J. Am. Chem. SOC. 1993,115, 6001-6012
600 1
Photochemistry of Phosphate Esters: a-Keto Phosphates as a Photoprotecting Group for Caged Phosphate Richard S. Givens,' Phillip S. Athey,+ Bogdan Matuszewski,* L. William Kueper, 111, Jie-you Xue,g and Thomas Fister Contribution from the Department of Chemistry, The University of Kansas, Lawrence, Kansas 66045 Received January 8, I993
Abstract: Irradiation of two families of a-keto phosphates yielded rearrangement products and deprotected phosphates as the major products. For both sets of reactants, the triplet excited state of the ketone reacted with quantum efficiencies that ranged from 0.10 to 0.38. Desyl phosphates yielded 2-phenylbenzo[b]furan independent of the nature of the solvent whereas phosphate esters of a-hydroxy-p-methoxyacetophenone rearranged to esters of p-methoxyphenylacetic acid. In all cases, the phosphate group with the remaining ligands intact was released in nearly quantitative yield. The desyl group was further developed as a cage ligand for CAMP. Upon photolysis, the desyl caged ester of CAMP (13) quantitatively released the nucleotide with a quantum efficiency of 0.33 f 0.01 and a unimolecular rate constant of 7.1 X 108 s-I. Additional synthetic, product, and mechanisticstudies are reported for the two series of a-keto phosphates. In earlier publications,l we reported that benzyl and 1- and 2-naphthylmethylphosphates undergo efficient, photochemically induced nucleophilic substitution reactions in which phosphate functions as a nucleofuge. We provided evidence through oxygen18 labeling, stereochemical, and substituent studiesthat photolysis of the carbon-oxygen bond of benzylic phosphates produced a reactive benzyl cation-phosphate anion pair.l*+ A comparison of phosphate with other photoactive nucleofugal groups by Pincock et a1.a established its efficacy as a good photolabile leaving group. Furthermore, we have shown that efficient reactions of the resultant carbocation with nucleophiles or nucleophilic solvents generally produce good yields of substitution products. We now report details on the photochemistry of two families of a-keto phosphates, 2-oxo- 1.2-diphenylethyl phosphateld(e.g., desyl phosphates la+) and 2-(4-methoxyphenyl)-2-oxoethyl phosphate (e.g., a-phenacyl phosphate 2a), and application of these reactions for the photorelease of a nucleotide, CAMP.
and Wilson in 1964.2 Among the minor photoproducts were the photocyclization product 2-phenylbenzo[b]furan ( 3 ) and its photodimers 4 (eq 1). The efficiency for conversion of the desyl
\
I , as diastereomers
Through these investigations, we have shown that a-keto phosphates are kinetically superior alternatives to o-nitrobenzyl esters3v4for the photorelease of biologically important phosphates. The desyl chromophore was first investigated as a possible photoprotecting group for carboxylic acids (e.g., le) by Sheehan
