The Polytopal Rearrangement at Phosphorus

ESSAY

10

THE POLYTOPAL REARRANGEMENT AT PHOSPHORUS F. H. WESTHEIMER

I. Introduction II. Chemistry A. Evidence for Pseudorotation B. Application of Polytopal Rearrangements to Chemistry C. Stereochemistry D. The Rearrangement III. Enzymology A. Enzymatic Displacements at Phosphorus . . . . B. Ribonuclease C. ΑΤΡ-α-S D. Tetraamminecobalt(III)-ATP E. ATP-/3-S F. ATP-y-S-y-,80 G. , 6 0, , 7 0, 180 H. Summary for Enzymology References

229 230 230 232 237 241 246 246 247 251 256 258 259 263 265 266

I. INTRODUCTION

Although the world of phosphorus compounds is small relative to that of carbon, its stereochemistry is much the richer. This arises in part because tricovalent, tetracovalent, pentacovalent, and hexacovalent compounds of phosphorus are all stable, and all exhibit interesting stereochemistry. However, the special interest in the stereochemistry of phosphorus depends not on its various valence states but on the polytopal rearrangement that occurs readily and rapidly in its pentacovalent compounds. This essay is concerned with that polytopal rearrangement and, in particular, with the question of whether that rearrangement does or does not occur in any of the many enzymatic processes that involve displacements at phosphorus. It might as well be stated at the outset that, so far at least, no evidence has been found for such rearrangements in biochemical systems. That statement, however, is itself an important gen229 REARRANGEMENTS IN GROUND AND EXCITED STATES, VOL. 2 Copyright © 1980 by Academic Press, Inc. AH rights of reproduction in any form reserved. ISBN 0-12-481302-X

230

F. H. WESTHEIMER

eralization; enzymatic reactions at phosphorus may have evolved so as to avoid the rearrangement altogether. The polytopal rearrangement at phosphorus, in its simplest and most typical form, consists of the transformation of one trigonal bipyramidal pentacovalent compound of phosphorus into another by the simultaneous exchange of the two apical substituents to equatorial positions and two equatorial substituents into apical positions; the result is the formation of a new trigonal bipyramid. If at least four of the substituents on the phosphorus atom are the same (as, for example, in CH3PF4), and if these four substituents are involved in the rearrangement, the resulting molecule appears to have been rotated by 90°; for this reason, the process is often called a "pseudorotation." A particular detailed mechanism for the pseudorotation requires that the molecule pass through the geometry of a square pyramid. The fifth substituent on phosphorus—the equatorial substituent that is not involved in the exchange process—is called the "pivot" for the pseudorotation; it remains equatorial in thefinalproduct. In the detailed mechanism outlined above, the "pivot" appears at the apex of the tetragonal pyramid that occurs as the intermediate or transition state of the rearrangement [Eq. (1)]. ■

(1)

*

2

3

Just as only a few carbon compounds are perfect tetrahedra, so only a few compounds of phosphorus are idealized trigonal bipyramids, and some, in fact, are most stable as square pyramids. These matters are discussed later in this essay (Section II,C). II. A. Evidence for

CHEMISTRY

Pseudorotation

The evidence for pseudoration is of three types: (a) nuclear magnetic resonance, (b) chemical reactivity and product formation, and (c) stereochemistry. They will be reviewed in that order, although historically, as one might expect, the three were intertwined. Fortunately, a number of excellent reviews (1-6) have been published on the subject, so that only a brief outline of the evidence need be given here before the research on enzymatic processes is discussed. In particular, McEwen and Berlin (7) published two outstanding volumes in which they collected and

10. THE POLYTOPAL REARRANGEMENT AT PHOSPHORUS

231

commented on significant papers in the field before 1975. Their critical compendium serves as the authoritative reference in the area of the stereochemistry of phosphorus. Nuclear Magnetic Resonance In 1953, Gutowsky, McCall, and Slichter (8) discovered that the 19F NMR spectrum of PF5 consisted of a doublet. Thefivefluorineatoms are all equivalent on the NMR time scale, and the signal is split by the spin of the 31P nucleus. In 1960, Berry (9) suggested the mechanism of Eq. (1) for an exchange process that makes the fluorine atoms equivalent. Subsequently, Muetterties (10-12) and Schmutzler (13-16) and their co-workers examined the NMR spectra of numerous alkyl- and arylfluorophosphoranes and concluded that the spectra could be understood by assuming that the compounds are trigonal bipyramids in whichfluorineatoms preferentially occupy apical positions, whereas alkyl and aryl groups occupy equatorial positions (the "polarity rule")· The pseudorotation process suggested by Berry could then occur readily in compounds of the structure RPF4, since an alkyl or aryl substituent, R, could function as the pivot for the pseudorotation process, and pairs of fluorine atoms could be exchanged between apical and equatorial positions. In compounds of the structure R2PF3, however, pseudorotation would be inhibited, since only one of the alkyl or aryl substituents could remain, as the pivot, in an equatorial position. Exchange of substituents in pairs would necessarily force one of the R groups into an unfavorable apical position. Pseudorotation had been invoked to explain the racemization of phosphine oxides (77), to explain displacements on phosphoramidates (18), and to explain the chemistry of cyclic phosphate esters (19,20), before the variable-temperature NMR spectra of oxyphosphoranes were investigated and analyzed (21). The first oxyphosphorane so studied (4) had previously been synthesized (22); the three methoxy groups were reported to give rise to only one doublet in the NMR spectrum and so appeared to be equivalent. A prior study of the hydrolysis of cyclic phosphoranes (79) had, however, suggested that afive-memberedring must occupy one apical and one equatorial position in a trigonal bipyramid so as to minimize angle strain (the "strain rule"). This restraint had also been invoked in prior work with fluorophosphoranes. When the structure of the phosphorane 4 is written as a trigonal bipyramid in accordance with the polarity and strain rules, it becomes apparent that the molecule should be 4 'frozen" in the conformation of structure 5. Reinvestigation of the NMR spectrum of this compound at low temperatures (21) showed that the predictions of theory were, in fact, fulfilled. At -45°C the doublet from the nine hydrogen atoms of the three methoxy

232

F. H. WESTHEIMER CH3

/°--C-CH3 (CH 3 0),P x II \ ^,C-COCH, HA C 6 H 5

CH3

O'^y-COCH, CH 3 0-<

-A-^Àï- H |0CH3 OCH 3

C

eH9


J-^rV—H 11 OCHj * OCH 3

groups collapses, and at -56°C, in CDC13 as solvent, the three methoxy groups give rise to three separate doublets, in accord with predictions from structure 5. The energy of activation for the pseudorotation process (23,24) is 11 kcal/mol. Whereas the difference in polarity between fluorine and carbon is large enough to hold compounds like (CH3)2PF3 in a fixed conformation, even at elevated temperatures, the difference in polarity between oxygen and carbon is merely enough to establish a barrier of the order of 10-15 kcal/mol. Following this initial NMR demonstration that the polarity and strain rules apply to oxyphosphoranes as well as to fluorophosphoranes, a finding that in retrospect seems all too obvious, a large number of investigations (7) thoroughly confirmed the initial observations and greatly expanded them in many directions. In particular (25,26), the NMR spectrum of 6 shows that the compound undergoes not only a pseudorotation process, but also a reversible ionization to phosphonium salt and enolate anion. The two processes result in different and easily identified spectral changes. The ionization of phosphoranes has independently been observed (27-29) but is not relevant to the polytopal rearrangement here under discussion. The spectral changes ascribed to pseudorotation are not caused by such ionization; the studies of the chemistry of 6 make this distinction quite clear. Since the initial studies of the energy of activation for pseudorotation of 5, 6, and related compounds (23,24), much further research has illuminated the field. In particular, Trippett and his collaborators (30-34) have defined and determined the "apicophiUcities" of various groups, i.e., their tendencies to occupy the apical position in trigonal bipyramids. Relative apicophiUcities turn out to depend not only on electronegativitity, but also on the ability of atoms to backbond to phosphorus; Trippett's papers should be consulted for details. B. Application of Polytopal Rearrangements to Chemistry

Before the use of NMR to demonstrate pseudorotations in oxyphosphoranes, the concept had proved essential to an understanding of the

10. THE POLYTOPAL REARRANGEMENT AT PHOSPHORUS

233

rates and products in the hydrolysis of cyclic esters of phosphoric acid (19,20,35). Methyl ethylene phosphate (7) undergoes acid hydrolysis about 1 million times as fast as does trimethyl phosphate, to produce roughly equal amounts (36,37) of methyl hydrogen hydroxyethylphosphate (8) and dihydrogen ethylene phosphate (9) [Eq. (2)]. Similarly, dihydrogen ethyl-

ΓΛ

0

0


OCH3

X

7

— ^ ^


H0CH 2 CH 2 0P-0CH 3

'0H

8

ΓΛ %'° 0AOH

+

(2)

