Nucleophilic Substitution at Tricoordinate Sulfur—Alkaline Hydrolysis of Optically Active Dialkoxysulfonium Salts: Stereochemistry, Mechanism and Reaction Energetics

Optically active dialkoxyisopropylsulfonium salts were obtained by methylation (ethylation) of optically active alkyl isopropanesulfinates using methyl (ethyl) trifluoromethanesulfonate. Alkaline hydrolysis of a series of methoxy(alkoxy)sulfonium salts afforded the two sulfinate products methyl isopropanesulfinate and alkyl isopropanesulfinate, both formed with a slightly prevailing inversion of configuration at the sulfur atom. DFT calculations revealed that this substitution reaction proceeded stepwise according to an addition-elimination (A–E) mechanism involving the formation of high tetracoordinate SIV sulfurane intermediates. In addition, the DFT calculations showed that recombination of the hydroxy anion with the methoxy(alkoxy)sulfonium cation—leading to the parallel formation of the two most stable primary sulfuranes, with the hydroxy and alkoxy groups in apical positions and their direct decomposition—is the most energetically favorable pathway.

Introduction
The majority of reactions with heterorganic compounds (H=P, S, Si, Se . . . ) are typical bimolecular nucleophilic substitution reactions of the S N 2-H type, occurring at heteroatom centers. The stereochemical course and mechanisms of these reactions have been a subject of extensive studies in the second half of the past century. It has been found that in the overwhelming majority of cases, they proceed with inversion of configuration at the stereogenic phosphorus and sulfur atoms. Although the observation of inversion may be rationalized by the concerted S N 2-H mechanism via a transition state, an alternative addition-elimination mechanism (A-E) involving the formation of trigonal bypiramidal intermediates (P V -phosphorane and S IV -sulfurane) has also been considered.
From that time, the other nucleophilic substitution reactions occurring with retention of configuration at the heteroatom centers (see, for example, [3,4]) have been rationalized in terms of the Westheimer's concept and the addition-elimination mechanism, involving the formation of high-coordinate intermediates and their pseudorotation. However, it should be pointed out here that these transiently formed intermediates have not been isolated or detected by spectroscopic methods due to their instability. Moreover, in this early period of studies on nucleophilic substitution reactions at heteroatoms, it was not possible to make a distinction between the SN2-H and the A-E mechanisms. A solution of this fundamental question was especially interesting for the identity substitution reactions at the chiral phosphorus and sulfur atoms. Taking advantage of the results of theoretical studies on substitution reactions at heteroatoms (P, S, Si) in simple achiral model compounds [5][6][7][8], we performed [9] the DFT calculations for the three symmetrical displacement reactions in the optically active phosphorus and sulfur compounds, as shown below ( Figure 1).
The kinetic measurements provided direct evidence that each elementary substitution step in these reactions occurs with a full inversion of configuration at the phosphorus and sulfur [10][11][12]. Now it has been found [9] that the methoxy-methoxy exchange reactions in phosphinate 4 and sulfinate 6 proceed in a stepwise fashion via the corresponding P V -phosphorane and S IV -sulfurane, respectively. The latter intermediates with the two methoxy groups in apical positions are the most stable, do not undergo pseudorotation and decompose directly to the starting compounds 4 and 6, but with opposite configurations at P and S. On the other hand, the chloride-chloride exchange at phosphorus in 5 is a typical concerted SN2-P process. From that time, the other nucleophilic substitution reactions occurring with retention of configuration at the heteroatom centers (see, for example, [3,4]) have been rationalized in terms of the Westheimer's concept and the addition-elimination mechanism, involving the formation of high-coordinate intermediates and their pseudorotation. However, it should be pointed out here that these transiently formed intermediates have not been isolated or detected by spectroscopic methods due to their instability. Moreover, in this early period of studies on nucleophilic substitution reactions at heteroatoms, it was not possible to make a distinction between the S N 2-H and the A-E mechanisms. A solution of this fundamental question was especially interesting for the identity substitution reactions at the chiral phosphorus and sulfur atoms. Taking advantage of the results of theoretical studies on substitution reactions at heteroatoms (P, S, Si) in simple achiral model compounds [5][6][7][8], we performed [9] the DFT calculations for the three symmetrical displacement reactions in the optically active phosphorus and sulfur compounds, as shown below ( Figure 1). Scheme 1. Stereochemical course of substitution at the phosphorus in cyclic five-membered thiophosphoryl chlorides.
From that time, the other nucleophilic substitution reactions occurring with retention of configuration at the heteroatom centers (see, for example, [3,4]) have been rationalized in terms of the Westheimer's concept and the addition-elimination mechanism, involving the formation of high-coordinate intermediates and their pseudorotation. However, it should be pointed out here that these transiently formed intermediates have not been isolated or detected by spectroscopic methods due to their instability. Moreover, in this early period of studies on nucleophilic substitution reactions at heteroatoms, it was not possible to make a distinction between the SN2-H and the A-E mechanisms. A solution of this fundamental question was especially interesting for the identity substitution reactions at the chiral phosphorus and sulfur atoms. Taking advantage of the results of theoretical studies on substitution reactions at heteroatoms (P, S, Si) in simple achiral model compounds [5][6][7][8], we performed [9] the DFT calculations for the three symmetrical displacement reactions in the optically active phosphorus and sulfur compounds, as shown below ( Figure 1).
The kinetic measurements provided direct evidence that each elementary substitution step in these reactions occurs with a full inversion of configuration at the phosphorus and sulfur [10][11][12]. Now it has been found [9] that the methoxy-methoxy exchange reactions in phosphinate 4 and sulfinate 6 proceed in a stepwise fashion via the corresponding P V -phosphorane and S IV -sulfurane, respectively. The latter intermediates with the two methoxy groups in apical positions are the most stable, do not undergo pseudorotation and decompose directly to the starting compounds 4 and 6, but with opposite configurations at P and S. On the other hand, the chloride-chloride exchange at phosphorus in 5 is a typical concerted SN2-P process. The kinetic measurements provided direct evidence that each elementary substitution step in these reactions occurs with a full inversion of configuration at the phosphorus and sulfur [10][11][12]. Now it has been found [9] that the methoxy-methoxy exchange reactions in phosphinate 4 and sulfinate 6 proceed in a stepwise fashion via the corresponding P V -phosphorane and S IV -sulfurane, respectively. The latter intermediates with the two methoxy groups in apical positions are the most stable, do not undergo pseudorotation and decompose directly to the starting compounds 4 and 6, but with opposite configurations at P and S. On the other hand, the chloride-chloride exchange at phosphorus in 5 is a typical concerted S N 2-P process.
In the hope of gaining further insight into the mechanism of displacement reactions at the sulfur atom, we decided to study the stereochemistry-mechanism relationship in the alkaline hydrolysis of optically active dialkoxysulfonium salts 8. This sulfur structure was selected for our study for two reasons. First, it contains two potential leaving groups bonded to sulfur and their leaving ability and apicophilicity are almost the same. Secondly, it was interesting to compare our results on the hydrolysis of 8 with those reported by DeBruin and Mislow [13,14] on the alkaline hydrolysis of diastereomeric dialkoxyphosphonium salts 9 and alkoxy(alkylthio)phosphonium salt 10 ( Figure 2). In the hope of gaining further insight into the mechanism of displacement reactions at the sulfur atom, we decided to study the stereochemistry-mechanism relationship in the alkaline hydrolysis of optically active dialkoxysulfonium salts 8. This sulfur structure was selected for our study for two reasons. First, it contains two potential leaving groups bonded to sulfur and their leaving ability and apicophilicity are almost the same. Secondly, it was interesting to compare our results on the hydrolysis of 8 with those reported by DeBruin and Mislow [13,14] on the alkaline hydrolysis of diastereomeric dialkoxyphosphonium salts 9 and alkoxy(alkylthio)phosphonium salt 10 ( Figure 2). In the present paper, we report the complete results of our investigations on the sulfonium salts 8, including the asymmetric synthesis of enantiomerically enriched isopropanesulfinic acid esters and their conversion to the corresponding dialkoxysulfonium salts 8; then, the stereochemical course of the alkaline hydrolysis of the salts 8 and the DFT studies aimed at the univocal determination of the mechanism of the investigated title reaction.

Synthesis of Optically Active Dialkoxysulfonium Salts 8
Racemic dialkoxysulfonium salts were obtained for the first time by Kobayashi and coworkers [15,16] in the reaction of sulfinic acid esters with strong alkylating reagents. The authors found that only alkanesulfinates-in contrast to arenesulfinates-form the corresponding dialkoxysulfonium salts, which are relatively stable in a solution of nitromethane. Having this in mind, we decided to prepare in the first step a series of optically active alkyl isopropanesulfinates 11 as precursors of the sulfonium salts 8. The sulfinates 11 were easily obtained according to our method [17], which in this case consisted in the asymmetric condensation of racemic isopropanesulfinyl chloride with achiral alcohols in the presence of (−)-N,N-dimethyl-α-phenylethylamine as an asymmetric reagent (see Scheme 2). All the dextrorotatory sulfinates 11a-f so-formed were enriched in the (R)-enantiomers and their optical purities were in the range of 10-27% (see Table 1).