ester to 3 was shown to be highly dependent on the nature of the substituents on the benzyl aromatic ring, with the m-methoxy substitution giving the highest yields of the benzofuran. The suggested mechanism for the formation of benzofuran began with n,# excitation of the ketone followed by intramolecular cyclization toformoxabicyclo[2.1 .O]pentaneintermediate5. Benzyl desyl sulfide was also shown to cyclize exclusively to 2-phenylbenzo[b]thiophene upon irradiation,6 in agreement with the mechanism suggested earlier by Sheehan et a1.s The photochemistryof other, closely related a-keto derivatives is exemplified by the series of substituted phenacyl chlorides 2b. In 1961, Anderson and Reese7 reported that phenacyl chlorides containing electron-donating groups at the para and ortho positions (e.g., 4-OH, 4-OCH3, and 2-OCH3), upon photolysis, rearranged primarily to esters (e.g., 6, eq 2), whereas electronwithdrawing substituents led exclusively to reduction product 4-methoxyacetophenone (2e). Few other reports relevant to the photochemistryof 4-methoxyphenacyl derivatives appeared until
'Current address: Dow Chemical Company, Freeport, TX 77541. t Current address: Merck Research Laboratories,West Point, PA 19486. i Current address: Department of Chemistry,Nankai University, Tianjin, Peoples Republic of China. (1) (a) Givens,R. S.;Matuszewski, B. J. Am. Chem.Soc. 1984,106,6860. (b) Givens, R. S.;Matuszewski, B.; Athey, P. S.;Stoner, R. M. J. Am. Chem. Soc. 1990,112,6016. (c) Givens, R. S.; Singh, R. Tetruhedron Leu. 1991, 48,701 3. (d) A preliminaryaccount has appeared;see: Givens, R. S.;Athey, P. S.;Matuszewski,B.; Kueper, L. W., III;Xue, J.-y. J. Am. Chem.Soc. 1992,
(4) (a) Engels,J.;Schlaeger,E.J. Med. Chem.1977,20,907. (b)Nerbonne, J. M.; Richard, S.;NargGt, J.; Lester, H. A. Nurure 1984, 310, 74. (c) Nargeot, J.; Nerbonne, J. M.;Engels, J.; Lester, H. A. Proc. Nut/. Acud. Sci. U S A . 1983, 80, 2395. (d) Kaplan, J. H.; Forbush, B.; Hoffman, J. F. Biochemistry, 1978, 17, 1929. (e) McCray, J. A.; Trentham, D. R.; Reid, G. P.; Walker, J. W. J. Am. Chem. Soc. 1988,110,7170. ( f ) McCray, J. A.; Trentham, D.R. Annu. Rev. Biophys. Biophys. Chem. 1989,18,239. (8) Corrie, J. E. T.; Trentham, D. R. Caged Nucleotides and Neurotransmitters. In Bioorganic Photochemistry;Morrison, H., Ed.; Wiley: New York, 1993;
(a) Pincock, J. A.; Arnold, B.; Jurgens, A. Can. J. Chem. 1985, 63, 3140. (b)Arrs, J.; Braddily, J.; Buchanan, J.G.;Thain, E. M. J. Chem.Soc., Chem. Commun. 1956, 4968. (c) Baldwin, J. E.; McConnaughie, A. W.; Moloney, M. G.; Pratt, A. J.; Shim, S . B. Tetruhedron1990,46,6879-6884. (3) Givens, R. S.; Kueper, L. W., 111. Chem. Reu. 1993, 93, 55-66.
(5) (a) Sheehan, J. C.; Wilson, R. M. J. Am. Chem. Soc. 1964,86,5277. (b) Sheehan, J. C.; Wilson, R. M.; Oxford, A. W. J. Am. Chem. Soc. 1971,
0
la: R = R = CHzCH3 b: R = CH(CH3)2 ; R = H or Na' c: R = R'= H or Na' d: a-D,R= R' = CHzCH3
114,8708. (2)
OCH, 2a
Vol. 2.
93, 7222. (6) Sheehan, J. C.; Umezawa, K. J. Org. Chem. 1973,38, 3771. (7) Anderson, J. D.; Reese, C. B. Tetrahedron Lett. 1962, 1.
OOO2-7863/93/1515-6001$04.00/00 1993 American Chemical Society
Givens et al.
