Strongly Acidic Auxin Indole-3-Methanesulfonic AcidSYNTHESIS OF ['4C]INDOLE-3-METHANESULFONIC ACID AND STUDIES OF ITSCHROMATOGRAPHIC, SPECTRAL, AND BIOLOGICAL PROPERTIES
Received for publication May 16, 1984 and in revised form September 8, 1984
JERRY D. COHEN*, BRUCE G. BALDI', AND KRYSTYNA BIALEKUnited States Department ofAgriculture, Agricultural Research Service, Plant Hormone Laboratory,Beltsville Agricultural Research Center- West, Beltsville, Maryland 20705 (J.D.C.), and Departments ofHorticulture (B.G.B.) and Botany (K.B.), University ofMaryland, College Park, Maryland 20742
ABSTRACT
A radiochemical synthesis is described for I"Cqindole-3-methanesul-fonic acid (IMS), a strongly acidic auxin analog. Techniques were devel-oped for fractionation and purification of IMS using normal and reversephase chromatography. In addition, the utility of both Fourier transforminfrared spectrometry and fast atom bombardment mass spectrometryfor analysis of IMS has been demonstrated. IMS was shown to be anactive auxin, stimulating soybean hypocotyl elonption, bean first inter-node curvature, and ethylene production. IMS uptake by thin sections ofsoybean hypocotyl was essentially independent of solution pH and, whenapplied at a 100 micromolar concentration, IMS exhibited a basipetalpolarity in its transport in both corn coleoptile and soybean hypocotylsections. '4C]IMS should, therefore, be a useful compound to studyfundamental processes related to the movement of auxins in plant tissuesand organelles.
Only a few auxin-like compounds are known which do nothave a carboxylic acid or a function easily converted to a car-boxylic acid. The synthesis of one such compound, IMS,2 wasfirst reported by Wieland et al. (28) and it was subsequentlyshown to be biologically active in a variety of test systems (1, 10,26, 27). Sulfonic acids are considered strong acids with pKavalues well below 0 (typically, negative 6.5 [18]) and as such existessentially fully nonprotonated under physiological conditions.In this respect, IMS may be unique among the known indoleauxins (2, 25).A 'chemiosmotic' hypothesis for explaining the uptake and
polar movement ofIAA has been proposed (1 1, 12, 20, 21). Thistheory suggests that transmembrane and polar transport ofauxinsare driven by an electrochemical potential which tends to resultin accumulation of the weak acid. The specific efflux of theanion by localized carriers is postulated to account for theobserved basipetal polarity. IMS should, because of its stronglyacidic properties, be a useful compound for testing these ideas.Studies utilizing IMS could also allow a distinction to be made
' Present address: Institute of Biological Chemistry, Washington StateUniversity, Pullman, WA 99164.
2 Abbreviation: IMS, indole-3-methanesulfonic acid.I Mention of a trademark, proprietary product, or vendor does not
constitute a guarantee or warranty of the product by the United StatesDepartment of Agriculture, and does not imply its approval to theexclusion of other products or vendors that may also be suitable.
between the mechanisms for moving auxin anions and proton-ated acidic auxins across biomembranes. The process of polarmovement is thought to involve efflux of the anion of auxin,and the protonated form should enter the cell (1 1, 12).
In this initial study we describe an easy method for synthesizing['4C]IMS, detail some useful techniques for chromatography andanalysis of this sulfonic acid, and report on some of its interestingbiological properties. It is hoped that availability of this com-pound will stimulate additional investigations which will refinecurrent theories about the mechanisms of auxin uptake andtransport.