Scheme 2.
Synthesis of optically active dialkoxysulfonium salts 8. In the present paper, we report the complete results of our investigations on the sulfonium salts 8, including the asymmetric synthesis of enantiomerically enriched isopropanesulfinic acid esters and their conversion to the corresponding dialkoxysulfonium salts 8; then, the stereochemical course of the alkaline hydrolysis of the salts 8 and the DFT studies aimed at the univocal determination of the mechanism of the investigated title reaction.

Synthesis of Optically Active Dialkoxysulfonium Salts 8
Racemic dialkoxysulfonium salts were obtained for the first time by Kobayashi and coworkers [15,16] in the reaction of sulfinic acid esters with strong alkylating reagents. The authors found that only alkanesulfinates-in contrast to arenesulfinates-form the corresponding dialkoxysulfonium salts, which are relatively stable in a solution of nitromethane. Having this in mind, we decided to prepare in the first step a series of optically active alkyl isopropanesulfinates 11 as precursors of the sulfonium salts 8. The sulfinates 11 were easily obtained according to our method [17], which in this case consisted in the asymmetric condensation of racemic isopropanesulfinyl chloride with achiral alcohols in the presence of (−)-N,N-dimethyl-α-phenylethylamine as an asymmetric reagent (see Scheme 2). All the dextrorotatory sulfinates 11a-f so-formed were enriched in the (R)-enantiomers and their optical purities were in the range of 10-27% (see Table 1). In the hope of gaining further insight into the mechanism of displacement reactions at the sulfur atom, we decided to study the stereochemistry-mechanism relationship in the alkaline hydrolysis of optically active dialkoxysulfonium salts 8. This sulfur structure was selected for our study for two reasons. First, it contains two potential leaving groups bonded to sulfur and their leaving ability and apicophilicity are almost the same. Secondly, it was interesting to compare our results on the hydrolysis of 8 with those reported by DeBruin and Mislow [13,14] on the alkaline hydrolysis of diastereomeric dialkoxyphosphonium salts 9 and alkoxy(alkylthio)phosphonium salt 10 ( Figure 2). In the present paper, we report the complete results of our investigations on the sulfonium salts 8, including the asymmetric synthesis of enantiomerically enriched isopropanesulfinic acid esters and their conversion to the corresponding dialkoxysulfonium salts 8; then, the stereochemical course of the alkaline hydrolysis of the salts 8 and the DFT studies aimed at the univocal determination of the mechanism of the investigated title reaction.

Synthesis of Optically Active Dialkoxysulfonium Salts 8
Racemic dialkoxysulfonium salts were obtained for the first time by Kobayashi and coworkers [15,16] in the reaction of sulfinic acid esters with strong alkylating reagents. The authors found that only alkanesulfinates-in contrast to arenesulfinates-form the corresponding dialkoxysulfonium salts, which are relatively stable in a solution of nitromethane. Having this in mind, we decided to prepare in the first step a series of optically active alkyl isopropanesulfinates 11 as precursors of the sulfonium salts 8. The sulfinates 11 were easily obtained according to our method [17], which in this case consisted in the asymmetric condensation of racemic isopropanesulfinyl chloride with achiral alcohols in the presence of (−)-N,N-dimethyl-α-phenylethylamine as an asymmetric reagent (see Scheme 2). All the dextrorotatory sulfinates 11a-f so-formed were enriched in the (R)-enantiomers and their optical purities were in the range of 10-27% (see Table 1).
A subsequent reaction of the sulfinates 11a-e with methyl trifluoromethanesulfonate, carried out in nitromethane, afforded the required optically active alkoxy(methoxy) isopropylsulfonium salts 8a-e. As the methylation takes place at the sulfinyl oxygen atom in the sulfinates 11, the configuration at the chiral sulfur atom in the sulfonium salts 8 remains unchanged and is also defined as R in accordance with the Cahn, Ingold and Prelog rules [18]. The values for the optical rotation of the isolated sulfonium salts 8a-e were very low expressing, indicating that their intrinsic asymmetry was extremely small. Selected data on alkyl sulfinates 11, sulfonium salts 8 and sulfinates 11, obtained after their alkaline hydrolysis, are summarized in Table 1. Table 1. Selected data on alkyl sulfinates 11, dialkoxysulfonium salts 8 and sulfinates 11 formed after alkaline hydrolysis of 8.

Stereochemical Course of the Alkaline Hydrolysis of Dialkoxysulfonium Salts 8
Due to their limited stability, the freshly prepared and isolated salts 8 were promptly dissolved in water and treated with a diluted solution of sodium hydroxide at room temperature. The hydrolysis reaction was terminated when the reaction solution became basic. The hydrolysis products (usually two sulfinates 11) obtained were separated and characterized. The outcome of the alkaline hydrolysis of (+)-(R)-ethoxy(methoxy)isopropylsulfonium trifluoromethanesulfonate 8b, shown in Scheme 3, best illustrates the experiments that were also performed with the other sulfonium salts 8.
A subsequent reaction of the sulfinates 11a-e with methyl trifluoromethanesulfonate, carried out in nitromethane, afforded the required optically active alkoxy(methoxy)isopropylsulfonium salts 8a-e. As the methylation takes place at the sulfinyl oxygen atom in the sulfinates 11, the configuration at the chiral sulfur atom in the sulfonium salts 8 remains unchanged and is also defined as R in accordance with the Cahn, Ingold and Prelog rules [18]. The values for the optical rotation of the isolated sulfonium salts 8a-e were very low expressing, indicating that their intrinsic asymmetry was extremely small. Selected data on alkyl sulfinates 11, sulfonium salts 8 and sulfinates 11, obtained after their alkaline hydrolysis, are summarized in Table 1. Table 1. Selected data on alkyl sulfinates 11, dialkoxysulfonium salts 8 and sulfinates 11 formed after alkaline hydrolysis of 8.

Stereochemical Course of the Alkaline Hydrolysis of Dialkoxysulfonium Salts 8
Due to their limited stability, the freshly prepared and isolated salts 8 were promptly dissolved in water and treated with a diluted solution of sodium hydroxide at room temperature. The hydrolysis reaction was terminated when the reaction solution became basic. The hydrolysis products (usually two sulfinates 11) obtained were separated and characterized.
The outcome of the alkaline hydrolysis of (+)-®ethoxy(methoxy)isopropylsulfonium trifluoromethanesulfonate 8b, shown in Scheme 3, best illustrates the experiments that were also performed with the other sulfonium salts 8. Thus, the (+)-(R)-ethoxy(methoxy)isopropylsulfonium salt (8b) prepared by methylation of (+)-(R)-sulfinate 11b gave upon alkaline hydrolysis two sulfinates: ethyl isopropanesulfinate (−)-(S)-11b and methyl isopropanesulfinate (+)-(R)-11f. The first sulfinate, isolated from a mixture by preparative gas chromatography, was formed with an inverted configuration at the sulfur with respect to the starting configurationindicating that hydrolysis of the sulfonium salt (+)-(R)-8b occurred with inversion at the sulfur atom. The second hydrolysis product, dextrorotatory methyl isopropanesulfinate 11f, has the same absolute configuration (R) as other alkyl isopropanesulfinates 11 produced by our asymmetric synthesis. A comparison of the chirality at sulfur in the starting sulfonium salt 8b with that of the sulfinate (+)-(R)-11f provides evidence that the latter ester is also formed with the inversion of its configuration at the sulfur. However, it should be noted that both esters 11b and 11f, isolated after alkaline hydrolysis and separated by preparative gas chromatography, are highly racemized. Thus, the (+)-(R)-ethoxy(methoxy)isopropylsulfonium salt (8b) prepared by methylation of (+)-(R)-sulfinate 11b gave upon alkaline hydrolysis two sulfinates: ethyl isopropanesulfinate (−)-(S)-11b and methyl isopropanesulfinate (+)-(R)-11f. The first sulfinate, isolated from a mixture by preparative gas chromatography, was formed with an inverted configuration at the sulfur with respect to the starting configuration-indicating that hydrolysis of the sulfonium salt (+)-(R)-8b occurred with inversion at the sulfur atom. The second hydrolysis product, dextrorotatory methyl isopropanesulfinate 11f, has the same absolute configuration (R) as other alkyl isopropanesulfinates 11 produced by our asymmetric synthesis. A comparison of the chirality at sulfur in the starting sulfonium salt 8b with that of the sulfinate (+)-(R)-11f provides evidence that the latter ester is also formed with the inversion of its configuration at the sulfur. However, it should be noted that both esters 11b and 11f, isolated after alkaline hydrolysis and separated by preparative gas chromatography, are highly racemized.
To check whether the applied experimental procedure and the results of the alkaline hydrolysis of the sulfonium salt (+)-(R)-8b are reproducible, the (−)-(S) enantiomer of 8b was prepared and hydrolyzed under the same conditions. It is interesting to note that the sulfonium salt (−)-(S)-8b was obtained by the ethylation of methyl isopropanesulfinate (+)-(R)-11f using ethyl trifluoromethanesulfonate. The results of the alkaline hydrolysis of the salt (−)-(S)-8b are shown in Scheme 4.
To check whether the applied experimental procedure and the results of the alkaline hydrolysis of the sulfonium salt (+)-(R)-8b are reproducible, the (−)-(S) enantiomer of 8b was prepared and hydrolyzed under the same conditions. It is interesting to note that the sulfonium salt (−)-(S)-8b was obtained by the ethylation of methyl isopropanesulfinate (+)-® -11f using ethyl trifluoromethanesulfonate. The results of the alkaline hydrolysis of the salt (−)-(S)-8b are shown in Scheme 4. To get more information on the effect of an alkoxy substituent on the course of the investigated reaction in particular, a series of optically active methoxy(alkoxy)isopropylsulfonium salts (+)-(R)-8a-e was subjected to mild alkaline hydrolysis. In accordance with the results described above on the hydrolysis of the salt (+)-(R)-8b, all the reactions performed-with one exception-afforded sulfinate esters 11b-e with the defined stereochemistry depicted in Scheme 5.