6002 J. Am. Chem. Soc., Vol. 115, No.14, 1993 0
0
2e: X = H
2a: X = 02P(OEt),
b: X=C1 c: X =02CR d: X = OP03H2
CH30
+Hx 69 R = CH3 b R = C(CHh
Sheehan et al.5 demonstrated their usefulness as photoprotecting groups for simplecarboxylicacid derivatives. In a comprehensive study, a series of phenacyl-protectedesters was shown to liberate the free acid and 4-methoxyacetophenone (2e) as exclusive photoproducts in either ethanol or dioxane. Concurrent with our earlier studies on la+, Epstein et a1.8 reported the photochemistry of diethyl 4-methoxyphenacyl phosphate (2a) in dioxane. In accord with Sheehan's studies? Epstein reported that 4-methoxyacetophenone was the exclusive product which resulted from the homolysis of the a-carbon-phosphate bond followed by hydrogen abstraction from the solvent by the a-keto radical, paralleling the photochemistryof the corresponding carboxylate esters. Epstein further suggested that the 4-methoxyphenacyl group might serve as a photoprotecting group for nucleotides. Baldwin et a1.k reported that 4-methoxyphenacyl dihydrogen phosphate (2d) also gave 4-methoxyacetophenone as the only ketone product. However, when compared with the benzoin and 2-nitrobenzyl dihydrogen phosphates, he found that 2d was the least efficient in the release of inorganic phosphate (Pi). Thesestudiesare the result of an intense interest by mechanistic biochemists in the general design and application of photoprotecting groups for phosphates, a subject of a series of recent reviews by Corrie and Trentham,4'9 Lester et al.? and Givens and K ~ e p e r . ~ These reviews provide a detailed analysis of the parameters which influence the photochemistry of protecting or cage groups including the irradiation conditions, solvents, and structure. According to L e ~ t e r ,ideal ~ cage groups should possess the following five properties: (1) good solubility in water at high ionic strength, (2) rapid and efficient photoreactivity, (3) good thermal and solvolytic stability, (4) high absorptivity at wavelengthsgreater than 310nm,and(5) biologicalstability (inertness) of the released protection group. One of the few functional groups that meets most of the criteria is the o-nitro-a-phenylethyl group (e.g., 7 , eq 3).4 Upon irradiation, caged o-nitro-a-phenylethyl
group is the slow release of the ligand from a relatively long-lived aci-nitro intermediate. For o-nitro-a-phenylethylphosphates (eq 3), the rate of release of the phosphate is limited by the rate of hydrolysis ofthe enol ether formed upon photolysis (e.g.,release of ATP from o-nitro-a-phenylethyl ATP occurs through acinitro intermediate 8; the intermediate decays with a rate of only 86 s-1 at pH 7.1).* A second major disadvantage derives from the highly absorbing products that often interfere with spectroscopic analysis of the reactions of the released nucleotide and the extent of conversion of the caged nucleotide. Finally, a-nitrosoacetophenone is not biochemically benign (a violation of Lester's fifth rule) and may interfere with the normal biochemical functions under investigation.
RMulb
Synthesband ExploratoryPhotochemistry. Desyl Phosphates. Desyl diethyl phosphate (diethyl 2-oxo- 1,2-diphenylethyl phosphate, la) was synthesized by modification of the known procedures for esterification of diethyl phosphorochloridate with the appropriate alcohol.10 Irradiation of la at 300 nm in acetonitrile, methanol, or benzene resulted in the formation of 2-phenylbenzo[b]furan (3) as a primary product as established by HPLC analysis, isolation, and spectroscopic identification (eq 4). The time-dependent disappearanceof l a was not significantly 0
0
PhV P O R Ph OR' la: R = R = CH2CH3 e: R = R = H or Na'
8'
derivatives of phosphates, amides, carboxylic esters, and ethers usually release the corresponding nucleofuge in good yield^.^ However, a well-known limitation of the o-nitro-a-phenylethyl (8) (a) Epstein, W. M.; Garrossian, M. J. Chem. Soc., Chcm. Commun. 1987,532. (b) Baldwin, J. E.; McConnaughie,A. W.; Moloney, M. G.;Ratt, A. J.; Shim, S. B. Tefruhedron 1990, 19, 6879. (9) (a) Gurney, A. M.; Lester, H. A. Physiol. Reo. 1987, 67, 583. (b) Lester, H. A.; Nerbonne, J. M. Annu. Reu. Biophys. Bloeng. 1982.11, 15 1.
HOPOR OR'
+ 1
9a: R = R = Et 9 b R = CH(Me)Z; R' = H or Na' 9c: R = R = H or Na'
influenced by the nature of the solvent. At low conversions (72 >60
6b
13
13
a
S-T = singlet-triplet crossing.