MATERIALS AND METHODS
Synthesis of Indole-3-methanesulfonic Acid. The procedurefollowed was essentially as described by Coker (7) at one-thirdthe scale reported and using KBr to generate the potassium salt(Fig. 1). The resulting light yellow crystals were found to be ofinsufficient purity so the product was chromatographed on a 2.3x 40 cm column of Sephadex LH-203 using 40% 2-propanol/water containing 10 mm triethylamine-acetate (pH 7.0) for elu-tion. The product was recrystallized from water as the potassiumsalt to yield white plate-like crystals. Silica gel TLC (ethanol: 1 NHCI; 95:5 RF = 0.75) showed only one spot by Ehmann's indolereaction (9), UV quenching, or H2SO4 charring. Product identitywas confirmed by spectrometry on a Nicolet 60SX Fouriertransform IR spectrometer (Fig. 2), by its 200 MHz NMRspectra, and by its mass spectrum on a V.G. ZAB 2F massspectrometer using fast atom bombardment to induce ion for-mation (Fig. 3). As an additional confirmation of identity, IMSwas synthesized from gramine and sodium sulfite (10) at thegram scale (Fig. 1) and the products obtained from the two
(1) Na2so3
N CH20H
Na2SO3
(2) (CH2NCHH3)2
H
CH2S O3H
NH
(CH3)2NH
(3)
FIG. 1. Diagram of the synthetic routes to IMS (structure 3) fromindole (structure 1) and from gramine (structure 2).
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FIG. 2. The Fourier transform IR absorption spectra of the syntheticIMS, K+ salt obtained in KBr on a Nicolet 60SX FT-IR. The samplewas prepared by sulfomethylation of indole (see text).
synthetic methods were compared. The product produced fromgramine proved to be identical to that produced by sulfomethy-lation of indole based on their chromatographic properties andinfrared spectra.
'4C-Labeled IMS was prepared similarly by sulfomethylationof indole using ['4C]formaldehyde (New England Nuclear). In-dole (6 mg, 0.5 mmol), sodium sulfite (126 mg, 1.0 mmol), and200 Ml water were added to a 300-Ml Reacti-vial (Pierce). The vialwas heated to 100°C in a dry block heater and ['4C]formaldehyde(50 MuCi, 10 mCi/mmol) added in 100 ,l of water. After 6 h thereaction mixture was partitioned between water and ether andthe water phase purified by ion pairing with 10 mM triethylamine-acetate (pH 7.0) on a 1 x 20 cm Sephadex LH-20 (Pharmacia)column in 40% 2-propanol/water. The yield was 35% and theradiochemical purity by TLC, 98.2%.Chromatography of IMS. Because of their low pKI0, sulfonic
acids are typically difficult to purify by chromatographic methods(24). We have developed several systems which can be usedeffectively with IMS. As discussed above, ion pair chromatogra-phy using triethylamine-acetate (pH 7.0) in 40% 2-propanol/water with Sephadex LH-20 is useful for preparative and semi-preparative separation. TLC on silica gel 60 (E. Merck, No.5763) can be done using absolute ethanol: 1 N HCI; 20:1 (solventA). Unfortunately, this solvent does not separate IAA (RF = 0.80)well from IMS (RF = 0.75). A second dimension of chromatog-raphy using chloroform:methanol:H20; 85:14:1 (solvent B) will,however, resolve IAA (RF = 0.45) from IMS (RF = 0.00). Alter-
natively, IMS has an RF = 0.4 and IAA RF = 0.9 on silica gelusing ethyl acetate:methyl ethyl ketone:ethanol:water (5:3:1:1)(solvent C) as solvent (16). Finally, reverse phase HPLC can bedone using polystyrene type supports such as Hamilton PRP-1(17). Using a 250 x 4.6 mm column of 10 Mm PRP-1 (HPLCTechnology, Lomita, CA) with 20% acetonitrile:H20 (adjustedto pH 1.0 with HCI) as mobile phase, IMS elutes with a retentionvolume of 5.2 ml.
