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3 Sample EROS Runs

In order to show the user the range of applications that can be made with EROS and to make her/him more familiar with running EROS, a series of sample runs are provided.
These studies include
- an introduction into the problem - a discussion of the essential elements of the reaction rules and how they are derived
- the input specifications
- the corresponding reaction rules
- the input file for running EROS

The examples start with a simple reaction without any reactivity evaluation (3.1), then cover combinatorial chemistry experiments, again without any evaluation of the reactions (3.2). A further example illustrates how EROS can be used for the exhaustive generation of isomers, (3.3).
Then, reactions are presented that include an evaluation of chemical reactivity. First, a simple laboratory reaction is presented (3.4). Then, the degradation of chemicals in the environment is dealt with (3.5). Next, the breakdown of a molecule in the mass spectrometer is modeled to simulate a mass spectrum (3.6).
The final example shows the combination of two reaction environments, a laboratory synthesis followed by the mass spectroscopy of the starting materials and reaction products (3.7).

3.1 The Synthesis of an Amide

Essentials to learn:
how the EROS system works
reactions as bond rearrangements
the use of phases

Description:
This example shows how the EROS system can model a simple reaction like the synthesis of an amide. In this example, acetic acid reacts with methylamine to give an amide. Figure 3-1 shows which bonds are broken and which ones are made in this reaction.

Figure 3-1. Amide synthesis from acetic acid and methylamine.

Clearly, the reaction proper does not follow this pathway, as an acid and an amine gives a salt in a proton transfer reaction. The reaction shown in Figure 3-1 only models the net result of an amide synthesis from an acid and an amine which has to be initiated by activation of the acid as an acid chloride or an ester. However, as no evaluation of chemical reactivity is attempted in this example, such a treatment of the overall changes in a sequence of reaction steps is allowed.
First, all the starting materials are put into phase 1. Then, the constraints in RULE_21 check if one of the chemicals is an acid. If there is an acid, it is moved into phase 2. In the same way, an amine is moved into phase 3.
Then, RULE_32 makes the rearrangement of atoms and bonds and transfers the products into phase 4 (mode: INERT). Phase 3 (mode: SURFACE) is in contact with phase 2 (mode: INERT) and generates the reaction by taking a molecule from phase 2 and a molecule from phase 3.
Finally, an output file is generated.

Figure 3-2. Scheme of phases needed in the reaction of acetic acid with methylamine.

Main features of the reaction rule file:

Rule header:
number of reactors: 1
number of phases: 4
mode of phase 1: MONOMOLEC
mode of phase 2: INERT
mode of phase 3: SURFACE
mode of phase 4: INERT

Reaction rules:

Check for acid group and transport acids into phase 2

Check for amino group and transport amines into phase 3

Amide formation

Reaction Generator:
Reaction levels: 1
Trace level: 0

Reaction rule file and input file:
The reaction rule and the CTX-input are contained as files (amide.tcl and amide1.ctx) on the CD-ROM for the distribution of the EROS system.

3.2 Combinatorial Chemistry

In this section, the use of EROS for modeling Combinatorial Chemistry experiments is explained.
In Combinatorial Chemistry two or more sets of molecules react with each other. Each molecule of the first group reacts with each molecule of the second group to give in all possible combinations of reactions all conceivable combinations of products. This can be achieved in a variety of experimental set-ups such as in parallel synthesis or liquid phase experiments.

3.2.1 The Synthesis of Esters and Amides

Essentials to learn:
how to set up the modeling of combinatorial chemistry experiments
the concept of phases in parallel synthesis
a reaction rule that can be applied both to the synthesis of amides and esters

Description:
The first example is the combinatorial synthesis of esters and amides.
The following reactions can be handled
acids or acid chlorides and amines react to amides
acids or acid chlorides and alcohols react to esters

First, the starting materials are put into phase 1 that has the mode MONOMOLEC.
From this phase, acids and acid chlorides are moved into phase 2 (mode: INERT). This is caused by the constraints in RULE_21 and RULE_22 in the reaction rule file. The constraints check if the molecule has an acid group or an acid chloride group. The amines and alcohols are moved into phase 3 (RULE_23 and RULE_24 in the reaction rule file; the constraints check for the presence of an NH2 - group or an OH - group). The phase 3 has the mode SURFACE and the phase 4 the mode INERT (Figure 3-3).