Scheme 5.
Stereochemical outcome of the alkaline hydrolysis of sulfonium salts 8a-e.
It was found that the proportions of the two sulfinates formed in the above reactions varied and were dependent on the size of the alkoxy substituent. The content of methyl sulfinate 11f in the isolated mixture of both the esters that was formed was determined by 1 H NMR spectroscopy and gas chromatography. Table 2 shows the results. Table 2. Content of methyl isopropanesulfinate 11f in a mixture with alkyl isopropanesulfinates 11a-e. An inspection of the results depicted above revealed that (a) both sulfinates 11f and 11b, being the hydrolysis products of the sulfonium salt (−)-(S)-8b, are formed with a prevailing inversion of their configuration, (b) the alkaline hydrolysis of the salt (−)-(S)-8b is accompanied by other side processes responsible for a great extent of racemization of the sulfinates 11b and 11f and (c) the experimental procedure for the alkaline hydrolysis of the 8 salts and the isolation of the 11 sulfinates is reproducible.
To get more information on the effect of an alkoxy substituent on the course of the investigated reaction in particular, a series of optically active methoxy(alkoxy)isopropylsulfonium salts (+)-(R)-8a-e was subjected to mild alkaline hydrolysis. In accordance with the results described above on the hydrolysis of the salt (+)-(R)-8b, all the reactions performed-with one exception-afforded sulfinate esters 11b-e with the defined stereochemistry depicted in Scheme 5.
To check whether the applied experimental procedure and the results of the alkaline hydrolysis of the sulfonium salt (+)-(R)-8b are reproducible, the (−)-(S) enantiomer of 8b was prepared and hydrolyzed under the same conditions. It is interesting to note that the sulfonium salt (−)-(S)-8b was obtained by the ethylation of methyl isopropanesulfinate (+)-® -11f using ethyl trifluoromethanesulfonate. The results of the alkaline hydrolysis of the salt (−)-(S)-8b are shown in Scheme 4. To get more information on the effect of an alkoxy substituent on the course of the investigated reaction in particular, a series of optically active methoxy(alkoxy)isopropylsulfonium salts (+)-(R)-8a-e was subjected to mild alkaline hydrolysis. In accordance with the results described above on the hydrolysis of the salt (+)-(R)-8b, all the reactions performed-with one exception-afforded sulfinate esters 11b-e with the defined stereochemistry depicted in Scheme 5.

Scheme 5.
Stereochemical outcome of the alkaline hydrolysis of sulfonium salts 8a-e.
It was found that the proportions of the two sulfinates formed in the above reactions varied and were dependent on the size of the alkoxy substituent. The content of methyl sulfinate 11f in the isolated mixture of both the esters that was formed was determined by 1 H NMR spectroscopy and gas chromatography. Table 2 shows the results. It was found that the proportions of the two sulfinates formed in the above reactions varied and were dependent on the size of the alkoxy substituent. The content of methyl sulfinate 11f in the isolated mixture of both the esters that was formed was determined by 1 H NMR spectroscopy and gas chromatography. Table 2 shows the results. It turned out that with an increase in the steric bulk of the alkoxy group in sulfonium salts 8, the formation of methyl sulfinate 11f gradually decreased. In the case of the alkaline hydrolysis of methoxy(neopentoxy)sulfonium salt 8e, methyl sulfinate 11f was not formed. A small amount of methyl sulfinate 11f that formed upon the hydrolysis of the sulfonium salts 8c and 8d was the main reason that our attempts to obtain this ester in a pure state by preparative gas chromatography were unsuccessful. Moreover, it is reasonable to assume that the other methyl sulfinates 11f are formed in these hydrolysis reactions with an inversion of their configuration and that are highly or completely racemized due to the side reactions discussed below. As sulfinates 11b-e were the major hydrolysis products of salts 8b-e, they were obtained in a pure state by preparative gas chromatography-although with a low yield. All these esters were highly racemized but formed with a predominant inversion of their configuration at the sulfur. To rationalize the stereochemical results and other observations on the alkaline hydrolysis of dialkoxysulfonium salts 8, it is necessary to first solve the question of the mechanism of this reaction. The hydrolysis of the sulfonium ion was performed in water. The solvation effect was estimated within an SCRF-CPCM approximation, in which the solvent was treated as the continuous medium, characterized by its dielectric constant.
DFT calculations for the hydrolysis of the dialkoxysulfonium salt excluded the concerted one-step S N 2-type substitution and confirmed the (A-E) mechanism. The attack of the hydroxyl ion on the four walls of the sulfonium trigonal pyramid led to the tetracoordinate sulfurane structures A, B and C, in which their relative energies were calculated (Scheme 6). Structure D could not be located as during the geometry optimization, the system transforms (via a spontaneous pseudorotation) into the more stable structure E, with both alkoxy groups occupying apical positions (see path 4 in Scheme 7 and the corresponding discussion below).
fonium salts 8, the formation of methyl sulfinate 11f gradually decreased. In the case of the alkaline hydrolysis of methoxy(neopentoxy)sulfonium salt 8e, methyl sulfinate 11f was not formed. A small amount of methyl sulfinate 11f that formed upon the hydrolysis of the sulfonium salts 8c and 8d was the main reason that our attempts to obtain this ester in a pure state by preparative gas chromatography were unsuccessful. Moreover, it is reasonable to assume that the other 11f methyl sulfinates are formed in these hydrolysis reactions with an inversion of their configuration and that are highly or completely racemized due to the side reactions discussed below. As sulfinates 11b-e were the major hydrolysis products of salts 8b-e, they were obtained in a pure state by preparative gas chromatography-although with a low yield. All these esters were highly racemized but formed with a predominant inversion of their configuration at the sulfur.

DFT Study on the Mechanism-Stereochemistry Relationship in the Alkaline Hydrolysis of Sulfonium Salts 8 and a Question of the Racemization of Sulfinate Products
To rationalize the stereochemical results and other observations on the alkaline hydrolysis of dialkoxysulfonium salts 8, it is necessary to first solve the question of the mechanism of this reaction. The hydrolysis of the sulfonium ion was performed in water. The solvation effect was estimated within an SCRF-CPCM approximation, in which the solvent was treated as the continuous medium, characterized by its dielectric constant.
DFT calculations for the hydrolysis of the dialkoxysulfonium salt excluded the concerted one-step SN2-type substitution and confirmed the (A-E) mechanism. The attack of the hydroxyl ion on the four walls of the sulfonium trigonal pyramid led to the tetracoordinate sulfurane structures A, B and C, in which their relative energies were calculated (Scheme 6). Structure D could not be located as during the geometry optimization, the system transforms (via a spontaneous pseudorotation) into the more stable structure E, with both alkoxy groups occupying apical positions (see path 4 in Scheme 7 and the corresponding discussion below). Scheme 6. Four possible S IV -sulfurane structures formed by the addition of a hydroxy ion to (+)-(R)-8b.
The Gibbs free energy of the alkaline hydrolysis of ethoxymethoxysulfonium salt (Equation (1)) is ΔGr = −63.4 kcal/mol. The energy of the ion recombination is partially responsible for such a great negative value. The formation of tetracoordinate sulfurane proceeds practically without an energy barrier (the barriers associated with the rearrangement of solvent molecules cannot be estimated within the SCRF approximation). The Gibbs free energy of the alkaline hydrolysis of ethoxymethoxysulfonium salt (Equation (1)) is ∆G r = −63.4 kcal/mol. The energy of the ion recombination is partially responsible for such a great negative value. The formation of tetracoordinate sulfurane proceeds practically without an energy barrier (the barriers associated with the rearrangement of solvent molecules cannot be estimated within the SCRF approximation).
We analyzed by DFT calculation the possible substitution routes, which are presented in Scheme 7.
Path 1 is the simplest route, in which the hydroxyl ion approaches the sulfonium cation from the opposite side to one of the alkoxy groups, forming low energy sulfuranes A or B in which two electronegative substituents-OH and OR-occupy the apical positions. In line with the commonly accepted substitution scheme, the departure of the apical group should be facilitated. This scheme implies an inversion of the configuration at the central sulfur atom, leading to (−)-(S)-11b and (+)-(R)-11f as a result of the evolution of MeOH and EtOH, respectively. This process requires the transfer of a proton from the hydroxyl group to the leaving alkoxyl group. Such a transfer is not possible directly. However, in a water solution, it can be easily mediated by solvent molecules. To prove this, we calculated the proton transfer through a bridge of two water molecules. The process, resulting in the substitution of the ethoxy group, is illustrated in Scheme 8. The substitution of the methoxy group proceeds analogously; the calculations confirmed that this process proceeds virtually without an energy barrier.

