Exploring and Re-Assessing Reverse Anomeric Effect in 2-Iminoaldoses Derived from Mono- and Polynuclear Aromatic Aldehydes

A curious and noticeable structural feature in Schiff bases from 2-aminoaldoses is the fact that imino tautomers arranged equatorially in the most stable ring conformation exhibit a counterintuitive reverse anomeric effect (RAE) in the mutarotational equilibrium, i.e., the most stable and abundant anomer is the equatorial one (β). As shown by our very recent research, this effect arises from the total or partial inhibition of the exo-anomeric effect due to the presence of an intramolecular hydrogen bond between the anomeric hydroxyl and the iminic nitrogen in the axial anomer (α). When the Schiff base adopts either an enamine structure or the imino group is protonated, the exo-anomeric effect is restored, and the axial α-anomer becomes the most stable species. Although the intramolecular H-bonding should appropriately be interpreted as a genuine stereoelectronic effect, the magnitude of the RAE could be affected by other structural parameters. Herein and through a comprehensive analysis of benzylidene, cinnamylidene, naphthalene, phenanthrene, and anthracene aldehydes, we show the robustness of the RAE effect, which is similar in extent to simple aldehydes screened so far, irrespective of the size and/or hydrophobicity of the substituent at the nitrogen atom.


Introduction
Imines or Schiff bases constitute privileged scaffolds dating back to the early days of synthetic organic chemistry that can easily be generated by condensation of carbonyl groups and primary amines.This transformation takes place through the intermediacy of a carbinolamine that undergoes further dehydration, leading to a double carbon-nitrogen bond [1].Over the years, imines derived from carbohydrates have been extensively studied in view of a broad range of applications, such as recognition of naturally occurring amino acids using fluorescence and absorption measurements.Titration of D-glucosamine salicylidenimine (1) with all of the 20 naturally occurring amino acids resulted in large fluorescence enhancements in the case of aromatic amino acids only, thus enabling the recognition of such amino acids down to 1.5-3 ppm through switch-on fluorescence behavior [2,3].Sugar imines and L-amino acids self-assemble by generating 1:1 hydrogen-bonded complexes and forming amphiphilic nanofibers through π-π interactions [4], although other non-covalent interactions may be involved as well.The recognition of M 2+ ions in solution and selective recognition of Cu 2+ in HEPES (a zwitterionic sulfonic acid) buffer are based on the formation of glyco-imino-conjugates [5,6].On the other hand, several imino conjugates of aldoses and D-glucosamine (2), which are transition state analogues, are potent inhibitors of glycosidases extracted from soybean and jack bean meal [7].
buffer are based on the formation of glyco-imino-conjugates [5,6].On the other hand, several imino conjugates of aldoses and D-glucosamine (2), which are transition state analogues, are potent inhibitors of glycosidases extracted from soybean and jack bean meal [7].
The first imine derived from 2-amino-2-deoxyaldoses (1) was reported in as early as 1913 [8][9][10].Later on, Wacker and Fritz in 1967 [11] and Panov et al. in 1973 [12] prepared a series of imines (3) derived from 2-amino-2-deoxy-D-glucopyranose (2) with benzaldehydes and their per-O-acetyl derivatives (4), and demonstrated by 1 H-NMR spectroscopy the appearance of equatorial anomers (β).In fact, with only one exception, all the known Schiff bases obtained from 2 and substituted benzaldehydes devoid of hydroxyl groups at the ortho position crystallize as β-anomers [13] (Chart 1).Certainly, this is a surprising and unexpected behavior because, in general, other aminoaldose derivatives show an axial stereochemistry (α-anomer), as a consequence of the anomeric effect.For imines of 2-aminoaldoses in solution, however, an equilibrium between α-and β-anomers can be detected where the latter largely predominates (Scheme 1).This behavior can be regarded in terms of a reverse anomeric effect (RAE), with values in the range of 1.9-2.3kcal/mol.This stabilization of the equatorial anomer neutralizes and exceeds the anomeric effect.Theoretical calculations show that this stereoelectronic effect results from the reduction (or elimination) of the stabilizing exo-anomeric effect in the axial anomer (5), owing to the formation of a hydrogen bond between the anomeric hydroxyl and the imine nitrogen.Moreover, solvent effects (modeled as discrete solvation) support the preferential formation of the equatorial anomer (β) [13].The present work sheds light into the influence exerted by the aromatic residue on the anomeric effect through a full set of spectral analyses in solution and computational assessment.