Figure 3-3. Combinatorial synthesis of amides and esters.

The amines and alcohols from phase 3 (mode: SURFACE) react with the acids and acid chlorides of phase 2 (mode: INERT) that is in contact with phase 3 (RULE_32 in the reaction rule file). The constraints in this rule search for the reaction substructures that are needed to give amides and esters. Then a simple rearrangement of atoms and bonds take place (Figure 3-4). The products arrive in phase 4 that has the mode INERT. Phase 4 is the output phase. The content of the reaction output file are all reactions made in this EROS run, the moves of the starting materials from phase 1 to phase 2 and 3, too.

Figure 3-4. Bond rearrangement and constraints on the atoms of the reaction type used.

The output file for the structures comprises all products from the output phase 4.
The two CTX output files can be watched with the CACTVS browser csbr.

Main features of the reaction rule file:

Rule header:
number of reactors: 1
number of phases: 4
mode of phase 1: MONOMOLEC
mode of phase 2: INERT
mode of phase 3: SURFACE
mode of phase 4: INERT
(see chapter 1.3.1.3 and 3.1)

Reaction rules:

Check for acid group and transport acids into phase 2

Check for acid chloride group and transport acid chlorides into phase 2

Check for amine group and transport amines into phase 3

Check for hydroxyl group and transport alcohols into phase 3

Condensation (see Figure 3-4)

Reaction Generator:
Reaction levels: 1
Trace level: 0

Reaction rule file and input file:
The reaction rule and the CTX-input are contained as files (amide.tcl and amide2.ctx) on the CD-ROM for the distribution of the EROS system.

3.2.2 The Synthesis of Pyrazoles

Essentials to learn:
How to code extensive bond rearrangements

Description:
In combinatorial chemistry different sets of starting materials are combined in all conceivable variations to synthesize a wide range of compounds.
Pyrazoles can be obtained from 1,3-diketones and substituted hydrazines.

Figure 3-5. Synthesis of pyrazoles.

In terms of number of bonds broken and made, this reaction involves quite an extensive electron rearrangement. Such extensive reaction schemes can also be coded in a reaction rule.
In a combinatorial chemistry experiment two or more sets of starting materials react in such a way as to react each molecule from set one with each molecule of set two, etc. In our example, a set of two 1,3-diketones will be reacted with three substituted hydrazines.
The two sets of starting materials are shown in Figure 3-6.


Figure 3-6. Starting materials of the combinatorial synthesis of pyrazoles.

After input of these five molecules, they are assigned to two different phases depending on the substructure they contain: The 1,3-diketones are assigned to phase 2 whereas the hydrazines are put into phase 3. Then, from each phase one molecule is taken out one at a time and reacted with a molecule of the other phase. As unsymmetrical 1,3-diketones can give rise to two different pyrazoles, (see Figure 3-5) both ways of combining 1,3-diketones with a hydrazine are explored. The products are stored in phase 4 (Figure 3-7).

Figure 3-7. Scheme of phases for the synthesis of pyrazoles.

The results of this program run are shown in Figure 3-8.

Figure 3-8. Products of the combinatorial synthesis of pyrazoles.

Main features of the reaction rule file:

Rule header:
number of reactors: 1
number of phases: 4
mode of phase 1: MONOMOLEC
mode of phase 2: INERT
mode of phase 3: SURFACE
mode of phase 4: INERT
(see chapter 1.3.1.3 and 3.1)

Reaction rules:

Check for the substructure of 1,3-diketones (see Figure 3-5) and transport 1,3-diketones into phase 2

Check for the substructure of hydrazines (see Figure 3-5) and transport hydrazines into phase 3

Pyrazole formation (see Figure 3-5)

Reaction Generator:
Reaction levels: 1
Trace level: 0

Reaction rule file and input file:
The reaction rule and the CTX-input are contained as files (pyrazole.tcl and pyrazole.ctx) on the CD-ROM for the distribution of the EROS system.

3.2.3 1,4-Benzodiazepines

Essentials to learn:
How to set up the modeling of multistep parallel synthesis

Description:
Whereas the previous example of the synthesis of pyrazoles deals with a one-step reaction, the example given here deals with a two-step synthesis. In particular, an experiment by Ellman and coworkers [24] for the synthesis of 1,4-benzodiazepines (see Figure 3-9) will be analyzed.