Molecules 2022, 27, x FOR PEER REVIEW 7 of 15
We analyzed by DFT calculation the possible substitution routes, which are presented in Scheme 7.

Scheme 7.
Possible pathways for the replacement of the methoxy and ethoxy group in sulfonium cation 8.
Path 1 is the simplest route, in which the hydroxyl ion approaches the sulfonium cation from the opposite side to one of the alkoxy groups, forming low energy sulfuranes A or B in which two electronegative substituents-OH and OR-occupy the apical positions. In line with the commonly accepted substitution scheme, the departure of the apical group should be facilitated. This scheme implies an inversion of the configuration at the central sulfur atom, leading to (−)-(S)-11b and (+)-(R)-11f as a result of the evolution of MeOH and EtOH, respectively. This process requires the transfer of a proton from the hydroxyl group to the leaving alkoxyl group. Such a transfer is not possible directly. However, in a water solution, it can be easily mediated by solvent molecules. To prove this, we calculated the proton transfer through a bridge of two water molecules. The process, resulting in the substitution of the ethoxy group, is illustrated in Scheme 8. The substitution of the methoxy group proceeds analogously; the calculations confirmed that this process proceeds virtually without an energy barrier. The alternative possibility one could imagine is the departure of the alkoxy group from the equatorial position. In such a case the proton transfer is possible, but this reaction is associated with a rather high energy barrier (transition states TS1(eq) and TS2(eq) for the substitution of OEt and OMe, respectively); see Scheme 9 and Figure 3. The alternative possibility one could imagine is the departure of the alkoxy group from the equatorial position. In such a case the proton transfer is possible, but this reaction is associated with a rather high energy barrier (transition states TS1(eq) and TS2(eq) for the substitution of OEt and OMe, respectively); see Scheme 9 and Figure 3. The alternative possibility one could imagine is the departure of the alkoxy group from the equatorial position. In such a case the proton transfer is possible, but this reaction is associated with a rather high energy barrier (transition states TS1(eq) and TS2(eq) for the substitution of OEt and OMe, respectively); see Scheme 9 and Figure 3.  The free energy profiles for the discussed variants of reaction path 1 are presented in Figure 4. Note that the barriers associated with changes in solvation cannot be easily calculated, but they are expected to be very low; thus, the main reaction profile is shown as virtually barrierless. Some pathways involving the formation of MeOH are omitted for clarity as they are almost identical to those involving the formation of EtOH. The sum of the free energies of the substrates ((+)-(R)-8b and OH − , as well as (H2O)2 in the case of water dimer-mediated substitution), was taken as zero.
Path 2 discusses the possibility of the Berry pseudorotation (ψ) of A and B to give sulfurane with Pr i and OR (R = Me, Et) groups in apical positions (F). However, structure F could not be found, as in the pseudorotational transition states (TS-A and TS-B), a spontaneous proton transfer from the OH to the equatorial alkoxy group occurs, resulting in the departure of the ROH and the formation of the corresponding sulfinate 11. The products of the transformation of A and B according to path 2 have opposite configurations to those produced in path 1 and, if created, this process would lead to com- The alternative possibility one could imagine is the departure of the alkoxy group from the equatorial position. In such a case the proton transfer is possible, but this reaction is associated with a rather high energy barrier (transition states TS1(eq) and TS2(eq) for the substitution of OEt and OMe, respectively); see Scheme 9 and Figure 3.  The free energy profiles for the discussed variants of reaction path 1 are presented in Figure 4. Note that the barriers associated with changes in solvation cannot be easily calculated, but they are expected to be very low; thus, the main reaction profile is shown as virtually barrierless. Some pathways involving the formation of MeOH are omitted for clarity as they are almost identical to those involving the formation of EtOH. The sum of the free energies of the substrates ((+)-(R)-8b and OH − , as well as (H2O)2 in the case of water dimer-mediated substitution), was taken as zero.
Path 2 discusses the possibility of the Berry pseudorotation (ψ) of A and B to give sulfurane with Pr i and OR (R = Me, Et) groups in apical positions (F). However, structure F could not be found, as in the pseudorotational transition states (TS-A and TS-B), a spontaneous proton transfer from the OH to the equatorial alkoxy group occurs, resulting in the departure of the ROH and the formation of the corresponding sulfinate 11. The products of the transformation of A and B according to path 2 have opposite configurations to those produced in path 1 and, if created, this process would lead to The free energy profiles for the discussed variants of reaction path 1 are presented in Figure 4. Note that the barriers associated with changes in solvation cannot be easily calculated, but they are expected to be very low; thus, the main reaction profile is shown as virtually barrierless. Some pathways involving the formation of MeOH are omitted for clarity as they are almost identical to those involving the formation of EtOH. The sum of the free energies of the substrates ((+)-(R)-8b and OH − , as well as (H 2 O) 2 in the case of water dimer-mediated substitution), was taken as zero.
Path 2 discusses the possibility of the Berry pseudorotation (ψ) of A and B to give sulfurane with Pr i and OR (R = Me, Et) groups in apical positions (F). However, structure F could not be found, as in the pseudorotational transition states (TS-A and TS-B), a spontaneous proton transfer from the OH to the equatorial alkoxy group occurs, resulting in the departure of the ROH and the formation of the corresponding sulfinate 11. The products of the transformation of A and B according to path 2 have opposite configurations to those produced in path 1 and, if created, this process would lead to complete racemizationwhich is contrary to the experimental results. The calculated energy profile for path 2 also shows that the free energy barrier of this transformation is relatively high, making this reaction pathway rather unlikely.
Path 3 assumes the attack of the OH (−) opposite the i Pr group, with an initial formation of the high energy structure C with apical i Pr and OH groups and equatorial alkoxy groups. Departure from the equatorial position is accompanied by a high energy barrier (see discussion of path 1)-so, another possibility is pseudorotation (TS-C), which gives a structure with alkoxy groups in the apical positions €. The E structure is the most stable among the sulfurane structures considered. Scheme 10 shows two isoenergetic conformers of E, differing in the orientation of the OH bond, which interacts with the alkoxy oxygen to facilitate the departure of the OMe or OEt groups, respectively. The transition states TS1 and TS2-leading to the elimination of EtOH or MeOH, respectively-are, by only about 1 kcal/mol, higher in free energy than the corresponding E1 and E2 sulfuranes. However, the transition state for Berry pseudorotation (TS-C) is also relatively high in energy ( Figure 5). the most stable among the sulfurane structures considered. Scheme 10 shows two isoenergetic conformers of E, differing in the orientation of the OH bond, which interacts with the alkoxy oxygen to facilitate the departure of the OMe or OEt groups, respectively. The transition states TS1 and TS2-leading to the elimination of EtOH or MeOH, respectively-are, by only about 1 kcal/mol, higher in free energy than the corresponding E1 and E2 sulfuranes. However, the transition state for Berry pseudorotation (TS-C) is also relatively high in energy ( Figure 5). Path 4 involves the attack of the OH (−) opposite the lone pair at the sulfur. This attack leads to a high energy transition state TS-D, which rearranges to E and finally decomposes with the evolution of ROH-as in path 3. However, the barrier associated with the formation of TS-D is so high that it prevents the reaction from proceeding along this pathway. The free energy profiles for the discussed paths 2-4 (for the case the most stable among the sulfurane structures considered. Scheme 10 shows two isoenergetic conformers of E, differing in the orientation of the OH bond, which interacts with the alkoxy oxygen to facilitate the departure of the OMe or OEt groups, respectively. The transition states TS1 and TS2-leading to the elimination of EtOH or MeOH, respectively-are, by only about 1 kcal/mol, higher in free energy than the corresponding E1 and E2 sulfuranes. However, the transition state for Berry pseudorotation (TS-C) is also relatively high in energy ( Figure 5). Path 4 involves the attack of the OH (−) opposite the lone pair at the sulfur. This attack leads to a high energy transition state TS-D, which rearranges to E and finally decomposes with the evolution of ROH-as in path 3. However, the barrier associated with the formation of TS-D is so high that it prevents the reaction from proceeding along this pathway. The free energy profiles for the discussed paths 2-4 (for the case Path 4 involves the attack of the OH (−) opposite the lone pair at the sulfur. This attack leads to a high energy transition state TS-D, which rearranges to E and finally decomposes with the evolution of ROH-as in path 3. However, the barrier associated with the formation of TS-D is so high that it prevents the reaction from proceeding along this pathway. The free energy profiles for the discussed paths 2-4 (for the case where (+)-(R)-11f + EtOH are produced) are presented in Figure 5. The corresponding profiles for the pathways leading to (−)-(S)-11b + MeOH are almost the same. The relative energies of the discussed isomeric sulfuranes and transition states are shown in Table 3.
Based on the DFT calculations, it can be concluded that path 1, involving the recombination of ions with the simultaneous formation of sulfuranes A and B (according to the A-E mechanism) followed by the dissociation of an alcohol molecule (MeOH or EtOH), is the most thermodynamically favorable. The other paths proceed through high energy transition states and are therefore less feasible.
In view of this important statement, the reason why both the hydrolysis productsmethyl and ethyl sulfinates 11-are highly racemized, must be due to the other sideprocesses that accompany the substitution reaction. For a long time, it has been known that the alkoxysulfonium salts R 2 S + -OR behave as ambident electrophiles [19]. Depending on the nature of a nucleophile, one observes a nucleophilic attack not only on sulfur ions but also on the carbons of alkoxy groups, resulting in de-alkyation. In the case of dialkoxysulfonium salts 8, which are more reactive than their monoalkoxy analogues, the bidirectional attack of the hydroxyl anion has an influence on the stereochemical outcome of the substitution reaction, as well as on the proportions of the two 11 sulfinates formed (see Scheme 11). Whereas the attack of a hydroxyl anion on the sulfur results in the formation of the methyl and ethyl sulfinates 11f and 11b-with the inversion of their configuration at S-the demethylation reaction (the attack of the OH − at the methoxy carbon) leads to ethyl sulfinate 11b, with a retained configuration at the sulfur. where (+)-(R)-11f + EtOH are produced) are presented in Figure 5. The corresponding profiles for the pathways leading to (−)-(S)-11b + MeOH are almost the same. The relative energies of the discussed isomeric sulfuranes and transition states are shown in Table 3. Based on the DFT calculations, it can be concluded that path 1, involving the recombination of ions with the simultaneous formation of sulfuranes A and B (according to the A-E mechanism) followed by the dissociation of an alcohol molecule (MeOH or EtOH), is the most thermodynamically favorable. The other paths proceed through high energy transition states and are therefore less feasible. Table 3. Relative enthalpies and Gibbs free energies (kcal/mol) of sulfuranes (Scheme 2; ΔHr, ΔGr-relative to the sum of the free energies of the substrates ((EtO)iPrSOMe (+) + OH (−) ), ΔHrel, ΔGrel-relative to the most stable sulfurane isomer (E2)) in water.  Table 3. Relative enthalpies and Gibbs free energies (kcal/mol) of sulfuranes (Scheme 2; ∆H r , ∆G r -relative to the sum of the free energies of the substrates ((EtO)iPrSOMe (+) + OH (−) ), ∆H rel , ∆G rel -relative to the most stable sulfurane isomer (E2)) in water.