Synthesis of 2-Amino-2-Deoxyaldose Imines
We employed as starting aminoaldoses the hydrochlorides of D-glucosamine (2) and 2-amino-2-deoxy-α-D-glycero-L-gluco-heptopyranose (6) [14][15][16], which were condensed with aldehydes derived from aliphatic and mono-and polynuclear aromatic hydrocarbons (Chart 2).Thus, the present study involves new Schiff bases obtained by reaction of benzaldehydes (7-18) with 2 and heptose 6, because for the latter, only the heptosimine derivative 37 had been described [17].Since the lone electron pair on the nitrogen atom For imines of 2-aminoaldoses in solution, however, an equilibrium between αand β-anomers can be detected where the latter largely predominates (Scheme 1).This behavior can be regarded in terms of a reverse anomeric effect (RAE), with values in the range of 1.9-2.3kcal/mol.This stabilization of the equatorial anomer neutralizes and exceeds the anomeric effect.Theoretical calculations show that this stereoelectronic effect results from the reduction (or elimination) of the stabilizing exo-anomeric effect in the axial anomer (5), owing to the formation of a hydrogen bond between the anomeric hydroxyl and the imine nitrogen.Moreover, solvent effects (modeled as discrete solvation) support the preferential formation of the equatorial anomer (β) [13].The present work sheds light into the influence exerted by the aromatic residue on the anomeric effect through a full set of spectral analyses in solution and computational assessment.
Molecules 2024, 29, x FOR PEER REVIEW 2 of 44 buffer are based on the formation of glyco-imino-conjugates [5,6].On the other hand, several imino conjugates of aldoses and D-glucosamine (2), which are transition state analogues, are potent inhibitors of glycosidases extracted from soybean and jack bean meal [7].
The first imine derived from 2-amino-2-deoxyaldoses (1) was reported in as early as 1913 [8][9][10].Later on, Wacker and Fritz in 1967 [11] and Panov et al. in 1973 [12] prepared a series of imines (3) derived from 2-amino-2-deoxy-D-glucopyranose (2) with benzaldehydes and their per-O-acetyl derivatives (4), and demonstrated by 1 H-NMR spectroscopy the appearance of equatorial anomers (β).In fact, with only one exception, all the known Schiff bases obtained from 2 and substituted benzaldehydes devoid of hydroxyl groups at the ortho position crystallize as β-anomers [13] (Chart 1).Certainly, this is a surprising and unexpected behavior because, in general, other aminoaldose derivatives show an axial stereochemistry (α-anomer), as a consequence of the anomeric effect.For imines of 2-aminoaldoses in solution, however, an equilibrium between α-and β-anomers can be detected where the latter largely predominates (Scheme 1).This behavior can be regarded in terms of a reverse anomeric effect (RAE), with values in the range of 1.9-2.3kcal/mol.This stabilization of the equatorial anomer neutralizes and exceeds the anomeric effect.Theoretical calculations show that this stereoelectronic effect results from the reduction (or elimination) of the stabilizing exo-anomeric effect in the axial anomer (5), owing to the formation of a hydrogen bond between the anomeric hydroxyl and the imine nitrogen.Moreover, solvent effects (modeled as discrete solvation) support the preferential formation of the equatorial anomer (β) [13].The present work sheds light into the influence exerted by the aromatic residue on the anomeric effect through a full set of spectral analyses in solution and computational assessment.
Also, the structural variation has been extended to imines derived from cinnamylidene aldehydes (19)(20)(21)(22), in which the ethylene linker separates the bulky aromatic ring from the sugar moiety, which could alleviate the steric hindrance while facilitating the transmission of electronic effects (i.e., the classical vinylogy principle).Furthermore, imines derived from naphthalene, phenanthrene, and anthracene aldehydes have been obtained, which lack a hydroxyl group adjacent to the aldehyde group (23)(24)(25)(26)(27)(28)(29).These compounds allow us to evaluate the potential steric effects associated with their volume, along with the influence of increasing the hydrophobicity of the iminic functionality on the tautomeric equilibrium.
Chart 2. Aldehydes used in the preparation of imines.
Chart 2. Aldehydes used in the preparation of imines.
Also, the structural variation has been extended to imines derived from cinnamylidene aldehydes (19)(20)(21)(22), in which the ethylene linker separates the bulky aromatic ring from the sugar moiety, which could alleviate the steric hindrance while facilitating the transmission of electronic effects (i.e., the classical vinylogy principle).Furthermore, imines derived from naphthalene, phenanthrene, and anthracene aldehydes have been obtained, which lack a hydroxyl group adjacent to the aldehyde group (23)(24)(25)(26)(27)(28)(29).These compounds allow us to evaluate the potential steric effects associated with their volume, along with the influence of increasing the hydrophobicity of the iminic functionality on the tautomeric equilibrium.
