Reactivity-based identification of oxygen containing functional groups of chemicals applied as potential classifier in non-target analysis

In this work, we developed a reactivity-based strategy to identify functional groups of unknown analytes, which can be applied as classifier in non-target analysis with gas chromatography. The aim of this strategy is to reduce the number of potential candidate structures generated for a molecular formula determined by high resolution mass spectrometry. We selected an example of 18 isomers with the molecular formula C12H10O2 to test the performance of different derivatization reagents, whereas our aim was to select mild and fast reaction conditions. Based on the results for the isomers, we developed a four-step workflow for the identification of functional groups containing oxygen.

SI1. Details on the suppliers of the isomers with empirical formula C12H10O2.S1 SI3.Supplementary discussion -a mechanistic discussion of reactions with the selected derivatization reagents.

Methylation of [OH, COOH, COC, CHO] via TMSH (19)
As a derivatization reagent, TMSH 19 is used as a "methyl group donor" 1 .The reagent proved to have a simple reaction procedure at room temperature and a high effectiveness for the methylation of most functional groups (hydroxyl-, aldehyde-and keto-groups as well as carboxylic acids).Generally, the derivatization of one functional group via methylation led to an ∆M of +14 Da and a new peak at m/z 200 Da.Double methylation was possible when two functional groups were present in the analyzed structure, such as for biphenyls 1, 3, 4, 16, and 17.In this case, two methyl groups were added, leading to an ∆M of +28 Da and the product peak at m/z 214 Da.
Methylation generally occurs under strong basic conditions.For example, the hydroxyl group reaction mechanism can be found in Supplementary Figure S3-1 According to Gries et al. 2 , the methylation of carboxylic acids via TMSH 19 (Supplementary Figure S3-2) follows a similar reaction mechanism as the previously explained methylation of hydroxyl groups.
Again, the hydroxyl anion serves as a proton acceptor, while the produced nucleophile attacks the trimethyl sulfonium cation.The formed carboxylic ester 28 leads to a product peak at m/z 200 Da.In the case of carboxylic acid 10, a second peak at m/z 214 Da, representing double methylation, was even observed.A possible explanation would be a tautomerization, producing a free hydroxyl group which can then subsequently be methylated as well, leading to the product 32.As tautomerization for carboxylic acids is generally less favored, only a fraction of the initial compound showed doublemethylation, producing two peaks in the chromatographic spectrum at 10.894 min and 11.060 min, respectively. 3 Supplementary Figure S3-2.Proposed reaction mechanism for the methylation of carboxylic acids (COOH) via TMSH 19 on the example of naphthalen-2-yl acetic acid 10.
Interestingly, aldehyde and keto groups were also methylated in some form.For aldehyde functionalities, the product peak was measured at m/z 232 Da, leading to an addition of +46 Da.Here, the free hydroxyl anion can start a nucleophilic attack at the electrophilic carbon in the aldehyde functionality, leading to the presence of two hydroxyl groups, which both can be methylated by TMSH 19, following the reaction mechanism in Supplementary Figure S3-3 A similar mechanism may also hold for ketones, keeping in mind that the first step, the addition of the OH -to the carbonyl carbon, is less efficient as compared to aldehydes because of steric and electronic effects of the additional alkyl group.This lower reactivity of ketones may require higher TMSH concentration to yield sufficient turnover rates.Ketones that can be converted into corresponding enols such as substrate 6 can be methylated in analogy to alcohols (Supplementary Figure S3-4 According to Chen et al. 9 , methylation of aryl methyl ketones via enaminone-formation should also be possible using DMF-DMA 22 assisted by Pd/C-catalyzed hydrogenation.However, no methylation has been observed for the applied reaction conditions in this work.

Hydrolysis of [COOR] via NH4OH (23)
As a selective procedure for carboxylic ester functionalities, a simple base-catalyzed hydrolysis reaction can be used, as seen in Supplementary Figure S3-11.The reaction follows an SN2 pathway, with product formation being irreversible.At first, the free hydroxyl anion can attack as a nucleophile at the electrophilic center of the carboxylic ester, producing a tetrahedral intermediate.The intermediate collapses to regain the CO double bond by releasing an alkoxide as a leaving group and forming a . Free hydroxyl anions from the derivatizing reagent remove the acidic proton from the functional group.The produced nucleophile can form a transition state with the remaining trimethyl sulfonium cation.A thioether bond is formed by nucleophilic attack, which is subsequently cleaved to form the methylated product 31 and dimethyl sulfide as a by-product.By derivatization of the second hydroxyl group, the double methylated product 26 is formed. 1 Supplementary Figure S3-1.Proposed reaction mechanism for the methylation of hydroxyl groups (OH) via TMSH 19 on the example of biphenyl-2,2'-diol 1.

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Double methylation led to an ∆M of +46 Da and subsequently to an m/z of 232 Da in product 34.The base peak at m/z 201 Da is possibly caused by the loss of CH3O + from the methyl esters present.The differences measured between the two aldehydes 9 and 13 can be explained due to steric hindrance.Supplementary FigureS3-3.Proposed reaction mechanism for the methylation of aldehyde groups (CHO) via TMSH 19 on the example of 4-methoxynaphtalene-1-carbaldehyde 9.
) and are expected to generate adducts with Δm = 14 Da (product 35).In case of product 35, further ketoenol tautomerization allows for a second methylation leading to product 36 with m/z of 200 Da.For Aldehyde functionalities, double methylation leading to a mass-to-charge ratio of 232 Da has been found, as for methylation with TMSH 19.During derivatization with DMF-DMA 22, the produced MeOH from the formation of the alkoxyimmonion cation 38 in 1) can start a nucleophilic attack on the electrophile carbon of the aldehyde group in 2).The formed carboxylate anion can then react after a similar formylation reaction mechanism as hydroxyl and carboxyl acid functionalities.Again, the base peak at a mass-to-charge ratio of 201 Da can be based on the loss of a CH3O + fragment from the methyl ester groups produced (Supplementary FigureS3-10).Supplementary FigureS3-10.Proposed reaction mechanism for the methylation of aldehyde groups (CHO) via DMF-DMA 22 on the example of 4-methoxynaphtalene-1-carbaldehyde 9.

Table S2 .
Derivatizing reagents with their corresponding CAS number and supplier.