Sulfurane S IV
∆H r ∆G r ∆H rel ∆G rel As the hydrolysis reaction of the methoxy(alkoxy)sulfonium salts 8 proceeds under basic conditions and as the departing methoxy and alkoxy anions are present in the reaction mixture, a high extent of racemization of both the produced sulfinates (methyl sulfinate 11f and alkyl sulfinates 11A-E) may also be due to the symmetric exchange reactions at the chiral sulfur in the 11 esters (MeO/MeO, RO/RO). The racemization of the sulfinate 11f is schematically depicted in Equation (2): More information on this type of identity substitution reactions at S and P may be found in our recent paper [9].
ing on the nature of a nucleophile, one observes a nucleophilic attack not only on sulfur ions but also on the carbons of alkoxy groups, resulting in de-alkyation. In the case of dialkoxysulfonium salts 8, which are more reactive than their monoalkoxy analogues, the bidirectional attack of the hydroxyl anion has an influence on the stereochemical outcome of the substitution reaction, as well as on the proportions of the two 11 sulfinates formed (see Scheme 11). Whereas the attack of a hydroxyl anion on the sulfur results in the formation of the methyl and ethyl sulfinates 11f and 11b-with the inversion of their configuration at S-the demethylation reaction (the attack of the OH − at the methoxy carbon) leads to ethyl sulfinate 11b, with a retained configuration at the sulfur. Scheme 11. Bidirectional attack of the hydroxyl anion on sulfonium salt 8b and its stereochemical consequences.
As the hydrolysis reaction of the methoxy(alkoxy)sulfonium salts 8 proceeds under basic conditions and as the departing methoxy and alkoxy anions are present in the reaction mixture, a high extent of racemization of both the produced sulfinates (methyl sulfinate 11f and alkyl sulfinates 11A-E) may also be due to the symmetric exchange reactions at the chiral sulfur in the 11 esters (MeO/MeO, RO/RO). The racemization of the sulfinate 11f is schematically depicted in Equation (2) More information on this type of identity substitution reactions at S and P may be found in our recent paper [9].

General Experimental Details
Commercial-grade reagents and solvents were used without further purification. THF and diethyl ether were distilled over NaH/benzophenone prior to use. Column chromatography was performed using a silica gel (70-230 mesh). The routine 1 H NMR Scheme 11. Bidirectional attack of the hydroxyl anion on sulfonium salt 8b and its stereochemical consequences.

General Experimental Details
Commercial-grade reagents and solvents were used without further purification. THF and diethyl ether were distilled over NaH/benzophenone prior to use. Column chromatography was performed using a silica gel (70-230 mesh). The routine 1 H NMR spectra were recorded at 60 and 90 MHz unless stated otherwise. Chemical shifts are quoted in parts per million (ppm) and reported relative to TMS as an internal standard. Boiling and melting points are uncorrected.

Asymmetric Synthesis of Alkyl Isopropanesulfinates 11a-f-General Procedure
To a solution of isopropanesulfinyl chloride (0.01 mol) in ether (50 mL) cooled to −70 • C, a mixture of proper alcohol (0.01 mol) and (−)-N,N-dimethyl-α-phenylethylamine (0.09 mol) dissolved in ether (40 mL) was added. The reaction mixture was stirred for 1 h. When the temperature of the reaction solution reached 20 • C, the etheral solution was washed with water (1 × 30 mL), diluted with 3% hydrochloric acid (1 × 30 mL) and with a solution (3%) of sodium hydrogen carbonate (1 × 10 mL). The reaction solution was then dried over MgSO 4 and the ether was evaporated under reduced pressure. The sulfinate 11 was purified by chromatography (silica gel; eluent: n-hexane/ethyl ether, 4:1). The values for the optical rotation and optical purity of the sulfinates 11, prepared as above, are given in Table 2. To a stirred solution of alkyl isopropanesulfinate 11 (0.001 mol) in nitromethane (20 mL), methyl trifluoromethanesulfonate (0.002 mol) was added. After 30 min, nitromethane and an excess of methylating agent were removed under reduced pressure at a temperature below 30 • C to obtain the sulfonium salts 8. Owing to their limited stability, the sulfonium salts 8 so-obtained were quickly characterized by 1 H NMR spectroscopy (see below) and subjected to alkaline hydrolysis. The sulfonium salts 8A-E prepared as described above were quickly dissolved in water (10 mL) and treated with a 0.02 N solution of sodium hydroxide in the presence of phenolphthalein. When hydrolysis was complete (yellowish color of the reaction solution), the water phase was extracted with n-hexane (3 × 10 mL) and chloroform (2 × 10 mL). The combined organic solutions were dried over MgSO 4 and evaporated under reduced pressure. The hydrolysis products (two sulfinate esters 11) were separated by preparative gas chromatography. Their optical rotations and proportions are summarized in Tables 1 and 2.

