Which the Phosphorylating Entity Derives from a Diester of Phosphoric Acid, and (2) Downloaded by Guest on September 25, 2021 1390 CHEMISTRY: A

VOL. 45, 1959 CHEMISTRY: A. TODD 1389 14 Jervis, R. E., D. R. Muir, J. P. Butler, and A. R. Gordon, J. Am. Chem. Soc., 75, 2855 (1953); Davies, J. A., R. L. Kay, and A. R. Gordon, J. Chem. Phys., 19, 149 (1951). 15 Sadek, H., and R. M. Fuoss, J. Am. Chem. Soc., 76,5897 (1954). 16 Evers, C. E., and A. G. Knox, J. Am. Chem. Soc., 73, 1739 (1951). 17 Gilkerson, W. R., J. Chem. Phys., 25, 1199 (1956). 18 Accascina, F., A. D'Aprano, and R. M. Fuoss, J. Am. Chem. Soc., 81, 1058 (1959). 19 Fuoss, R. M., J. B. Berkowitz, E. Hirsch, and S. Petrucci, these PROCEEDINGS, 41, 27 (1958). 20 Ref. 1, Table 17.1, p. 241. 21 Mead, D. J., R. M. Fuoss, and C. A. Kraus, Trans. Faraday Soc., 32, 594 (1936); Tucker, L. M., and C. A. Kraus, J. Am. Chem. Soc., 69, 454 (1947). 22 Burgess, D. S., and C. A. Kraus, J. Am. Chem. Soc., 70, 706 (1948); Luder, W. F., and C. A. Kraus, ibid., 69, 248 (1947). 23 McDowell, M. J., and C. A. Kraus, J. Am. Chem. Soc., 73, 329 (1951); Reynolds, M. B., and C. A. Kraus, Ibid., 70, 1709 (1948). 24 Fuoss, R. M., and C. A. Kraus, J. Am. Chem. Soc., 79, 3304 (1957). SOME ASPECTS OF PHOSPHATE CHEMISTRY* By SIR ALEXANDER TODD UNIVERSITY CHEMICAL LABORATORY, CAMBRIDGE, ENGLAND Communicated July 17, 1959 During the past fifteen to twenty years a very large amount of work has been done on organic phosphates and polyphosphates. Some have been developed for in- dustrial or military purposes, e.g., in the insecticide and war-gas fields, but a con- siderable proportion has been a consequence of systematic attack on nucleotide chemistry, mainly in Cambridge and in other schools developed by former members of the Cambridge laboratory. In addition, biochemical studies on phosphates have increased greatly in recent years and, somewhat belatedly, physicochemical in- vestigations are now being undertaken. Although much more physicochemical work will be needed to provide accurate quantitative data, it is now possible to make a number of generalisations on the evidence to hand, and to make a variety of predictions which are, I believe, of general interest, not only from a chemical, but also from a biological standpoint. For much that has hitherto seemed obscure in biochemical processes involving phosphate and polyphosphate esters is now recog- nised as being merely a manifestation of properties which can be demonstrated in the laboratory and it is certain that as we extend our purely chemical knowledge of the phosphates the remaining apparent anomalies will also disappear. In this paper I intend to consider firstly the methods by which phosphorylation may be brought about (i.e., formation of esters, amides, and anhydrides of phosphoric acid derivatives), and secondly the use of phosphoric acid derivatives as alkylating agents capable of forming inter alia carbon-carbon linkages. Phosphorylation.-If we omit from consideration the long established and fre- quently unsatisfactory method of phosphorylating alcohols by direct reaction with phosphoryl chloride in presence of a base, modern methods of phosphorylation fall into two broad groups-(1) those which yield as initial products triesters of phosphoric acid (or, in the case of anhydrides, fully esterified polyphosphates) and in which the phosphorylating entity derives from a diester of phosphoric acid, and (2) Downloaded by guest on September 25, 2021 1390 CHEMISTRY: A. TODD PROC. N. A. S. those based on agents prepared from monoesters of phosphoric acid and yielding as initial products diesters of phosphoric acid. Although it has been customary in the past to regard these two groups as similar, it may well be that they are often quite different from one another, and, as will be shown later, a distinction between them helps to clarify a number of otherwise puzzling observations. Group 1 Methods: The first major class of phosphorylation procedures falling into this group can be loosely described as the anhydride class, i.e., that in which a tetraester of pyrophosphoric acid, the mixed anhydride of a phosphodiester with a stronger acid, or a diester of, e.g., phosphorochloridic acid is employed as phosphorylating agent, usually in presence of a base. In all such cases there are two possible mechanisms to consider: (a) the anhydride may first ionise before it phosphorylates or (b) the intact molecule may be the active reagent. This is, of course, equally true of carboxylic acid anhydrides when used as acylating agents, but, whereas in, say, the mixed anhydride of trifluoracetic acid and acetic acid the two mechanisms lead to different results, in the case of the phosphoric anhydrides both give effectively the same result. Under normal conditions of phosphorylation in presence of a base a mixed pyrophosphate (1) appears always to yield predom- inantly, if not exclusively, a phosphate (2) derived from the weaker of the two acids composing the pyrophosphate. Similarly, a mixed anhydride of a phosphodiester and a sulphonic acid or trifluoroacetic acid will be a phosphorylating agent while a corresponding mixed anhydride with, e.g., acetic acid will be an acylating rather than a phosphorylating agent. 