CH,0H

ene phosphate undergoes acid-catalyzed hydrolysis and oxygen exchange at comparable rates, and both of these rates exceed by factors of many millions the corresponding rates for dimethyl phosphate, despite the fact that oxygen exchange does not involve ring cleavage (38). The speed of these reactions is presumably related at least in part to the strain in the fivemembered rings. Thermochemical measurements (39-41) have shown that five-membered cyclic phosphates generate considerably more heat on hydrolysis than do their acyclic analogues. Furthermore, X-ray analyses (42,43) of methyl ethylene phosphate and similar cyclic phosphates (41) show that the O-P-O bond angle at phosphorus is "pinched" from the normal tetrahedral angle to 95°-97°. Obviously, the opening of the ring can proceed with relief of strain, but how can the rapid hydrolysis of methyl ethylene phosphate to dihydrogen ethylene phosphate, without ring opening, make use of the strain energy? This rhetorical question may be satisfactorily answered if the nucleophile attacks a face of the tetrahedral phosphorus atom to occupy an apex of a trigonal bipyramidal intermediate and if the leaving group leaves from an apex ofthat intermediate. The trigonal bipyramidal intermediate must, at least in some cases, survive long enough to undergo pseudorotation, in accordance with the strain and polarity rules cited here (19,20). Thus, methyl ethylene phosphate should add water under acid catalysis to yield 10 [Eq. (3)]. This may undergo acid-catalyzed ring cleavage to 8 but cannot lose methanol, since the methoxy group is not in an apical position. If, however, 10 undergoes rapid pseudorotation, it can produce 11; the methoxy group is then apical and can therefore readily leave to produce 9 and methanol. The intermediates 10 and 11 are strain-free and so facilitate the rapid reactions that are observed (20).

234

F. H. WESTHEIMER

o ΓΛ

+

°>'°

^ 0

XOCH

^

Η,Ο

(3)

^Γ

CH^O

C H0

"OH

3 10

3 '

υπ

,,

/^"OH OH

A comparison of the results for the hydrolysis of methyl ethylene phosphate (7) with those for the hydrolysis of ethyl (44) and of methyl (19,35) propylphostonate (12), strongly supports the interpretation that was advanced for this original example. The trigonal bipyramidal intermediate 13 postulated in the hydrolysis of the ester 12 should be ς 'frozen" in the conformation as drawn; it cannot pseudorotate without surmounting the barrier imposed by either the strain rule or the polarity rule. Thus, the methoxy group cannot readily assume an apical position, and the hydrolysis of 12 should take place exclusively with ring opening. This prediction was experimentally verifed (19); at room temperature, acid-catalyzed ring opening proceeds to the extent of more than 99.5% [although somewhat more reaction to liberate methanol occurs at higher temperatures (45)].

0

0CH 3 12

Γ0Η 13

0

0C 2 H 5 14

Furthermore, compound 14, although it must exhibit about as much strain as do 7 and 12, hydrolyzes at a rate comparable to that of its acyclic analogue (35). Thisfindingconforms to theory. Consideration of the rules already stated for forming trigonal bipyramidal intermediates shows that the addition of water to 14 necessarily leads to an intermediate of high energy. If the ring is placed in diequatorial position, the intermediate violates the strain rule; if it is placed in the equatorial-apical position, a carbon substituent must assume an apical position and so violate the polarity rule. Presumably the latter structure is the actual intermediate, and the relief of strain in forming the intermediate approximately balances the barrier to placing a nonpolar substituent in an apical position. The hydrolyses, as formulated, are assumed to proceed by way of hydroxyphosphoranes as intermediates. The reality of such compounds, and their reactivity, have recently been confirmed (46-49).

235

10. THE POLYTOPAL REARRANGEMENT AT PHOSPHORUS

Ho.io

o.oi

O

2

4

6

8

IO

I2

o.ooi

I4

pH Fig. 1 Hydrolysis and exocyclic cleavage of methyl ethylene phosphate.

The pH-rate and pH-product profiles for the hydrolysis of methyl ethylene phosphate are relevant to the later discussion; they are presented in Fig. 1. In particular, in mildly alkaline solution, ring opening from 7 to yield the anion of 8 predominates over loss of methanol to yield the anion of ethylene phosphate (9). At high pH, the phosphorane 10 presumably is ionized to 10a. Although 10a could undergo pseudorotation, this process must compete with cleavage, and the rate of cleavage of the anion should greatly exceed that of the neutral phosphorane. The reaction in mild alkali, therefore, proceeds without pseudorotation. Similarly, ethylene phosphate itself shows exchange of oxygen with the solvent only in acid solution (38). The negatively charged oxygen atoms of the dianion 10b are electron donating and so preferentially occupy equatorial positions. Since only one of the two can remain, as pivot, in an equatorial position, a pseudorotation would necessarily force the other into an apical position. The rules for pseudorotation correctly suggest, then, that a dianionic phosphorane should also undergo cleavage in preference to pseudorotation. A number of other, similar investigations have confirmed the applica-

^RV

CH,0-^==^

OH ΙΟα

~0

"0

m: «er-

OH

10b

236

F. H. WESTHEIMER

tion of these rules and of pseudorotation to the hydrolysis of phosphates. Many of the most elegant demonstrations have been concerned with stereochemistry, and a few such examples are discussed in the next section. In another example, however, in which the pseudorotation rules correctly predicted the products formed (50), the alkaline hydrolysis of 15 was shown to proceed with loss of the acetoin residue, whereas that of 16 proceeded predominately with loss of methanol. Furthermore, 17 (R = i - Pr) was hydrolyzed (51-53), and the products formed were explained on the basis of the pseudorotation rules; the complex chemistry of 0 II C H 3 - C - C H - C H 33 I 0

0 II C H3 3 - C - C H - C H3, I 0

0 II HOCH 2 CH 2 SP(OR) 2

n^V™3

*P(0CH3)2

OCH3 15

16

17

many related compounds was similarly rationalized. The theory was used to interpret the products formed in the reactions of 18 (54). The pH-rate profiles for the exchange of oxygen between phosphinates or phosphine oxides and solvent are generally in accord with the pseudorotation mechanism and provide examples in which pseudorotation is presumably rate limiting (55f56). Most of the data fit nicely into the patterns predicted by theory. An exception arises with compound 19; CH2=c-C02" ° x „ - „ . P0 2 OCH 3

18

HII 0

19

oxygen exchange occurs at a moderate rate, whereas the usual rules for pseudorotation suggest that the reaction should occur only with great difficulty (57). Perhaps the extreme distortions from both trigonal bipyramidal and square pyramidal geometry, imposed by the bicyclic system, make this a special case. Perhaps only a small energy barrier separates highly distorted trigonal bipyramids and highly distorted square pyramids and thus allows a polytopal rearrangement. This example, because it does not readily conform to theory, is of special importance. All of these examples and the enzymatic examples of hydrolysis considered later rest on the implicit assumption that each hydrolytic reaction proceeds by the addition of a nucleophile to the central phosphorus atom

10. THE POLYTOPAL REARRANGEMENT AT PHOSPHORUS

237

to yield a pentacovalent intermediate. This assumption is probably valid for all of the cases considered here, but several possible alternatives must be ruled out to make the arguments complete. First, some phosphate monoester monoanions, ROP03H~, dissociate to alcohol and P0 3 " (monomeric metaphosphate ion) (58-65). This reaction is the phosphorus analogue of the SN1 reaction, or, perhaps more precisely, monomeric metaphosphates bear the same relationship to phosphate esters that acylium ions bear to carboxylic acid esters. Second, the attack of a nucleophile on an ester might lead to a trigonal bipyramidal transition state, strictly analogous to the trigonal bipyramidal transition state in the displacement reaction of carbon compounds. When pseudorotation is observed, that fact oflFers persuasive evidence that a metastable intermediate is formed in the course of the reaction, but what of the examples in which pseudorotation is not observed? Might not these be examples in which the trigonal bipyramidal configuration corresponds to an energy maximum rather than to a minimum? The evidence suggests that phosphoranes are most readily formed when they contain small rings (66-69)', possibly in some acyclic systems, only a transition state intervenes in the hydrolytic reaction, although a true phosphorane intermediate has been demonstrated in the hydrolysis of methyl diphenylphosphinate (70,71). Third, perhaps the hydrolyses of some phosphates take place by way of hexacoordinated phosphate anions. This possibility, first suggested by Ramirez and his co-workers (72,73), provides the pathway for the hydrolysis of pentaphenoxyphosphorane (74). Since hexacoordinated anions of phosphorus can readily be formed under conditions similar to those used in hydrolytic reactions (29), the possibility of hexacoordinated intermediates cannot be dismissed lightly. Finally, in some instances, the cleavage of a phosphate ester takes place with C-O rather than with P-0 cleavage (75). Although all of these complications may arise in one reaction or another, they are probably not important in any of the chemical examples cited here. It remains to be seen, however, whether the enzymatic processes that proceed without pseudorotation occur by way of trigonal bipyramidal transitions states, without the formation of phosphoranes. C.

