J. Org. Chem., Vol. 58, No. 25, 19’75
!&T\TOR-2-FORMYLPYRIDOXAL 5 ’ - P H O S P H A T E
ZNHCH,), 19625-78-6; 4a (11, = CMea), 21995-77-7; 4a (R, = tert-butyloxycarbonyl-kleucyl), 42222-15-1 ; 4a (R, = carbobenzoxy-kseryl), 42222-16-2; 4a (& = carbobenzoxy-k glutamyl), 42222-17-3; 4a (113 = benzoyl-kleucyl), 42222-19-5; 4a ( 1 1 3 = carbobenzoxyglycylglycyl), 42222-24-2; 4a (R, = phthaloylglycyl), 21995-78-8; 4a (It3 = carbobenzoxy-1, glutaminyl), 42222-17-3; 4b ( I t 3 = ZNHCHZ),42222-01-5; 4b (It3 = ChIe,), 42222-02-6; 4e (11, = ZNHCHz), 42222-03-7; 4f (Ii3 = H ) , 42222-04-8; 4f (113 = CMe,), 42222-05-9; 4f (It3 = Me), 42222-14-0; Sa, 42222-06-0; 5b, 42222-07-1 ; Sf, 42222-08-2; 6a (11, = H), 42222-09-3; 6f (113 = H), 42222-10-6; 7 (11, =
4295
ZNHCHz), 21995-76-6; Sf, 4767-01-5; 11, 42222-13-9; formic acid, 64-18-6; acetic acid, 64-19-7; carbobenzoxy-P-cyanoalanine, 3309-41-9; carbobenzoxytriglycine ethyl ester, 250335-7; carbobenzoxyglycyl-bserine methyl ester, 10239-27-7; phthaloyldiglycirie ethyl ester, 2641-02-3; carbobenzoxy-I, glutaminyl-ttyrosine methyl ester, 42222-23-1; berizylamine, 100-46-9; pivalic acid, 75-989 ; carbobenzoxyglycine, 1138-80-3 ; carbobenzoxy-I,ssparagine, 2304-96-3; benzoyl-hleucine, 146683-7; glycine ethyl ester hydrochloride, 623-33-6; Irserine methyl ester hydrochloride, 5680-80-8; ktyrosine methyl ester hydrochloride, 3417-91-2.
Synthesis of 2-Nor-2-formylpyridoxal 5’-Phosphate, a Bifunctional Reagent Specific for the Cofactor Site in Proteins ANNAPOCKER Department of Riochemistrv, University of Washington, Seattle, Washington 98196 Received June 6,1973 A facile and general procedure was developed for the preparation of vitamin Be amine oxides and their corresponding 2-carbinol rearrangement products. These compounds served as intermediates in the synthesis of the 6’-phosphate, dicarboxaldehyde 2-nor-2-formylpyridoxal 5’-phosphate and of 2-nor-2-formyl-4-deoxypyridoxo1 an isomer of pyridoxal 5’-phosphate. Both analogs were prepared for the purpose of exploring the spacial configuration of polypeptide chains at the pyridoxal j’-phosphate site in glycogen phosphorylase (EC 2.4.1.1) and other enzymes.