Also, the structural variation has been extended to imines derived from cinnamylidene aldehydes (19)(20)(21)(22), in which the ethylene linker separates the bulky aromatic ring from the sugar moiety, which could alleviate the steric hindrance while facilitating the transmission of electronic effects (i.e., the classical vinylogy principle).Furthermore, imines derived from naphthalene, phenanthrene, and anthracene aldehydes have been obtained, which lack a hydroxyl group adjacent to the aldehyde group (23)(24)(25)(26)(27)(28)(29).These compounds allow us to evaluate the potential steric effects associated with their volume, along with the influence of increasing the hydrophobicity of the iminic functionality on the tautomeric equilibrium.
Chart 2. Aldehydes used in the preparation of imines.
Clearly, the synthesis of α-anomers 30, 31, 52 and 54 is interesting, because only one related case having this abnormal configuration has been described in our previous study, involving the reaction of 2 with 18 [13].All reactions took place in hydroalcoholic media by treating 2-aminoaldose hydrochlorides with sodium hydroxide or sodium bicarbonate to release the free bases of the α-anomers (59).Interconversion then occurs between the two anomers (59, 60), which condense with the aryl aldehyde present (61, 62) (Scheme 3).Reactions are often heated at ~60 °C for a few minutes, because the aromatic aldehyde can be poorly soluble at room temperature.
A preliminary assessment of the above-mentioned examples indicates that, in general, reactions conducted at room temperature with concomitant imine crystallization in Scheme 2. Generation of substituted pyrazines from 6 under basic conditions.By using the cinnamylidene aldehydes 19-22, the corresponding β-anomers of Schiff bases 45-49 could be obtained as well (Chart 4).Compounds 45 [19], 46 [20], and 48 [20] were previously described.
Clearly, the synthesis of α-anomers 30, 31, 52 and 54 is interesting, because only one related case having this abnormal configuration has been described in our previous study, involving the reaction of 2 with 18 [13].All reactions took place in hydroalcoholic media by treating 2-aminoaldose hydrochlorides with sodium hydroxide or sodium bicarbonate to release the free bases of the α-anomers (59).Interconversion then occurs between the two anomers (59, 60), which condense with the aryl aldehyde present (61, 62) (Scheme 3).Reactions are often heated at ~60 °C for a few minutes, because the aromatic aldehyde can be poorly soluble at room temperature.
Clearly, the synthesis of α-anomers 30, 31, 52 and 54 is interesting, because only one related case having this abnormal configuration has been described in our previous study, involving the reaction of 2 with 18 [13].All reactions took place in hydroalcoholic media by treating 2-aminoaldose hydrochlorides with sodium hydroxide or sodium bicarbonate to release the free bases of the α-anomers (59).Interconversion then occurs between the two anomers (59, 60), which condense with the aryl aldehyde present (61, 62) (Scheme 3).Reactions are often heated at ~60 • C for a few minutes, because the aromatic aldehyde can be poorly soluble at room temperature.
A preliminary assessment of the above-mentioned examples indicates that, in general, reactions conducted at room temperature with concomitant imine crystallization in short times (less than 15 min) led to α-anomers.In slow reactions taking long (several hours) before crystallization, the β-anomer is usually isolated.

Structural Characterization
In the infrared (FT-IR) spectra of imines 30-42 and 50-58, the absorption of the C=N bond at ~1635-1650 cm −1 stands out.Both 1 H and 13 C NMR spectra support the assigned structures (Tables S1-S6 and S10-S12).Thus, the α-anomeric configuration of 30, 31, 52 and 54 is inferred from the low value of J1,2 (3.8 Hz) and by the downfield shift of H-1 and the upfield shift of the C-1 atom [13], relative to the corresponding signals for β-anomers.The rest of the imines show high coupling constants J1,2 (~7-9 Hz), consistent in all cases with the β-anomer.In addition, the IR spectra of cinnamylidene derivatives 45-49 also show the absorptions arising from the stretching vibration of the ethylene double bond at ~1620 cm −1 .In the proton spectra, the signals of the iminic proton and those of the ethylene fragment should be mentioned, i.e., a doublet at ~7.1 ppm and double doublet at ~6.9 ppm.The large coupling constants between such protons (JCH=CH~16 Hz) indicate that the stereochemistry around the carbon double bond is trans (E).The high coupling constants J1,2 (~8.6 Hz) and the chemical shift of the anomeric carbon (δC1~95 ppm) point to an equatorial (β) disposition of the anomeric hydroxyl in all cases (Tables S7-S9).

