Branched Chain Amino Acid Biosynthesis Inhibitors

The discovery of herbicides that inhibit the production of the branched chain amino acids leucine, isoleucine and valine was a major break through in weed control technology.  Dupont commercialized the first sulfonylurea (SU) herbicide, chlorosulfuron, in the early 1980s followed shortly by American Cyanamid’s (now part of BASF) introduction of the first imidazolinone (IMI) for soybeans, imazaquin.  These new herbicide families selectively controlled many important weed species at very low use rates, they had excellent crop safety over a wide range of crop growth stages with very low mammalian toxicity.  Since animals do not have ALS they posed little or no risk to human health.

Chlorosulfuron was marketed as “Glean” for POST weed control in wheat.  Glean was first evaluated at several ounces per acre, but field evaluations quickly indicated that this herbicide was very effective at much lower use rates.  Eventually, use rates of only a few grams per acre were found to be sufficient for annual broadleaf weeds, while Canada thistle control could be achieved with 1.5-2.0 ounces per acre.  Glean provided excellent control of several weed species that were very difficult to control with auxinic  (2,4-D, MCPA, dicamba) or contact PSII inhibitors (bromoxynil) that were commonly used for broadleaf weed control in small grains.  One of the key weed species that drove the rapid adoption of this new technology was kochia (Kochia scoparia).  Initial kochia control with Glean was greater than 95%.  The rapid increase in ALS resistant kochia, which occurred a few years after Glean’s introduction, would foretell the Achilles’ heel of ALS inhibitors.

Initial field and laboratory studies with sulfonylurea herbicides revealed some remarkable attributes to this new chemistry.  Laboratory enzyme assays provided a good explanation for the extremely low use rates.  ALS I50 values (herbicide concentration required to reduce enzymatic activity by 50%) were in the low nM (10-9 molar) range.  Sulfonylurea herbicides are rapidly absorbed by roots and foliage, translocate in both xylem and phloem and have residual soil activity.  The residual soil activity was long enough to require significant plant back restrictions for sensitive broadleaf species, such at sugarbeets and potatoes.  Herbicide selectivity was found to be a function of metabolism rate; therefore, slight changes in chemical structure allowed for the development of herbicides that could be used selectively in previously sensitive broadleaf crops, like sugarbeet (triflusulfuron, UpBeet®) and potato (rimsulfuron, Matrix®).  This dependence on herbicide metabolism for selectivity means that under adverse weather conditions crop injury can occur.   

DuPont developed non-transgenic sulfonylurea-tolerant-soybean (STS) in the 1990s, marketed as Reliance STS or Synchrony STS varieties.  A POST application of chlorimuron and thifensulfuron was used to provide broad-spectrum weed control.


Figure 2.  Basic SU structure (A) and three common SU herbicides (B).

Table 1.  Important properties of SU herbicides from Figure 2. 

 Herbicide  Water Solubility (ppm)  pKa  Log Kow  General use Pattern
 Chlorimuron  11 at pH 5 450 at pH 6 1200 at pH 7  4.2  2.5 at pH 5 0.36 at  pH 7  PPI, PRE and POST in soybean, POST in peanuts and non-crop.
 Halsulfuron  15 at pH 5 1630 at pH 7  3.5

 1.67 pH 5 -0.018 pH 7

 PPI and PRE in corn with MON 13900 safener, POST in corn and grain sorghum, POST to warm and cool season turf, PRE and POST in dry beans. Excellent for yellow nutsegde control.

 Sulfometuron  10 at pH 5 300 at pH 7  5.2  na  PRE and POST applications in forestry and numerous PRE and POST applications to non-crop sites.

The IMIs have many of the same characteristics as the SUs.  They are absorbed by both roots and shoots and have excellent translocation in both the xylem and phloem.  Like the SUs, there is significant residual soil activity to the point that there are 24 to 36 month plant back restrictions for some sensitive species.  IMI use rates are  higher than the SUs and this is consistent with the ALS I50 values that are in the lower µM (10-6 molar) range or 1000 times lower affinity for ALS than the SUs.  These herbicides are still very potent ALS inhibitors.  The primary markets for these herbicides are soybeans, alfalfa, peanuts and dry beans; however, imazathabenz (Assert®) controls wild oat and several important broadleaf weeds in wheat and barley.  Imazapyr (Habitat®, Powerline®) is used for pipelines, rights-of-way, conifer release, brush and tree control.  Imazapyr has an ideal combination of log Kow and pKa for long distance phloem transport.  This could explain why imazapyr is the only herbicide to control the invasive tree species, tamarisk, when applied as a foliar treatment. 