13
50
duction product 2e. Comparison of the relative efficiencies for irradiation of 2a in methanol-O-d with those in methanol revealed a small isotope effect of 1.3 and 1.8 for formation of 2e and 6a, respectively (Table IV). However, when the irradiation was carried out in methanold4, a large solvent isotope effect of 5.4 was found for the appearance of Ze,whereas the isotope effect for 6a remained essentially unchanged at 1.4. Finally, the quantum efficiencies for the appearance of 3 and cAMP (12) were determined by HPLC analysis in the same manner as described for the analysis of l b and IC and by 31P N M R analysis. The quantum efficiencies for the disappearance of 13 and the appearance of 12 were nearly identical, whereas the measured appearance efficiency of 3was usually much lower, primarily due to the low solubility of 3 (Table V). A standard SV quenching experiment of 13 with sodium 2-naphthalenesulfonate gave a triplet lifetime of 0.52 f 0.1 ns using a rate constant for diffusion2’ of 4.4 X 101OM-ls-l for the binary solvent of 1:1 aqueous buffer and dioxane. The stability of desyl cAMP (13) in aqueous solutions at pH 1.8,7.2, and 8.4 was established through half-life determinations (Table VI). In basic media, 13 is unstable and undergoes hydrolysis of the cyclic phosphate forming desyl adenosine 3’- or 5’-monopho~phate.2~At neutral and low pHs, the ester is stable to hydrolysis within the time frame of the photochemical experiments. The phosphorescence spectrum of 13 indicated that the lowest triplet-state energy was 73 f 1 kcal/mol, in agreement with the other desyl mono-, di-, and triphosphates reported in this study.
Discussion Our recent studieda+ of the photochemistry of arylmethyl phosphates and our earlier report of the photochemistry of &n (23) Corriec has suggested that neighboring group participation by the day1carbonylmay enhance the rate of hydrolysis of a-keto phosphates under mildly basic conditions.
a-keto phosphate” have shown that these reactions occur by a photochemically induced dissociation through ionic intermediates. This was first established for benzylic and arylmethyl phosphatesI8-c and is now extended to the desylld and 4-methoxyphenacyl7 phosphates reported here. As reported earlier, our studies of desyl phosphate derivatives have demonstrated a recurring pattern of reactivity in which the desyl moiety is converted to 2-phenylbenzo[b]furan (3) irrespective of the solvent or the structure of the desyl phosphate ester.la.d The release of the phosphate with its attendant ligands is also independent of the solvent. The overall process is highly efficient and emanates from the triplet state of the desyl group. While the mechanistic details for the cyclization of the desyl moiety are not known, a reasonable pathway would be that shown in Scheme V in which photolysis initially generates a desyl cationphosphate anion pair. This is in accord with the mechanistic picture we had previously developed from our extensive investigations of benzilic phosphates.1a-c We were quite puzzled, then, when reports appeared* on the photolysis of closely related a-keto phosphates 2a and 2d. EpsteinS8 and Baldwinsb both reported that the fate of the 4-methoxyphenacyl moiety upon photolysis of 4-methoxyphenacyl phosphate esters 2a and 2d was to form a reduction product, 4-methoxyacetophenone (b). No rearrangement products of the type reported for other phenacyl derivatives’ were indicated. 4-Methoxyphenacyl Phosphate Photochemistry. 4-Methoxyphenacyl phosphate 2a was reexamined in a search for other reaction products indicative of a carbocation intermediate. In fact, a relatively efficient carbocation rearrangement of 2a to tert-butyl4-methoxyphenylacetatewas discovered to occur for irradiations in tert-butyl alcohol/benzene, a poor hydrogen-atomdonating medium (eq 2). Photoreduction product 2e was also found, albeit as the minor product. Finally, a small amount of a-tert-butoxy-p-methoxyacetophenonewas observed and is most probably derived from a competing ground-state solvolysisprocess. The reaction was quenched with naphthalene, consistent with the
6006 J . Am. Chem. Sa.,Vol. 115, No. 14, 1993
Givens et al.
Scheme N. Mechanism for the Reduction Product from 2a in MeOH or Dioxanea 3
Et)2
hv hv
--
S-T
@a 2a
H
0
(OEt),
2c
S-T = singlet-triplet crossing.