Structural Characterization
In the infrared (FT-IR) spectra of imines 30-42 and 50-58, the absorption of the C bond at ~1635-1650 cm −1 stands out.Both 1 H and 13 C NMR spectra support the assign structures (Tables S1-S6 and S10-S12).Thus, the α-anomeric configuration of 30, 31, and 54 is inferred from the low value of J1,2 (3.8 Hz) and by the downfield shift of H-1 a the upfield shift of the C-1 atom [13], relative to the corresponding signals for β-anome The rest of the imines show high coupling constants J1,2 (~7-9 Hz), consistent in all ca with the β-anomer.In addition, the IR spectra of cinnamylidene derivatives 45-49 a show the absorptions arising from the stretching vibration of the ethylene double bond ~1620 cm −1 .In the proton spectra, the signals of the iminic proton and those of the ethyle fragment should be mentioned, i.e., a doublet at ~7.1 ppm and double doublet at ~6.9 pp The large coupling constants between such protons (JCH=CH~16 Hz) indicate that the ste ochemistry around the carbon double bond is trans (E).The high coupling constants (~8.6 Hz) and the chemical shift of the anomeric carbon (δC1~95 ppm) point to an equator (β) disposition of the anomeric hydroxyl in all cases (Tables S7-S9).

Structural Characterization
In the infrared (FT-IR) spectra of imines 30-42 and 50-58, the absorption of the C=N bond at ~1635-1650 cm −1 stands out.Both 1 H and 13 C NMR spectra support the assigned structures (Tables S1-S6 and S10-S12).Thus, the α-anomeric configuration of 30, 31, 52 and 54 is inferred from the low value of J 1,2 (3.8 Hz) and by the downfield shift of H-1 and the upfield shift of the C-1 atom [13], relative to the corresponding signals for β-anomers.The rest of the imines show high coupling constants J 1,2 (~7-9 Hz), consistent in all cases with the β-anomer.In addition, the IR spectra of cinnamylidene derivatives 45-49 also show the absorptions arising from the stretching vibration of the ethylene double bond at ~1620 cm −1 .In the proton spectra, the signals of the iminic proton and those of the ethylene fragment should be mentioned, i.e., a doublet at ~7.1 ppm and double doublet at ~6.9 ppm.The large coupling constants between such protons (J CH=CH ~16 Hz) indicate that the stereochemistry around the carbon double bond is trans (E).The high coupling constants J 1,2 (~8.6 Hz) and the chemical shift of the anomeric carbon (δ C1 ~95 ppm) point to an equatorial (β) disposition of the anomeric hydroxyl in all cases (Tables S7-S9).
The pyranose structure of 30-42 and 50-58 could further be confirmed by transforming some unprotected compounds into the corresponding per-O-acetyl derivatives 63-84 (Charts 6 and 7), which were obtained in good yields by treatment with acetic anhydride in pyridine at ambient temperature [11,26].The pyranose structure of 30-42 and 50-58 could further be confirmed by transforming some unprotected compounds into the corresponding per-O-acetyl derivatives 63-84 (Charts 6 and 7), which were obtained in good yields by treatment with acetic anhydride in pyridine at ambient temperature [11,26].Acetylation of 54 led to an approximately equimolar mixture of both β-(79) and αanomers (80), thereby evidencing that during the acetylation process, 54 had enough time to partially transform into its β-anomer (53) (Scheme 4).Acetylation of 52 also led to an anomeric mixture dominated by the α-anomer 78, which was obtained in pure form by fractional crystallization.The corresponding β-anomer (77) could easily be obtained from 51.
Molecules 2024, 29, x FOR PEER REVIEW 7 of 44 Acetylation of 54 led to an approximately equimolar mixture of both β-(79) and αanomers (80), thereby evidencing that during the acetylation process, 54 had enough time to partially transform into its β-anomer (53) (Scheme 4).Acetylation of 52 also led to an anomeric mixture dominated by the α-anomer 78, which was obtained in pure form by fractional crystallization.The corresponding β-anomer (77) could easily be obtained from 51.In order to minimize the anomerization reaction, we attempted the acetylation of compounds 30 and 31 at a lower temperature (<−10 °C); however, in both cases, the βanomer was obtained (63 and 64, respectively).
Acetylation of 54 led to an approximately equimolar mixture of both β-(79) and αanomers (80), thereby evidencing that during the acetylation process, 54 had enough time to partially transform into its β-anomer (53) (Scheme 4).Acetylation of 52 also led to an anomeric mixture dominated by the α-anomer 78, which was obtained in pure form by fractional crystallization.The corresponding β-anomer (77) could easily be obtained from 51.In order to minimize the anomerization reaction, we attempted the acetylation of compounds 30 and 31 at a lower temperature (<−10 °C); however, in both cases, the βanomer was obtained (63 and 64, respectively).
Acetylation of 54 led to an approximately equimolar mixture of both β-( 79) and αanomers (80), thereby evidencing that during the acetylation process, 54 had enough time to partially transform into its β-anomer (53) (Scheme 4).Acetylation of 52 also led to an anomeric mixture dominated by the α-anomer 78, which was obtained in pure form by fractional crystallization.The corresponding β-anomer (77) could easily be obtained from 51.In order to minimize the anomerization reaction, we attempted the acetylation of compounds 30 and 31 at a lower temperature (<−10 °C); however, in both cases, the βanomer was obtained (63 and 64, respectively).