Plant Material. Soybean seedlings (Glycine max L. cv Hark;Comm. Ag. Devel., Iowa State Univ., Ames, IA) were grown at25°C for 5 to 7 d in rolled paper towels in the dark. Duringexamination a phototropically inactive green safelight was used.Seedlings 10 to 15 cm in length were selected and 1-cm segmentswere cut from just below the apical hook. Seedlings of maize(Zea mays L. cv Silver Queen; Meyer Seed Co., Baltimore, MD)were grown for 4 d under the same conditions as were used forsoybeans. First internodes were obtained from light grown plantsof Phaseolus vulgaris L. cv Bush Burpee Stringless (Meyer) aspreviously described (3).Movement of Auxins. Ten seedling segments (soybean) or five
coleoptile sections (maize) were placed on an 0.75% agar (Fisher)block 0.35 x 1.0 x 1.0 cm in size. To measure basipetal move-ment, an agar block of the same dimensions containing the 14Cindole acid was placed on top of the sections and they wereincubated for 3 h in the dark at 25C. Apical transport wasmeasured similarly, except the donor block was on the bottomand receiver on the top. Agar blocks and sections were collectedand placed in scintillation vials containing ACS counting solu-tion (Amersham). After overnight extraction in the dark, thevials were counted on a Beckman 9000 liquid scintillationcounter using the external standard system to obtain dpm. Inaddition to [14C]IMS, carboxyl-['4C]IAA (59 mCi/mmol, Amer-sham) was used for comparison. Because of its higher specificactivity, it was necessary to dilute the radioactive IAA to 10mCi/mmol for experiments that required a 100-Mm concentra-tion. All experimental treatments were done in duplicate ortriplicate and each experiment was repeated at least once (n 24). In some experiments with soybean, a parallel experiment wasrun and the tissue or block was extracted with 70% acetone.Following extraction, carrier IMS (10 ,g) was added, the solventwas removed, and the sample was analyzed by 2-dimensionalTLC (solvent A in first dimension followed by solvent B insecond dimension). The TLC plate was first examined under UVlight and then by fluorography after spraying with fluoroHance(Research Products).
Accumulation of Auxins. The method followed was essentially
r38016
0
C)C
(D
FIG. 3. The fast atom bombardment mass spectra ofIMS, K+ salt obtained using a V.G. ZAB 2F mass spectrom-eter. Spectra were obtained at 8 kv using xenon and withFomblin for mass calibration. Major significant ions shownare m/z 288 (molecular ion for the dipotassium salt), m/z250 (molecular ion, potassium salt), m/z 211 (molecularion, free acid), m/z 168 (IMS, K+- [SO3H+]), and 130(quinolinium ion). Ions at m/z of 93, 185, and 277 arefrom residual glycerol.
196 COHEN ET AL.
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as described by Edwards and Goldsmith (8) except for the use ofsoybean hypocotyl tissue and the '4C-labeled auxins. The uptakeperiod was for 5 min and the radioactivity was determined asabove.
Ethylene Production. Ten 1-cm soybean hypocotyl sectionswere incubated with shaking in serum-stoppered 50 ml Erlen-meyer flasks containing 3 ml of a solution of 10 mm Mes (pH6.7), 50 mm sucrose, and the indicated acidic indole. After 6 hthe gas phase was sampled (0.5 ml) and the ethylene levelmeasured by GC. The experiment was done twice with a total of5 replications.
Biological Activity. The growth promoting properties of IMSwere tested by floating ten 1-cm soybean hypocotyl sections in a6-cm Petri dish on 10 ml of water containing the indole atvarious concentrations. IMS (potassium salt) is highly watersoluble so that organic solvents were not used as an aid indissolution. IAA was tested as a control and small amounts ofethanol (<1%) were used. The length of sections was measuredafter a 24-h incubation at 25°C. The experiment was conductedthree times and the data represent the average of a total of 30sections for each treatment. High resolution growth recording ofbean first internode curvature was performed as previously de-scribed (3).