The introduction of st,able covalent bridges bet,ween amino acid rrsiduos has been recently utilized as a dircct, chemical t m l to provide informat’ion about the spacial orientat,ion of polypeptide chains in proteins. It occurred to us that this approach, couplcd with a syst,cmat,ic application of affinity labcling techniques, may rovcal tho architecture of important sites on cnzymes and pcrhaps cont ributc to the elucidation of thc catalytic mechanism. 2-Sor-2-formylpyridoxal 5’phosphate (9) and 2-nor-2-formyl-4-dcoxypyridoxol5’phosphatc (4)2 wtrc synthrsizcd as part of a project designed t? probe t,hc t’hrce-dimonsionalstructure around thc pyridoxal 5’-phosphate pocket in glycogen phosphorylase (EC 2.4.1.1)3and a number of othor ctnzymes. Earlier invcstigations indicatod that the absence of the 2-methyl group has little cffcct on thc activity of the c o f a ~ t o r . Consequently, ~~~ an additional funct’ionalit’ywas anchored at this position in the expectation t’hat the enzyme will conserve a high dcgree of specificity for this analog and thus promote the formation of a bisazomethine cross-linkage a t the PLP site. The synthetic pathway, as depicted in Scheme I, is based on an irnprovcd two-step synthesis of 2-pyridine carbinol analogs serving as intermediates for the final products doscribed herein. When m-chloroperbenzoic acid5 is employed in either protic or aprotic solvents in the cold, a smooth N-oxidation of vitamin Be compounds proceeds. Although several syntheses of pyr( 1 ) 1’. Wold, Methods k’nrumol.. 26B,623 (1972). (2) Abbreviations used are as follows: u-hydroxy indicates the introduction of a 2‘-hydroxyl group and 2-nor-2-formyl, the reyilacement of a 2-methyl with a 2-formyl: I’LP. pyridoxal 5’-phosphate; m-CPBA, m chloroperbenzoic acid; P P A , polypliosphoric acid; T H F , tetrahydrofuran: (TI?AhO,trifluoroacetic anhydride. (3) For previous work on this subject see A. Pocker and E . 15. Fischer, Biochemistry, 8 , 5181 (1969); S. Shaltiel, J. L. Iledrick, A. Pocker, and E. H . Fischer, ibid., 8 , 5189 (1969). (4) E. E. Snell, Vitam. Ilorm. (Neiu Y o r k ) , 2 8 , 265 (1970); W. Dowhan, Jr., a n d E. 12. Snell. J. U i o l . Chem., 246, 4629 (1970). ( 5 ) .J. Cymerman Craig and K. K. Purushothaman, J . Ore. Chem., 88, 1721 (1970).
idoxol N-oxide had been publi~hed,~?’ none would seem to equal the general applicability or good yields obtained with this reagent’. Ethanol was frequently used as solvent from which the amine oxidos crystallized out during t’he course of oxygenation. I’rotwtion of a formyl group was effected by hemiacetal formation; ot’herwisc extensive oxidation to the corrcsponding carboxylic acid is the prcfcrrod pat,hway as noticed in the quantitative conversion of pyridoxal 5’phosphate to 4-pyridoxic acid 5’-phosphate. On the other hand, carbinol groups arc: attackcd only on a limited scale and the rclativoly small improvcmcnts in yield do not warrant tho work involved in protection and deprotcction. Thus several compounds in the vitamin BO group were rcadily converted to thc amine oxides with thc cxccption of the 1’12 diethyl acetal, which under a variety of conditions gave rise to low yields and a number of by-product’s. In a second st’ep, an intramolecular rearrangement8 of the amine oxides to the corresponding 2-pyridine carbinols was effected by trifluoroacetic anhydride [(TFA)20],a rcagent which was not previously utilized for such conversions on a preparative scale.Q We found t,his anhydride to be superior to acetic anhydride in that it rcquired mild conditions for acylation and gave rise to fewer by-products. With w-hydroxypyridoxol readily available, attempts were made to oxidize 7c to 8 by activated manganese dioxide. The complex mixture of products thus obtained was fractionated on an ion-exchangc column and was found to include w-hydroxypyridoxal (7a) and 2nor-2-formylpyridoxol as well as other acidic components, but only small amounts of the desired dicarbox(6) T. Sakuragi and F. A. Kummerow, J . Oro. Chem., 24, 1032 (1959). (7) G. R. Bedford. A. R. Katritaky, and 13. M. Wuest. J . Chem. Soc., 4601 (1963). (8) S. Oae, Tetrahedron. 20, 2677 (1966). (9) Trifluoroacetic anhydride \vas previously employed on a preparative scale in a Beckmann rearrangement [see W. 1). Einmons. J . Amer. Chem. Soc., 79, 6522 (1957)l and in mechanistic studies of N-0 bond cleavage of amine oxides [T. Koenig. ibid., 88, 4045 (1966)l.
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J. Oiy. Chem., Vol. 38, N o . 25, 1978
I
0 14
aldehyde. Consequently, 8 was obtained by the selective oxidation of w-hydroxypyridoxal methyl hemiacetal (7d) followed by acid hydrolysis. Phosphorylation of the 5-hydroxymethyl function was effected with l1 polyphosphoric acid by well-established to afford 9. A similar synthesis gave 2-nor-2-formyl-4-deoxypyridoxol 5'-phosphate 4, a cofactor analog possessing a divergent orientation of reactive groups around the pyridine ring. Likewise, the preparation of whydroxypyridoxal j'-phosphate (12), previously obtained via an elaborate procedure, l2 is readily accomplished starting from 7a (unpublished results). This approach constitutes a general route for the conversion of 2mcthylpyridoxol analogs to their 2-formyl derivatives and opcns the way to further modifications of this position. Verification of the structure of the new compounds proceeded in a number of ways. Whereas 3-pyridinols display in general only one absorption band at acid pH, the amine oxides consistently show two strong bands, as illustrated in Table I. Therefore, ultraviolet spectra served as a diagnostic tool to differentiate the (10) E. A. Peterson, H. A. Sober, and A. Meister, Biochsm. Prep.. 8 , 29 (1953). (11) M.Iwanani, T.Numata, and M. Muraknmi, Bull. Chem. SOC.J a p . , 41, 161 (1968). (12) Y . Nakai. F. Nasugi, N. Ohishi, and S. Fukui, Vitamins (Kvoto), 88, 189 (1968).
