Pergamon 0031-9422(95)00460-2 Phytochemistry, Vol. 40, No. 4, pp. 10271031, 1995 Copyright ~3 1995 Elsevier Science Ltd Printed in Great Britain. All rights reserved 0031 9422/95 $9.50 + 0.00 A VERSATILE AND EFFICIENT METHODOLOGY FOR THE PREPARATION OF CHOLINE ESTER AUXIN CONJUGATES BAL,AZSA. BOzSt3, RICHARD A. FLUCK,* RACHEL A. JAMETON, PHYLLIS A. LEBER~" and JEFFREY G. VARNES Department of Chemistry; *Department of Biology, Franklin & Marshall College, Lancaster, PA 17604-3003, U.S.A. (Received in revised form 24 April 1995) Key Word Index--Auxin conjugate; indole-3-acetic acid; acetylcholine analogue; choline ester. Abstract--A two-step synthetic strategy affords indole-3-acetylcholine (IAC) from indole-3-acetic acid (IAA) via the intermediacy of 2-dimethylaminoethyl indole-3-acetate. Thus, treatment of indole-3-acetate with 1-dimethylamino-2- chloroethane, followed by methylation with methyl iodide, yields 55-70% recrystallized IAC from IAA. Using the same methodology, six analogues of IAC have been prepared in overall yields ranging from 40 to 60%. INTRODUCTION Derivatives of choline serve important functions in plant physiology. Phosphatidylcholine is a key component of plant cellular membranes, and acetylcholine has been found in the tissues of many plants [1]. Indole-3-acetic acid (IAA) is a potent plant growth hormone, and exam- ples of IAA conjugates with plant activity abound in the literature I-2]. According to the 'hormonal homeostasis theory', IAA conjugates play a vital role in regulating the level of free IAA available to a growing plant I-3]. IAA conjugates have been implicated in other plant hormone metabolic functions as well: (1) transport of IAA, (2) storage and subsequent re-use of IAA, and (3) protection of IAA from enzymic destruction I-4]. RESULTS AND DISCUSSION We have recently reported that indole-3-acetylcholine iodide (1), a novel analogue of acetylcholine and an IAA conjugate, promotes pea stem elongation at concentra- tions as low as 1/~M 1"5]. We now wish to report on the synthetic strategy and methodology that we have devised for the preparation of 1. Our synthetic strategy (Scheme 1) identifies 2-dimethylaminoethyl indole-3-acetate (2) as a key intermediate that can be accessed via esterification of indole-3-acetic acid (IAA). Although our initial syn- thetic route to the intermediate involved boron trifluor- tAuthor to whom correspondence should be addressed. ~:The hydrochloride salt of compound 2 has been cited in one previous reference: Thuillier, G. and Rumpf, P. (1960) Bull. Soc. Chim. Fr. 1789. 'Indolylacetylcholine', presumably the bromide salt, was prepared according to the method of Fourneau and Page (1914) Bull. Soc. Chim. Fr. 15, 544; Keyl, M. J., Michaelson, I. A. and Whittaker, V. P. (1957) J. Physiol. 139, 434. However, the only characterization of the compound reported was the ion exchange chromatographic retention volume. ide-catalysed Fischer esterification of IAA with 2- dimethylaminoethanol [5], we have obtained a signifi- cant yield improvement by treatment of indole-3-acetate with 1-dimethylamino-2-chioroethane (DMAC). The new methodology affords routine overall yields of 55-70% recrystallized IAC (1) from IAA. The structure proofs of 2 and 1, both hitherto un- reported in the literature,:~ rest primarily on NMR spec- troscopy, both 1H and ~3C (Tables 1 and 2). The NMR assignments for the aromatic portion of IAA were based on indole. The acid portion of esters 1 and 2 was modelled by IAA; the alcohol portion of 2 by 2-dimethyl- aminoethanol (DMAE), and the corresponding portion of 1 by choline iodide (ChI). The only significant devi- ation between the aH NMR spectrum of 2 and that of DMAE involves the 0.65 ppm downfield shift of the OCH2 hydrogens (63.45 in DMAE versus 64.1 in 2). In the ~3C NMR spectrum of 2 the CH20 carbon is shifted downfield by 3.0ppm whereas the CH2N ÷ carbon is shifted upfield by 4.2 ppm. The methylene carbons in both DMAE and 2 have been differentiated by means of a 2D HETCOR pulse sequence. The ~H NMR spectrum of 1 is not appreciably differ- ent from those of the two constituents IAA and ChI except that the CH20 hydrogens in 1 are shifted down- field 0.65 ppm relative to the corresponding hydrogens in ChI. Two carbons in the alcohol portion of 1 have 13C NMR chemical shifts slightly different from the ana- logous carbons in ChI. The CH20 carbon in 1 is shifted downfield by 3.0 ppm whereas the CH2N+ carbon in 1 is shifted upfield by 3.1 ppm. The carbons adjacent to the quaternary nitrogen have been unambiguously iden- tified by an equal intensity triplet structure due to split- ting by laN. The two-step methodology outlined in Scheme 2 for the synthesis of IAC from IAA is extremely versatile. Thus, we have extended this methodology to the prep- 1027