is comparable to the corresponding rate constant for intramolecular hydrogen abstraction by other 4-methoxy-substituted phenone triplets (Le., p-methoxyvalerophenone, k, = 7.7 X 105 s-l) in methan01.2~ Wagner,” Yang,25 and Pitts26 have noted that the contribution of thelower lying (~,r*)3 state to the excitedstate reactivity of p-methoxyaryl ketones renders it much less reactive, more than an order of magnitude lower than a pure n,# triplet, in hydrogen abstraction reactions. In our case, however, thelower hydrogenabstractionreactivity isoffset by thecompeting rearrangement photochemistry, even in methanol. The formation of 4-methoxyacetophenone (k) in tert-butyl alcohol/benzene is more difficult to accord with this mechanism. One possibility is a competing, minor direct homolysis of the C - O phosphate bond to form a triplet radical pair which could abstract a hydrogen atom from the solvent or any other adventitious hydrogen donor to provide 2e. In the absence of any hydrogen donor, recombination or coupling reactions might compete but were not investigated. Clearly, the dominant process in both polar solvents examined here is the rearrangement to 4methoxyphenylacetate which occurs in methanol with a rate constant of 5.1 X 105 s-1, as determined from the quenching data. The acetophenonephenylacetate rearrangement has excellent literature precedence in a number of solvolytic studies of 8-phenethyld e r i ~ a t i v e s(e.g., ~~ 14, eq 9) and in a report of related photochemical reactions of
qxsoH -1 Q 1 SOH +0CH3
Scheme V. Carbene Route to the Formation of 2-Phenylbenzo[b]furan (3)o
R=OCH,. X Z O T S
/
14, R = OCH3, OH,CI X = OTs, OBs, Br
L OPO3Et2
h,d L = H,D
I - LOPOsEq?
3 a
(9)
os
S-T = singlet-triplet crossing.
triplet-state assignment determined for the analogous desyl derivative. Thus, our results contrast with those reported for photolysis of 2a in p-dioxane, a good hydrogen-atom-donating solvent, where photoreductionis apparently the major or possibly the exclusive process.* We also reexamined the photoreaction of 2a in methanol, an alternative to p-dioxane as a good hydrogen-atom source. Here, again, we found that photoreduction competed with the rearrangement but the process remained a minor one (Table IV). The quantum efficiencies for b a n d 6 in methanol were an order of magnitude higher than in tert-butyl alcohol/benzene, due in part to the combination of lower polarity and poorer hydrogenatom-donating power of the tert-butyl alcohol/benzene solvent. From the quenching data, a pseudo-first-order rate constant of 2.1 X 10s s-1 was calculated for the formation of 2e by an intermolecular hydrogen-atom abstraction from methanol, which
a-phenacyl chlorides7 (e.g., 2b). Anderson and Reese7 observed that 2- and 4-methoxyphenacyl chlorides gave appreciable yields of ethyl 2- and 4-methoxyphenylacetates when irradiated in ethanol. These examples of neighboring group participation in a photochemical reaction also exhibited the same sensitivity to substituent effects on migratory aptitude of the aryl group, paralleling that in the ground-state reactions.29 While no mechanistic studies are available for phenacyl chlorides, both radical and ionic intermediates have been suggested. Evidence for radical intermediates has been reported for homolysis of a-carbonyl photolysis reactions in some related bicyclic a-chloro @,yenoneswhere participation of the carboncarbon double bond is observed. For the endo isomer, where back-side r-participation is stereoelectronically not feasible, evidence for radical intermediates was forthcoming from photolysis in the presence of iodide as a chlorine-atom-trappingagent. The observation of 12- indicated a successful trap.29 Exo isomer 15, which produced no 12-, gave a single, major rearrangement product resulting from r-bond participation (eq 10). A general mechanistic picture for the rearrangement of a-keto phosphates is shown in Scheme 111. The origin of the reduction product in methanol and dioxane must be derived from a second pathway, either (1) hydrogen(24)(a) Wagner, P.J.; Kemppainen, A. E.; Schott, H. N. J. Am. Chem. SOC.1973,95,5604.(b) Wagner, P.J.; Kamppainen, A. E. J. Am. Chem. SOC.1968.90, saga. (25)Yang, N . C.; Dusenbery, R. L. J. Am. Chem. Soc. 1968,90,5899. (26)Pitts, J. N.; Johnson, H. W.; Kuwana, T. J. Phys. Chem. 1%2,66, 2456. (27)Streitwieser, A. Soluolytic Displacement Reactions; Mcgraw Hill: New York, 1962. (28)(a) Cram, D.J. J. Am. Chem. Soc. 1949,71,3863;1952,74,2129; 1964,86,3767.(b) Olah, G. A.; Singh, E.P.J. Am. Chem. Soc. 1984,106, 3265. (c) Fornorini, S.;Muraglia, V. J. Am. Chem. SOC.1989,111, 873. (29) (a) Givens, R. S.; Strekowski, L.; Devonshire, R. L. J. Am. Chem. Soc. 1974,Sa, 1637. (b) Givens. R. S.: Strekowski, L. . I Am. . Chem. Soc. im,97,5867.