Mutarotation of Imines
We performed a study on the mutarotation of imines in DMSO, whose origin could be ascribed not only to the existence of an anomeric equilibrium, but also to other phenomena such as tautomeric equilibria, sugar ring-size variation, conformational equilibria, typical carbohydrate rearrangements, reactions with solvent molecules, etc.The mutarotational behavior of imines 32-36 in solution is identical to that described for other imines of 2 derived from benzaldehydes [13], and imines 37-41 derived from 6 behave in the same way (Scheme 5).These imines only equilibrate with their respective α-anomers (93-104), while α-imines 30 and 31 do so with their β-anomers 93 and 94, with the anomeric ratio remaining unaffected for some months (Chart 10).
The mutarotational behavior of imines derived from 2 and 6 with aldehydes bearing fused aromatic rings is similar as well (110-114) (Chart 11).Most of them appear as the β-anomer (50, 51, 53, 55-58), although we were able to isolate two α-anomers (52 and 54).These compounds in solution slowly equilibrate with their β-anomers (88 and 89), which represent the dominant species (Figure 1).When the latter are allowed to evolve in solution, the final percentages of each anomer at equilibrium are approximately the same.
These compounds in solution slowly equilibrate with their β-anomers (88 and 89), which represent the dominant species (Figure 1).When the latter are allowed to evolve in solution, the final percentages of each anomer at equilibrium are approximately the same.Table 1 shows the percentage variation for the β-anomer of some imines using DMSO-d6 and pyridine-d5 as solvents.In both cases, the results obtained are similar, although in pyridine the equilibration occurs faster.Probably the basic nature of this solvent is behind the rapid anomerization, since it is known that this phenomenon is sensitive to general acidic and basic catalysis [31].This rapid anomerization in pyridine explains the failure to prepare per-O-acetylated α-anomers from α-imines, such as 30 and 31, or the formation of mixtures of both anomers (as happens with 52 and 54).The absence of typical oxazolidine signals at ~5-6 ppm and ~90-97 ppm [32][33][34][35][36][37], rules out the possibility of an equilibrium involving such five-membered heterocycles, which result from addition of the anomeric hydroxyl to the imine bond.These compounds in solution slowly equilibrate with their β-anomers (88 and 89), which represent the dominant species (Figure 1).When the latter are allowed to evolve in solution, the final percentages of each anomer at equilibrium are approximately the same.Table 1 shows the percentage variation for the β-anomer of some imines using DMSO-d6 and pyridine-d5 as solvents.In both cases, the results obtained are similar, although in pyridine the equilibration occurs faster.Probably the basic nature of this solvent is behind the rapid anomerization, since it is known that this phenomenon is sensitive to general acidic and basic catalysis [31].This rapid anomerization in pyridine explains the failure to prepare per-O-acetylated α-anomers from α-imines, such as 30 and 31, or the formation of mixtures of both anomers (as happens with 52 and 54).The absence of typical oxazolidine signals at ~5-6 ppm and ~90-97 ppm [32][33][34][35][36][37], rules out the possibility of an equilibrium involving such five-membered heterocycles, which result from addition of the anomeric hydroxyl to the imine bond.Table 1 shows the percentage variation for the β-anomer of some imines using DMSOd 6 and pyridine-d 5 as solvents.In both cases, the results obtained are similar, although in pyridine the equilibration occurs faster.Probably the basic nature of this solvent is behind the rapid anomerization, since it is known that this phenomenon is sensitive to general acidic and basic catalysis [31].This rapid anomerization in pyridine explains the failure to prepare per-O-acetylated α-anomers from α-imines, such as 30 and 31, or the formation of mixtures of both anomers (as happens with 52 and 54).The absence of typical oxazolidine signals at ~5-6 ppm and ~90-97 ppm [32][33][34][35][36][37], rules out the possibility of an equilibrium involving such five-membered heterocycles, which result from addition of the anomeric hydroxyl to the imine bond.

