Tyrian Purple: 6,6’-Dibromoindigo and Related Compounds
ABSTRACT The genesis of the purple dye from shellfish, its composition, origin, intermediates, analysis and synthesis of the components, 6,6'-dibromoindigo, 6- bromoindigo and 6,6'-dibromoindirubin are reviewed
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ABSTRACT: Objective: Indigo and indigoid dyes are natural dyes and have been known since Bronze Age (B.C. 2000). Nowadays, indigoid dyes are widely used in the industry of textile, cosmetic, food and medicine. The aim of this study was to estimate the DNA damage of indigotin, 6-bromo indigotin, indirubin and 6-bromo indirubin by in vitro alkaline single-cell gel electrophoresis (SCGE-Comet) in the peripheral lymphocytes. Methods: The cytotoxic effects of indigo and indigoid dyes were assessed by trypan blue exclusion. The cells were incubated with 10, 25, 50 μg/mL of the test substances for 30 min at 37°C. Comet assay was used to evaluate the genotoxic effect of test substances on human peripheral lymphocytes. Results: Our results revealed that indigotin and 6-bromo indigotin increased the DNA migration in a dose-dependent manner. DNA damage was higher in cells that had been incubated with 50 μg/mL indigotin and 6-bromo indigotin (p<0.05). Conclusion: Our results indicate that indigo and indigoid dyes would be genotoxic at higher concentrations. It is probable that a genotoxic effect might occur due to the fact that the individuals who have worked with these dyestuffs, both in the past and today, used highly concentrated dyes. Key words: Indigotin, 6-bromo indigotin, indirubin, 6-bromo indirubin, comet assay, DNA damageJournal of Marmara University Institute of Health Sciences. 07/2012; 2(3):108-112.
- Medecine sciences: M/S 06/2004; 20(5):516-8. · 0.56 Impact Factor
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ABSTRACT: Abstract Anthranilic acid (2-aminobenzoic acid, Aa) is the biochemical precursor to the amino acid tryptophan, as well as a catabolic product of tryptophan in animals. It is also integrated into many,alkaloids isolated from plants. Aa is produced industrially for production of dyestuffs and pharmaceuticals. The dissertation gives a historical background,and a short review on the reactivityof Aa. The synthesis of several types of nitrogen heterocycles from Aa is discussed. Treatment of anthranilonitrile (2-aminobenzonitrile, a derivative of Aa) with organomagnesium,compounds,gave deprotonation and addition to the nitrile triple bond to form amine-imine complexed,dianions. Capture of these intermediate with acyl halides normally gave aromatic quinazolines, a type of heterocyclic compounds that is considered to be highly interesting as scaffolds for development,of new drugs. When the acyl halide was a tertiary 2-haloacylhalide, the reaction instead gave 1,4- benzodiazepine-3-ones via rearrangement. These compounds,are isomeric to the common benzodiazepine drugs (such asdiazepam, Valium®) which are 1,4- benzodiazepine-2-ones. Capture of the dianionswith aldehydes or ketones, led to 1,2- dihydroquinazolines. Unsubstituted imine anions could be formed by treatment of anthranilonitrile with diisobutylaluminium hydride. Also in this case capture with aldehydes gave 1,2-dihydroquinazolines.
Molecules 2001, 6, 736-769
Tyrian Purple: 6,6’-Dibromoindigo and Related Compounds
Christopher J. Cooksey
59 Swiss Avenue Watford WD18 7LL, UK; tel +44 1923 241 688; fax +44 870 054 7454
Received: 15 August 2001 / Accepted: 20 August 2001 / Published: 31 August 2001
Abstract: The genesis of the purple dye from shellfish, its composition, origin,
intermediates, analysis and synthesis of the components, 6,6’-dibromoindigo, 6-
bromoindigo and 6,6’-dibromoindirubin are reviewed
Keywords: 6,6’-dibromoindigo, 6-bromoindigo, 6,6’-dibromoindirubin, tyrindoxyl,
tyriverdin, tyrindoleninone, tyrindolinone, synthesis, structure, properties
6,6’-Dibromoindigo is a major component of the historic pigment Tyrian purple, also known as
Royal purple, shellfish purple and Purple of the Ancients. Arguably, it is the oldest known pigment,
the longest lasting, the subject of the first chemical industry, the most expensive and the best known.
The colour is derived exclusively from marine shellfish of the Muricidae and Thaisidae families. The
long history, stretching back well into the pre-chemical era, and embracing chemistry, biology and
sociology, contains not a few misconceptions and erroneous conclusions. This review attempts to set
the record straight.
This molluscan dye has been known since pre-Roman times and in the Mediterranean region there
is evidence for the industry around the 13th century B.C.  at Sarepta, now Sarafand, Lebanon. The
ancient industry was distributed world-wide . Surviving details of the ancient process are
insufficient to explain the chemistry involved and this is the subject of continuing speculation. But it is
clear that the dye does not exist in the mollusc and is generated from precursors, sometimes termed
chromogens, contained in the hypobranchial gland.