Stereochemistry

The SN2 reaction leads to an inversion of configuration at the reacting carbon atom. The chemistry of trigonal bipyramidal compounds is necessarily more complex. In particular, a reaction that occurs with a single pseudorotation in a trigonal bipyramidal intermediate leads to retention of configuration. [To achieve an interconversion of R and S isomers in a

238

F. H. WESTHEIMER

trigonal bipyramidal structure requires five successive pseudorotations (76).] If a reaction that proceeds by way of a phosphorane leads to retention of configuration, a rearrangement is presumably involved. Conversely, if a reaction occurs cleanly with inversion of configuration, a polytopal rearrangement is presumably not involved. 1. Hydrolysis of Chiral Phosphonium Salts Many stereochemical studies concern the hydrolysis of phosphonium salts. Such salts, if they contain chiral phosphorus atoms, can be resolved (77-79) by conventional means and then reduced, either electrochemically (80,81) or with Si2Cl6 (or similar reagents) (82,83), to yield thermally stable, optically active phosphines. Much of the stereochemistry of these phosphines is based on the entirely reasonable assumption that their reactions with alkyl halides, to yield optically active phosphonium salts, and with hydrogen peroxide, to yield optically active phosphine oxides (76), proceed with retention of configuration.* In addition, since the configurations of a number of relevant compounds have been determined by X-ray crystallography (84-88), at least some of the stereochemical assignments are securely anchored. The mechanism of the alkaline hydrolysis of acyclic phosphonium salts was established by VanderWerf and McEwen and their co-workers (89,90), who showed that the reaction is kinetically first order in phosphonium salt and second order in hydroxide ion and proceeds with inversion at phosphorus. The corresponding reaction with alkoxide instead of hydroxide is relatively slow, first order in alkoxide, and results predominantly in racemization. These facts lead inexorably to the conclusion that the proton on hydroxide ion is essential to the hydrolysis reaction and suggest the following mechanism: CH

®/ C H 2C 6 H 5 CÎH>

H

CH 2 -C 6 H 5 H3^d^C«H5

1

5

OH

20

C H

* 5 (4)

21

CH 2 -C 6 H 5

rv5 ^2M5

22

C

^3 ,C6H5

/S*

^2 n 5

0

+ WH

*

23

* In an early article on chiral phosphines, Homer (81) wrote that he had achieved a chemical cycle to verify these assumptions. The cycle, however, was not sound, and in private correspondence he has withdrawn it.

239

10. THE POLYTOPAL REARRANGEMENT AT PHOSPHORUS

The stereochemical series involved here was established (89,90) by preparing chiral methylethylphenylphosphine and converting it with retention both to the chiral salt 20 and to the chiral oxide 23. (Presumably, phosphoranes other than 21 and 22, with groups other than benzyl in the apical position, are also formed during hydrolysis but do not compete successfully in the irreversible reaction, since benzyl anion is a much better leaving group than the alternatives.) When, however, cyclic phosphonium salts are hydrolyzed, the stereochemical result depends on the ring size (97-96). In particular, the eis- and fraAw-phospholanium salts 24-27 undergo alkaline hydrolysis to the corresponding oxides with complete retention of configuration. This result is readily rationalized by the rules already stated, as follows. Hydroxide ion attacks the salt to yield a trigonal bipyramidal intermediate with hydroxide ion attached at an apical position. The benzyl group, however, cannot initially assume the other apical position, since the ring is constrained to span one apical and one equatorial position. In order to place a benzyl group in an apical position so that it can leave the phosphorane, the molecule must undergo a pseudorotation. Conversely, the observation that the reaction takes place with retention of configuration is strong evidence that a pseudorotation occurs during the overall reaction. CH3

A-T

CH3

WCH2)" R

CH2C6H5

i^H (®jCH2)n R

CH2C6H5

24 R = C H 3 ; /? = 1

25 R = CH 3 ; /7 = 1

26 R=C 6 H 5 ;/7 = 1

27 R = C6H5',/7=1

28 R = C 6 H 5 ; / 7 = 3

29 R =C 6 H 5 ;/7 = 3

If the ring is larger and, in particular, if it is seven-membered, as in 28 and 29, the reaction with alkali leads to inversion. Presumably, these rings preferentially occupy diequatorial positions in phosphorane intermediates; because of the large ring size, such structures will be strain-free. An extraordinarily large number of examples has been developed on this model; all can be accounted for, and many can be predicted by the rules for reaction summarized here. A few examples are discussed below. The salts 30 and 31 are attacked by hydroxide ion with loss of stereospecificity. This result can be accounted for if the phosphoranes, formed as intermediates during hydrolysis, undergo three successive pseudorotations (96) more rapidly than they lead to the formation of product.

240

F. H. WESTHEIMER

CH 3 CH 3 CH 3 CH,

cH,y

f -C f•6 i Hn 5

CH,..

CH,

H

-CH 3

u

CH

CH 2 C 6 H 5

CH 3

+

6n5 CH2CgH5

30

2. Hydrolysis of Esters The attack of nucleophiles on phosphinate esters generally takes place with inversion of configuration at phosphorus. However, the methanolysis of the esters 32 and 33 takes place with retention of configuration, requiring, then, a pseudorotation in the pentacovalent intermediate. CH, CH 3 H. > 3 ' 3 CH 3 CH,

P=0 C H3 , / 0CD3 32

H

ÇH 3 CH 3

CH 3

-P=0 CH, / 3 0CD3 33

Many other examples involving the hydrolysis of phosphonium salts, phosphate, phosphonate, and phosphinate esters have been uncovered (97-103). Corresponding research on thiophosphates is principally the work of Michalski and Mikolajczyk (104-105). The entire body of research has been analyzed (76,103) and is presented in McEwen and Berlin (7). For the purposes of this essay, the point is accepted as proved that reactions at tetracovalent phosphorus generally proceed by way of pentacovalent intermediates formed in accordance with the polarity and strain rules; nucleophiles enter and leave from apical positions and pseudorotation occurs at the central phosphorus atom when such rearrangement may proceed without surmounting excessively high energy barriers and when it is required to produce the products observed. 3. More Precise Definition of the Stereochemistry of Phosphoranes Before proceeding further, some consideration must be given to the assumptions that have explicitly or implicitly been made in the brief discussion above. First and foremost, the pentacovalent compounds with which we have dealt have been written as trigonal bipyramids (10). Although most of the phosphoranes that have been examined have indeed

241

10. THE POLYTOPAL REARRANGEMENT AT PHOSPHORUS

proved to be trigonal bipyramids, or slightly distorted trigonal bipyramids, a number of recent X-ray studies have revealed other structures. From X-ray data, acyclic phosphoranes and most phosphoranes with only one ring are known to approximate trigonal bipyramids in structure, whereas some of the spiranes exist as square pyramids (66,106-121). For example, pentaphenoxyphosphorane, as one would expect, is an almost perfect trigonal bipyramid (121), as is the monocyclic phosphorane 34 (66,116). On the other hand, the spiranes 35, 36, and 37 approximate square pyramidal structures, and 38 and 39 are intermediate between the two idealized geometries. ÇH 3 ÇH 3

—hcH 3

H-

CH,

®SK®

- % ~

CH 3 · CH

X

3L Q

CF CF, 34

R = ^-Pr

ockf*

36

35

9βΗ5

0

C

eH9^

^CH(CF3)2 CH 3

n / C6eH 5

3

f

H -CF,

@x@ 0C6H5

CF, 37

38

39

D. The Rearrangement

The mechanism for the polytopal rearrangement at phosphorus shown in Eq. (1) has proved to be correct, but such was not immediately obvious and required proof. All of the possible modes by which exchange can take place between substituents of a trigonal bipyramid have been classified mathematically (112-126);fiveand only five classes of rearrangements are possible. These may be represented by matrices (122,123), which show the number of steps required to convert any particular isomer to any other particular isomer. Several detailed mechanisms may be associated with any one of the five distinct and independent matrices. The determination of mechanism must then begin with finding the correct class of rearrangement, that is, the correct matrix to be assigned the transformations discussed above. The correct class has proved to be that for pairwise ex-

242

F. H. WESTHEIMER

change of two equatorial and two apical ligands (an ae,ae exchange) and conforms to the mechanism that has been explained and used in this essay. The interconversion of the isomeric phosphoranes can be represented by geometric diagrams as well as by the correct matrix, and several such representations have been suggested (25,727-/29); all of them, as well as the appropriate matrix, are, of course, equivalent. It is doubtful whether all the chemical and NMR data could be accommodated by any mechanism other than the accepted one. In any case, the validity of the pseudorotation mechanism has been thoroughly verified. Britton and Dunitz surveyed the various mathematical possibilities (130), and concluded that all of the transformations except the ae,ae exchange require a transition state in which all five substituents attached to phosphorus lie in a plane. Such a geometric restraint is obviously out of the question, so that, on the basis of simple qualitative reasoning, one can decide in favor of the mechanism chosen. Experimentally, the most persuasive work is that of Whitesides and his co-workers (131,132). They examined in detail the NMR spectra of 40, 41, and 42. At high temperatures, the four fluorine atoms of 40 are equivalent, F