RESULTS AND DISCUSSION
The synthesis of ['4C]IMS is simple, can be done with standardlaboratory apparatus, and takes less than 1 d. In trial runs, yieldsas high as 70% were obtained, but with [14C]formaldehyde, onlyabout half of this amount was obtained based on the suppliersstated initial quantity of ['4C]formaldehyde. The reasons for thislower yield are unknown, but formaldehyde is volatile and somecould be lost by a variety of routes. Relative to commerciallyavailable labeled indoles, the use of ['4C]formaldehyde to form[14C]IMS is, nevertheless, quite economical. Higher specific ra-dioactivity IMS should be obtainable via the same route by usinghigher specific radioactivity [14C]formaldehyde, using [3H]form-aldehyde or by generation of [35S]sodium sulfite from [35S]sul-furic acid. The last possibility could, theoretically, yield IMS ofa specific radioactivity of up to 1500 Ci/mmol from the readilyavailable starting materials. The specific radioactivity of the IMSused in this report was, however, quite satisfactory for the studiesthat were done.The structure of IMS was confirmed by its spectral properties.
Its IR spectra (Fig. 2) showed a strong absorbance characteristicof N-H of indole at 3385 wavenumbers and strong absorbancenear 1200 wavenumbers indicative of a sulfonate salt (23). Thesedeductions were confirmed by comparison to spectra obtainedof salts of other sulfonic acids (2-[N-morpholino]ethane-sulfonicacid and p-toluenesulfonic acid) and to that of IAA. The NMRspectra in D20 were as expected for a 3-substituted indole.Attempts to obtain mass spectra by electron impact or chemicalionization techniques using a Finnigan 4000 series mass spec-trometer were not successful with IMS. However, use of the fastatom bombardment capabilities of the V.G. ZAB 2F mass spec-trometer resulted in significant ionization of IMS and yieldedexcellent mass spectra of a mixture of mono- and di-potassiumsalts (Fig. 3).IMS promotes growth of isolated soybean hypocotyl sections
to about the same extent as IAA (Fig. 4). However, the maximumresponse to IAA occurs at 10-' M and to IMS at I0 M.Concentrations of IMS up to 10-2 M still promote elongation,while 10-3 M IAA inhibits growth (Fig. 4). Curvature of beanfirst internode sections is also promoted by IMS, although ahigher concentration of IMS is required relative to IAA. Thekinetics for curvature following IMS treatment were measuredusing an angular transducer (3, 19) and were similar to that seenfollowing a unilateral application of IAA (Fig. 5). A rapid re-
5,IAA
ji4-E
3
'r.r
H20 -6 -5 -4 -3
IMs
illil- 6 - 5 -4 -3 -2Lo Cocnrin M
Log 10 Concentration, M
FIG. 4. Growth of 1 cm etiolated soybean hypocotyl segments floatedon solutions containing IAA or IMS at the indicated concentrations for24 h. Values given are increased over initial length and bars indicate theSE which occurred.
E1.000
E0.80
30.6-
°0.4
0.2-
.2040 60 8 0 12020i 40 670 80 1600 120
Time (min.)FIG. 5. The rates of curvature induced by a unilateral application of
IAA and IMS to bean first internode sections. The rate of horizontaldisplacement was measured using an angular transducer (3) after appli-cation of 1 nmol/section IAA or 10 nmol/section IMS. At least 10 stemsections were used for each treatment.
60
w
V401
C)
20
IAA-..
/ IMS*I, .
0--O T--0
-6 -5 -4 -3 -2Log1o Concentration, M
FIG. 6. Ethylene production after 6 h of treatment of soybean hypo-cotyl sections with the indicated concentration of IAA or IMS. Ethylenewas measured by GC using flame ionization detection.
sponse was observed with maximum curvature occurring at 20to 25 min following auxin application. This early rise in curvaturerate was followed by a longer term stimulation of curvature (Fig.5). Most prior reports of biological activity of IMS are consistentwith our findings (1, 26, 27), although early reports (i.e. 28)indicated IMS to be inactive. This inconsistency may have beenrelated to the higher concentrations of IMS required to showoptimal activity (26).
