 65th birthday

238 U. Pffiller et al.

Ar-NH2 NaNO2/HC~ Ar-(CNI! + 2_ 3_

4 Fig. 1

C H 3 ~ H 3

55 Ar Ar

5 _6 Fig. 2

Terner, Schnur and Gurland, 1963; Schott, 1964; Horobin, 1971, 1975; Jordan, 1971). Lansnink (1968) isolated two chemically pure blue fractions by preparative thin-layer chromatography from commercial SSB, and detected at least 18 frac- tions and impurities. He named the less polar substance SSB-I (content 20%, Rf=0.49) and the more polar SSB-II (60%, Rf=0.19). Pffiller, Franz and G16ckner (1977) substantiated these results, improved the analytical detection of dye components by two-dimensional thin-layer chromatography, and devel- oped a highly efficient chromatographic method for large-scale isolation of SSB-I and SSB-II. By combining these methods, the authors detected more than 40 colored and colorless by-products in SSB, independent of the origin.

As a result of his study including absorption and infrared spectroscopy, Lansnink (1968), proposed that the structure 4 (Fig. 1) seems justified for SSB-II and that SSB-I possibly is a dye with two primary amino groups in 1,8-position (5, Fig. 2). However, for elucidation of the structure of such dyes, the absorption and infrared spectroscopy are unspecific and ambigous.

Sch~ifer (1970) used formula 6 (Fig. 2) for SSB without further particulars on the orientation of the substituent Ar (Ar=4-phenylazo-naphthalenyl-azo-). Lansnink (1968) obtained SSB-I and II in mg amounts and we isolated the two blue components in 1-10 g amounts per run (Franz, Pftiller and Gl6ckner, 1977). Now it was possible to do a detailed study on the chemical structures of the blue SSB components for a more complete understanding of their histo- chemical properties.

Sudan Black B 239

Material and Methods

For the isolation of SSB-I and II according to Franz, Pfiiller and Gl6ckner (i977), we used SSB (technically) from VEB Chemiekombinat Bitterfeld. The obtained dye components were purified twice by column chromatography. The degree of purity was 99% and >98% for SSB-I and SSB-II, respectively, as has been shown by two-dimensional thin-layer chromatography (PftiIIer~ Franz and G16ckner, 1977). 1,8-naphthylendiamine (VEB Chemiekombinat Bitterfeld) was purified by crystallization (ethanol/water 1:2, mp 65-66~ The perimidine derivate 1 was prepared according to the literature (BASF, 1900) and purified by crystallization (ethanol) and precipitation (with water from ethanol); mp 106~ According to Turner (1949), the dye 2 was prepared and then separated from 8% o-isomere by column chromatography; mp 124~

The chromatographic separations were performed on neutral alumina (activity II-III, VEB Laborchemie Apolda) for column chromatography and on silicagel sheets 'silufol' fiom Kavalier, Prague. The C,H,N analyses were carried out with a microanalyzer, model F & M 185. Spectrophoto- metrical measurements were performed with a universal spectralphotometer, IR spectra with the UR 10, mass spectra with a Varian MAT 7ll, and NMR spectra with the 80 MHZ-Tesla-NMR spectrometer BS 487 or with the Bruker-NMR spectrometer HFX 90. Internal standard hexamethyl- disiloxane (HMDS) was used. Melting points are uncorrected.

Results

Some Chemical Characteristics of SSB-I and SSB-H

The SSB componen t s show a different behavior at higher temperatures. SSB-I melts at 180-186~ under slight decomposi t ion and SSB-II was completely de-

composed under these condit ions. SSB-I can be subl imated without decomposi- t ion at 140 160~ and 10 ~ tort . At 1 0 - t torr, the decomposi t ion of SSB-I begins and the c o m p o u n d 2 is detectable in the sublimate. It is very difficult to remove the last residues of solvents (benzene, chloroform) and amines, espe- cially amine 1. The amine 1 can be detected in SSB-II by N M R spectroscopy wi thout its separat ion in D6-acetone as solvent. To remove the solvents, the dyes were stored 5 days under n i t rogen in the dark in a desiccatore with paraffin and polyvinylpyrrol idone. Then they were dryed at 50~ and 10-1 torr. After

that, these C ,H ,N values were obta ined by microanalysis (Table 1). The values for n i t rogen reported by Lansn ink (1968) are too low. Perhaps

the dyes were not free f rom amines and solvents. We found a simple color react ion for the dis t inct ion of bo th SSB components . SSB-II reacts with a