Figure 3-9. Synthesis of 1,4-benzodiazepines by Ellman and coworkers.[24]

Three sets of starting materials are necessary to perform this sequence, 2-aminobenzophenones, amino acids, and alkylating agents. For modeling this experiment with EROS, the two 2-aminobenzophenones, three amino acids, and three alkylating agents shown in Figure 3-10 were chosen.



Figure 3-10. Starting materials of the combinatorial synthesis of 1,4-benzodiazepines.

The entire reaction sequence is modeled by six phases (see Figure 3-11), one phase for storing all starting materials, three phases for storing the three different sets of these starting materials, and two phases for performing the two major reaction steps, the combination of the 2-aminobenzophenone with an amino acid followed by cyclisation to 1,4-benzodiazepines, and the alkylation step at nitrogen-1 of this ring system.

Figure 3-11. Scheme of phases for the synthesis of 1,4-benzodiazepines.

Figure 3-12 shows the structures obtained in this combinatorial chemistry experiment. All 18 conceivable structures (2 x 3 x 3) were obtained in this run.

Figure 3-12. Products of the combinatorial synthesis of 1,4-benzodiazepines.

Main features of the reaction rule file:

Rule header:
number of reactors: 1
number of phases: 6
mode of phase 1: MONOMOLEC
mode of phase 2: INERT
mode of phase 3: SURFACE
mode of phase 4: INERT
mode of phase 5: SURFACE
mode of phase 6: INERT
(see chapter 1.3.1.3)

Reaction rules:

Check for the substructure and transport 2-aminobenzophenones into phase 2:

Check for the substructure and transport amino acids into phase 3:

Attention: Don´t use asparagine (Asn) or glutamine (Glu) as amino acids, because the alkylating agents react with the free amide groups of Asn and Glu in the non-alkylated benzodiazepines, too.

Check for a chlorine, bromine or iodine atom with a bond to an aliphatic carbon atom and transport of alkylating agents into phase 4

Ring closure and formation of an unsubstituted 1,4-benzodiazepine:

Alkylating reaction:

Reaction Generator:
Reaction levels: 2
Trace level: 0

Reaction rule file and input file:
The reaction rule and the CTX-input are contained as files (diazep.tcl and diazep.ctx) on the CD-ROM for the distribution of the EROS system.

3.3 Exploration of all Potential Reaction Products: Chlorination of Benzodioxine

Essentials to learn:
Exhaustive exploration of all reaction products
Suppression of duplicate reaction products

Description:
This example shows how EROS can be applied to the exhaustive generation of isomers for a given problem. This is illustrated here with the generation of all mono-, di-, all the way to octasubstituted chloro-dibenzodioxines.

Figure 3-13. Chlorination of benzodioxine.

The first reaction step generates monochlorobenzodioxines, the second dichlorobenzodioxines, etc. Each reaction step is taken care of by an individual phase of the reactor used; the first two phases are needed for the initial storage of the starting materials.

Figure 3-14. Scheme of phases for the chlorination of tetra-chlorinated benzodioxines.

No evaluation of the chlorination of benzodioxine and its substituted derivates is performed as only all possible substituted derivates should be obtained. It is not aspired to estimate the relative ratios of these chlorinated compounds under certain reaction conditions.
The following result is obtained:

2

monochlorobenzodioxines

10

dichlorobenzodioxines

14

trichlorobenzodioxines

22

tetrachlorobenzodioxines

14

pentachlorobenzodioxines

10

hexachlorobenzodioxines

2

heptachlorobenzodioxines

1

octachlorobenzodioxines

This example also shows that the methods for unique identification of a compound, in our case, a hashcode algorithm,[25] work correctly as the one and the same compound can be obtained by different reaction pathways but it will be output only once.