Theoretical Methods
All quantum mechanical calculations were performed using the Gaussian 16 suite of programs [20]. The geometries of the model compounds were optimized using two DFT methods: the hybrid B3LYP density functional model [21] corrected for dispersion interactions using the Grimme D3(0) empirical term [22], with the Def2-TZVP basis set [23] was used in aqueous solutions; SCRF calculations in water were performed using the CPCM model with UFF atomic radii, implemented in Gaussian 16 [20]. All stationary points were identified as stable minima or transition states by frequency calculations. Geometry optimizations were performed using normal convergence criteria as defined in Gaussian. The integration grid was set to ultrafine. The vibrational analysis provided thermal enthalpy


Revealing the Chemical Composition of Birch Pollen Grains by Raman Spectroscopic Imaging

The investigation of the biochemical composition of pollen grains is of the utmost interest for several environmental aspects, such as their allergenic potential and their changes in growth conditions due to climatic factors. In order to fully understand the composition of pollen grains, not only is an in-depth analysis of their molecular components necessary but also spatial information of, e.g., the thickness of the outer shell, should be recorded. However, there is a lack of studies using molecular imaging methods for a spatially resolved biochemical composition on a single-grain level. In this study, Raman spectroscopy was implemented as an analytical tool to investigate birch pollen by imaging single pollen grains and analyzing their spectral profiles. The imaging modality allowed us to reveal the layered structure of pollen grains based on the biochemical information of the recorded Raman spectra. Seven different birch pollen species collected at two different locations in Germany were investigated and compared. Using chemometric algorithms such as hierarchical cluster analysis and multiple-curve resolution, several components of the grain wall, such as sporopollenin, as well as the inner core presenting high starch concentrations, were identified and quantified. Differences in the concentrations of, e.g., sporopollenin, lipids and proteins in the pollen species at the two different collection sites were found, and are discussed in connection with germination and other growth processes.

Introduction
Birch pollen is of specific interest because it is one of the most dominant tree pollen types found in Northern and Central Europe, and causes the majority of allergic rhinitis [1]. Numerous environmental factors have been and are being discussed in relationship to the increase in allergic diseases [2][3][4]. According to a publication of the German Federal Environmental Agency [5], experimental and in situ studies show an influence of different environmental factors-such as the atmospheric CO 2 concentration and air temperatureon pollen and biomass production, respectively, by allergenic plant species. Compared to pre-industrial CO 2 concentrations, both the current and a predicted 21st century CO 2 concentration increased pollen and allergen production of Ambrosia artemisiifolia L., an invasive plant with high allergenic potential [6][7][8]. Ziska  higher biomass production of Ambrosia plants at urban compared to rural sites, with urban sites having higher CO 2 (30 to 31% and 3%, respectively) and air temperature values (1.8 to 2 • C and 1.8 • C, respectively) than rural sites [9,10]. In Europe, the air concentrations of pollen from numerous plant species-some of which are highly allergenic-have increased over the past 30 years, especially in urban areas [11]. To investigate the aforementioned changes, systematic studies implementing analytical methods are needed in order to record biochemical changes in a qualitative and quantitative way. Raman spectroscopy allows the non-destructive identification and characterization of airborne particles such as pollen, and is in this context of special interest, as it also allows molecular imaging on a sub-grain level.
By combining molecular information with subcellular optical resolution, vibrational spectroscopies can fill the gap between destructive modalities, which only provide chemical information from bulk samples, and analytical modalities, e.g., brightfield microscopy, that can give morphological information but lack the chemical specificity. Over the past decade, various studies concerning pollen were performed using Raman and Infrared (IR) spectroscopy [12][13][14][15][16][17]. A small subset of studies dealt with different enhancement techniques, such as surface-enhanced Raman spectroscopy [18][19][20] or resonance-enhanced Raman spectroscopy [21]. The majority of studies focused on the taxonomic classification and their relationships [22][23][24][25][26], and there were few studies on the differentiation due to, e.g., growth and environmental conditions [27][28][29] or nutritional parameters [30]. Recently, efforts towards a rapid and automatic identification of pollen were made based on the reduction of the spectral dataset into specific band regions [31], or on the utilization of a high-throughput IR system [32]. An automated high-throughput Raman system was specifically developed to fulfill major demands for the reliable and automatic detection and classification of pollen, which enabled a comprehensive dataset of ca. 22,000 individual pollen grains from 18 families, 36 genera, and 37 species [33]. An interesting on-side study was performed by Bohlmann et al. in a rural forest site in Finland by investigating the optical properties of pollen in the atmosphere using a multiwavelength Raman polarization lidar (the light detection and ranging method). They presented the potential of the particle depolarization ratio to track pollen grains in the atmosphere [34]. In a following study from the same group, the authors used the same method with a novel algorithm to characterize and classify pure pollen, and thereby estimated their optical properties [35]. A comprehensive insight into the biochemistry of pollen was reported by Kendel et al. using Fourier Transform (FT-)Raman and FT-IR spectroscopy for the analysis of different pollen species [36], thus demonstrating the potential of these complementary techniques. While FT-Raman spectroscopy provided mainly information on the strong Raman scatterers sporopollenin and carotenoids in the grain walls, FT-IR spectra tend to be more sensitive to the components of the grain interior, such as lipids and carbohydrates. In an even larger-scaled study, Bagcioglu et al. used seven different IR and Raman methodologies to highlight different chemical aspects of 15 conifer pollens [37]. They discussed the differences between the species in sporopollenin as the major constituent of the grain walls, as well as the presence of lipids and proteins in the corpus of the conifer pollen grains. The study did not include imaging approaches, and only measured the Raman spectra at two defined positions on a pollen grain. As the authors state, the layered structure leads to the chemical heterogeneity of the pollen grains; hence, the spectra differ when the depth of the laser focus is changed. Almost all Raman or IR studies lack imaging approaches, and are therefore confined to single spectrum representing an average spectrum or even only a small area of a pollen grain. The local molecular variations are especially prominent in Raman spectra recorded on confocal microscope setups with a high NA objective [31,37]. While IR technologies typically lack sufficient spatial resolution to differentiate different pollen layers, and hence are based on average spectra [17,38], Raman imaging can provide detailed molecular maps of a pollen grain with valuable information on sub-grain resolution [24].
In this study, pollen grains of different birch species, a typical tree species native to Germany, were subjected to Raman spectroscopic imaging. Birches are frequently found in mixed forests, tree alleys, parks, and open spaces at field margins, and are therefore ubiquitous. The monitoring of changes in the grains of different birch species over the years with changing climatic conditions (e.g., changes in the mean annual temperature or the amount of precipitation) can be important, and important for the consideration of the allergenic potential. Therefore, the aim of this study is the evaluation of the Raman spectroscopic imaging of single birch pollen grains, with the main focus being the investigation of their layered structure by imaging and analyzing their spectral Raman signatures. The results of seven different birch pollen species collected at two different locations in Germany are presented. The hierarchical cluster analysis (HCA) of the Raman images visualizes the grain wall in several layers, as well as the inner core. Besides sporopollenin as the main constituent of the outer layer (exine), carbohydrate bands reveal the presence of starch in the inner vegetative cell. A multiple-curve resolution algorithm further revealed differences in the concentration of sporopollenin, carbohydrate, lipid and protein components in the pollen species.