(CH 3 ) 2 N-P^'

CH3 [Cyi

CH(CH3)2 41

40

Ar=XO

CH(CH3)2

42

Ar= ^ O V C H ( C H 3 ) 2 CH(CH 3 ) 2

regardless of the mechanism for exchange, whereas at low temperatures the molecules will be frozen in the configuration shown above, so that there are two equatorial and two apical fluorine atoms. Furthermore, the dimethylamino group is presumably frozen with respect to rotation about the P-N bond. At intermediate temperatures, however, the structures (and hence the NMR spectra that are predicted) depend on the mechanism for the exchange process. If, for example, one equatorial and one apical fluorine atom could be interchanged while the others remained in place, the spectrum at intermediate temperatures would show more different

10. THE POLYTOPAL REARRANGEMENT AT PHOSPHORUS

243

fluorine signals than those that would be found for the ae,ae exchange, in which all four fluorine atoms of 40 exchange simultaneously in pairs. Whitesides and his collaborators worked out the detailed NMR spectra anticipated for various mechanisms and, on the basis of the observed spectra, arrived at the same conclusion as had Dunitz. The spectra of 41 and 42 must likewise be interpreted on the basis of an ae,ae exchange and no other. However, these spectra are even more revealing. First, at low temperatures, where the molecules are frozen in a single conformation, each of the four p-methyl groups of the biphenyl residues of 41 gives rise to a separate signal, and each of the two methyl groups of the isopropyl residue gives rise to a separate doublet (split by the adjacent hydrogen atom). This is what would be expected if the molecule existed as a trigonal bipyramid; the methyl groups of the isopropyl residue are then diastereotopic. On the other hand, if the geometry of the molecules were square pyramidal and if the isopropylphenyl group were rotated to a symmetric configuration (i.e., either between the dimethylbiphenyl groups, or midway between the tolyl groups of one of the dimethylbiphenyl groups), the methyl groups of the isopropyl groups would not be diastereotopic, and furthermore only two different kinds of aromatic methyl groups would appear in the NMR spectrum. In a congruent study, an optically active bis(biphenylene)-2-biphenylylphosphorane (43) was prepared so that a square pyramidal structure for this compound (and hence presumably for 39) can be ruled out (133-135). These considerations are especially important because many spirophosphoranes have shown square pyramidal rather than trigonal bipyramidal structures.

The temperature dependence of the NMR spectrum of 41 can then be analyzed on the assumption that the compound is bipyramidal. As with 40, the spectra require that the reaction proceeds by the simultaneous pairwise exchange of substituents (132). Significantly, the NMR spectra at intermediate temperatures are inconsistent with a direct transformation

244

F. H. WESTHEIMER

from the one trigonal bipyramid to another but can be quantitatively accounted for, provided that a square pyramidal structure, such as that shown as structure 2, exists in the rearrangement as a true intermediate rather than as a transition state. (The temperature-dependent NMR spectra of 42, although consistent with the same interpretation, do not demand it.) Of course, one cannot generalize from the example of 41 to all phosphoranes; probably, some phosphoranes undergo pseudorotation in which the square pyramidal structure is a transition state and some in which it is an intermediate. Detailed Mechanism of the Pseudorotation Although the work of Whitesides and his collaborators (131,132), and of Britton and Dunitz (130) (and the vast literature on phosphoranes) make it clear that the polytopal rearrangement occurs by the mathematical possibility in which two pairs of apical-equatorial substituents simultaneously undergo exchange, a question still arises as to the detailed mechanism by which that transformation occurs. Two such mechanisms have been advanced. The first, suggested by Berry (9), is that implied by Eq. (1). This involves bending two of the equatorial bonds away from each other, so as to increase the angle between them from 120° to 180°, and simultaneously bending the bonds to the apical substituents toward each other but in the direction away from the third ("pivot") equatorial substituents, so as to decrease the angle between them from 180° to 120°. The square pyramidal structure is a natural intermediate in this overall process. The Berry mechanism follows the path of one of the normal vibrations of a trigonal bipyramid (136,137). This fact, although suggestive, is not conclusive with respect to the detailed geometry of the transition state. A second detailed mechanism, suggested by Ramirez and Ugi and their co-workers (138,139), is described as a "turnstile." It requires bending equatorial-apical pairs by a small but undetermined angle toward each other so as to produce a distorted structure, intermediate between a trigonal bipyramid and a square pyramid, and then rotating a pair of substituents and the residual triple simultaneously but in opposite directions. This produces a new structure geometrically similar to the first postulated intermediate but related to a different trigonal bipyramid. The final motion is then one in which the bond angles relax to those appropriate to the final phosphorane. The most important point to note about these mechanisms is that they both conform to the same matrix for the polytopal rearrangement and that they both necessarily yield the same products. Neither mechanism can lead to any phosphorane that the other does not, or racemize or invert configuration if the other does not; they are mathematically equivalent.

10. THE POLYTOPAL REARRANGEMENT AT PHOSPHORUS

245

Ramirez and Ugi have suggested that extra possibilities for rearrangement might be presented by the turnstile as contrasted to the Berry mechanism, provided that the original, slightly distorted structure corresponds to an energy minimum and undergoes a further transformation without actually completing the rearrangement to the logical phosphorane. This distinction is without merit. The pathway outlined by the Berry process goes through a square pyramidal structure which may often represent an energy minimum and which, as Whitesides et al. (132) have shown, represents an energy minimum in the pseudorotation of 41. The two mechanisms differ only in that the postulated intermediate for the Berry mechanism, a square pyramid, is clearly defined and has been realized experimentally in at least one instance, whereas the intermediate for the turnstile is of undefined geometry. The two possible detailed mechanisms not only always lead to the same products, but differ only in small changes in bond lengths and angles at the transition state. Indeed, since compounds that do not conform to the formula PX5 are distorted from trigonal bipyramidal symmetry (and may even approach square pyramids), the idealized pathways outlined above cannot usually be followed, and each compound will exhibit its own, slightly different path.* Whitesides et al. (132) have even suggested that the geometries of the idealized transition states for the two detailed mechanisms may be sufficiently similar that they are indistinguishable within the limits set by the uncertainty principle. However, to the extent that a distinction can be made between the two processes (and it is a distinction almost without a difference) the Berry pathway is preferred. This conclusion arises from two considerations. First, the pathway for the pseudorotation of 41 has already been shown to proceed by way of a square pyramidal intermediate (132). Second, in recent work, Holmes and his collaborators have collected and codified all the X-ray data available for phosphoranes and have examined these structures in detail (106,107). They fall almost precisely on the "Berry coordinate"; that is, the known structures of stable phosphoranes are distorted from idealized trigonal bipyramids or from idealized square pyramids in * The claim (139,140) that the compound below can undergo a turnstile rotation but not a Berry-type process is not apparent from inspection of models, despite the authors' statements to the contrary. The detailed pathway of the polytopal rearrangement for such a constrained molecule is undoubtedly idiosyncratic.

CF.

3

246

F. H. WESTHEIMER

the way that would be required if the motion, initiated by the normal vibrational mode that begins the Berry-type process, were continued. Of course, no law of nature demands that the structures of stable compounds lie along the reaction coordinate of a dynamic process, but such is certainly reasonable. The same molecular forces that determine the geometry of stable compounds dominate the configurations of molecules in motion along a reaction coordinate. Furthermore, Holmes's research (106-111) with phosphoranes parallels that of Dunitz (141), who analyzed the structures of various organic compounds that appear to lie along the reaction coordinate of the carbonyl addition reaction. The X-ray data considered by Dunitz have apparently "photographed" the reaction coordinate for the process. By analogy, Holmes's data probably provide a picture of the polytopal rearrangement with which we are concerned. III. ENZYMOLOGY A.