In addition to the stimulation of cell elongation, IMS alsoinduces increased ethylene production by soybean hypocotyls(Fig. 6). As was seen with growth stimulation, the effect of IMSon ethylene production occurred at significantly higher concen-trations than was observed for a similar response with IAA. Ithas been suggested that this concentration difference was due toreduced uptake of the strongly anionic compound (25). IMS
FIG. 7. The pH dependence of IAA ( A, *) and IMS (0, A, 0)uptake by thin (Imm) soybean hypocotyl sections in different buffers.Uptake period was for 5 min. Buffers used were 20 mm citrate (0, 0),20 mm Mes (A, A), and 20 mm Mops (0, U). Each incubation vialcontained I ml of buffer solution, 30 1-mm sections, and 100 nm of theindole acid (10 and 59 mCi/mmol for IMS and IAA, respectively).
uptake into thin slices of soybean hypocotyls was measured as afunction of solution pH (Fig. 7). IMS uptake was similar to thatof IAA at pH 6.0 and at neutral pH. Acidic pH had little effecton IMS uptake while IAA uptake was strongly promoted inacidic buffers (Fig. 7). The pH effect on IAA uptake into soybeanhypocotyl sections is similar to that reported for corn coleoptiles(8) and the results are typical of what would be expected foruptake of a weak organic acid (13). Uptake of a strongly acidicanion like IMS should not exhibit any pronounced pH effect inthe physiological range and this hypothesis agrees with ourexperimental results. The possibility exists that some fraction ofthe retained counts could be located external to the plasma-lemma. Such a possibility makes it difficult to ascribe the quan-titative difference in biological activity between IMS and IAA totheir observed rates of uptake at physiological pH values.The data on uptake are consistent with the possibility that
uptake was entirely extracellular with little or no permeability ofthe plasma membrane for the anions of either IAA or IMS in 5min. Some fraction of the observed uptake of IMS and also theuptake observed for IAA at neutral pH values could also be dueto a basal rate ofanion influx into the cell. It may be hypothesizedthat an anion carrier on the plasmalemma could operate in bothdirections and effectively function as an efflux carrier only whenthe internal anion concentration is greater than that in theexternal milieu or under conditions when uptake is not limiting,such as at very high auxin concentrations. If the anion carrier islocated preferentially on the basal end of some cells (14), andthe above hypothesis also is correct, then IMS should be trans-ported in a polar fashion when applied at high concentrations.This is what is observed when ['4C]IMS is used in the agar blockassay for polar transport (Table I). IMS shows a slight basipetalpolarity similar to that of IAA when applied to soybean hypo-cotyls at 100 gm; however, at lower concentration (1 uM) onlyIAA shows basipetal polarity. With corn coleoptiles the effect iseven more dramatic. At 100 gM a substantial basipetal polarmovement ofIMS is seen, although the differential is not as greatas for IAA. At 1 uM, however, the polarity with IMS is in the
Table I. Measurement ofthe Movement ofIAA and IMSMovement of IAA and IMS was through 1-cm tissue segments of
soybean hypocotyl (10 sections/treatment) or maize coleoptile (5 sec-tions/treatment) after 3 h incubation using the donor/receiver agar blockmethod. Basipetal and apical refer to the direction of movement of theapplied indole auxin, i.e. basipetal transport is measured by having thereceiver at the base of the tissue sections.
Percentage of Counts' at Following Indole AcidConcn.