Molecules 2001, 6
The process is described by Pliny , writing in the 1st century AD:
The vein [hypobranchial gland] already mentioned is then extracted and about a
sextarius [ca. 7 lb] of salt added to each hundred pounds of material. It should be
soaked for three days, for the fresher the extract, the more powerful the dye, then boiled
in a leaden vessel. Next, five hundred pounds of dye-stuff, diluted with an amphora
[about 8 gallons] of water, are subjected to an even and moderate heat by placing the
vessels in a flue communicating with a distant furnace.
Meanwhile, the flesh which necessarily adheres to the veins is skimmed off and a
test is made about the tenth day by steeping a well-washed fleece in the liquefied
contents of one of the vessels. The liquid is then heated till the colour answers to
expectations. A frankly red colour is inferior to one with a tinge of black. The wool
drinks in the dye for five hours and after carding is dipped again and again until all the
colour is absorbed.
In more recent times, a distinctly different process for obtaining the purple has been described, first
by Cole in 1685 , in which the contents of the hypobranchial gland are spread on to cloth and the
colour develops in response to air and light.
The Ancient process
Meyer Reinhold  has reviewed in detail the importance of the purple in ancient Greek and Roman
times and a large body of literature has accumulated which has been summarised by Dedekind ,
Becker  and in less detail by Born [8-9]. It is generally believed that the dyeing process involved
generation of the purple dye from the precursor(s), followed by reduction to a leuco compound and
subsequent oxidation on the cloth to give the colour in the same way as modern vat dyes. Much effort
has been devoted to the discovery of potential reagents which could have been used at that time to
reduce the dibromoindigo to the leuco form. In Pliny’s description, the word plumbum has no adjective
and depending on whether nigrum (“black”) or album (“white”) is added, could be translated as either
lead or tin. Experiments with lead and tin usually in strongly alkaline solution [10-12] have met with
variable success but tin is the stronger reductant . Other suggestions for reducing agents are
mercaptans ; methane thiol is a potential byproduct but the amount present is small and would be
insufficient to convert all the dye into the leuco form. Experiments with dodecanethiol in 1M NaOH at
78-88 ºC were successful . Honey has been suggested, although the reducing properties of glucose
are not very powerful. Experiments with iron reducing systems, mentioned by the ancients, did not
succeed, but may have referred to other dyes which were cheap alternatives to the purple.
The alternative suggestion is of a biochemical reduction, analogous to that for indigo in the woad
vat, which has recently been shown [15-17] to utilise Isatis clostridium, but was reportedly
unsuccessful for dibromoindigo. However, based on the successful woad vat parameters (pH 7.8;
Molecules 2001, 6
50 ºC), and the long (10 day) fermentation period, this process has been successful  in producing a
dye bath from Murex trunculus and reducing synthetic dibromoindigo using cockles .
The direct process
William Cole  clearly described this process in which the contents of the hypobranchial gland of
Nucella lapillus (at Minehead in the UK) are spread on to linen...
he "found this species on the shores of the Bristol Channel, which on cracking and
picking off the shell, exhibited a white vein lying transversely in a little furrow or cleft
next the head of the fish; which must be digged out with the stiff point of a horse hair
pencil being made short and tapering; which must be so formed by reason of the
viscous claminess of that white liquor in the vein so that by its stiffness it may drive in
the matter into the fine linnen or white silk ....... if placed in the Sun will change into
the following colours, i.e., if in the winter about noon, if in the summer an hour or two
after sunrise and so much before setting (for in the heat of the day the colours will come
on so fast, that the succession of each colour will scarce be distinguishable) next to the
first light green will appear a deep green; and in a few minutes this will change into a
dull sea green; after which, in a few minutes more, it will alter into a watchet blue; from
that in a little time more it will be purplish red; after which, lying an hour or two
(supposing the Sun still shining) it will be of a very deep purple red; beyond which the
Sun can do no more."
Every author who describes this process remarks on the distinctive colour changes that take place in
the sunlight. In Mediterranean sunshine, the process is complete in less than ten minutes [20, 21]. The
second notable characteristic is the powerful odour which accompanies the colour development: it is
likened to garlic or assafoetidea and is quite distinct from the odour of decomposing shell-fish. Other
examples are given by Letellier  and Schunck . Réaumur  confirmed the need for oxygen
in the purple forming process, Duhamel  confirmed that light was necessary, but it was left to
Lacaze-Duthiers  to demonstrate the potential use in photography.
In South America, the direct application route has a long history. Here the species used is Purpura
pansa and in contrast to the Mediterranean molluscs, it does not need to be sacrificed to yield the dye.
