Cameron Cromartie and Todd 9. Colouring Matters of the Aphididce. Part XVI.1 Reconsideration of the Structure of the Erythroaphins. By D. W. CAMERON,R.I. T. CROMARTIE,and LORDTODD. Further investigation has shown that the erythoaphins contain only two ‘Iaromatic ” CH groups and that they contain in addition two MeCH.0- I groupings. Previous structures tentatively assigned to these pigments are therefore untenable. IN 1948 we first reported the existence of a remarkable group of colouring matters to which we gave the general name “ aphins.” 2 These substances occur in the hanolymph of many dark species of Aphididw or are derived from substances present in the hzmolymph. For each insect species there are four aphins to be considered (a)the protoaphin a yellow glucosidic pigment present in the hzmolymph of the living insect; (b) the yellow xantho- aphin which is sugar-free and is formed from protoaphin by a specific enzyme system present in the insect and bringing about the change on the death of the insect; (c) the orange chrysoaphin produced when xanthoaphin is kept in solution or more rapidly by means of acid or alkali; and (d) the red erythroaphin formed in a similar way from chrysoaphin and representing the relatively stable end-product of the series.The con- versions (b) *(c) --t (d) occur spontaneously in extracts of the insects and can be conveniently followed by spectroscopic observation. All the aphins so far studied in detail belong to one or other of two series corresponding pigments in each series being apparently Part XV Johnson Todd and Watkins J.1956 4091. a DuewelI Human Johnson,MacDonald and Todd Nature 1948 182 769. [19641 Colowing Matters of the Aphidid@. Part XVI. stereoisomeric with one another. The two series we describe as the aphins-fb and the aphins-sl these designations being derived from the name of the insect species from which they were first isolated-aphins-fb from the common bean aphid Aphis fabae and aphins-sl from the willow aphid Tuberolachnus salignus. There exist therefore a protoaphin-fb and a protoaphin-sZ each the parent substance of a distinct series of pigments. In our earlier studies we examined several other aphid species in a preliminary way and some were shown to yield aphins-fb. In particular however erythroaphin-py from Safipaphis pyri appeared possibly different from both erythroaphins-fb and -sL3 This has not been substantiated by subsequent work4 which suggests identity with the -sl isomer.The isolation of protoaphin-ph from A. philadelphi has also been described but for lack of material no further work on this series has been carried out and there are no grounds for suggesting that it differs from the -fb or -sl series. The pigments obtained from Hamame- Zistes species, though probably closely related to the erythroaphins are structurally different and are not considered further here. In endeavouring to elucidate the structure of the aphins and the nature of the observed interconversions it was decided to concentrate attention first on the stable erythroaphins.An extended series of investigations led in 1955 to the advancement of two possible structures (I) and (11) for erythr~aphin.~ The difference between the -fb and the -sl isomers was attributed to the stereochemistry at the junction of the heterocyclic rings with the rest of the molecule? it being considered that both rings were cis-fused in erythro- aphin-fb one cis- and the other tram-fused in the sl-isomer. Of the two possibilities (I) was preferred since although acetaldehyde was obtained from the erythroaphins by acid treatment the conditions necessary for its formation were very much more vigorous than would have been expected on the basis of formula (11)which contains two dioxolan rings. Neither structure could however be regarded as established and indeed although all the complex reactions of the erythroaphins could be explained on the basis of either there were a number of detailed points for which they provided no very satisfactory explanation.The difficulty with which acetaldehyde could be produced was easier to explain on the basis of (I)than (11),but on the other hand all attempts to prepare derivatives of ethylene glycol from erythroaphin derivatives failed and oxidation by the Kuhn-Roth procedure always gave ca. 3.8 mol. of acetic acid results more in keeping with structure (11)than (I). Again on either formula the amount of coronene as compared with that of perylene derivatives produced on zinc dust