The mode of action of Glyphosate-based herbicides is unique. Glyphosate inhibits the action of the enzyme 5-enolpyruvoylshikimate-3-phosphate synthetase (EPSPS) which is involved in the biosynthesis of essential aromatic compounds, such as the aromatic amino-acids. Due to the inability of the plants to produce these compounds they die. This pathway is not present in birds, fish, insects or mammals and so they remain unaffected.

The EPSPS is involved in catalysing the transfer of the carboxyl vinyl part of phosphoenolpyruvate (PEP) to the 5-OH of S3P producing EPSP and inorganic phosphate. The EPSPS substrates are multiply charged charged anions and for maximum binding all anionic sites must be exposed.Similarly glyphosate is optimally active in dianionic form. Glyphosate competitively inhibits PEP and uncompetitively inhibits S3P. Neither PEP or glyphosate has any significant interaction with EPSPS unless S3P is present. In the presence of S3P glyphosate binds irreversibly to the enzyme.

Glyphosate displays a unique affinity for EPSPS and does not interfere with the action of any other enzyme which uses PEP as a substrate which means that glyphosate cannot behave simply as a ground state mimic for PEP. Minor changes in the structure of glyphosate can reduce its ability to inhibit the activty of EPSPS. There is mechanistic evidence that the catalysis proceeds via the protonation of the PEP double bond and so it is thought that glyphosate behaves as a transition state analogue of the transient PEP oxonium ion.

The glyphosate binding site is very specific and it is obvious that glyphosate and the PEP oxonium ion are chemically not very similar. The glyphosate can, however, (due to its flexibility of chain) adopt a conformation of low energy which is quite similar in three dimensional structure to the PEP oxonium ion. This occurs when the phosphonic acid of PMG is overlapped with that in the PEP oxonium ion and the two carboxyl groups are placed in corresponding positions (as shown below).

As a result of the high specifity of the binding site, in EPSPS, glyphosate and it's EPSPS inhibitting derivatives form a very small group of compounds. In fact there are only two, very similar, analogues of glyphosate which are as effective at inhibitting the action of EPSPS to the same extent as glyphosate. These are N-hydroxy-glyphosate and N-amino-glyphosate.

It has been hypothesised that the only glyphosate-based herbicidal compounds which act via EPSPS inhibition are those which breakdown, for what ever reason, to form the glyphosate anion. This means that any alteration of the glyphosate backbone can destroy or seriously inhibit the EPSPS inhibitting ability (herbicidal activity) of the molecule.Thus, backbone modified glyphosate derivatives are very rare in the patents literature and generally only those molecules which deliver glyphosate through various hydrolytically unstable amides and esters make up the majority of the patents involving glyphosate analogues.