Table 1. C,H,N values of SSB-I and SSB-II

SSB-I SSB-II

C H N C H N

Calcd., % 76.29 5.30 t8.41 76.29 5.30 /8.41 Found, % 76.43 5.18 I8.15 76.61 5.08 18.10

17.96 18.26 Found by Lansnink (1968) - - t4.35 14.60

240

Table 2. Assignments of characteristic IR data of SSB-I and SSB-II

U. Pftiller et al.

Wavelength in cm-~ Assigned group

SSB-I SSB-II

650 1090 aromatic groups

2930 2930 2976 2970 2890 2862 1525 1525 1365-1395 1385 1395

CH 3-

3040-3070 3045 3072

1525 1525 1585 1595 1600 1650

aromatic groups

3395 3395 Ar-NH-R 1585 t585 -NH- 1600 1590 1620 1650 1295 1293 C-N 1305 1310

osmium tetroxide solution and immediately shows a color change from blue or violet-brown (on chromatograms) to yellow; after 10-15 min, SSB-I also shows this color reaction.

Absorption Spectroscopy

Our results and studies correspond to the spectroscopic data given by Lansnink (1968). We also found changes in light absorption, dependent on media pH values. The extinction values measured by us are generally somewhat higher.

IR Spectroscopy

IR absorptions and intensities are listed in Table 2 and the spectra are re- produced in Figure 3.

A correct assignment of primary and secondary amino groups is difficult because interference is possible between aromatic and amino groups.

Mass Spectroscopy

The spectra of both blue components show marked molecular peaks, especially SSB-I at 456 m/e + (tool wt of SSB: 456.6). It is possible to give fragmentation

Sudan Black B

14 11 9 ~5 3,5 3 ()aj , , . , ..,. , ~ , - 7 , , , F1oo

" " -" ~ . i ' N " ' ""'.

~".. . . . = + ~ : ~ - f \ .

:! {L

(cm-1) 1- 2'600 3000 3400 l

I I i L I = _____L_J_ O

Fig. 3: IR spectra of SSB-I ( - - ) and SSB-II ( . . . . . ) (2-3 mg dye per 150 mg KBr)

100

,tO ~1 18 77 182 336

+'~ + I + 11t m _ . . I , . I).il ! , . . . ,= , J , . . lli Z ko

~100

i,~ 20

/*3 183

11.5

" Ill L 15/. I ,I,il ,I,IL ,,,l

z6 '~ " I~o . . . .

198

413

II,

~ig. 4: Mass spectra of SSB-I and SSB-II

247 413

200 300 400 role

441

?

24 t

M § /-,56

I , L

schemata for SSB-I and II which correspond to mass spectral data. Figure 4 shows the characteristic mass peaks of SSB-I and II.

N M R Spectra

The N M R spectra of SSB-I and II are very complicated in their aromatic region, Therefore, it was impossible to analyze the spectra completely. Neverthe- less, some important information concerning the structure of SSB-I and [I was obtained from these spectra (Figs. 5 and 6).

242 U. Pfiiller et al.

Fig. 5a and b: 1H-NMR spectrum of SSB-II in CDC|3 a Standard spectrum, b Four times expanded spectrum of aromatic region

The spectrum of SSB-II is shown in Figure 5. It is not contradictory to the proposed structure (4). The singlet of the methyl groups appears at 1.34 ppm. The integrated intensity of the aliphatic and aromatic protons is about 6: 16. In the aromatic region (Fig. 5b), the signals at 6.39 ppm, 7.29 ppm, and 8.28 ppm were assigned by the aid of INDOR technique to the protons H9, Ha, and H~ of the dihydroperimidine ring. Likewise, INDOR irradiations indicated that the four lines at 6.28 ppm, 6.38 ppm, 7.92 ppm, and 8.02 ppm belong to an AB spin-system. But it could not be decided whether these lines represent the 4- and 5-protons of the dihydroperimidine ring or the 2'- and 3'-protons of the 4-phenylazonaphthalenylazo grouping.