Main features of the reaction rule file:

Rule header:
number of reactors: 1
number of phases: 10
mode of phase 1: INERT
mode of phase 2 - 9: SURFACE
mode of phase 10: INERT
(see chapter 1.3.1.3)

Reaction rules:

Reaction Generator:
Reaction levels: 8
Trace level: 0

Advanced Options (optional):
To break off the reaction network transfer an integer variable named phase_inc with the value of 1 to the rule file (see chapter 2.3). The output file contains only not fully chlorinated benzodioxines.
For example: To get all pentachlorobenzodioxines set phase_inc to the value of 1, set the last phase generating reactions to the value of 6, and set the standard output phase to the value of 7 (see Figure 3-14).

Reaction rule file and input file:

The reaction rule and the CTX-input are contained as files (dioxin.tcl and dioxin.ctx) on the CD-ROM for the distribution of the EROS system.

3.4 Multistep Laboratory Reaction: Bromination of Phenol

Figure 3-15. Bromination of phenol.

Essential to learn:
How to derive rules for calculating reaction rates

Description:
This reaction is run in a single vessel, the concentrations of starting materials are such that multiple reactions between the different starting materials might occur. The following specifications are made: one reactor, one phase, reaction mode: MIX.
Next, a reaction rule for the bromination of phenol had to be developed and stored in the knowledge base of the EROS system. The reaction center, i.e., the bonds broken and made in the reaction, was specified as shown in Figure 3-16.

Figure 3-16. Bond rearrangement in the reaction of phenol with Br2.

The following restrictions were imposed onto the carbon atom: it has to be part of an aromatic system, where an oxygen or nitrogen atom has to be conjugated to in a distance of two or four bonds. These constraints are valid for carbocyclic aromatic systems. To also use the reaction rule for heteroaromatic systems, the constraints would have to be adapted.
In order to make quantitative predictions, mechanisms for the estimation of the relative rates of bromination at the various positions of phenol have to be given. The following observations were used: Bromination of phenol gives about 80% p-bromophenol and 20% o-bromophenol,[26] allowing the conclusion that bromination in para-position is eight times faster than bromination in ortho-position (there are two ortho-positions !). As no absolute second order rate constants were available, bromination in ortho-position was set to 0.01 l/s•mole and in para-position to 0.08 l/s•mole. In order to account for the influence of a bromine substituent onto the rate of further bromination recourse was made to the following observation: The rate of nitration of bromobenzene is 3% of the rate of nitration of benzene.[27] It was therefore assumed that with each bromine substituent also the rate of bromination drops to 3% the rate without this additional bromine substituent.
With these rate constants the integration of the differential equations in the kinetic modeling was performed by the Gear algorithm.[21] Figure 3-17 shows the sequence of reaction products obtained in this reaction modeling. Figure 3-18 reproduces the results of the kinetic modeling of this system of reactions.

Figure 3-17. Reaction scheme of the bromination of phenol.

Figure 3-18. Concentration vs. time plot of the bromination of phenol.

Main features of the reaction rule file:

Rule header:
number of reactors: 1
number of phases: 1
mode of phase 1: MIX
kinetic of reactor 1: gear

initial concentrations:

Reaction rules:

Bromination (see Figure 3-16)
Deactivating substituents are not taken into account.

Reaction Generator:
Reaction levels: 3
Trace level: 0

Reaction rule file and input file:

The reaction rule and the CTX-input are contained as files (brphen.tcl and brphe.ctx) on the CD-ROM for the distribution of the EROS system.

3.5 Degradation of Chemicals in the Environment: Degradation of Atrazine and Prometon

Figure 3-19. Degradation of s-triazines

Essentials to learn:
how to derive rates for evaluating reaction rates from half-life times
exhaustive exploration of degradation products
how to deal with the multiple application of a chemical into the environment

Description:
The major degradation reactions of s-triazine herbicides such as prometon or atrazine under anaerobic conditions in soil are reductive dealkylation and hydrolysis. The concentration of the herbicides is generally at rather low concentration so that no reaction between the triazines will occur. Thus, no reactions of this chemical with other molecules of its kind have to be considered, but only those with chemicals having high concentration in the environment such as water or oxygen, or, in this case, bacteria that perform reductive dealkylation which is modeled by using hydrogen as a starting material. The general specifications for modeling these reactions were therefore: one reactor, one phase in the mode: monomolecular (which, in this case, corresponds to a pseudo-monomolecular process).
The two reaction types shown in Figure 3-20 were included in the knowledge base. Figure 3-21 shows the sequence of degradation reactions obtained for s-triazine herbicides with these two reaction types.

