Radical-Induced Dephosphorylation of Fructose Phosphates in Aqueous Solution+

Henryk Zegota++ and Clemens von Sonntag* Max-Planck-Institut für Strahlenchemie, Stiftstr. 34-36, D-4330 Mülheim 1 Z. Naturforsch. 36b, 1331-1337 (1981); received May 29, 1981 D-Fructo8e-l-phosphate, D-Fructose-6-phosphate, y-Irradiation, Radical Reactions, Mass Spectrometry Oxygen free N20-saturated aqueous solutions of D-fructose-1-phosphate and D- fructose-6-phosphate were y-irradiated. Inorganic phosphate and phosphate free sugars (containing four to six carbon atoms) were identified and their G-values measured. D-Fructose-1-phosphate yields (G-values in parentheses) inorganic phosphate (1.6), hexos-2-ulose (0.12), 6-deoxy-2,5-hexodiulose (0.16), tetrulose (0.05) and 3-deoxytetrulose (0.15). D-Fructose-6-phosphate yields inorganic phosphate (1.7), hexos-5-ulose (0.1), 6-deoxy-2,5-hexodiulose (0.36), 3-deoxy-2,5-hexodiulose and 2-deoxyhexos-5-ulose (together 0.18). On treatment with alkaline phosphatase further deoxy sugars were re- cognized and in fructose-1-phosphate G(6-deoxy-2,5-hexodiulose) was increased to a G-value of 0.4. Dephosphorylation is considered to occur mainly after OH attack at C-5 and C-l in fructose-1-phosphate and at C-5 and C-6 in fructose-6-phosphate. Reaction mechanisms are discussed.

Introduction react with the solute molecules by abstracting The radiation chemistry of sugar phosphates in carbon-bound hydrogen atoms. Because of the high aqueous solutions has found already considerable reactivity of the OH radicals hydrogen abstraction attention [1-10]. Most of the work has been carried occurs nearly at random [13]. Therefore, D-fructose- out with D-ribose-5-phosphate. The same processes 1-phosphate (1) and D-fructose-6-phosphate (2) will that govern phosphate elimination in D-ribose-5- H0-CH2 (P)-O-CH2 phosphate have been shown also to operate in DNA where they lead to strand breaks [11, 12]. Y-TCH2-0-® YYCH20H In 10-2 M aqueous solutions of the sugar phos- OH OH phates, practically all the radiation energy is D-Fructose-1-phosphate D-Fructose-6-phosphate absorbed by the solvent water. In the radiolysis of water the following species are formed (reaction (1)): 1 2 both give rise to a set of five different primary H20 -y OH, H, eaq-, H202, H30+, OH" (1) radicals. Some of these radicals will eliminate in-

In the presence of N20 the hydrated electrons (eaq-) organic phosphate in the subsequent free radical are converted into OH radicals (reaction (2)): reactions. In the present paper resulting phosphate free products (and some products retaining the eaq" + N20 -> OH + OH- + N2 (2) phosphate group) will be presented and mechanistic The OH radicals and H atoms (G(OH + H) - 6)** aspects will be discussed.

Results * Reprints requests to Prof. Dr. C. von Sonntag. Dilute solutions (10-2 M, pH 8.2) of the sugar 0340/5087/81/1000-1331/$ 01.00/0 phosphates were saturated with oxygen-free N20 + Part XX of the Series "Radiation Chemistry of for 45 min and y-irradiated at a dose rate of Carbohydrates". For part XIX see M. N. Schuch- mann and C. von Sonntag, Int. J. Radiat. Biol. 34, 3 X lOis eV • g-1 • h-i at doses of 1.2 x 1019 eV • g-1 399 (1978). and 2.4 X 1019 eV • g-1 at room temperature. On ++ Permanent address: Institute of Applied Radiation irradiation the pH drops by one unit. The phosphate Chemistry, Technical University, Lodz, Poland free carbohydrate products were either reduced

with NaBD4 and trimethylsilylated (TMS) [14] or Table I. Products and their G values from the radiolysis of fructose-phosphates in aqueous solutions saturated with oxygen-free N2O.