Enzymatic Displacements at Phosphorus

The outline above of pseudorotation at phosphorus has suggested the important part that this polytopal rearrangement plays in the chemistry of phosphorus compounds. A major question arises as to whether such rearrangements occur during enzymatic reactions of phosphates. The previous section has shown how stereochemical considerations might be used to help elucidate this matter. When a simple displacement occurs at a chiral phosphorus atom, its configuration is reversed. When the reaction is accompanied by a single pseudorotation, configuration is retained, and, when multiple pseudorotations occur, with various exits from the rapidly equilibrating system of pseudorotamers, the chiral center may be more or less racemized. Of course, other mechanisms can be responsible for these results or could complicate them. Two inversions lead to retention of configuration without requiring pseudorotation. If and when a phosphate residue is transferred to an active-site residue on an enzyme and then transferred again to the enzymatic substrate, the possibility of two inversions is necessarily present. An example is presented later in this essay. (Section III,G). A reaction that leads to inversion of configuration requires an odd number of inversions, but in principle that number could be three rather than one, or the process might involve one step with retention (perhaps by way of a pseudorotation) and another with inversion. However, although these complications must be considered, the simplest explanation of an inversion is that a displacement has occurred by the normal SN2 mechanism. All of the single-step enzymatic reactions at phosphorus that

10. THE POLYTOPAL REARRANGEMENT AT PHOSPHORUS

247

have so far been investigated have resulted in inversion, and the presumption is therefore that no pseudorotation has occurred. B. Ribonuclease

The first method used to determine the stereochemistry of reactions at phosphorus is based on the chemistry of chiral phosphorothioates. Surprisingly, most enzymes of phosphate metabolism act on such compounds, although somewhat more slowly than on their natural substrates. In 1968, Eckstein (142,143) prepared the two diastereomeric 2',3'-cyclic uridine phosphorothioates 44 and 45. The triethylammonium salt of the endo isomer (sulfur endo to the ribose ring) has been crystallized, and its configuration has been determined by X-ray crystallography (144). This isomer, but not its exo diastereomer, is a substrate for ribonuclease (145). In aqueous methanol, the enzymatic reaction leads to the production of 46, one of the diastereomeric methyl esters of uridine-3'-phosphorothioic acid. This compound was also crystallized as its triethylammonium salt, and its absolute configuration was determined by X-ray crystallography (146). In the enzymatic degradation of ribonucleic acid, ribonuclease acts on the polymer to cleave a 5'-phosphodiester bond and yield a 2' ,3'-cyclic diester of a pyrimidine nucleotide. The methanolysis reaction is the microscopic reverse of a similar closure of a 3'-ester of uridylic acid to a cyclic phosphorothioate. If one may generalize from the methyl ester to a nucleoside ester (147) and assume that the geometry of the pathways is the same for these compounds of sulfur as for the normal substrates of ribonuclease, then the reaction of 44 to 46 [Eq. (5)] defines the stereochemistry of thefirststep in the action of ribonuclease. The reaction occurs with inversion of configuration about the chiral phosphorus atom. The stereochemistry of this reaction is entirely secure, since both the configuration of the starting material and that of the final product have been determined by X-ray methods. The second step of the action of ribonuclease is similar to the first. The two steps differ in that (the reverse of) the first step may involve the opening of the cyclic phosphate with methanol, whereas the second step involves the opening of the same molecule with water. On the basis of the reasonable assumption that the stereochemistry of the reaction with water is the same as that with methanol, the second step in the action of ribonuclease must also proceed with inversion. Nevertheless, the stereochemistry of the second step of ribonuclease has been determined independently—in fact, it was the first enzymatic reaction at phosphorus for which the stereochemistry was found—but here the demonstration of stereochemistry is more complicated and rests in part on the rules for pseudorotation.

248

F. H. WESTHEIMER

HO —CH 2

44

45 aqueous CH^OH ribonuclease

(5)

HO-CH2

46

The independent method for determining the stereochemistry of the second step was advanced in 1970 by Usher, Richardson, and Eckstein (148). Their chemistry involved the enzymatic hydrolysis of the endo isomer of cyclic uridine phosphorothioate (44) in water enriched in 18 0. On the assumption that the enzymatic reaction will be stereospecific, the resulting 3'-phosphorothioate will consist of a single stereoisomer, with a chiral thiophosphate residue. This compound (47) was then recyclized by reaction with diethyl phosphorochloridate, and the two resulting diastereomeric 2',3'-cyclic phosphorothioates were separated chromatographically. These compounds were analyzed for 18 0. The isotope was found concentrated in the exo isomer 45; the endo isomer 44 was nearly free of 18 0. The two successive reactions—one enzymatic and one

249

10. THE POLYTOPAL REARRANGEMENT AT PHOSPHORUS

chemical—resulted, then, in the regeneration of the same diastereomer with which the reaction had begun (plus the other diastereomer enriched in 18 0). It follows that the two steps (enzymatic cleavage and chemical closure) proceed with the same stereochemistry—either both inversions or both retentions. The choice has been made on the basis of the rules for pseudorotation. The endo compound 44 was hydrolyzed with alkali, in water enriched with 18 0, to yield a mixture of the 2' and 3' isomers of uridine phosphorothioate [Eq. (6)]. The Chromatographie separation of these struc-

H0-CH2

HO-CH2 H20

ribonuclease ( or "OH ) (C 2 H 5 0) 2 POCI

44

47

(6)

(C 2 H 5 0) 2 POCI

45

tural isomers can be carried out without difficulty. Closure of the 3' isomer with diethyl phosphorochloridate led to the same mixture of 44 (essentially unenriched with 180) and 45 (enriched with 180) as that from the sample of 47 produced enzymatically. The reaction with alkali and that with ribonuclease then proceed with the same stereochemistry. However,

250

F. H. WESTHEIMER

the chemical reaction almost certainly proceeds with inversion or, more precisely, without pseudorotation. The argument, already presented with respect to the hydrolysis of ethylene phosphate, will be repeated here for completeness (37). Attack of alkali on 44 should lead to either of two trigonal bipyramids. In both, the hydroxide ion will occupy an apical position; the other apical position will be occupied either by the 2'-oxygen atom or by the 3'-oxygen atom; in any case, the ring must occupy one apical and one equatorial position in accordance with the strain rule. We are here concerned only with the intermediate 48, in which attack occurs to cleave the bond in the 2' position. In 48, both the oxygen and sulfur atoms carry negative charges. Both atoms, therefore, are strongly electron donating, and both should occupy equatorial positions as shown. Neither can be placed in an apical position without paying a penalty in energy as defined by the polarity rule. In other words, 48 cannot readily undergo pseudorotation. This prediction has already been confirmed with respect to the chemistry of ethylene phosphate (38) and of methyl ethylene phosphate (57); hydrolysis in acid is accompanied by pseudorotation, whereas hydrolysis in base involves only a direct ring opening, with no pseudorotation. On the basis of the close analogy between the cyclic phosphorothioate and ethylene

:Ù OH 48

phosphate, one may reasonably conclude that the reaction of 44 with alkali proceeds without pseudorotation and therefore with inversion at phosphorus. But this conclusion immediately solves the stereochemistry of the entire sequence and shows that all the reactions involved—the enzymatic opening of 44 and chemical closure of 47, as well as the alkaline opening of 44—proceed with stereochemical inversion at phosphorus and so presumably without pseudorotation (149). Perhaps this result might have been predicted. If alkaline cleavage oc-

251

10. THE POLYTOPAL REARRANGEMENT AT PHOSPHORUS

curs without pseudorotation and if the mechanism for the action of ribonuclease requires base catalysis, perhaps one might conclude that the enzymatic and the chemical reactions have similar stereochemical consequences. However, one need only state this argument to realize that one cannot be content with it and that the experimental proof offered by Usher, Richardson and Eckstein (148) was essential. This, in fact, was the research that opened the field of the stereochemistry of enzymatic reactions at phosphorus, and the developments (many of which have come to fruition only very recently) were undoubtedly stimulated by this seminal contribution. a ATP-a-S Many enzymatic reactions of phosphate chemistry involve ATP. The molecule (49) may undergo cleavage at three different sites (750): kinases, ATPases v

y UDP-glucose

. ... -

OH /

OH > OH

I

11 I I

I

pyrophosphorylase NH2

Π3

HO—P+0 — 4 - 0 — P + O - J - P — 0 —CH« {

4 9

OH

" 7

OH

PRPP synthetase

One would like to know the stereochemistry of all of the transformations, or at least of representative examples of each type. If ATP is substituted on the a or ß-phosphorus atom with sulfur, that phosphorus atom is then chiral, and the enzymatic reactions (if and when they occur) are stereochemically defined. In order that the γ-phosphorus atom of ATP be chiral, it must be further substituted, as, for example, with sulfur, 16 0, and 18 0. All of these possibilities have been achieved. The two diastereomers of ΑΤΡ-α-S have been prepared (151,152). Adenosine 5'-phosphorothioate was synthesized by the action of PSC13 on levorotatory (natural) adenosine, (153). The phosphorothiate was converted chemically to ADP-a-S by activating the thiophosphate residue with diphenyl phosphorochloridate and then condensing the resulting intermediate with inorganic phosphate. A mixture of the two possible diastereomers was formed. A similar condensation with pyrophosphate gave a mixture of the diastereomers of ATP-a-S.