Figure 3-20. Degradation reactions of s-triazines in soil.

Figure 3-21. Reaction scheme of the degradation of s-triazines in soil.

The concentration dependence of the materials produced in this sequence of steps of pseudo first order rates is shown in Figure 3-22.

Figure 3-22. Concentration vs. time plot of the degradation of prometon.

a) Single application of a herbicide

Main features of the reaction rule file:

Rule header:
number of reactors: 1
number of phases: 1
mode of phase 1: MONOMOLEC
kinetic: gear

initial concentrations:

Reaction rules:

Hydrolysis

Reductive dealkylation

Hydrolysis of cyanuric acid

Decarboxylation

Reaction Generator:
Reaction levels: 9
Trace level: 0

Reaction rule file and input file:
The reaction rule and the CTX-input files are contained as files (triazine.tcl, atraz.ctx and promet.ctx) on the CD-ROM for the distribution of the EROS system.

Note: The displayed reaction time in the concentration-time plot may be less than the wished reaction time (see chapter 5.3).

b) Multiple application

This example also shows how to deal with reactions of chemicals that are regularly applied such as drugs or, as with this example, plant protection agents. It is assumed that prometon is regularly applied once a year. The development of the degradation products over time is shown in Figure 3-23.

Figure 3-23. Concentration vs. time plot of the degradation of prometon applied every year.

Main features of the reaction rule file:

If you transfer a variable named multi to the rules (e.g.: multi#1, the value will not be recognized) the application of the triazine is enabled every year. Together with this the reaction time is set to 1.10376e8 seconds (3.5 years) and the conversion limit is set to 1.01, so that it cannot be reached at all.
In both cases, single application (without multi) and multiple application (with multi#1), you can specify the reaction time and the initial concentration of the triazine, which is added every year in the case of multiple application.
Therefore set the concentration e.g. with conc:0.001 with the advanced options. Due to a slight inconsistency in the Tcl interface (plus signs are not accepted in a number; see chapter 5.1.4) and the fact that Tcl does not have different data types, specify huge numbers as character strings, which are given to the rules. This is true for the reaction time (rxtime \$2.1e8). The backslash in front of the $ sign is necessary because of the shell wrapper which is called from the GUI. Additionally you can set the conversion limit in the case of a single application with e.g. conv_lim:0.5.

3.6 Simulation of Mass Spectra

The mass spectrum of N-methyl-N-propyl-butylamine

Essentials to learn:
How to simulate mass spectra
The reaction rules for mass spectra simulation
The evaluation of a reaction network based on the probability of reaction steps

Description:
The mass spectra that can be simulated with EROS7 refer to 70eV EI mass spectra. This is so because the evaluation mechanisms in EROS7 have been derived from a database of 70eV EI mass spectra. At present, the quality of the simulated mass spectra is, in most cases, still quite unsatisfactory. This is mainly due to the limited set of reaction types included in the rule file. In fact, apart from ionization, only four fragmentation types (Figure 3-24) are included in the rule file.

Figure 3-24. Fragmentation types included in the rule file.

These fragmentation reactions are mainly valid for aliphatic compounds; specific fragmentation reactions of aromatic systems are conspicuously absent. Even for aliphatic systems only the major fragmentation reactions are considered; some important reactions such as hydrogen rearrangements or inductive cleavage are not yet accounted for. The reason is that good evaluation mechanisms for hydrogen rearrangements have not yet been developed. Even for the fragmentation types shown in Figure 3-24, the evaluation is based on work that has been performed five years ago.[5]
Having said this, it is nevertheless true that for many monofunctional aliphatic compounds the major peaks in the mass spectrum can be reproduced. Even the peak intensities often are quite in agreement with experiment giving support to the mathematical basis for calculating peak intensities on the basis of estimations of probabilities for the individual fragmentation steps.
The example discussed here deal with the simulation of the mass spectrum of a simple monofunctional aliphatic compound, the trialkylamine N-methyl-N-propyl-butylamine.
The resulting simulated mass spectrum is shown in Figure 3-25.

Figure 3-25. Simulated and experimental mass spectrum of N-methyl-N-propyl-butylamine.

If you switched on writing the file with the mass spectra, an additional button will appear to view the simulated mass spectra in the result section of the EROS GUI.