The multiplet centered at 8.92 ppm was assigned to the protons Hs, and Ha, respectively, which may be expected to absorb at a lower field than the other protons of the molecule. The broad signal of the NH protons appears between 4.0 and 4.8 ppm. The singlet of the methyl groups was also found in the spectrum of SSB-I (Fig. 6). The integrated intensity of the aliphatic and aromatic protons is the same as for SSB-II. In the 13C-NMR spectrum

Sudan Black B 243

Fig. 6a and b: tH-NMR spectrum of SSB-I in CDC13. a Standard spectrum, b Four times expanded spectrum of aromatic region

7

Fig. 7

of SSB-I, the quarternary carbon Cz was identified by its chemical shift (6n~tDS = 63,04 ppm) and by a proton off-resonance decoupling experiment. These NMR parameter are clear evidence for the existence of the dihydroperimidine ring even in this dye component. On the other hand, the orthoisomeric structure (7, Fig. 7) is confirmed by the shifts of the NH protons. Kurkovskaja et al. (1973) found that in fi-naphthylamino-azobenzenes signals due to both free

244 U. Pffiller et al.

Table 3. Chemical shifts of the assigned NMR signals of SSB-I, SSB-II, the perimidine derivate 1 and.the azo dye 3

H 3 ~ C H 3 ~1 =Ar HNI 3N, H N, B'

9 ~ R~ 2 ' ~ 7 '

R6 r N = N - ~

Compound R ~HMDS (ppm) R PHMDS (ppm)

2,3-dihydro- R4=H -CI-I 3 1.34 - 2,2-dimethyl- R6=H H4. 9 6.31 - 1H-perimidine NH 3.95

4-phenylazo- napthalen- amine-1

1 - N = N - H~ = I - N H 2 H~

H; NH2

6.55 7.75 8.90 4.35

7 SSB-I R,=Ar R6=H

-CH3 1.54 H7 6.95 H8 7.18 H 9 6.31 H 1 4.0 H a 10.36

4 SSB-II R4=H -CH3 1.34 H; \ R6 =Ar H7 8.28 H~ f

H s 7.29 H 9 6.39 NH 4.0-4.8

8.92

and intramolecular bonded (to azo group) N H protons can be observed at 5 and 10 ppm, respectively. In conformity with this, the signal at 10.36 ppm was assigned to the bonded and the broad signal at 4 ppm to the free N H proton.

The chemical shifts of the assigned signals in SSB-I and II in the 2,3-dihydro- 2,2-dimethyl-lH-perimidine (1), and in the 4-phenylazo-naphthalenamine-1 (3) are listed in Table 3.

C h e m i c a l - Analytical Investigations

The dyes SSB-I and I I were subjected to reductive cleavage and the resulting amines identified by thin-layer chromatography and by comparison with au-

245

SSB-I 7

\ /

4 u

SSB-II g

10

11 NH2 Fig. 8

thentic samples. In both cases, we found aniline (8), 4-naphthalenediamine (9), and the aminodihydroperimidines 10 and 11 from SSB-I and II, respectively (Fig. 8).

Reduetive Cleavage

a. A solution of 30 mg dye component in ethanol was added to 2 ml Na2S20~ solution (8% in water) at 60~ under magnetic stirring. The colorless solution was made alkaline and extracted with 2 3 ml ether. This extract was used for TLC investigations.

b. 0.2 m1 acetic acid were added to a solution of 20 mg SSB in 3 mi 90% ethanol and then at 50~ under stirring zinc powder. The resultant colorless solution was treated as described in a.

The TLC separation and detection were carried out according to Bassl et al. (1967). (TLC=thin layer chromatography)

The amines 10 and 11 were synthesized by reductive cleavage of o- and p-phenylazo-2,3-dihydro-lH-perimidine (12 and 13) which were prepared ac- cording to Vyskocil (1964) and separated by preparative thin-layer chromato- graphy (Fig. 9). Vyskocil synthesized the phenylazo dyes of 1,8-naphthylenedia- mine (14).