Results and Discussion
The grains of seven different birch species sampled in the Forest Botanical Garden and Arboretum of the University of Göttingen and the Botanic Garden Jena of the Friedrich Schiller University Jena were subjected to Raman imaging. Figure 1a shows a brightfield image of a Betula (B.) ermanii pollen grain, with the typical triangular-like shape of birch pollen due to three visible protrusions. Intensity-based Raman maps at three distinct Raman bands for the same pollen reveal a more detailed structure of the grain than is simply observed in the brightfield image. Figure 1b displays the Raman intensity map of the CH stretching region between 2800 and 3010 cm −1 , which is present in nearly all biological compounds. A higher brightness in the Raman map indicates a higher Raman signal intensity of the band at the specific location. For example, while the core of the pollen grain appears bright, indicating a higher concentration of CH, the three protrusions have a reduced intensity, indicating that the CH concentration is lower in comparison to the core. On the other hand, the protrusions are prominently visible in Figure 1c, in which the sample distribution of the 1570 to 1620 cm −1 band region is shown. This spectral band indicates the presence of sporopollenin with its strongest band at 1604 cm −1 , and thus marks the exine of the pollen grain. Contrarily, Figure 1d highlights the inside of the pollen grain by mapping the distribution of the spectral region between 1620 to 1680 cm −1 , where typically the amid I band of proteins and the C=C stretch vibration of lipids around 1654 cm −1 is present. Figure 1e represents the mean spectrum with its standard deviation of the entire recorded Raman image of the pollen grain with previously assigned band positions.
It is apparent that the fingerprint region between 400 and 1800 cm −1 includes a more comprehensive band profile than that which is presented in the intensity maps. A better insight can be gained by analyzing Raman images with spectral clustering algorithms, in which spectra are grouped based on a particular metric, e.g., Euclidian distance or Manhattan distance, of similar band profiles and averaged into cluster mean spectra, while the spatial distribution based on the chemical information is revealed in a color-coded cluster image ( Figure 2). Five clusters are characterize the pollen grain in sufficient detail, in which four layers surrounding the inner core are revealed. The core of the pollen grain is represented by the red cluster 5. The corresponding cluster spectrum is dominated by carbohydrate bands originating from starch at 481, 861, 937, 1084 and 1263 cm −1 [28]. Starch, with amylopectin and amylose as its two naturally occurring components, is known to form granules in the vegetative cell of pollen [25]. Further bands, 1450 and 1657 cm −1 , can be assigned to CH 2 /CH 3 deformation, as well as amid I and C=C stretch vibrations, which are commonly representative of proteins and lipids. The orange cluster 4 is highly different, with the most intense bands at 1604 and 1169 cm −1 , which can be associated with sporopollenin as the building block of the exine. The cluster is located at the three protrusions seen in the brightfield image ( Figure 1a). Clusters 1 to 3 seem to be mixtures of clusters 4 and 5 in varying ratios. The purple cluster 3 shows a still-high content of sporopollenin, and can be found on the outside of the grain, most likely representing the exine in combination with the orange cluster. The light-blue cluster 2 already has more influence from proteins and lipids, as the band at 1650 cm −1 is more pronounced than that in cluster 3. Even more pronounced bands at 1650 cm −1 and at 1004 cm −1 , typical for phenylalanine ring breathing, are visible in the dark-blue cluster 1, where the carbohydrate vibrations are also stronger. Both light-and dark-blue clusters form layers surrounding the red inner core. Therefore, it is reasonable to assume that they represent the intine. Roughly summarized, clusters 1 and 2 relate to the intine, cluster 3 and 4 to the exine, and cluster 5 relates to the vegetative cell. However, the clear separation of the exine, intine and interior is not straightforward, as the spectral profiles overlap due to their similar chemical structures, e.g., of carbohydrates. For example, the intine is typically composed of cellulose and pectin, which are-akin to starch-polysaccharides, and are composed of monosaccharides. Due to their having the same functional groups, the differences in the band profiles are only minor wavenumber shifts. Because cellulose bands cannot be identified in the recorded spectra, they might be strongly overlapping with the abundant starch bands in the vegetative cell, making it potentially challenging to correctly differentiate the intine [39]. Furthermore, although the measurements were preformed confocally, information of components related to sporopollenin might still be detected in cluster 1 and 2 of the intine due to a high scattering cross section of the compounds. Of note, three clusters also provide enough separation to distinguish the exine, intine and the interior; however, the authors think that five clusters provide a better and more detailed representation (see Supporting Information Figure S1 for three clusters).
Summarizing the spectral data, birch pollen grains have high sporopollenin and carbohydrate contents and low amounts of components related to proteins and lipids. There are no bands of carotenoids visible, which even in a low amount would produce a clear signal due to resonance enhancement. This observation is consistent with literature, in which birch pollen-as an anemophilous species-is known to have high carbohydrate but low carotenoid and protein contents [36]. The mean spectra in Figure 2b are based on the HCA clustering of, in total, 40 pollen grains from two different collection sites and seven different birch species of the genus Betula (B.). The corresponding cluster maps are presented in Figure 3.
typically the amid I band of proteins and the C=C stretch vibration of lipids around 1654 cm −1 is present. Figure 1e represents the mean spectrum with its standard deviation of the entire recorded Raman image of the pollen grain with previously assigned band positions. It is apparent that the fingerprint region between 400 and 1800 cm −1 includes a more com insight can be gained by analyzing Raman images with spectral clustering algorithms, in which spectra are grouped based on a particular metric, e.g., Euclidian distance or confocally, information of components related to sporopollenin might still be detected in cluster 1 and 2 of the intine due to a high scattering cross section of the compounds. Of note, three clusters also provide enough separation to distinguish the exine, intine and the interior; however, the authors think that five clusters provide a better and more detailed representation (see Supporting Information Figure S1 for three clusters).