252

F. H. WESTHEIMER

The individual diastereomers can be obtained enzymatically. When a mixture of the diastereomers (50 and 51) of ADP-a-S is allowed to react with phosphoenolpyruvate and pyruvate kinase, an enzymatic reaction occurs with only one of the diastereomeric diphosphates (arbitrarily called the "A" isomer), to yield only one of diastereomers (52) of ATP-a-S (again called the "A" isomer) and leave behind the " B " isomer of ADP-a-S [Eq. (7)]. Conversely, when the mixture of diastereomers of ADP-a-S is treated with creatine phosphate and creatine kinase, a reaction occurs with the " B " isomer of the diphosphate, to yield the " B " isomer (53) of ΑΤΡ-α-S and leave behind the "A" isomer of ADP-a-S. The Chromatographie separation of ADP-a-S and ΑΤΡ-α-S is readily accomplished, completing the preparation of the pure diastereomers of the α-thio analogues of both ADP and ATP. These compounds are then available for further enzymatic reactions. Subsequently, the absolute configuration of these molecules has been determined, and the stereochemistry of their reactions at phosphorus explored. NH2 AOP-a-S(A) (unreocltd)

ATP-a-S(B) 53

2

' 0 , P N H - C - N - C H 9C 0 9" 3

-

| CH3

;

crtotmt kinase

2

ADP-e-S(A)

2

2

OPO, "

ATP-o-S(A)

, 3 0

5

+ ADP-a-S(B) 51

C H 2= C - C 0 2pyruvot· kinas«

2 5

+

(7)

ADP-a-S(B) (unr«act«d)

The absolute configurations of the "A" and " B " isomers of ATP-a-S were assigned (154) on the basis of their reactions with snake venom diesterase. This enzyme attacks the " B " isomer 400 times more rapidly than it attacks the "A" isomer. It also preferentially attacks theR isomer (see below) of 54, U-p(S)-A. On the basis of the reasonable and usual assumption that a given enzyme preferentially uses substrates of the same stereochemistry, the configuration at phosphorus of the enzymatically active isomer of ΑΤΡ-α-S must be the same as that of U-p(S)-A. The Cahn-Ingold-Prelog rules then specify that the configuration (755) at phosphorus of the active isomer of ΑΤΡ-α-S is R. The absolute configuration of the active isomer of U-p(S)-A (54), was determined by connecting it to that of the endo isomer (44) of cyclic uridine phosphorothioate, which had been established by X-ray crystallography (144). The chemistry is illustrated in Eq. (8). The inversion at phosphorus postulated in the conversion of 54 to 44 parallels those already demonstrated for other reactions catalyzed by ribonuclease. The assignment of configuration to the " B " isomer (and by elimination to the "A" isomer) of ΑΤΡ-α-S is then secure.

253

10. THE POLYTOPAL REARRANGEMENT AT PHOSPHORUS

RNAase \ p ^ O — adenosine

A0

(}

s +

44 adenosine snake venom diesterase uridine

+

A-5-P(S)

1. RNA Polymerase Of the two diastereomers of ΑΤΡ-α-S, only the "A" isomer 52 (that is, only the isomer with the 5 configuration at phosphorus) reacts with RNA polymerase (756). When UTP and ΑΤΡ-α-S are added to that enzyme in the presence of a template of synthetic poly[d(A-T)] (the alternating polymer of deoxyadenylic acid and thymidylic acid), a new polymer (55) is formed, consisting of alternating units of thioadenylic acid and urydilic acid[. . . A(S)UA(S)UA(S)U . . .] [Eq. (9)]. This polymer is a substrate for ribonuclease and, in accordance with the specificity of RNAase, is cleaved at the 5' position of the nucleotide adjacent to the pyrimidine residue to yield 56, a cyclic dinucleotide thiophosphate which may be abbreviated as A-pU > pS. This, in turn, is cleaved by spleen phosphodiesterase to yield the familiar endo isomer (44) of cyclic uridine phosphorothioate. These transformations allow the determination of the stereospecificity of RNA polymerase. Since the cleavage catalyzed by the spleen diesterase does not attack any bond attached to the chiral phosphorus atom, no stereochemical change can be effected during the final hydrolysis. On the assumption that the action of RNAase inverts configuration at phosphorus, a consideration of the known configurations of the starting material and of the final product then shows that one additional inversion has occurred. Since RNA polymerase effects an inversion at phosphorus, presumably the reaction it catalyzes does not involve a pseudorotation.

254

F. H. WESTHEIMER

RNA ...A(S)UA(S)UA(S)U... S

+

UTP +

/

X

0 P 0 2" - 0 P 0 3

polymerase 55

poly(dAT) template

(9) ribonuclease

spleen diesterase

44 +

adenylic

acid

2. UDPG Phosphorylase Frey and his co-workers (157,158) demonstrated that the reaction catalyzed by uridine diphosphoglucose phosphorylase also proceeds with stereochemical inversion at phosphorus. They carried out the chemical synthesis of uridine diphosphate-a-S by procedures that paralleled those published (151,152) for adenosine diphosphate-a-S. The mixture of diastereomeric diphosphates reacts with phosphoenolpyruvate in the presence of pyruvate kinase to yield UTP-a-S(A) and leave behind UDP-a-S(B), paralleling the preparation of the corresponding isomer of ΑΤΡ-α-S. Although the absolute configuration of these compounds need not be known for the demonstration (see below) of inversion catalyzed by UDPG phosphorylase, the assignment can nevertheless be made with confidence on the basis of the assumption that pyruvate kinase always selects for the same configuration at phosphorus.* * The naming of the "A" and "B" isomers is, of course, arbitrary. However, the "A" isomer of UTP-a-S is that produced from the "A" isomer of UDP-a-S by the action of pyruvate kinase. Since the reaction does not involve bonds to the chiral phosphorus atom, the configurations at phosphorus of the "A" isomers of UDP-a-S and of UTP-a-S are the same.

10. THE POLYTOPAL REARRANGEMENT AT PHOSPHORUS

255

Frey also prepared a mixture of the A and B diastereomers of UTP-a-S by chemical synthesis, again paralleling the prior work with ΑΤΡ-α-S. The "A" and "B" isomers can be cleanly distinguished by proton-decoupled 31 P NMR spectroscopy. The signal from the α-phosphorus atom of UDP-a-S(A) appears as a doublet (split by the β-phosphorus atom), whereas the spectrum of the mixture of "A" and "B" isomers shows two doublets. This allowed an assignment for the signals from the "B" as well as the "A" isomer. Similarly, the doublet from the spectrum of the "B" isomer of UDP-a-S was obtained. Comparison with the two doublets from the mixture of stereoisomers allowed the identification of the signal from the t4A" isomer as well. The stage was then set for a determination of the stereochemistry of the enzymatic reaction with UDPG phosphorylase. When a mixture of the "A" and "B" stereoisomers of UTP-a-S and glucose 1-phosphate was treated with UDPG phosphorylase, reaction occurred with only one of the stereoisomers of UTP-a-S. The isomer left behind was identified by its proton-decoupled 31P NMR spectrum as the "A" isomer; therefore, the "B" isomer (57) had reacted. The product (58) was hydrolyzed by brief heating at pH 2.5. This reaction cleaves the glucose residue at the glycosidic bond and produces UDP-a-S [Eq. (10)]. The product was identified as the "A" isomer (59) by NMR spectroscopy. Overall, then, the "B" isomer of UTP-a-S reacts under the influence of uridine diphosphate glucose phosphorylase to give a molecule with the configuration at phosphorus of the "A" isomer; the process therefore involves a single inversion of configuration at phosphorus.

UTP-a-S(B)

UDPG

+

phosphorylast

57

(10) CH2OH

0

HN 0

i

> Λ^υ

BH OP0 2 "— 0 » - P - ^ 0 — C 2

58

II IOO°C

HN "

0H

0 H

UDP-a-S(A) 59

+

glucose

256 D.

F. H. WESTHEIMER

Tetraamminecobalt (III) -A TP

In order to investigate the stereochemistry (and hence the possibility of polytopal rearrangement) at the central phosphorus atom of adenosine triphosphate, an ATP with a chiral ß-phosphorus atom is needed. This could be ATP-ß-S. Much of the enzymology so far developed, however, involves tetraamminecobalt(III)-ATP. Enzymes utilizing ATP as substrate require magnesium ion for activity, and a strong presumption has been established that a complex of magnesium ion and ATP is the actual substrate (159,160). By means of 31P NMR spectroscopy one can distinguish among the three phosphorus atoms of ATP, and NMR data can be used to suggest, from the chemical shifts that occur on complexing ATP with cations, which of the phosphate groups is directly attached to metal ions. With ATP and Mg2+, the predominant complex involves the ß- and γ-phosphate groups. In such a complex, the phosphorus atom is chiral; it is attached to two different phosphate groups and to two anionic oxygen atoms, which differ in that only one of them is adjacent to the magnesium ion. Complexes of magnesium ion, however, are entirely too labile to be of use in the determination of stereochemistry. A few ions form kinetically stable chelates. The half-time for the dissociation at room temperature of tetraamminecobalt(III) complexes with oxygen ligands is of the order of days, and similar stabilities have been observed for complexes of chromium(III) (161,162). The ß-phosphorus atoms in ATP complexes of Co(III) and Cr(III), then, will be stable and chiral and, provided that the complexes will serve as substrates for enzymes, will allow the determination of the stereochemical consequences of the relevant enzymatic processes. Recently, Cleland and his collaborators (163-170) prepared and separated the two diastereomeric complexes in which tetraamminecobaltic ion is attached at the ß- and γ-phosphate residues of ATP. One of these ß,y complexes proved to be active with hexokinase (168). This finding strongly implies that a ß,y complex with magnesium ion is the true substrate for hexokinase. Although the absolute configurations of these complexes need not be known in order to demonstrate inversion or retention in enzymatic processes, they have nevertheless been determined (169). The adenosine re0P0 2 "- -0 — adenosine O-FT0 (NH34Co 34

\

0

/

0 — PO," 60

(NH3)4Co

0 0 —P0 2 ~ 61

(11)

257

10. THE POLYTOPAL REARRANGEMENT AT PHOSPHORUS

sidue was removed {167,169) from the tetraamminecobalt(III) complex 60 to produce the cobalt(III) complex (61) of inorganic triphosphate, in which the ß-phosphate residue is still chiral, unaffected by the hydrolysis [Eq. (11)]. The zwitterionic diacid of this triphosphate was subjected to X-ray crystallography; the S isomer corresponds to the isomer of 60 that is inactive with hexokinase.* Phosphoribosylpyrophosphate Synthetase Phosphoribosylpyrophosphate (PRPP) synthetase catalyzes the pyrophosphorylation of ribose 5-phosphate by the metal complexes of ATP, as shown in Eq. (12). Li, Mild van, and Switzer (171,172) found that 0

2-

0 \ Θ

odenosine —OPOo-O^-P'

P— 0

OH

+

I

i

0 3 PO—CH "CH2

OH

V

^ M'2+(+)

OH

62

(12)

PRPP synthetase

2- Ο,ΡΟ—C 2adenosine — OP0 3

^9

W

0

/

OH

OH

\^ ^

?