Synthesis of SSB-I and SSB-H

The final proof for the chemical structure of SSB components has been deter- mined by alternate synthesis according to the following schema (Fig. 10).

In this connection, it was impossible to condense SSB-I with acetone in the presence of dilute sulfuric acid to form SSB-II. That means SSB-I does not have two primary amino groups in 1,8-position as proposed by Lansnink for this dye component. The coupling of 1 and 3 yielded the isomeric SSB dyes (SSB-I to SSB-II as 1:2). On the other hand, 1,8-naphthalenediamine (14) and 3 yield the isomeric dyes 15 and 16 in a similar proportion as well as a number of by-products. The compounds 15 and 16 were separated by column chromato- graphy and condensed with acetone to form two blue dyes; 15 yields SSB-I and 16 SSB-II. The reduction of the synthesized isomeric dyes presents the picture already described.

246

12 13

U. Pffiller et al.

Fig. 9

L

4 A r

acetone I H §

+ Ar-4~ 3_ 3__ A r - t ~ ",

acetone / H + Fig. 10

Synthesis of SSB-Components

The SSB components were synthesized in a similar manner as described by Vyskocil (1964) for dyes 12 and 13. Compound 3 was prepared according to Tr6ger and Schaefer (1926). Dyes 15 and 16 were separated by column chromato- graphy on alumina (I0 cm, ~1 . 5 cm) with carbon tetrachloride, benzene, and chloroform/ethanol as eluents. Then the dyes were solved in 2 ml 70% ethanol and 0.3 ml acetone. After addition of three drops 20% sulfuric acid, the mixture was stirred 0.5 h at 60~ cooled, neutralized with soda solution, and extracted with ether. The ether extracts were used for chromatographical comparison with SSB-I and II.

Preparation of Stabilized SSB Solutions for Histochemical Use

SSB weighing 0.2 g (SSB-I, SSB-II, or mixtures) were dissolved in 100 ml 70% ethanol which contained maximally 0.1% hydroquinone dimethylether or 2.6-di-tert.-butyl-phenol and 0.01% EDTA. A SSB solution containing a

Sudan Black B 247

special chelating antioxydant (<0.05%) is advantageous for binding air and metallic cations, e.g., a solution of the antidote unithiol (sodium-l,2-dimercapto- propansulfonate, prepared according to Busev, 1972). In this way, prepared solutions can be stored in brown, tightly sealed bottles at 4~ for at least 6 months. Sediments are not observed and the photometrically estimated dye content amounts to 0.19%. It is possible to prolong the storing time, when the consumed solution volume is compensated by addition of glass balls. Histo- chemical investigations with the new dye solutions were not carried out hitherto.

Discussion

Our chemical and spectroscopic studies have shown that the two blue main components of SSB are ortho-para-isomeric tetrazo dyes with the structure 4 and 7 for SSB-II and I, respectively. Both isomeric dyes, especially SSB-II, are basic compounds which can bond to cationic exchangers in the H+-form and to appropiate negatively charged cell and tissue structures. SSB-I is obviously less basic because an intramolecular hydrogen bond may exist between one secondary amino group and the ortho-diazo group. This phenomenon is represented by molecular models in Figure 11.

These findings provide a more complete understanding of the less polar character of SSB-I, its higher stability against light, acids, air, and its higher volatility, which make it possible to sublimate SSB-I. The SSB-I showed, in the absorbed state, e.g., on chromatograms, no color change as did SSB-II from blue to violet-brown. The color change, of course, is reversible and in solvent vapors the blue color immediately appears. In solutions, the behavior of both SSB components is similar. Thus, it appears that SSB-II is the more basic dye, SSB-I shows stronger hydrophobic interactions and no great tendency to form intermolecutar hydrogen bonds. Therefore, SSB-I shows in comparison with SSB-II higher Rf values, better solubility in nonpolar solvents, better ex- tractibility from inorganic and organic absorbents, and a minor ability to form associates with polar compounds.

If both dye components are present in the solution, their behavior against cell and tissue structures are more similar to each other. That means each dye component can act like a solubilizer for the other.