0 0 0 0 PhCH20 11 11 OPh base PhCH2O 11 - i OPh 'P-0--P< + R0H - > \P-OR + 0)-P PhCH20/ OPh PhCH20/ \OPh (1) (2) In the event of ionisation (or effective ionisation) of such an anhydride as (1) it is clear that the phosphorylium cation (the phosphorylating entity) would derive from the weaker of the two acids, the anhydride oxygen remaining attached to the phosphorus of the stronger acid as part of its anion. In the intact anhydride molecule, however, both the phosphorus atoms carry a measure of positive charge, that belonging to the stronger acid being the more positive of the two. An approaching nucleophile might therefore be expected to attack both phosphorus atoms. But, since the P= 0 group has no additive properties in the phosphates, any such attack can result in attachment of the nucleophile only if there is simultaneous expulsion of a group on the other side of the phosphorus atom. Hence, although a nucleophile can, and doubtless does, approach both phosphorus atoms, the overwhelming proportion of successful attacks will be those which cause simultaneous expulsion of the most stable anion, i.e., that of the stronger acid, and hence for the most part phosphorylation by the weaker of the two acids is observed.' In sharp contrast mixed anhydrides of carboxylic acids when reacting as intact molecules normally acylate with the stronger of the two acids forming the anhydride, since the C = 0 group has additive properties and an approaching nucleophile will therefore tend to attach itself to the more positive of the two carbonyl carbons present, i.e., that belonging to the stronger acid. This behaviour of the phosphoric anhydrides (which probably applies also to Downloaded by guest on September 25, 2021 VOL. 45, 1959 CHEMISTRY: A. TODD 1391 those of other strong inorganic oxy-acids) is well illustrated in the so-called exchange reaction of pyrophosphates2' wherein if a pyrophosphate A-B derived from two different phosphodiesters A and B is brought into contact with a salt of a third phosphodiester, C, then, provided the order of stability of their ions is B> C> A, we will observe the reaction A - B + C A - C + B. This reaction, in a somewhat modified form to be discussed later, is probably the basis of many biological synthetic processes involving polyphosphates. In the above methods of phosphorylation using acid anhydrides the driving force can be regarded as the expulsion of a stable anion. But there is a second type of method known in which a phosphorylating reagent is produced by protonation, usually of a compound containing the grouping - N = C - OP(O) (OR)2, i.e., an imidoyl phosphate. Reagents of this nature include the simple imidoyl phosphates and the hypothetical intermediates in the reaction of phosphates with carbodi- imides,5 ketenimides, and cyanamide derivatives. A typical example of such a reagent is the protonated imidoyl phosphate (3) which reacts readily with a phosphodiester anion as shown to yield a pyrophosphate. The reactive intermediate in the reaction between a carbodiimide and a phosphodiester is believed to have structure (4). In each case the electronic displacements indicated lead to ready attack on phosphorus by an anion with expulsion of the nitrogenous esterifying group as an amide. + + R-C=GNHR' RHN-C=NHR O==P 7P0\OR"0RP R'O OR' R'O OR' (3) (4) It is of interest to note that, using diesters of phosphoric acid as starting materials in these imidoyl phosphate methods, pyrophosphate preparation is very much fa- voured, whereas esterification of alcohols to yield phosphotriesters is usually difficult. This would suggest that actual fission of intermediates like (3) to give phosphorylium cations does not occur; this would be in line with common laboratory experience that compounds such as tetrabenzyl and tetraethyl pyrophosphate which probably react as intact molecules are very indifferent phosphorylating agents for weak nucleophiles like alcohols, whereas reagents like dibenzyl phos- phorochloridate are effective for this purpose. Since imidoyl phosphates can be rendered effective phosphorylating agents by addition of a proton, it should be possible to develop phosphorylating agents from other types of phosphate esters by electron withdrawal. Such a process would be, of course, an oxidative phosphorylation and its reality has been demonstrated in a number of laboratory examples. Oxidative phosphorylation has not yet been exhaustively studied from a preparative standpoint, but- its general scope and limitations should be like those of the imidoyl phosphate procedures.

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