2700
J . Am. Chem. SOC. 1984, 106, 2700-2701
Scheme I .
Probable Mechanism of' the Reaction Catalyied by 5-~nolpyruvoylsIiikinlate-3-phosphate Synthetase
r
700-
L
4
3
Scheme 11. Predicted I'ate of the Stoichiometric Deuterium and Tracer Tritium Labels in the 1;nzyme-Catalyzed Condensation of Phosphoenolpyruvate with Shikimate 3-Phosphatea D
(TIH
BOH t
@
oL-6 -
H$D
60
+
coo-
6
BULK
Scheme 111. Stereoanalytical Scheme for Determining if the Tritium Label in [ 9-2H , 3 H ] Chorismate is E or Z a
coo -
'6ID
SO
coo-
6H
coo-
7
TRACE
b, c
-D equally on both sides i n bulk
tracer T m a i n l y 2
a It is assumed for illustration only that the addition step is anti and the elimination step is syn; S O H is the shikimic acid 5-hydroxyl function.
methyl-d, sulfoxide and left at room temperature for 12-24 h, until the 'H N M R showed the presence only of the aryl enol ether 8.15 This material was then reduced with cis stereochemistry using Wilkinson's catalystI7 to yield the two diastereoisomeric aryl lactyl ethers. Birch reduction'* afforded (R,S)-lactate in which the configuration at carbon 2 is related to that at carbon 3 (Scheme 111). The @)-lactate was removed by oxidation with (S)-lactate dehydr~genase'~ and the remaining (R)-lactate subjected to Kuhn-Roth oxidation20 to yield chiral [2H,3H]aceticacid. The configuration of the acetic acid was then analyzed.I0 It was found that when (Z)-phosph0[3-~H,~H]enolpyruvate was the substrate in the reaction with shikimate-3-phosphate (3) to make 5, the acetate configuration after conversion to chorismate and stereoanalysis as outlined in Scheme 111was S ("F"value" 0.39). When (E)-phosph0[3-~H,~H]enolpyruvate was the substrate, the final acetate was R ("F"value" 0.62).21 These data show that (Z)-phosphoenolpyruvate results in (Z)-chorismate (the isotopic labels in the enolpyruvoyl moiety remain "in place"), which requires that in the 5-enolpyruvoylshikimate-3-phosphatesynthetase reaction, the addition step (Scheme I) has an opposite stereo(1 5) The usual method of aromatization using acetic anhydride/pyridine5 was found, using a stereospecifically deuterated sample of [9-2H]-chorismate,'6 to result in the complete stereochemical randomization of the isotopic label at carbon 9. This problem is happily avoided by the simple method reported here. (16) Hoare, J. H.; Berchtold, G. A. J . Am. Chem. SOC.,following paper in this issue. (17) In methanol/benzene (50:50,v/v). H 2 at 4 atm, room temperature, 15 h. Yield -80%. The product was purified by ion-exchange chromatog raphy. (18) Lithium in ammonia, followed by mild acid hydrolysis. Yield 75-80%. (19) Using 3-acetylpyridine adenine dinucleotide as cofactor at pH 9.5, to drive the reaction in the direction of lactate oxidation. The (R)-lactate was purified by ion-exchange chromatography. (20) Control experiments using an unlabeled sample of lactic acid in D20 showed that the product acetate was