Conformational Analysis
The high coupling constants J2,3 ≈ J3,4 ≈ J4,5 ≥ 9 Hz fully agree with a D-gluco configuration in 4 C1 conformation for all the imines derived from 2 and 1 C4 (L-gluco) for those based on heptose 6 (Figure 2) [38,39].NOE experiments [40,41] carried out on compound 69 gave rise to the enhancements shown in Figure 3, which confirmed the proximity of H-2, the iminic hydrogen, and one of the ortho hydrogens at the aromatic ring, all consistent with a 1 C4 (L-gluco) conformation and coincidental with that determined through NMR spectroscopic data.Similar NOE enhancements were observed for 82 having a 4 C1 (D-gluco) conformation.The absence of a NOE effect between the H-1 and H-2 protons is in agreement with their β-configuration.Such NOE effects, together with those determined in other imines from 2 [13], strongly support some key structural features, namely the planarity of the arylimino group, its (E)-configuration, and the fact that the half-plane containing the entire conjugated unsaturated system is approximately perpendicular to the plane of the pyranose ring (Figure 4).NOE experiments [40,41] carried out on compound 69 gave rise to the enhancements shown in Figure 3, which confirmed the proximity of H-2, the iminic hydrogen, and one of the ortho hydrogens at the aromatic ring, all consistent with a 1 C 4 (L-gluco) conformation and coincidental with that determined through NMR spectroscopic data.Similar NOE enhancements were observed for 82 having a 4 C 1 (D-gluco) conformation.The absence of a NOE effect between the H-1 and H-2 protons is in agreement with their β-configuration.

Conformational Analysis
The high coupling constants J2,3 ≈ J3,4 ≈ J4,5 ≥ 9 Hz fully agree with a D-gluco configuration in 4 C1 conformation for all the imines derived from 2 and 1 C4 (L-gluco) for those based on heptose 6 (Figure 2) [38,39].NOE experiments [40,41] carried out on compound 69 gave rise to the enhancements shown in Figure 3, which confirmed the proximity of H-2, the iminic hydrogen, and one of the ortho hydrogens at the aromatic ring, all consistent with a 1 C4 (L-gluco) conformation and coincidental with that determined through NMR spectroscopic data.Similar NOE enhancements were observed for 82 having a 4 C1 (D-gluco) conformation.The absence of a NOE effect between the H-1 and H-2 protons is in agreement with their β-configuration.Such NOE effects, together with those determined in other imines from 2 [13], strongly support some key structural features, namely the planarity of the arylimino group, its (E)-configuration, and the fact that the half-plane containing the entire conjugated unsaturated system is approximately perpendicular to the plane of the pyranose ring (Figure 4).Such NOE effects, together with those determined in other imines from 2 [13], strongly support some key structural features, namely the planarity of the arylimino group, its (E)configuration, and the fact that the half-plane containing the entire conjugated unsaturated system is approximately perpendicular to the plane of the pyranose ring (Figure 4).

Conformational Analysis
The high coupling constants J2,3 ≈ J3,4 ≈ J4,5 ≥ 9 Hz fully agree with a D-gluco configuration in 4 C1 conformation for all the imines derived from 2 and 1 C4 (L-gluco) for those based on heptose 6 (Figure 2) [38,39].NOE experiments [40,41] carried out on compound 69 gave rise to the enhancements shown in Figure 3, which confirmed the proximity of H-2, the iminic hydrogen, and one of the ortho hydrogens at the aromatic ring, all consistent with a 1 C4 (L-gluco) conformation and coincidental with that determined through NMR spectroscopic data.Similar NOE enhancements were observed for 82 having a 4 C1 (D-gluco) conformation.The absence of a NOE effect between the H-1 and H-2 protons is in agreement with their β-configuration.Such NOE effects, together with those determined in other imines from 2 [13], strongly support some key structural features, namely the planarity of the arylimino group, its (E)-configuration, and the fact that the half-plane containing the entire conjugated unsaturated system is approximately perpendicular to the plane of the pyranose ring (Figure 4).Again, the large coupling constants between the ethylene protons (J CH=CH ~16 Hz) measured for 45-49 and their acetyl derivatives (73)(74)(75) indicate that the stereochemistry of the double bond is trans (E).Furthermore, the coupling constant between the imine proton and the neighboring ethylene proton (J CH-CH=N ~8.8 Hz) evidences that both protons maintain an antiperiplanar relationship (Figure 4).It is interesting to note that the H-2 signal in imines is usually the most deshielded resonance, appearing at ~2.9 ppm, except in the case of anthracenes 56-58 and their acetylated derivatives 82-84, which are shifted downfield (∆δ H-2 ~0.3 ppm); and the same happens to the iminic hydrogen of these compounds (∆δ CH=N ~0.8-1 ppm).Such variations are not shown by phenanthrene derivatives 55 and 80, which behave similarly to naphthalenes 50-54 and 76-80.The origin of the observed variations lies most likely in the spatial arrangement of the anthracene nucleus, whose proximity to the H-2 and CH=N protons would cause this deshielding.
Steric effects are noticeable in imines derived from anthracene, involving both the iminic hydrogen and the nitrogen atom.Steric tension can in part be relieved by rotating the aromatic system, although this reduces the delocalization through the imine double bond (Figure 5).dene derivatives (R=H or Ac).