1.0MM 100 JAMTissue Receiver Tissue Receiver
Soybean HypocotylIMS
BasipetalApical
RatioIAA
BasipetalApical
Ratio
Maize ColeoptileIMS
BasipetalApical
RatioIAA
BasipetalApical
Ratio
12.1 ±0.811.6 ± 0.5
1.04
22.0 ± 0.88.5 ± 1.52.59
2.2 ± 0.92.5 ± 0.20.88
6.6 ± 0.61.6 ± 0.1
4.16
4.8 ± 0.64.7 ± 0.2
1.02
2.0 ± 0.31.1 ± 0.1
1.82
0.2 ± 0.15.7 ± 1.3
0.03
12.6 ± 1.20.1 ± 0.04126.00
11.9 ± 2.011.4± 3.3
1.04
5.8 ± 0.57.5 ± 1.30.77
2.5 ± 1.12.7 ± 0.10.93
2.4 ± 0.41.6 ± 0.3
1.50
1.4 ± 0.20.9 ± 0.03
1.56
4.8± 1.10.5 ± 0.1
9.60
4.1 ±0.6 1.3±0.15.6 ± 0.9 0.03 ± 0.020.73 43.33
' Percentage of total count recovered from donor, receiver, and tissueat end of 3-h incubation.
apical direction (Table I). Although these data are consistentwith the above hypothesis on uptake, they are not conclusive. Itis probable that several alternate explanations could serve equallywell to explain the observed data.
Quantitatively, the amount ofIMS that moved through tissuesegments in the apical direction is greater than is that of IAA(Table I). It should be noted that the apparent smaller ratio ofbasipetal to apical movement ofIMS as compared to LAA is theresult of a slightly increased rate of apical movement that resultsin the noted decreased ratio. The total amount of IMS movingin the basipetal direction in 3 h is greater than the amount ofIAA that moves (Table I).
In some experiments the soybean sections and agar receiverblocks were each extracted into 70% acetone/water and analyzedby 2-dimensional TLC using fluorography. Most of the radioac-tivity extracted from the agar block ran on TLC coincident withthe added carrier IMS and thus was either still IMS or someproduct with very similar chromatographic properties. Most ofthe radioactivity extracted from the plant tissue, however, re-mained at the origin in both solvent systems. A small amount ofradioactivity could be detected which co-migrated with the addedcarrier IMS; however, this was estimated to be less than 20% oftotal spot density detected on the fluorogram. Cleavage of sul-fonamides is considerably more difficult than the correspondingcarboxylic acid amides (5, 22). Attempts to effect cleavage ofthemajor radioactive product isolated from the tissue by using anionradicals derived from naphthalene and sodium metal, a tech-nique which has been shown to hydrolyze sulfonate esters andamides (4, 5, 15), failed to yield detectable IMS. Furtherattemptsdirected at production of additional amounts of this material bytreatment of soybean sections in solutions of IMS failed to
produce significant amounts of metabolites. It appears that theradioactive product found in the tissue after transport experi-ments: (a) is a metabolite of IMS, (b) is probably not an IMSconjugate, and (c) is produced in stoichiometric amounts atrelatively low levels of IMS treatment. Thus, at least quantita-tively, the metabolic fate of IMS appears to be different fromthat of exogenous IAA (6).
In conclusion, we have described an easy method for synthesisof ['4C]IMS yielding a product of high specific radioactivity andgood purity. We have also described several simple chromato-graphic techniques for its fractionation and demonstrated theutility of both Fourier transform IR spectrometry and fast atombombardment mass spectrometry for analysis of IMS. We havealso shown that IMS is a functional auxin, stimulating cellelongation growth as well as ethylene production. IMS is takenup by thin sections in a manner essentially independent ofsolution pH and, under some conditions, exhibits substantialpolarity in its transport. We feel that ['4C]IMS should, therefore,be a useful additional compound with which to study fundamen-tal processes related to auxin movement in plants.
Acknowledgments-We thank Dr. Autar K. Mattoo for his assistance with theautoradiography, Mr. Irving Newman for help with the ethylene determination,and Dr. Axel Ehmann (Shell Development Co.) for providing the FAB-MS analysis.Dr. J. George Buta provided valuable advice on chemical and spectral methods forwhich we are most grateful. Special thanks are due to Mrs. Delores Sessions andMrs. Sunny M. Keithley for their expert assistance in the preparation of thismanuscript.
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