Figure 2.
Representative cluster analysis of a Betula ermanii pollen grain. (a) HCA cluster image and (b) cluster spectra with the same color coding. HCA clustering was performed on, in total, 40 grains from seven different birch species. Five clusters described the pollen grain in detail, while clusters 1-4 represent four layers surrounding the inner core (cluster 5). The mean spectrum of cluster 4 reveals a high content of sporopollenin in the outside layer, which decreases in layers closer to the inner core (cluster 1-3). The mean spectra of cluster 5 are dominated by carbohydrate bands.
Summarizing the spectral data, birch pollen grains have high sporopollenin and carbohydrate contents and low amounts of components related to proteins and lipids. There are no bands of carotenoids visible, which even in a low amount would produce a clear signal due to resonance enhancement. This observation is consistent with literature, in which birch pollen-as an anemophilous species-is known to have high carbohydrate but low carotenoid and protein contents [36]. The mean spectra in Figure 2b are based on the HCA clustering of, in total, 40 pollen grains from two different collection sites and seven different birch species of the genus Betula (B.). The corresponding cluster maps are presented in Figure 3.  Analyzing the occurrences and distributions of the clusters, a clear difference in the orange cluster 5 and purple cluster 3, both representative for a high sporopollenin concentration, can be seen. Pollen grains from a (I-IV) and b (V) have clearly defined orange clusters on the outside of the grain, although in varying amounts. In most cases a purple-clustered layer separates the orange from the inner clusters of the grain. In contrast, in grains from b (VI, VII and VIII), less sporopollenin (orange cluster) can be seen. Here, the purple clusters do not form a layer but group on the outside in the protrusion of the Each tile represents one of five measured pollen grains for each species, whose names are displayed on top. As the tiles have the same dimensions, grains from B. pendula and pumila (a (I, II)) are the smallest, with a diameter of ca. 20 µm. B. pubescens (b (IV)) is around 25 µm, and the other grains are between 30 and 35 µm in diameter.
Analyzing the occurrences and distributions of the clusters, a clear difference in the orange cluster 5 and purple cluster 3, both representative for a high sporopollenin concentration, can be seen. Pollen grains from a (I-IV) and b (V) have clearly defined orange clusters on the outside of the grain, although in varying amounts. In most cases a purple-clustered layer separates the orange from the inner clusters of the grain. In contrast, in grains from b (VI, VII and VIII), less sporopollenin (orange cluster) can be seen. Here, the purple clusters do not form a layer but group on the outside in the protrusion of the grains, as the orange cluster does in the other pollen. This might indicate that the wall of these three birch pollen grains of b (VI, VII, VIII) have a reduced stability, as sporopollenin is an extremely resilient structure against physical and chemical stressors, especially against solar UV radiation [37]. Furthermore, in these three grains, the dark-blue cluster is more pronounced than in the other grains, in which more carbohydrate contributions are present in the corresponding mean spectrum, which surrounds the red cluster of the vegetative cell. The aforementioned difficulty of assigning certain clusters to specific layers is now even more obvious. While the exine of pollen grains a (I-IV) and b (V) can be assigned to the orange and purple cluster, the exine in the grains b (VI, VII and VIII) seem to be represented by the purple and light-blue clusters. It is noteworthy that as the pollen grains were measured dried, the folding of the pollen wall occurs. Based on an effect termed harmomegathy, the apertures and pores of pollen are closed off in order to minimize desiccation [40]. This could lead to a distorted view of the layered structure of pollen grains.
However, in Figure 3a, higher sporopollenin contents were observed in samples collected in the Botanic Garden Jena of the Friedrich Schiller University Jena compared to those of the Forest Botanical Garden and Arboretum of the University of Göttingen. Given the limited number of samples, it is not possible to verify whether this difference is indeed due to the different environmental conditions in these two locations. Nevertheless the difference in sporopollenin content can be seen when comparing pollen grains from Figure 3a (IV) and Figure 3b (VIII), which are from the same species of B. papyrifera, but were collected at the two different locations.
In order to gain quantitative metrics for the identification of components in the pollen grains, a multiple-curve resolution-alternating least squares (MCR-ALS) algorithm was used [41,42]. Here, input spectra and concentrations are fitted iteratively to the dataset in order to calculate the underlying pure component spectra and corresponding concentrations as a non-negative linear combination. Five spectra were identified to sufficiently describe the dataset, and are presented in the Supplementary Information Figure S2. Three components are presented in more detail in the following analysis. Figure 4 shows the results of a spectrum, which can be assigned to sporopollenin. Nearly all of the bands in the calculated spectrum in Figure 4a can be correlated to vibrations of phenylpropanoic acids, such as ferulic acid, p-coumeric acid or sinapic acid (1604, 1557, 1169, 979, 447 cm −1 ), which are the building blocks of sporopollenin [36,37]. However, the structural composition of sporopollenin is still not completely understood. The band at 546 cm −1 reveals acyl group vibrations and 1440 cm −1 of CH 2 /CH 3 deformation vibrations. Figure 4b   Another component found by the MCR-ALS algorithm can be assigned to carbohydrates ( Figure 5). The bands at 481, 861, 937, 1084, 1263 cm −1 , as stated above, as well as the bands at 1125 and 1338 cm −1 can most likely be assigned to the starch components amylopectin and amylose. Compared to the red cluster spectra in Figure 2b, the bands of carbohydrates seem to be more clearly pronounced, as shown in Figure 5a. Interestingly, the band at 1454 cm −1 is shifted to higher wavenumbers. This, as well, could indicate the presence of starch, as the CH2 twisting and CH bending vibrations of starch occur at 1462 cm −1 , while the CH2/CH3 deformation vibrations in lipids and proteins are typically at lower wavenumbers around 1445 cm −1 [28]. Another component found by the MCR-ALS algorithm can be assigned to carbohydrates ( Figure 5). The bands at 481, 861, 937, 1084, 1263 cm −1 , as stated above, as well as the bands at 1125 and 1338 cm −1 can most likely be assigned to the starch components amylopectin and amylose. Compared to the red cluster spectra in Figure 2b, the bands of carbohydrates seem to be more clearly pronounced, as shown in Figure 5a. Interestingly, the band at 1454 cm −1 is shifted to higher wavenumbers. This, as well, could indicate the presence of starch, as the CH 2 twisting and CH bending vibrations of starch occur at 1462 cm −1 , while the CH 2 /CH 3 deformation vibrations in lipids and proteins are typically at lower wavenumbers around 1445 cm −1 [28]. As expected, the carbohydrates are highly dense in the inner part of the pollen grain, as seen in Figure 5b, and indirectly reveal the location of the vegetative cell. Apparently, B. pumila (II) has only a really low amount of carbohydrates, which is also shown in Figure 5c in the averaged MCR-ALS coefficient value of 0.04. The concentration of carbohydrates does not correlate with the collection site, and varies more between the species than is seen in the sporopollenin content. The MCR-ALS coefficients for the B. papyrifera from the Botanic Garden Jena of the Friedrich Schiller University Jena are, at 0.38, higher than those from the Forest Botanical Garden and Arboretum of the University of Göttingen, with 0.29. However, the dataset is too small to give a conclusion about species-dependent correlations.
A new component, which was not revealed by the HCA cluster analysis, is pre- As expected, the carbohydrates are highly dense in the inner part of the pollen grain, as seen in Figure 5b, and indirectly reveal the location of the vegetative cell. Apparently, B. pumila (II) has only a really low amount of carbohydrates, which is also shown in Figure 5c in the averaged MCR-ALS coefficient value of 0.04. The concentration of carbohydrates does not correlate with the collection site, and varies more between the species than is seen in the sporopollenin content. The MCR-ALS coefficients for the B. papyrifera from the Botanic Garden Jena of the Friedrich Schiller University Jena are, at 0.38, higher than those from the Forest Botanical Garden and Arboretum of the University of Göttingen, with 0.29. However, the dataset is too small to give a conclusion about species-dependent correlations.
A new component, which was not revealed by the HCA cluster analysis, is presented in Figure 6. The spectrum is rich in lipid bands. More specifically, several bands can be assigned to phosphate group vibrations of phospholipids at 864, 960, and 1077 cm −1 . Furthermore, 1302 cm −1 is characteristic for lipids. However, influences of proteins can also be seen due to the presence of the phenylalanine ring breathing band at 1006 cm −1 , and the maximum at 2925 cm −1 in the CH stretching region also indicates protein contributions. Some bands can be related to both lipids and proteins. 1263 cm −1 can indicate fatty acids, and the amid III band of proteins and 1657 cm −1 C=C double-bond vibration of lipids and the amid I band of proteins. Again, the band at 1447 cm −1 presents CH 2 /CH 3 deformation vibrations. can be assigned to phosphate group vibrations of phospholipids at 864, 960, and 1077 cm −1 . Furthermore, 1302 cm −1 is characteristic for lipids. However, influences of proteins can also be seen due to the presence of the phenylalanine ring breathing band at 1006 cm −1 , and the maximum at 2925 cm −1 in the CH stretching region also indicates protein contributions. Some bands can be related to both lipids and proteins. 1263 cm −1 can indicate fatty acids, and the amid III band of proteins and 1657 cm −1 C=C double-bond vibration of lipids and the amid I band of proteins. Again, the band at 1447 cm −1 presents CH2/CH3 deformation vibrations. In all of the samples, the representation in the imaging domain reveals that this lipid-and protein-rich component is located in the inner layers of the grains in a rather discrete form, especially in pollen grains of B. alleghenensis (b III). Furthermore, B. pumila (b II), In all of the samples, the representation in the imaging domain reveals that this lipidand protein-rich component is located in the inner layers of the grains in a rather discrete form, especially in pollen grains of B. alleghenensis (b III). Furthermore, B. pumila (b II), which seems to exhibit nearly no carbohydrates, has a higher occurrence of this component. The presence or absence of the lipid-rich component is discussed as an indicator of the germination state of the pollen grain [43]. A high phospholipid content might indicate the accumulation of this membrane component, which is necessary for the growth of the pollen tube. Furthermore, it might reveal the position of the generative cell, which later in the process of germination separates into the sperm cells. The averaged representation in Figure 6c indicates that the pollen grains from the Botanic Garden Jena of the Friedrich Schiller University Jena have a higher lipid content than the grains from the Forest Botanical Garden and Arboretum of the University of Göttingen. Only B. papyrifera from Jena (IV) has a low amount, which correlates with B. papyrifera from Göttingen (VIII).

Sample Preparation for the Raman Analysis
The frozen grains were mortared using a pestle. Two milliliters of distilled water were added to the mortared pollen of each frozen birch catkin, and were suspended. Sieving (2 µm) was used to remove large non-specific components of the grain pollen suspension, and then the suspension was centrifuged at 14,000 rpm at 4 • C. The deposed pellet was stored at −20 • C until analysis for further processes. Using a microscope (400-fold), the purity of the pellet was verified (>95%). For the Raman measurement, the pollen grains were dried under ambient conditions on a CaF 2 slide. This was necessary due to the hydrophobic property of the exine, which is commonly due to the presence of pollenkitt.

Raman Spectroscopic Measurements
The Raman measurements were performed on a confocal Raman microscope alpha300 R (WITec, Ulm, Germany) equipped with a 785 nm narrow-band diode laser (Toptica Photonics, Gräfelingen, Germany). The spectrometer, equipped a 300 g/mm grating, allows us to cover the wavenumber region between 100 to 3300 cm −1 at a spectral resolution of approx. 6 cm −1 . Raman spectra were recorded with an integration time of 0.5 s using a 50× Zeiss objective with 0.9 NA. The prepared pollen grains were placed on a CaF 2 slide, and the Raman images were recorded on individual grains with a step size of 1 µm.
The data analysis of the recorded Raman images was performed in R with hyperSpec as a main package [44,45]. Several preprocessing steps were performed, including wavenumber calibration, with paracetamol spectra as references, and interpolation. The rubberband method was used to correct the background, and afterwards a cosmic spike correction algorithm [46] was used. An area normalization over the range of 400 to 3100 cm −1 was implemented. In order to reduce the dataset, spectra not exhibiting information from the pollen grains were removed, e.g., of the surrounding CaF 2 slide, and a correlation function with a threshold of 0.75 was implemented as a quality control to remove outliers. A Savitzky-Golay function from package parcma was chosen to smooth the spectra with a quadratic filter order and filter length of 9 [47].
An HCA based on a Pearson distance calculation and the Ward clustering algorithm of the entire dataset grouped the spectra into clusters of the same spectral profile. In a false-color representation, the calculated weighted mean spectra of the clusters can be presented in the imaging domain, revealing spectral differences within one pollen grain. Additionally, an MCR-ALS algorithm was chosen to calculate the pure components and their abundances in a quantitative approach [41]. In an alternating fashion, pure spectra and concentration profiles were optimized until a convergence criterion was achieved [42]. The five cluster mean spectra from the HCA were used as input starting spectra, and non-negativity constraints for both the spectra and the concentrations were set.

Conclusions
With this study, we were able to establish a method to characterize the components of pollen grains using Raman spectroscopic imaging. The different layers of pollen grains were identified and visualized based on their biochemical signatures. The established method allows the detailed analysis of single grains, which provides novel information for future studies about, e.g., differences in growth or germination processes. Raman spectroscopy can provide spatially resolved, label-free information non-destructively and rapidly, enabling the analysis of very rare palynological specimens. Moreover, through the development of more automated and compact systems, grains could also be sampled in field studies and tagged directly to individual plants for a more precise investigation of ecological conditions. Furthermore, general investigations are necessary, especially against the background of the changing climatic conditions. We must clarify how climate elements and factors change, and to what extent this leads to the detected differences.