Like the SUs, IMI selectivity is based on rapid metabolism.  Legumes were found to have inherently high tolerance to the IMI’s due to rapid hydroxylation followed by glucose conjugation.  In the days before glyphosate-tolerant soybeans, the IMIs were the most important active ingredient in soybean production.  Alfalfa is also very tolerant to imazethapyr (Pursuit®) and imazamox (Raptor®).  This allowed for the rapid establishment of pure alfalfa stands without the need for a nurse crop and provided growers with excellent forage production the year of establishment.  Because rapid metabolism is responsible for selectivity adverse weather conditions can result in crop injury if herbicide metabolism is slowed by cool temperatures.  

A series of IMI tolerant non-transgenic crops called Clearfield® have been developed by chemical mutagensis.  These included Clearfield corn, Clearfield canola, and Clearfield wheat.  In the case of Clearfield sunflowers the resistance gene was backcrossed into commercial sunflower varieties from IMI resistant wild sunflowers. 

Figure 3.  Basic structure of imidazolinone herbicides (A) and three common IMI herbicides (B)

Table 2. Important properties of common IMI herbicides from Figure 3.   

 Herbicide Water Solubility (ppm)  pKa  Log Kow  General Use Pattern
 Imazethapyr  1400 at pH 7  3.9  1.49  Surface pre-plant, PPI, Pre and POST to soybeans and dry beans, POST in alfalfa and PPI, PRE at cracking, and POST in peanuts. Controls many annual broadleaf weeds.
 Imazaquin  160,000 as ammonium salt at pH 7  3.8  0.34  Surface pre-plant, PPI, PRE and POST to soybeans. Controls certain annual grasses and many annual broadleaf weeds.
 Imazapyr  11,272 ppm  3.6  0.11  Both soil and foliar activity, used primarily in non-crop situations, pipline, rights of way, conifer release, bramble, brush and tree control. Controls many annual and perennial broadleaf and grass weeds.

In spite of issues with herbicide resistance, chemical companies continue to develop and commercialize new herbicide with the ALS target site.  Currently there are more than 50 herbicides that are commercialized world wide for selective weed control in a wide variety of crops.  In the late 1990’s, Dow AgroSceinces commercialized a new family of ALS inhibitors called the triazolopyrimidines.  There are currently three herbicides in the US market that belong to this family of ALS inhibitors; cloransulam, diclosulam, and flumetsulam.  These herbicides are readily translocated in the xylem and phloem, with better overall soil activity than foliar activity.  Selectivity is based on herbicide metabolism, for instance in corn the ½ life of flumetsulum is only 2 hours, while in sensitive weed species it can exceed 4 days. 

Figure 4.  Basic structure of trizazolopyrimidines (A) and structure of common herbicides (B).

Table 3. Important properties of trizazolopyrimidines herbicide registered in US (from Figure 4).


 Water Solubility (ppm)


 Log Kow

 General Use Pattern

 Chloransulam methyl  3ppm pH 5 184ppm pH 7 3450 pH 9  4.8  1.12 pH 5 -0.36 pH 7 -1.24 pH 8.5

 Can be applied PPI, surface pre-plant, PRE and POST for broadleaf weed control in soybeans.

 Diclosulam  117ppm pH 5 124ppm pH 7 4290ppm pH 9  4.1  1.42 pH 7

 Soil applied for broadleaf and nutsedge control in peanuts. Application rates range 17-26 g/ha

 Flumetsulam  49ppm pH 2.5 5600ppm pH 7  4.6  -1.2 pH 7

 Soil and foliar applications in corn and soybeans. Weed spectrum increases with soil applications. Very little grass activity.

The pyrimidinylthio-benzoates are another herbicide family with an ALS target site.  In US markets this herbicide family has only one member called pyrithiobac (Staple®).  The trade name indicates that this compound, marketed by DuPont, is used in cotton.  Pyrithiobac can be absorbed following both soil and foliar applications, but appears to translocate primarily in the phloem.  

Figure 5.  Single member of the pyrimidinylthio- benzoates sold in the US.

Table 4.  Important properties of the pyrimidinylthio-benzoate, pyrithiobac, sold in the US.

 Herbicide Water Solubility (ppm)  pKa Log Kow   General use Pattern

 760 at pH 4

 2.34  -0.84  PRE and POST applications in cotton

The final herbicide group that targets ALS belongs to a new chemical family called the sulfonylaminocarbonyl-triazoliniones.  Like the pyrimidinylthio-benzoates, this chemical family has a single product marketed in the US by Bayer Crop Science, propoxycarbazone-sodium salt (Olympus®).  This herbicide can be absorbed by both roots and foliage and translocates in the xylem and phloem.  This herbicide is one example of how much flexibility there is in designing ALS targeted herbicides with very specific selectivity.  In the US, Olympus provides selective control of winter annual grasses primarily Bromussp. in winter wheat.  Monsanto has an SU that provides very similar selectivity called sulfosulfuron (Maverick®).

Figure 6.  Single member of the pyrimidinylthio-benzoates sold in the US.  Propoxycarbaxone-sodium salt is marketed as Olympus for annual grass control in wheat.