a-Keto Phosphates as a Photoprotecting Group
J. Am. Chem. SOC., Vol. 115, No. 14, 1993 6007
Scheme M. Ion Pair Route to 2-Phenylbenzo[b]furana
15-endo
15-exo
55% ls,d
L = H,D
atom abstraction from the solvent by a carbonyl *,A* (mixed with n,?r*) triplet followed by homolysis of the carbon-oxygen bond of the phosphate or (2)the reverse order of these two steps in which homolysis precedes hydrogen-atom abstraction. The primary isotope effect of 5.4 for the photoreduction process which is observed only in methanol-dd and the absence of an isotope effect on the rearrangementprocess coupled with the lower overall efficiency for ketone disappearance in tert- butylalcohol/benzene suggests that the timing for the hydrogen abstraction is prior to C-0 bond rupture whereas the photosolvolysis occurs directly from the triplet excited state. These results require two separate mechanisms for rearrangement and reduction which are outlined in Schemes I11 and IV, respectively. Desyl Phosphates Photochemistry. While the acetophenone phosphate provided an interesting model for ‘caged” chromophores, we chose to pursue on our earlier investigation of the desyl derivatives. Our intent was to take advantage of the higher efficiency of the desyl phosphates, their simplified photoproduct mixture, and the minimal solvent effects on both the efficiency and the product mixture.’* An important consideration in the development of the desyl group as a useful cage was also the ease of synthesis of mixed esters containing the benzoin ligand. A possible entry to desyl esters was envisaged through adapting Ramirez and Marecek’sls synthesisof acetoin phosphates toour target esters. As illustrated in Scheme I, benzil was readily converted into diphenyl phosphorochloridate l l c in greater than 90%overall yield. The entire sequence was run without isolation or purification of intermediate dioxaphosphole. As shown earlier by Ramirez and Marecek,ls phosphorohloridates react sequentially with a variety of alcohols to yield mixed di- or triesters of acetoin phosphate, each addition requiring a progressively stronger base to effect the transformation. In the Ramirez strategy, the acetoin ligand is the removable protecting group, excised last by hydrolysis. Our strategy differs only m the activation requirement of the final step, namely, removal of the desyl group by photoactivated heterolysis. The advantages of this strategy derive both from the specificity of incorporating individual ligands sequentially and in the direct generation of the photolabile desyl (Le,, a-keto) ester. Furthermore, the sequence is economical because only 1 molar equiu of alcohol per ligand is required. Scheme I1 provides two examples, a simple, mixed isopropyl desyl phosphate diester (lb) and an unsubstituted desyl phosphate monoester (IC) which were synthesized using the dioxaphosphole strategy. Other synthetic strategies were also employed for mixed desyl esters, such as the reaction of diethyl phosphorochloridateswith benzoin.10 We were also able to effect esterification with an S N ~ strategy by reacting the tri-n-butylammonium phosphate salt of CAMP in NJV-dimethylacetamide (DMA) with desyl bromide (eq 7). The yields from this reaction are significantly lower (1 520%) than the other routes noted above but are similar to those obtained with other caged derivatization reactionsakf Fortunately, the nucleotide can be recycled to increase the product yield by this route. The photolysis of triester 2a in methanol, benzene, and acetonitrile gave excellent yields of 2-phenylbenzo[b]furan (3) and diethyl phosphate (9a) with an efficiency of nearly 30%.la.b Likewise, the salts of water-soluble esters l b and ICgave just two products in aqueous acetonitrile but at reduced efficiencies (eq 4 and Table 111). In fact, the reaction was shown to be pH sensitive, occurring most efficiently at pH