No. Product D-Fructose-1-phosphate (1) D-Fructose-6-phosphate (2)

3 Inorganic phosphate3 1.6 1.7 4 D-ara&irio-Hexos-2-ulose 0.12 — 5 D-Z?/a:o-Hexos-5-ulose — 0.10 6 6 -Deoxy -D -fÄreo - 2,5 -hexodiulose 0.16 0.40b 0.36 0.39b 7 3-Deoxy-2,5-hexodiulose — \ 0 18 0 36b-c 8 2-Deoxyhexos-5-ulose 0.01° ^ J 9 4-Deoxy-D -^rZi/cero-2,3-hexodiulose J * — 10 D-Arabinonic acid <0.01 — 11 D-grfycero-Tetrulose 0.05 — 0.0 lb 12 3 -Deoxytetrulose 0.15 0.03 a From ref. [10]; b After treatment with alkaline phosphatase; c Deoxy compoimds with the deoxy group in the middle of the molecule. converted into the methoxime-TMS derivative isomer described earlier [18, 20]. The precursor is [9, 15, 16]. Identification was by GC-MS. For the 3-deoxy-tetrulose (12). interpretation of the mass spectra of the deoxy After NaBD4 reduction the threitol and erythritol compounds and lactones the NaBD4-reduced TMS- TMS ethers show identical mass spectra with typical derivatives are most suitable [14] whereas osuloses fragment ions at m/e (%) 73(100), 103(20), 104(11), and diuloses are often better characterized by the 117(15), 118(9), 130(4), 133(4), 147(31), 205(15), methoxime-TMS derivative [16]. 206(14), 218(15), 232(d), 308(4). Quantitative measurements were done by GC 103 308 206 205 308 103 using 2-deoxy-D-en/iAro-pentitol or erythritol as (218) (218) internal standards and appropriate response factors CH2OTMS-CDOTMS-CHOTMS-CH2OTMS [9]. Some results were also obtained by treating the The precursor is D-grZycero-tetrulose (11). irradiated samples with alkaline phosphatase prior The material from the arabinitol TMS ether peak to derivation. Only the carbohydrates with six and shows the fragment ions at m/e (%) 73(100) 103(29), less carbon atoms, but not the dimer fraction, have 105(11), 147(20), 205(9), 207(5), 217(8), 219(7), been analyzed. The results are compiled in Table I. 307(3), 309(4), 319(2), 321(2). 105 409 207 307 309 205 411 103 D-Fructose-1-phosphate (319) (217) (219) (321) CD2OTMS-CHOTMS-CHOTMS-CHOTMS-CH2OTMS The major phosphate free product is 6-deoxy-D- £Areo-2,5-hexodiulose (6). Reference material was The precursor is arabinonic acid (10). available [17]. The other major product is D-arabino- After NaBD4 reduction, low yields of deoxyhexos-2-ulose (glucosone) (4). It was identified as hexitols with the deoxygroup in the middle of the its methoxime-TMS derivative. Reference material molecule were also observed but have not been was available [16]. characterized. On treatment with alkaline phosphatase G(6- Very early in the gas chromatogram of a NaBD4 deoxy-D-ZAreo-2,5-hexodiulose) increases and deoxy reduced and trimethylsilylated sample appears a compounds with the deoxy group in the middle of compound showing typical fragment ions (intensities the molecule become more noticible and two more im parentheses) at m/e (%) 73(100), 103(92), 104(30), compounds have been identified: 130(11), 147(27), 190(7), 220(26), 233 (M-90, 1%). The most prominent fragment ions of the first 103 220 103 compound are: m/e (%) 73(100), 103(13), 104(70), CH2OTMS-CDOTMS-CH2-CH2OTMS 130(5), 147(19), 206(5), 218(7), 220(23), 232(9), The mass spectrum is similar to that of its isotopic 308(3), 322(3), 335(1). 