T-^.-KV

Hy

1 1 -0^ ^0. ^M'2+<+>

63

PRPP synthetase could utilize one of the two diastereomers of the β,γcobalt complex of ATP. The diastereomer 62 is the opposite of that utilized by hexokinase. In fact, the mixture of diastereomers can be sepa* The identification of configuration at phosphorus is rigidly specified by the CahnIngold-Prelog rules. The rules however, seem idiosyncratic when applied to these molecules. Since the atomic number of magnesium is less than that of phosphorus, whereas that of other metals (cobalt, chromium cadmium, calcium) is greater than that of phosphorus, the designation, R or S, depends on the metal ion in the complex. Thus, whereas the cobalt complexes pictured above for 58 and 59 are both S, the corresponding magnesium complexes are R. Cleland and Cornelius (168) have offered an alternative system for describing these particular complexes, but here the conventional (if idiosyncratic) designations are used.

258

F. H. WESTHEIMER

rated by allowing one diastereomer (60) to react with glucose and hexokinase; the unreacted diastereomer then serves for the pyrophosphorylation of ribose 5-phosphate. The circular dichroism of the complex 63 was found to be opposite to that of the starting complex of ATP (62). On the basis of the reasonable assumption that the sign of the circular dichroism is determined by the chirality of the phosphorus atom, the reaction catalyzed by PRPP synthetase must proceed with inversion at phosphorus. £ ATP-ß-S The chirality of the ß-phosphorus atom in ATP has been developed on the basis of the chemistry of the cobalt and chromium complexes discussed above. An alternative method involves the diastereomers of ATP-/3-S, which (151,152) have been prepared starting with chemically synthesized ADP-/3-S [Eq. (13)]. That material can be subjected to enzymatic processes similar to those used for the synthesis of the diastereomers of ΑΤΡ-α-S. (These reactions, although dominant, are not ADP-0-S

CH3-C-OP032,"/

\ C H 2 = C - C 0 2 , Mg** ,

(13)

acetate kinase À

ATP-£-S(B)

ATP-0-SU)

64

65

stereochemically "clean.") In the presence of Mg2+, the "A" isomer (65) of ATP-ß-S is cleaved by myosin, whereas the " B " isomer (64) is essentially inert. Conversely, the " B " isomer, again in the presence of Mg2+, is active with hexokinase and with nucleoside diphosphate kinase, whereas the "A" isomer is essentially inert. The absolute stereochemistry of these isomers has been determined as follows. Jaffe and Cohn (173) utilized NMR spectroscopy to study the reactions of ATP-ß-S with hexokinase in the presence of various metal ions and discovered that the stereochemistry is metal ion dependent. In particular, hexokinase catalyzes the reaction between glucose and ATPß-S(B) in the presence of Mg2+, but the stereochemistry is reversed in the presence of Cd2+; here, by contrast, the "A" isomer is active and the " B " isomer inert. Investigation of the NMR spectra of the magnesium and

259

10. THE POLYTOPAL REARRANGEMENT AT PHOSPHORUS

cadmium complexes of ATP-0-S strongly suggests that, as expected, the magnesium ion is attached at the oxygen atom of the ß-phosphate residue, whereas the cadmium ion is attached at sulfur. This all makes sense on the basis of the assumption that hexokinase always uses compounds of the same geometry at phosphorus. Furthermore, the absolute stereochemistry of the Co(III) complex that is active with hexokinase is known from Cleland's work. The complexes that react with hexokinase are then 66 and 67. Both ATP-a-S(A) and ATP-/3-S(A) have the S configuration at phosphorus. The stereochemistry of ΑΤΡ-α-S and of ΑΤΡ-β-S can be "02P — 0 0 \

OAMP

P^ 2+ / ^ S Mg—0

"02P — 0 and

0 \

2+ / Cd — S

66

OAMP P^ %

67 A

B

f

t

2-

I

3

adenosine — O - P - O - P - O - P O »

A

Θ

A 68

summarized by structure 68, in which sulfur in an "A" position yields an "A" isomer, and sulfur in the "B" position yields a "B" isomer. F.

ATP-y-S-y-*0

Kinases transfer the terminal phosphate residue of ATP to and from various substrates. The terminal phosphorus atom has been made chiral in two ways. The chemistry of ATP-y-S-y-180 is treated in this section, and that of ΑΤΡ-γ-160,170,180 is treated in Section III,G. To prepare chiral ATP-y-S-y-l80, the diastereomeric cyclic thiophosphates of D-glyceric acid (69 and 70) were synthesized (174) and separated by column chromatography. This amounts to a resolution at the chiral phosphorus atom. Although the authors did not determine the absolute configuration of the isomer of the cyclic phosphorothioate with which they worked, each of the isomers was stereochemically pure. 0-H_COg

69

0

O-H-7

'

CO2

70

260

F. H. WESTHEIMER

One of these isomers was treated with LiOH in water enriched in 18 0. In accordance with the pseudorotation rules and with the analogy provided by the work of Usher, Richardson, and Eckstein (148), the reaction should take place with inversion by an 4'in-line'' mechanism, i.e., without pseudorotation. Although it is not known which of the two diastereomers corresponds to formula 69 and which to 70, the chemistry is illustrated here (without detriment to the final conclusion) with structure 69. This isomer, on hydrolysis, would produce 71 and 72, both chiral at phosphorus. C0 5 CH,— C^

/

\ P ^o

*0/Ns

OH 2-

72

71

1. Pyruvate Kinase, Hexokinase, and Glycerol Kinase The chiral phosphate residue of 71 can be converted to glycerol 3-phosphorothioate by three separate routes. First, enolpyruvate phosphorothioate can be prepared by the action of enolase (775) on 71, and the thiophosphate residue can be transferred with the aid of pyruvate kinase (776) to ADP to yield ATP-y-S-y-180 (73) [Eq. (14)]. Hexokinase (777) acts on 73 and glucose to yield glucose 6-phosphorothioate [Eq. (15)], and this C02H*-C-*0

,0

C02

I

enolase

C —0.

I

H2C

t

H 2Ί C OH

.0

V

ADP, pyruvate k i n a s e .

(14)

* \ adenosine — 0 P 0 2 — 0 P 0 2 — 0 *

/ 73

2-

261

10. THE POLYTOPAL REARRANGEMENT AT PHOSPHORUS

in turn enters the glycolytic pathway (750) to yield, eventually, dihydroxyacetone phosphorothioate. The latter was reduced with NADH in the presence of glycerolphosphate (178) dehydrogenase to yield glycerol phosphorothioate [Eq. (15)]. In this overall process, the thiophosphate ATP-/-S-7-,80

*2. CHoOPOSO HO—L \ V ^ O

glucose hexokinase

H O - V ^ ^ ^ \^V~H OH OH

73

Enzymes of glycolysis

(15) CH2OH | C«0 I * CH2OPOSOc~

NADH -*► glycerol dehydrogenase

CH2OH T H---C—OH | t CH2OPOSO 74

residue is transferred twice—once in the formation of ATP-y-S-y-180 and again in the phosphorylation of glucose catalyzed by hexokinase. The other reactions of glycolysis do not affect the chiral phosphorus atom. In an alternative process, glycerol was directly phosphorylated by ATP-y-S-y-180 [Eq. (16)]. This process also involves two reactions in OH ATP-/-S-X-,80

I

HOCH2-CH-CH2OH

73 glycerol

kinase

(16)

OH

2-*

f

OSOPO — C H 2 — C — C H p O H H 74

which the phosphorus atom is transferred—once in the preparation of ATP-y-S-y-180 and once in the phosphorylation of glycerol (179)—and (as in the previous example) the question must be decided as to whether these reactions proceed with retention, inversion, or racemization. Finally, 71 can be reduced enzymatically to glycerol 3-thiophosphate (175) [Eq. (17)]. This pathway does not involve any reactions at the phosphorus atom of the thiophosphate residue and so leaves the chirality of the phosphorus atom unchanged. Although the stereochemistry of the thiophosphate residues of the three