distillation was surprisingly low. Finally the visible and ultraviolet spectra of tetra-acetyldihydroerythroaphin,although almost identical in general pattern with that of 3,4,9,10-tetra-acetoxyperylene, showed a bathochromic shift of nearly 50 mp relative to the latter; lo a shift of this magnitude is difficult to reconcile with a structure in which there are no oxygen atoms attached directly to the perylene system other than in positions 3,4,9 and 10.Further difficulties arose when a closer study of the other aphin pigments was under- taken. In our earlier work we were able to give only tentative molecular formulae for these and not unexpectedly closer examination with larger amounts of material showed that revision was necessary. As will be reported in the following paper protoaphin has a formula C,,H,O1, xanthoaphin C,H,Olo and chrysoaphin C,H%O,.The formation of xanthoaphin from protoaphin is thus accompanied formally by the loss of 1 mol. of glucose; xanthoaphin loses 1 mol. of water in passing into chrysoaphin; and chrysoaphin yields erythroaphin CWH2,O again by loss of 1 mol. of water. Although it was possible 8 Johnson Quayle Robinson Sheppard and Todd J. 1961 2633. 4 Calderbank Ph.D. Thesis Cambridge 1954. 6 Duewell Human Johnson MacDonald and Todd J. 1950 3304. 6 MacDonald. J. 1954 2378. 7 Brown Calderbank Johnson Joshi Quayle and Todd J. 1955 959. * Brown Calderbank Johnson Quayle. and Todd J. 1955 1144. Brown Johnson Quayle and Todd J. 1954 107. 10 Calderbank Johnson and Todd J. 1964 1285. Colouring Matters of the Aphidid&. Part XVI. on the basis of formula (I) or (11) to formulate more or less plausible structures for xantho- aphin and chrysoaphin the derivation of a compatible structure for protoaphin known at the time to be naphthalenic was well-nigh impossible.Reconsideration of the evidence on which formulz (I) and (11) were based was clearly necessary. The erythroaphins are undoubtedly derivatives of 4,9-dihydroxyperyIene- 3,lO-quinone in which the chromophoric system is substituted by two symmetrically disposed non-aromatic groupings each containing five carbon and two ethereal-oxygen atoms. The erythroaphins react readily with amines to give diamino-derivatives in a manner apparently analogous to the well-known amination of quinones (including the perylenequinones). They can also be halogenated to dihalogenoerythroaphins which can react further with amines under mild conditions to give diaminodihalogenoerythro-aphins.11J2 The properties of all these erythroaphin derivatives are consistent with the substituents’ having been introduced directly on the perylene nucleus and in our earlier work these facts were taken as evidence that in erythroaphin itself there must be four substitutable “ aromatic ” CH groups.This has always been recognised by us to be the basic assumption in the arguments used in deriving structures (I) and (11); indeed if it is accepted (I) and (11) are the only possible formulations for erythroaphin. But it was merely an assumption and in taking up the problem afresh it was decided first to put it to the test by careful infrared and nuclear magnetic resonance spectroscopic studies.These established beyond doubt that the dihalogenoerythroaphins contain no aromatic CH group i.e. that there are only two free positions in the perylene nucleus of erythro- aphin. Moreover the nuclear magnetic resonance results indicated not only that erythro- aphin contains 4 side-methyl groups but that each of these is attached to a carbon carrying both a hydrogen and an oxygen atom i.e. that the molecule contains 4 groupings of the type CH,-CLH . Both these findings are incompatible with structures (I) and (11). ‘0-We have therefore carried out in the last few years a comprehensive attack on the aphin problem studying simultaneously all four types of pigment and we have arrived at a solution which explains all the facts rationally and without difficulty.These investigations are described in detail in the succeeding six papers (Parts XVII-XXII). As the researches involved are of some complexity these papers do not present a chronolo- gical account but rather a selection of results set out to deal with the structure and the absolute stereochemistry of the protoaphins erythroaphins xanthoaphins and chryso- aphins in that order. UNIVERSITY CAMBRIDGE. [Received May 2nd 1963.1 CHEMICALLABORATORY l1 Brown Johnson MacDonald Quayle and Todd J. 2952 4928. l2 Brown Calderbank Johnson MacDonald Quayle and Todd J. 1955 954.