104 220 308 322 206 425 103 Discussion (218) (232) (335) CDHOTMS-CH2-CHOTMS-CHOTMS-CDOTMS-CH2OTMS Reactions from the radicals at C-5 The precursor is 2-deoxyhexos-5-ulose (8). It has been shown in a number of cases that The other compound shows typical fragment ions phosphate is readily eliminated if the radical site is at m/e (%) 73(100), 103(9), 131(17), 147(23), 205(12), at ß position to the phosphate group [3, 7, 9, 11, 12, 206(7), 218(3), 232(28), 244(2), 245(3), 335(1). 19-21]. Phosphate elimination (reaction (3)) is 103 425 206 322 205 425 103 expected to occur from radical 13 which is generated (335) (232) (335) by hydrogen abstraction from C-5 of D-fructose-6- (244, 245) (244,245) phosphate. There is good evidence that radical CH2OTMS—CDOTMS-CDOTMS—CH2-CHOTMS-CH20TMS cations (c/. refs. [22, 23]) are intermediates in these The precursor is 4-deoxy-D-grfo/cero-2,3-hexodi- elimination processes. The reaction of the radical ulose (9). cation 14 with water gives rise to radical 15 (reaction (4)). D-Fructose-6-phosphate Again, the most prominent product is 6-deoxy-D- On the basis of results from ESR studies on fAreo-2,5-hexodiulose (6). Its yield is markedly similar radicals [24] radical 15 should be unstable higher than that from fructose-1-phosphate but and readily transform into radical 16 (reaction (5)) does not increase significantly on phosphatase which is reduced in a disproportionation reaction by treatment. Two further deoxy compounds have other radicals (-RH) yielding 6-deoxy-D-*Areo-2,5- been identified: 2-deoxyhexos-5-ulose (8), (described hexodiulose (6) (reaction (6)). above) and 3-deoxy-2,5-hexodiulose (7). The latter is characterized by the mass spectrum of the TMS ®-0-CH2 ®CH2 ethers of a NaBD-i-reduced sample. Typical fragment -®-0" I| OH ions are mje (%) 73(100), 103(9), 130(12), 131(18), •ÖT JQi (3) 147(25), 206(15), 218(5), 232(35), 244(2), 245(4), OH OH 308(12), 335(2). 13 14 103 425 206 322 308 206 103 (335) (232) (218) (244,245) (217) *CH2 H20 I OH CH2OTMS-CDOTMS-CHOTMS-CH2-CDOTMS-CH2OTMS 14 Tj* » <« H On phosphatase treatment, the yields of the deoxy HO ] "CH20H compounds carrying the deoxy group in the middle OH of the molecule is doubled. GC-analysis revealed 15 five peaks already present prior to phosphatase *CH2 treatment. No MS analysis has been done with this -H,0 fraction. It is believed that 4-deoxy-2,3-hexodiulose 15 (5) (9) might also be present in this fraction (see Discus- 0d V^ ^VC ~CH20H OH sion). This compound would not give rise to new GC peaks. 16 In the fraction of phosphate free products after CH3 NaBD4-TMS treatment mannitol and glucitol-TMS RH, -R ethers give rise to identical mass spectra with 16 (6) typical fragment ions at mje (%) 73(100), 103(15), CAj_^ch2OH OH 104(13), 117(5), 147(19), 206(21), 218(9), 308(6), 320(16), 332(1). 104 206 410 308 308 410 206 103 (320) (218) (218) (320) In D-fructose-1-phosphate the corresponding CHDCTMS-CHOTMS-CHOTMS-CHOTMS-CDOTM8-CH2OTMS radical at C-5, 17 will lead to the same product Their common precursor is D-Zyxo-hexos-5-ulose however retaining the phosphate group at C-l. The (5), 3-Deoxytetrulose (12, see above) has also been elimination of the OH group at C-6 of radical 17 found. (reaction (7)) is a process which has its analogs in *CH2 q •h2o 18 (7) HO-CH2 CH2-O-(P) i yo^ Oc H OH u® HO-CH2 OH 19 (8) 17 1— CH2-O-(P) OH