POCKER
0 15
amine oxides from their precursors as well as from their 2-carbinol derivatives. Moreover, the course of N-oxidation can be conveniently monitored by following the marked hypsochromic shift (ca. 20 nm) of the r-r* transition which occurs in ethanolic solution; the contribution of the reagent m-CPBA is minimal. Aromatic amine oxides are known to possess low acidic dissociation constants and are consequently singled out from all other intermediates by migration in an electrical field and on cationic exchangers. Furthermore, the conversion of the bases to the 2carbinol derivatives was established employing nmr spectroscopy by following the disappearance of the 2methyl proton signals in the cationic species and the appearance of the 2-methylene signals, the latter being clearly distinct from both the 4- or 5-methylene peaks as reported in Table II.13 The presence of the ring proton signal coupled with a positive Gibbs reaction given by all tho intermediates ruled out a pyridone structure. It has been established that both hydration and hemiacetal formation in pyridoxal derivatives increases with the acidity of the solution. Consequently, the aldehydic C-4 proton signal is shifted to a' higher field and appears as a doublet, the result of COUpling to one of the 5-methylene protons. The nonequivalence of the latter is apparent only at higher pH. (13) W. Korytnyk and R . P. Singh, J . Amer. C h m . Soc., 86, 2813 (1963). and references cited therein.
%NOR-2-FORMYLPYRIDOXAL
J. Org. Chem., Vol. 38,N o . 25, 19YS
5'-PHOSPHATE
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TABLE I ULTRAVIOLET ABSORPTIONSPECTRA OF INTERMEDIATES A N D FINAL PRODUCTS -0.1 Compd
Xmx
Pyridoxal N-oxide 4-Deoxypyridoxol AT-oxide (2) Pyridoxol 5'-phosphate N-Oxide (15) w-Hydroxypyridoxol N-oxide
29 1 2.58 28.5 258 293 260 300 262 288
-Phosphate buffer pH 7-
A' H(3 emx
4970 7900 6500 4100 5100 4800 5300 9100 8000
hmbx
Lmsx
--0.1 Amax
V . Na0I-Ierne.*
310 393 317
3600 2400 5900
312a 387 31P
6200 1800 7200
325 258 sh 331
4800 7950 5700
325" 2.iA sh 331Q
6700 7000 7500
305 5400 7300 315 390 700 390 100 303 6600 7400 313 282 8300 w-Hydroxy-Cdeoxypyridoxol (7b) 244 6900 252 3900 303 6900 313 8500 282 9200 w-Hydroxy-4-deoxypyridoxol 5'-phosphate (3) 244 6900 252 4000 368 8900 368 8300 315 sh 100 2-Nor-2-formylpyridoxal (8) 2R5 sh 6500 255 sh 6800 285 7800 410 ,5900 5500 41Bb 323 sh 2300 '2-Nor-2-formylpyridoxal 5'-phosphate (9) 2.55 sh 5400 2Fj5 sh 5200 296 6100 378 6400 378 5200 310 sh 3200 2-Nor-2-formy1-4-deoxypyridoxol R'-phosphate (4)c 308 3100 303 3800 285 7000 237 11#io0 237 9100 In 50 a Insignificant shifts occurring in the alkaline range indicate that no additional proton dissociation takes place above pH 7. mM glycerophosphate buffer (pH 7) containing 30 mM mercaptoethanol, the standard buffer for phosphorylase assay, a considerable blue shift to 385 nm occurs. The shift was not observed with glycerophosphate buffer alone. It is tentatively attributed to a selective thioacetal formation at the 2-carbonyl position, since none of the Ccarbonyl derivatives thus far investigated have displayed a shift of this magnitude. c Uncorrected for water content. o-Hydroxypyridoxal (7a)
TABLE I1 NMRSPECTRA OF THE SYNTHETIC INTERMEDIATES AS COMPARI