262

F. H. WESTHEIMER

OH 2* I ^"OSOPOCH2— C-CH 2 OH

OH

,*

I

various

I

enzymes

"0S0P0-CH 2 —C —C0 2 " H

71

74

(17)

H

samples of 74 can differ only in the relative positions of an 160 and an 180 atom, the determination of chirality can be made by cyclizing the glycerol phosphorothioates. The cyclization process, carried out chemically with diphenyl phosphorochloridate and pyridine, can be assumed to follow an ''in-line" mechanism; i.e., it must occur with inversion but without polytopal rearrangement at phosphorus, in analogy with work already cited (148). The glycerol phosphorothioate may in principle be present as either of two diastereomers. The configuration at the central carbon atom is, of course, fixed, but two possible stereochemical configurations are possible at phosphorus. Cyclization of the diastereomer shown as structure 75 [Eq. (18)] will lead to two diastereomeric cyclic thiophosphates (76 and OH 0V

T

,0-CHp- -C--CH20H

I * / P'"V 0

(C6H50)2P0CI

>s

A

75

H

,(C6H50)2P0CI pyridine

pyridine

(18)

V 0 -' Π7

CH 2 0H

Θ

o'

» 76

H 77

77) in (essentially) equal amounts, one formed by the loss of 160 and the other by the loss of 18 0. The two diastereomeric cyclic thiophosphates 76 and 77 can be separated chromatographically, and the 18 0 content of each established. One is enriched in 180 and the other is not, as expected for the products from chiral thiophosphate. The resulting data show that all three of the enzymatic processes that attack chiral phosphorus (those catalyzed by pyruvate kinase, hexokinase, and glycerol kinase) proceed with the

10. THE POLYTOPAL REARRANGEMENT AT PHOSPHORUS

263

same stereochemical consequences; i.e., all are inversions or else all are retentions. The logic of these transformations holds despite the ambiguity as to whether the actual configuration of 69, chosen here for purposes of illustration, is correct. If it is not, all of the configurations at phosphorus shown here would have to be reversed, but the qualitative conclusion would remain the same. The processes outlined here unfortunately have not yet led to a definitive solution to the problem of whether all of these reactions occur with retention or all with inversion. Knowles and his co-workers have, however, shown that, if one causes inversion, all do.* 2. Adenylate Kinase Chiral ATP-y-S-y-180 and ADP-y-S-y-180 have been prepared and their configurations determined (181,182). Adenylate kinase catalyzes the transfer of the terminal P018OS moiety from ATP to AMP. Comparison of the product with the ADP-y-S-y-180 of known configuration that had been prepared showed that the reaction takes place, in conformity with the other enzymatic reaction cited here, with inversion of configuration at phosphorus. G.

16

0,

17

0,

18

0

Although the many different phosphorothioates cited here are substrates for a wide variety of enzymes, perhaps some doubt may remain as to the legitimacy of stereochemical conclusions drawn from these analogues of the phosphates. The work of JafiFe and Cohn in particular (775), which shows that the stereochemical consequences of reactions with phosphorothioates depends on the particular metal ion used for activation, counsels caution. Recently, two independent syntheses (183,184) and two quite different methods of analysis have been published for phosphate esters in which the chirality at phosphorus depends on substitution with the three stable isotopes of oxygen. ,6

o

R-OP-0 ,8

0

78 * Knowles and Blättler (180) have now demonstrated, using chiral [160,170,180]phosphate, that glycerol kinase catalyzes phosphorylation with inversion at phosphorus. It follows, then, that the reactions catalyzed by hexokinase and pyruvate kinase also proceed with inversion.

264

F. H. WESTHEIMER

Alkaline Phosphatase Knowles and his co-workers have now used their chiral phosphate (183) to study the stereochemical pathway for alkaline phosphatase (185). This enzyme (186,187) catalyzes phosphate transfer in two steps (first, phosphorylation of the enzyme and, second, transfer of the phosphate group from the enzyme to solvent or other acceptor). Provided that the two steps proceed stereospecifically and with the same stereochemistry, the overall result must necessarily be retention, and indeed retention was observed. If both steps are inversions (in keeping with the other examples cited here), then neither involves a pseudorotation; this statement, however, remains to be proved. The chemical reactions used to synthesize chiral esters of [160,170,180]phosphate are shown in Eq. (19). In the course of the syntheses, the diastereomeric cyclic esters were separated, and one of them (79) was used to prepare both the phenyl ester 80 and the primary ester of 5-propanediol (81) with [160,170,180]phosphate. Me

MeNHN^Me

p ,7 bci,

Η 0

^-"6Η H

5

Et20 H

ROH 1 NEt3

(19)

Me

>

- \ ,8

R-0 2-

~"

0

80 81

2χ.Μβ

Pd/H2

l80^|N0^Zc6H5 >^70

R = C6H5 R = CH2CHOHCH3

H

H,V

R

- \ s"**Y*%

•S\-fL 79

"

In a separate experiment, the phenyl ester 80 was converted, by the action of alkaline phosphatase, to the ester (81) of S-propanediol [Eq. (20)]. The latter, then, has been prepared by two routes; in order to establish the stereochemical course of the enzymatic reaction, it is sufficient to find out whether the two samples of [160,170,180]phosphate ester of 5-propanediol have identical or enantiomeric configuration at phosphorus. (In fact, the stereoanalytical method used actually provides the absolute configuration at phosphorus.) Knowles's stereochemical analysis is logically sound, although complicated. The phosphate ester of propanediol can be cyclized, with expulsion of an oxygen atom. Since the isotope effects must be negligible, one-third

265

10. THE POLYTOPAL REARRANGEMENT AT PHOSPHORUS

C 6 H 5 -0. X

P.

,8

6 •"° » /

2-

i vo17

0

9H3

CH 3

H—Ç—OH

H»-C—OH

CHgO" iline alkaline phosphatase

*0

ΟΗ2Οχ

\

p

/

» V 17 ιβ'I 0 0

80

I

<2- (20) )

ΘΙ

of the material will lose each of the isotopes of oxygen. The resulting cyclic ester (82) is then converted to a mixture of diastereomeric methyl esters (83 and 84), and these esters are separated chromâtographically. The mixture of diastereomers (83 and 84) contains all six possible isotopic species, designated, by listing the isotopic substituents at phosphorus, as follows: (a) 160,17OCH3; (a') 170,18OCH3; (a") 180,16OCH3; (b) 16 18 0, OCH3; (b') 170,16OCH3; (b") I80,17OCH3. Provided, however, that the propanediol phosphate was chiral at phosphorus, one diasteromeric CH 3

CH 3

P

P

0

OR

II

82 R=H

I

84 R = C H 3

83 R=CH 3

methyl ester (e.g., 81) will contain the species a, a', and a", whereas the other (e.g., 82) will contain b, b', and b". The masses of the two sets of isotopic isomers are the same, but they can nevertheless be distinguished by metastable ion mass spectrometry. In a mass spectrometer, a derivative of each isomer loses formaldehyde from the P-OCH3 group. By observing the metastable ion mass spectrum, one can determine whether (for example) the [180]formaldehyde comes from a molecule that also contains 17 0 (i.e., a'), or whether it comes from a molecule that also contains 16 0 (i.e., b). This result serves to identify the enantiomer involved. Essentially this method (but with a few added complications) was used to determine the overall stereochemical pathway for alkaline phosphatase. Obviously, many other problems in the stereochemistry of phosphorus will be elucidated with chiral [ 16 0, 17 0, 18 0] phosphates. H. Summary for Enzymo/ogy

The data presented here show that thefirst(146) and second (148) steps of the action of ribonuclease, the reaction catalyzed by UDPG phos-

266

F. H. WESTHEIMER

phorylase, (157,158) the reaction catalyzed by RNA polymerase (156), the reaction catalyzed by phosphoribosylpyrophosphate synthetase (171,172), and that catalyzed by adenylate kinase (181,182) all proceed with inversion at phosphorus. Although six examples are insufficient to establish a firm rule, they are enough to provide the presumption that all enzymatic reactions at phosphorus proceed with inversion and that, therefore, they occur without pseudorotation at phosphorus. Although explanations of this generalization are bound to be speculative, especially since the "fact" itself has not been confirmed, some guesses about the results are perhaps warranted. If a pseudorotation were permitted during the reaction catalyzed by an enzyme, the enzyme would probably have to undergo a conformational change to accommodate the different geometry required for entering and leaving groups. Of course, many enzymatic reactions are accompanied by conformational changes, and so far at least the mechanistic basis for these conformational changes is obscure. Perhaps, however, the demand for a conformational change places one extra requirement on natural selection, and when the extra step is not absolutely needed—when a displacement without pseudorotation is possible—the process of evolution has selected the simpler route (187).

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10. THE POLYTOPAL REARRANGEMENT AT PHOSPHORUS

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270

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