The elements of this dyeing process were described by Juan and de Ulloa  in 1744:
On the coasts belonging to the province of Guayaquil the finest purple is found. The
animals from which it is derived are contained in shells, about the size of walnuts, and
live on rocks washed by the sea. They contain a juice or humour, which is taken out,
and yields the true purple. ... Cotton, thread, and other delicate materials are dyed with
it. It gives a lively and durable colour, which does not lose its lustre by frequent
washings, but is rather improved thereby, and does not fade through long-continued use
and exposure. Near the port of Nicoya in the province of Guatemala [now Costa Rica]
Molecules 2001, 6
the same kind of shellfish is found and is used for dyeing cotton ... Various processes
are employed for extracting the juice or humour . Some kill the animal. They take it out
of its shell, and, having laid it on the back of the hand, press and squeeze it with a knife
from the head to the tail, and then separate the expressed juice, the rest of the animal
matter being thrown away. They treat in this way a number of animals until they have
sufficient quantity of juice. They then draw through the thread which they wish to dye,
and no more is required ... Others express the juice without killing the animal. They do
not take it entirely out of the shell, but only press it so as to cause a certain quantity to
be ejected, with which the threads are dyed. The shells are then laid again on the stones
from which they were taken. They recover, and after some time give a fresh quantity of
juice, but not so much as the first time. If the operation is repeated three or four times,
the quantity is very small and the animal dies of exhaustion.
The features which distinguish this process from others are that cotton threads are dyed separately
and subsequently woven, and that the mollusc is apparently not harmed by periodic milking. A century
later, Squier  describes the process on the Pacific coast of Nicaragua:
The process of dyeing the thread illustrates the patient assiduity of the Indians. It is
taken to the seaside, when a sufficient number of shells are collected, which being dried
from the sea water, the work is commenced. Each shell is taken up singly, and a slight
pressure upon the valve which closes its mouth forces out a few drops of the colouring
fluid, which is then almost destitute of colour. In this each thread is dipped singly, and
after absorbing enough of the precious liquid, is carefully drawn out between the thumb
and finger, and laid aside to dry. Whole days and nights are spent in this tedious
process, until the work is completed. At first the thread is of a dull blue colour, but
upon exposure to the atmosphere acquires the desired tint. The fish is not destroyed by
the operation, but is returned to the sea, where it lays in a new stock of colouring matter
for a future occasion.
This operation has more recently been described by Nuttall  in 1909, Thomson  in 1995,
Rios-Jara, et al.  in 1994 and Michel-Morfin in 2000 [32, 33].
The identity of the purple
In the nineteenth century, there was gradual progress towards identifying the purple pigment as
dibromoindigo. Bizio  showed that the pigment from Murex brandaris and Murex trunculus had
the characteristics of indigoid pigments; Schunck  isolated 7 mg of the dye which he called
punicin from 400 Purpura capillus (after which his patience was exhausted). In 1880, Schunck 
obtained a sample of cotton dyed in this way at Realejo on the West Coast of Nicaragua, said to be
made with the extract of Purpura patula. It was a dull purple, harsh to the touch and emitted a peculiar
smell. Extraction with warm dilute hydrochloric acid followed by boiling ether brightened the colour.
Molecules 2001, 6
He then treated the cloth with boiling aniline, which after a second extraction removed all the colour.
After cooling the aniline solution, all the coloured material crystallised. In this way, he obtained 0.099
grams from 24 grams of cloth. The colouring matter had all the properties of punicin, which he had
earlier obtained from Purpura capillus.
In 1909 Friedlander  solved the identity problem by processing the hypobranchial glands of
12,000 Murex brandaris and obtained 1.4 grams of pigment. The hypobranchial glands were removed
from the animals, spread on to filter paper and the colour allowed to develop in sunlight. The filter
paper was macerated and heated for 0.5 hour with dilute sulfuric acid (1:2) and after filtration washed
with hot water. The residue was Soxhlet extracted with ethanol to remove impurities and then the
pigment extracted with ethyl benzoate from which it separated in shining crystals. A second
crystallisation from ethyl benzoate and finally from quinoline completed the purification. Elemental
analysis showed that it contained bromine, which was unexpected, with an empirical formula
C16H8Br2N2O2. An unsymmetrically substituted indigo is unlikely since the precursor would be a C16
compound, but the precursor is not coloured, and of the four symmetrical isomers of dibromoindigo,
two of which were then known, 5,5’- and 6,6’-, the latter was the most similar. Two syntheses
confirmed the conclusion. Confirmation that dibromoindigo is the major coloured component of
Tyrian purple has been repeated for many mollusc species over the years. The rapid advance in
analytical techniques in the second half of the 20th century has greatly reduced the effort required. A
summary of the techniques used is shown in Table 1.
Table 1. Methods used to identify dibromoindigo in molluscs and on artefacts.
Species / artefact
Driessen L A 1944 photodebromination of
elemental analysis; MS
van Alpen 1944textile
Baker & Sutherland 
Sasaki K 
Gibaja Oviedo & Salazar
de Cavero 
Taylor G W 
McGovern & Michel 
Sarepta pot shard
Daniels [46,47] 1985textile from Enkomi