I J 0 18 CH2-O-(P) 20 (9) OH

phosphatase 20 (10)

HO-CH2

19 (11)

OH the reactions of similar radicals [25, 26] and the to be well established in the light of results from subsequent reactions (reaction (9)) will be analogous DNA [11]. The subsequent water elimination reac- to those discussed above (reactions (4-6)). tions (13) and (14) have their analogs in reactions In competition to reaction (7) one would except a observed with other sugars [9, 30-32]. The driving rearrangement of radical 17 to radical 19 (reac- force of these reactions may be the gain in energy by tion (8)), a process which is well established [25, the formation of an allyl radical and water. An allyl 27-29]. The rearranged radical 19 is of the same radical is also formed by loss of water from the typ as radical 13, rapidly eliminates phosphate and

b in a reaction sequence similar to reactions 4-6 ch2 ho-ch2 i /cl oh h,0 |/0nph ultimately ends up as 6 (reactions (11)). hc (12) As is shown in Table I, G (6) from D-fructose- ch20h ch20h oh 6-phosphate (2) is not affected by phosphatase 14 21 treatment but G (6) from D-fructose-1 -phosphate ho-ch2 (1) strongly increases on phosphatase treatment 0H 22 (13) indicating the presence of 20. It is also noted that n -h20 o-^ch2OH the combined yields of 6 as expressed by the phos- 21 ho phatase treated samples are equal for both 1 and 2. ho-ch2 This is to be expected because the OH radicals will & 23 (11) >-^ch2oh attack the 5-position in both cases with similar ho probabilities. The low yield of 6 (G(6) = 0.4) does not entirely reflect the OH attack at this position ho-ch2 ho-ch2 •Rh i ^ oh because of side reactions, e.g. dimer formation [9]. 22 c15u 16) h2oh h2oh Without phosphatase treatment 3-deoxy-D- oh oh <7Zt/cero-2,5-hexodiulose (7) is formed from D-fruc- 24 tose-6-phosphate (see Table I). This product may ho-ch2 ho-ch2 •rh i ^ oh also have the radical at C-4 as its precursor. Reac- 23 _ U° ji (17. 16) tions (12)—(18) present a scheme according to which >—-4h2oh )—/\h2oh 10 oh product 7 might be formed. Reactions (12) appears 2,3-dihydroxypropyl radical as shown by ESR Reactions from other radical sites spectroscopy [33]. The disproportionation reactions 4-Deoxy-D-<7Zycero-2,3-hexodiulose (9) is only (15) and (17) yield enol ethers. In a previous study observed after phosphatase treatment, i.e. it is on D-ribose-5-phosphate [9] such intermediates formed as its 1-phosphate in the case of fructose-1- have been characterized by deuterium labelling phosphate (1) and as its 6-phosphate in the case of experiments. Similar experiments have not been fructose-6-phosphate (2). It is a very much expected carried out in the present study and conclusions product which arises from the well documented with respect to the enol ether intermediates are water elimination of 1,2-dihydroxyalkyl radicals drawn by analogy. [25, 34] followed by the reduction of the acylalkyl radical by other radicals ('RH) present (cf. reac- Reactions from the radicals at C-l in 1 tions (24) and (25)). Most likely the isomers of 9 and at C-6 in 2 carrying the deoxy function at C-3 and the carbonyl Another dephosphorylation process is the oxida- functions at C-2 and C-4 are also formed but their tion of the radicals a to the phosphate group (26 polyhydric alcohol derivatives were not sufficiently and 27) [9]. Other radicals or radiolytically formed separated by GC from other isomers and no mass H2O2 may serve as oxidants. In general, such a spectrum could taken from an isolated stereoisomer. reaction is more efficient in the presence of good oxidizing agents, e.g. Fe3+ ions [9]. Carbonium ions Carbon-carbon bond scission are likely intermediates. There is some material with less than six carbon The product 2-deoxyhexos-5-ulose (8) observed atoms, especially erythrulose and 3-deoxytetrulose in the radiolysis of fructose-6-phosphate might also from D-fructose-l-phosphate. have the radical at C-6 as a precursor. A process Carbon-carbon bond fragmentation can occur similar to that formulated for 7 might be considered after H abstraction only by a /^-fragmentation (reactions (21-23)). The intermediate 28 could also process. For these two compounds one might con- be reached by starting from the radical at C-4. sider radical 32 as the precursor (reaction (26-29)).

HO-CH2 H0-CH2 CL OH oxidation •CL OH HO"] ho; (19)

.CH-O-® H20, -HO-(P) CHO OH OH 26

27 (P)-0-CH

27 + H2O (21) CH20H OH 28

CHO RH ^©-O-CH 0 H,0 ^ I 28 C. (22, 23) -R hA^ lAcH20H -(p)-0H CH20H ®-O-CH2 ®-0-CH2 ®-0-CH2 OH -HOO OH -RH (24. 25) -R l FCH20H :—ifcH2°H t CHch22 0H OH C£