Both components are planare molecules with an aromatic bond system and, therefore, can show charge-transfer interactions with other molecules, especially acceptor compounds (Balasubramanyan, 1966).

The SSB components have a wide diversity of chemical and physical charac- teristics. They show different polar and ionic, hydrophobic and charge-transfer interactions with other molecules.

Based on these findings, we will attempt to explain the various histochemical behavior of SSB in staining procedures.

Due to the different basic and hydrophobic characters of SSB components and their different ability to form ionic and hydrophobic bonds, there are many possibilities for histochemical reactions and staining (Lansnink, 1968; Gl6ckner, Pffiller and Franz, 1977).

248 U, PfO.11er et al.

Fig. 11. Molecular models of SSB-I and SSB-II

SSB-I, in the first place, is a lysochrome lipid dye and can stain neutral lipids and other less polar compounds, as also observed by Lansnink. SSB-II is a more basic dye and can stain previously negatively charged structures such as nucleic acids, acidic mucopolysaccharides, etc. and, to a lesser extent, the neutral lipids. This may be the reason for unspecific stainings observed for SSB (Brock et al., 1952; Schott, 1964; Fredricsson, 1958; Fredricsson et al., 1958 ; Schott and Schoner, 1965). If SSB-I and II are present in combination, both components must be regarded as possibly mutual solubilizers. Numerous impurities (30-40%, more than 40 substances) in commercial SSB might be responsible to a great extent for its unspecific staining properties. It is presently unknown to what extent the by-products and impurities can influence the distri- bution of SSB-I and II in the histochemical object, the nature and stability of staining, the dye fixation, and the re-extraction of dye (differentiation). The solvents for SSB also greatly influence these phenomena (Schott, 1964; Lansnink, 1968). The action of air, cations, acids, bases, and light can also change the nature and character of deposited dye.

The stable sudanophilia may be attributed to the ionic binding of SSB, especially SSB-II, to negatively charged groups and to the fixation of by-pro- ducts (Casselman, 1954). Are these groups blocked by HgC12 or by esterification, the stable sudanophilia is not detectable, e.g., on leukocytes (Lillie and Burtner, 1953; Terner etat., 1963). Various procedures for detecting 'masked' sudanophilia in smears were described (Ackermann, 1952; Mironescu, 1964; Hermansky, 1965). The results were not very satisfactory or were variable. This may be caused by heterogenity of SSB (Hermansky, 1965).

Both SSB components show solvatochromic and metachromic effects as expected from the chemical structure (Lansnink, 1968; Diezel and Neimanis, 1957). Further studies in this field with pure SSB components are necessary.

SSB-! and II were oxidized by agents such as gold-III-chloride and periodic acid into yellow compounds, perhaps quinons. Osmium tetroxide can be used to detect SSB-I. The SSB-stained tissues can be fixed with very dilute solutions (0.01%) of gold-HI-chloride (G16ckner et al., 1977). Above all, SSB-I can form complexes with cations. This property might be important for stable fixation of SSB in tissues.

Sudan Black B 249

Schaefer and Fischer (1972) shown that peroxidase activity of cells and tissues might be the reason for stable sudanophilia in the lipid staining. Endoge- nic peroxidase catalyzed the reaction of SSB with phenolic compounds and spontaneously formed peroxides and deposits results. The authors believe that the diamine 6, formed by hydrolysis from SSB, and other amines couples with phenoles in the presentce of peroxides and peroxidases. Indeed, Schaefer and Fischer (1972) showed, that the Sudan Black B reaction, according to Sheehan and Story (1947), on granulocytes and myelocytes was stopped by horse faddish peroxidase added to the SSB solution. According to our results, not the amine 6, but other primary and secondary amines, e.g., the perimidine 1, cause the oxydative coupling reaction. In no case was the amine 6 hitherto isolated from SSB preparations. Peroxides and other components for coupling should not form in pure SSB solutions containing chelating agents and antioxydants. There- fore, this cause for stable sudanophilia may be excluded. This and other questions are now under study.

The present study examines the chemical structure of SSB components and the possibility of a relationship between structure and histochemical behavior. For a more complete understanding of this matter, further investigations are necessary.

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Received August 16, 1977