Again, the large coupling constants between the ethylene protons (JCH=CH ~16 Hz) measured for 45-49 and their acetyl derivatives (73)(74)(75) indicate that the stereochemistry of the double bond is trans (E).Furthermore, the coupling constant between the imine proton and the neighboring ethylene proton (JCH-CH=N~8.8Hz) evidences that both protons maintain an antiperiplanar relationship (Figure 4).It is interesting to note that the H-2 signal in imines is usually the most deshielded resonance, appearing at ~2.9 ppm, except in the case of anthracenes 56-58 and their acetylated derivatives 82-84, which are shifted downfield (ΔδH-2~0.3ppm); and the same happens to the iminic hydrogen of these compounds (ΔδCH=N~0.8-1ppm).Such variations are not shown by phenanthrene derivatives 55 and 80, which behave similarly to naphthalenes 50-54 and 76-80.The origin of the observed variations lies most likely in the spatial arrangement of the anthracene nucleus, whose proximity to the H-2 and CH=N protons would cause this deshielding.
Steric effects are noticeable in imines derived from anthracene, involving both the iminic hydrogen and the nitrogen atom.Steric tension can in part be relieved by rotating the aromatic system, although this reduces the delocalization through the imine double bond (Figure 5).Accordingly, a conformational analysis of the aryl moiety of 56 and 112 has been achieved, and the energy landscape of the arrangements generated around the NC-Carom bond calculated, i.e., by rotating the dihedral angle θN=C-C1-C2 from 0° to 360° with a step size of 15° each.The DFT study was performed using the 6-311G(d,p) [42,43] and def2-TZVP valence-triple-ζ [44] basis sets, with all geometries optimized in the gas phase at the B3LYP [45,46] and M06-2X [47] levels of theory without any geometrical restriction.The M06-2X/def2-TZVP combination has been reported to provide suitable geometry optimization in terms of cost and accuracy for carbohydrate derivatives [48][49][50].Solvent effects were simulated using the SMD method [51].Such results are shown in Figure 6 and Table 2.The graph is repeated every 180°, and the two minima obtained are almost identical for both anomers, the most stable conformers corresponding to dihedral angle values θN=C-C1-C2 from ~40° to 50° (Figure 7).In other words, the minima represent a compromise to reach electron delocalization while reducing steric hindrance.Calculations using the 6-311G(d,p) basis set afford similar results for both anomers.However at the def2-TZVP level, the angle rotated by the β-anomer to reach the first minimum is similar, but in the Accordingly, a conformational analysis of the aryl moiety of 56 and 112 has been achieved, and the energy landscape of the arrangements generated around the NC-C arom bond calculated, i.e., by rotating the dihedral angle θ N=C-C1-C2 from 0 • to 360 • with a step size of 15 • each.The DFT study was performed using the 6-311G(d,p) [42,43] and def2-TZVP valence-triple-ζ [44] basis sets, with all geometries optimized in the gas phase at the B3LYP [45,46] and M06-2X [47] levels of theory without any geometrical restriction.The M06-2X/def2-TZVP combination has been reported to provide suitable geometry optimization in terms of cost and accuracy for carbohydrate derivatives [48][49][50].Solvent effects were simulated using the SMD method [51].Such results are shown in Figure 6 and Table 2.

Theoretical Analysis of Imine Stability
A computational study to determine the relative stability of the different species involved in mutarotational equilibria appears to be a compulsory task.The simplest imine pair derived from benzaldehyde (30/93) was selected to shorten the computational cost.The number of possible conformations is exceedingly high: the three staggered conformations of three hydroxyls and the iminic substituent of the pyranose ring, together with  The graph is repeated every 180 • , and the two minima obtained are almost identical for both anomers, the most stable conformers corresponding to dihedral angle values θ N=C-C1-C2 from ~40 • to 50 • (Figure 7).In other words, the minima represent a compromise to reach electron delocalization while reducing steric hindrance.Calculations using the 6-311G(d,p) basis set afford similar results for both anomers.However at the def2-TZVP level, the angle rotated by the β-anomer to reach the first minimum is similar, but in the opposite direction (−40 • to −43 • ).The calculated conformation corresponding to the most stable point for other polynuclear imines shows dihedral angle values θ H2-C2-N=CH from ~0• to 7 • (vide infra); the identical conformation is inferred from NOE effects.