 Table 5.  Important properties of the only sulfonylaminocarbonyl-triazoliniones sold in the US.

 Herbicide Water Solubility (ppm)  pKa Log Kow   General use Pattern
 Propoxycarbazone-sodium salt


 2.1  -1.55  Annual grass and some broadleaf weed control in wheat. Use rates 30-70 g/ha

Symptoms of ALS inhibitors

In the early 1980s growers associated good herbicide activity with rapid symptom development, but these new ALS inhibitors were very slow acting.  If the weather was cool it could take 14 days to show good herbicide injury symptoms on susceptible weeds.  One key symptom of ALS herbicides is that plants stop growing within hours of application.  This kind of symptom is much more difficult to demonstrate unless treated and control plants are side by side. 

One common feature of all ALS herbicides is that they need to have phloem mobility.  Phloem mobility ensures that the herbicide will be translocated to root and shoot meristems.  These are the sites of rapid growth, high demand for branched chain amino acids, high protein synthesis and maximum ALS activity.  In fact ALS activity is almost undetectable in older leaves.  Chlorosis of the shoot meristems is one of the most consistent symptoms of ALS injury. 

Figure 7a.  Imazapic (Plateau, BASF) injury on leafy spruge,  notice that several apical meristems are dead, but lower leaves show very few symptoms.  This application was made in the spring, so there was very little downward (basipetal) translocation. Symptoms illustrated in these two figures would be typical of most ALS inhibitors. (photo by J. Sebastian, CSU)  

Figure 7b. Imazethapyr (Pursuit, BASF) injury symptoms in dry beans.  Normally, imazethapyr can be used selectively in dry beans; however, cool temperatures can sometimes cause significant injury due to reduced metabolism.  Severe chlorosis is associated with the youngest leaf, demonstrating upward (acropetal) translocation and accumulation in rapidly growing meristems. (photo by B. Wilson, UNL)

Herbicide Binding

How is possible that such a wide range of chemical compounds could have herbicidal activity against a single target site?  One explanation is that these herbicides are non-competitive inhibitors, meaning that the herbicide does not compete with pyruvate or 2-ketobutyrate for binding to ALS.  These herbicides bind to some area away from the active site of ALS and this changes the 3D configuration of ALS reducing its affinity for its normal substrates.  Variations on this theme were the standard explanation of how so many different chemical structures could have the same target site.

Thanks to researchers at the University of Queensland in Brisbane, Australia, we now have an elegant explanation for SU and IMI binding to ALS.  Based on x-ray crystallography of ALS from Arabidopsis  thaliana, these researchers were able to determine that SU and IMI herbicides block access to a channel that leads to the ALS active site.  When SUs bind to Arabidopsis ALS, the herbicide molecule bends at the sulfonyl group placing the two rings at almost a right angle to each other (Figure 8).  The sulfonyl group and the aromatic ring block the entrance to the channel, while the remainder of the molecule inserts into the channel.  When comparing yeast and Arabidopsis SU binding several important residues (amino acids) were found to be closer to the herbicide in Arabidopsis ALS.  One important amino acid that will be discussed when we examine ALS resistance is serine 653 (S653), which is a contact point in Arabidopsis ALS but not in yeast ALS.

For the imidazolinone, imazaquin, it is the dihyroimidazolone ring that blocks the channel leading to the active site, while the quinoline ring extents toward the protein’s surface (Figure  8).  Imazaquin interacts with 12 amino acids by noncovalent interactions, while the carboxylic acid forms a salt bridge with arginine 377 (R377).  The isopropyl and methyl groups on the imidazolinone ring are important for securing the herbicide to the protein, which explains why these side chains are important for good herbicidal activity.

Figure 8.  A) Chlorimuron-ethyl positioned in the channel to the active site of Arabidopsis ALS.  Notice how the molecule is bent around the sulfonyl group placing the two ring structures almost at right angles to each other.  B) Imazaquin blocking access to the ALS active site.  Imazaquin is closer to the surface of the enzyme compared to chlorimuron.  Figure provided by Dr. Ronald Duggleby, University of Queensland, Brisbane, Australia.  This figure was previously published in PNAS 103:569-573.

The SU and IMI herbicides have significantly different affinities for Arabidopsis ALS.  The I50 values for SUs are in the nM range, while IMIs require herbicide concentrations in the low µM range.  This new x-ray crystallography information illustrates the major differences between SU and IMI binding to ALS.  SU herbicides have at least 50 van der Waals contacts and six hydrogen bonds, while imazaquin had 28 van der Waals contacts and only one hydrogen bond.  In addition, SU binding is approximately 2 angstroms (one angstrom=0.1 nano meters) closer to the active site compared to the IMI, imazaquin.  These features combine to explain the excellent herbicidal activity of SUs at such low use rates.