30 31 9 -®

HO-CH2 HO-CH, LO OH . koSf (26) V^CH2-O-® V CH2-0-® OH OH

32 33

33 -> ^[HOCHa-CHOH-CO-CHaOH + servation that 10 is found with 1 but with 2 only 11 after phosphatase treatment. OO-CHaOP (27) Material balance /^-fragmentation In the present study only the disproportionation 3 3 products of the radicals have been identified, but HOCH2-CH-CO-CH2OH -f POCH2COOH not the combination products. For this reason no 34 (28) complete material balance can be reached. This can •RH,-R 3 4 v CH2OH-CH2-CO-CH2OH (29) only be achieved if dimerization processes are 12 inhibited, e.g. by O2 [6, 13]. Olefinic intermediates such as enol ethers etc. further reduces the product The C-4 radical from the isomer, D-fructose-6-phos- yields by scavenging radicals. Evidence for this has phate, might react in a different reaction path, recently been obtained from a study on the photo- eliminating phosphoric acid whereby forming an lysis of D-fructose in aqueous solutions [32]. The allyl radical. Such a type of reaction is observed importance of the sum of the phosphate eliminating with 2'-deoxy-5'-cytidylic acid [35]. A possible processes is therefore reflected by G( (inorganic phos- product is 6-deoxy-2,4,5-hexotriulose which might phate) which are around 1.6 for both 1 and 2. This have escaped detection, 6-deoxy-D-iÄreo-2,5-diulose indicates that more than one quarter of the radicals (6) being such a prominent product. Both would generated eliminate phosphate or their reactions give on NaBÜ4 reduction the same polyhydric lead to products with highly labile phosphate. alcohols albeit the former with one more D atom incorporated. If the 6-deoxy-2,4,6-hexotriulose Experimental would be only a minor contributor, it would not be D-Fructose-1-phosphate, Ba salt A grade (Calbio- easily recognisable in the mass spectrum. There is a chem), D-fructose-6-phosphate, Ca salt (Merck) and very small probability that OH radical abstract alkaline phosphatase in suspension (1 mg/1 ml) oxygen bound H atoms from carbohydrates [36]. (Boehringer) were commercially available. Irradia- tions and work up were as described previously Therefore it has to be expected that the yield of oxyl [9, 25]. radicals which we known to fragment quite readily [34] is quite small. The likely precursor of D-arabi- One of us, H. Z., would like to thank the Max- nonic acid (10) is the oxyl radical at 0-2 which frag- Planck-Gesellschaft for a stipend. We are very grateful for the help by the staff of the institutes ments by splitting the bond between C-l and C-2. GC- and MS-departments, especially Ms. U. Rauhut, In accordance with such a mechanism is the ob- Mrs. K. Sageb, Mr. H. Damen, and Mr. M. Scheppat. [1] N. K. Kochetkov, L. I. Kudryashov, M. A. Chle- [18] M. Dizdaroglu, H. Scherz, and C. von Sonntag, nov, and L. P. Grineva, J. 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Naturforsch. 30b, 609 (1975). 2 1981, 143. [7] L. Stelter, C. von Sonntag, and D. Schulte-Froh- [24] G. Behrens, G. Koltzenburg and D. Schulte- linde, Z. Naturforsch. 30b, 656 (1975). Frohlinde, unpublished results. [8] C. L. Greenstock and E. Shierman, Int. J. Radiat. [25] M. Dizdaroglu, D. Henneberg, G. Schomburg, Biol. 28, 1 (1975). and C. von Sonntag, Z. Naturforsch. 30b, 416 [9] L. Stelter, C. von Sonntag, and D. Schulte-Froh- (1975). linde, Int. J. Radiat. Biol. 29, 255 (1976). [26] M. Dizdaroglu, K. Neuwald and C. von Sonntag, [10] H. Zegota and S. Bachman, Proceedings of the Z. Naturforsch. 31b, 227 (1976). 4th Tihany Symposium on Radiation Chemistry, [27] E. S. Huyser, J. Org. Chem. 25, 1820 (1960). 1976, Budapest Akademiai Kiado, p. 827 [28] C. von Sonntag, K. Neuwald, and M. Dizdaroglu, (1977). Radiat. Res. 58, 1 (1974). [11] M. Dizdaroglu, C. von Sonntag, and D. Schulte- [29] M. Dizdaroglu, and C. von Sonntag, Z. Natur - Frohlinde, J. Am. Chem. Soc. 97, 2277 (1975). forsch. 28b, 635 (1973). [12] M. Dizdaroglu, D. Schulte-Frohlinde, and C. von [30] A. G. W. Bradbury and C. von Sonntag, Z. Na- Sonntag, Z. Naturforsch. 30c, 826 (1975). turforsch. 31b, 1274 (1976). [31] M. Dizdaroglu, D. Henneberg, K. Neuwald, [13] M. N. Schuchmann and C. von Sonntag, J. Chem. G. Schomburg, and C. von Sonntag, Z. Natur - Soc. Perkin Trans. 2. 1977, 1958. forsch. 32b, 213 (1977). [14] M. Dizdaroglu, D. Henneberg, and C. von Sonn- [32] C. Triantaphylides, H.-P. Schuchmann and C. tag, Org. Mass Spectrom. 8, 335 (1974). von Sonntag, Carbohydr. Res., in press. [15] R. A. Laine and C. C. Sweeley, Carbohydr. Res. [33] S. Steenken, private communication. 27, 199 (1973). [34] C. von Sonntag, Adv. Carbohydr. Chem. Bio- [16] M. Dizdaroglu, D. Henneberg, C. von Sonntag, chem. 37, 7 (1980). and M. N. Schuchmann, Org. Mass Spectrom. 12, [35] H.-P. Schuchmann, R. Wagner, and C. von Sonn- 772 (1977). tag, unpublished results. [17] M. Dizdaroglu, J. Leitich, and C. von Sonntag, [36] K.-D. Asmus, H. Möckel and A. Henglein, J. Carbohydr. Res. 47, 15 (1976). Phys. Chem. 77, 1218 (1973).