Main features of the reaction rule file:

Rule header:
number of reactors: 1
number of phases: 1
mode of phase 1: MONOMOLEC
kinetic: prob_kin

Reaction rules:

Ionization (see Figure 3-24)

Alpha cleavage (see Figure 3-24)

Onium reaction (see Figure 3-24)

Carbonyl elimination (see Figure 3-24)

McLafferty reaction (see Figure 3-24)

Reaction Generator:
Reaction levels: more than 3
Trace level: 0

Advanced Options:
In this example it is possible to use an additional reaction type by switching on hydrogen rearrangements with a row reactivity specification. It does not take the differences due to the size of the molecule in account. Click on the Advanced Options Button in the Data Input area of EROS7 - User Interface (EROS - GUI) and pass a variable called huml with the value 1 (type ‘huml#1’) to the rule file. (Reaction levels: more than 9).
Attention: If you switch on the hydrogen rearrangement many more reactions are simulated ans so the EROS run take quite longer!

Data Output:
Mass spectra: Name of the file that should contain the mass spectra (only produced, if the MS file is switched on).

Reaction rule file and input file:
The reaction rule and the CTX-input are contained as files (ms.tcl and ms1.ctx) on the CD-ROM for the distribution of the EROS system.

3.7 Working with Two Reactors: Acetal Formation from Propanal and Ethanol and Subsequent Simulation of the Mass Spectra

Essentials to learn:
Working with two reactors
Combining a laboratory reaction with the simulation of mass spectra

Description:
This example serves to illustrate how a combination of two reactors can be used. The first reactor performs a laboratory reaction, the second models the events in a mass spectrometer.
The reaction chosen is the formation of an acetal from propanal and ethanol (Figure 3-26).

Figure 3-26. Formation of an acetal and simulation of a mass spectrum.

The following reaction types have been coded for this synthesis.

Figure 3-27. Reaction types in the rule file for the first reactor.

Each reaction product (and each starting material) is individually handed over to the second reactor in order to have its mass spectrum simulated (see Figure 3-26).
The reaction types used for the simulation of mass spectra are the same as in the previous example (see Figure 3-24).
The sequence of reaction steps generated in the first reactor is shown in Figure 3-28.

Figure 3-28. Sequence of reaction steps in the acetal formation.

The mass spectra simulated for the two starting materials are shown in Figure 3-29 and compared with their experimental mass spectra as taken from the MassLib spectra database.[28]

Figure 3-29. Mass spectra of starting materials.

The comparison of the simulated with the experimental [28] mass spectra of the products in this reaction sequence is made in Figure 3-30.

Figure 3-30. Mass spectra of products.

Note, that the experimental mass spectra are not included on the CD.

Main features of the reaction rule file:

Rule header:
number of reactors: 2
number of phases in the first reactor: 1
number of phases in the second reactor: 1
mode of phase 1 in reactor 1: MIX
mode of phase 2 in reactor 2: MONOMOLEC
kinetic in reactor 1: gear

kinetic in reactor 2: prob_kin

Reaction rules:

Ionization (see Figure 3-24)

Alpha cleavage (see Figure 3-24)

Onium reaction (see Figure 3-24)

Carbonyl elimination (see Figure 3-24)

McLafferty reaction (see Figure 3-24)

Formation of the acetal (see Figure 3-27)

Reaction Generator:
Reaction levels: 5
Trace level: 0

Advanced Options:
In this example it is also possible to use an additional reaction type by switching on hydrogen rearrangement. Click on the Advanced Options Button in the Data Input area of EROS7 - User Interface (EROS - GUI) and pass a variable called huml with the value 1 (type ‘huml#1’) to the rule file. (Reaction levels: more than 10).

Data Output:
Mass spectra: Name of the file that should contain the mass spectra

Reaction rule file and input file:
The reaction rule and the CTX-input are contained as files (mixms.tcl and mixms.ctx) on the CD-ROM for the distribution of the EROS system.

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Prof. Dr. J. Gasteiger
Computer Chemie Centrum, Org. Chem., Uni. Erlangen
Nägelsbachstraße 25
D-91052 Erlangen

Gasteiger@CCC.Chemie.Uni-Erlangen.DE