Theoretical Analysis of Imine Stability
A computational study to determine the relative stability of the different species involved in mutarotational equilibria appears to be a compulsory task.The simplest imine pair derived from benzaldehyde (30/93) was selected to shorten the computational cost.The number of possible conformations is exceedingly high: the three staggered conformations of three hydroxyls and the iminic substituent of the pyranose ring, together with the nine (3 × 3) conformations adopted by the hydroxymethyl group at C-5, which amount to 3 6 = 729 conformations for each anomer.Some simplifications can be envisaged for the hydroxyl groups, taking into account that the most stable conformations will be those leading to intramolecular hydrogen bonding.We then considered several dispositions, and the most stable conformers correspond to 30 and 93, which differ only by the orientation of the anomeric OH group (Figure 8).This hydroxyl is oriented towards the electron pair of the nitrogen atom, enabling an intramolecular hydrogen bond in the α-anomer (30).In the β-anomer, that hydroxyl is arranged along the direction of the endocyclic oxygen (93).This methodology reduces drastically the number of structures to be calculated.

Theoretical Analysis of Imine Stability
A computational study to determine the relative stability of the different species involved in mutarotational equilibria appears to be a compulsory task.The simplest imine pair derived from benzaldehyde (30/93) was selected to shorten the computational cost.The number of possible conformations is exceedingly high: the three staggered conformations of three hydroxyls and the iminic substituent of the pyranose ring, together with the nine (3 × 3) conformations adopted by the hydroxymethyl group at C-5, which amount to 3 6 = 729 conformations for each anomer.Some simplifications can be envisaged for the hydroxyl groups, taking into account that the most stable conformations will be those leading to intramolecular hydrogen bonding.We then considered several dispositions, and the most stable conformers correspond to 30 and 93, which differ only by the orientation of the anomeric OH group (Figure 8).This hydroxyl is oriented towards the electron pair of the nitrogen atom, enabling an intramolecular hydrogen bond in the α-anomer (30).In the β-anomer, that hydroxyl is arranged along the direction of the endocyclic oxygen (93).This methodology reduces drastically the number of structures to be calculated.

Theoretical Analysis of Imine Stability
A computational study to determine the relative stability of the differe volved in mutarotational equilibria appears to be a compulsory task.The si pair derived from benzaldehyde (30/93) was selected to shorten the compu The number of possible conformations is exceedingly high: the three stagg mations of three hydroxyls and the iminic substituent of the pyranose ring, t the nine (3 × 3) conformations adopted by the hydroxymethyl group at C-5, w to 3 6 = 729 conformations for each anomer.Some simplifications can be envi hydroxyl groups, taking into account that the most stable conformations leading to intramolecular hydrogen bonding.We then considered several and the most stable conformers correspond to 30 and 93, which differ only b tion of the anomeric OH group (Figure 8).This hydroxyl is oriented toward pair of the nitrogen atom, enabling an intramolecular hydrogen bond in t (30).In the β-anomer, that hydroxyl is arranged along the direction of the e ygen (93).This methodology reduces drastically the number of structures to b All the potential species involved in mutarotational equilibria (30, 93, 115, and 116) [52], and the heterocycles that could have been formed by reaction of the imino group with the anomeric hydroxyl (117-118), have been taken into account and are depicted in Scheme 6.
Moreover, for bicyclic structures like 117-120, the two possible orientations of the hydrogen atom at the NH group, i.e., either axial (a) or pseudo-equatorial (e), have been considered as well (Chart 12 and Table 3).The tabulated data also collect the relative energies obtained by computation in the gas phase and using bulk solvation in DMSO, the solvent where NMR spectra are recorded (for optimized structures, see Figure 9).
All the potential species involved in mutarotational equilibria (30, 93, 115, and 116) [52], and the heterocycles that could have been formed by reaction of the imino group with the anomeric hydroxyl (117-118), have been taken into account and are depicted in Scheme 6. Scheme 6. Acyclic and cyclic structures considered in mutarotational equilibria.
Moreover, for bicyclic structures like 117-120, the two possible orientations of the hydrogen atom at the NH group, i.e., either axial (a) or pseudo-equatorial (e), have been considered as well (Chart 11 and Table 3).The tabulated data also collect the relative energies obtained by computation in the gas phase and using bulk solvation in DMSO, the solvent where NMR spectra are recorded (for optimized structures, see Figure 9).Results obtained with the two hybrid functionals, B3LYP and M06-2X, are quite similar.Both in the gas phase and DMSO, the imine having an anomeric α-configuration is slightly more stable than its β-counterpart, although the difference is, indeed, so small that they can be ranked with identical stability.Both anomers (30 and 93) are interconverted through an acyclic aldehydic form.We estimated the energy of the two conformations adopted by the side chain along with the orientations of the aldehyde group that would lead to each anomer (115 and 116) [52].The pronounced energy difference with respect to the corresponding pyranoid forms, in general ≥7 kcal/mol, explains why the acyclic forms hardly reach detectable concentrations in NMR experiments.It is well known, for example, that although the acyclic form of D-glucose is present in aqueous solutions, more than 99% actually exists as pyranose structures [38,39].All the potential species involved in mutarotational equilibria (30, 93, 115, and 116) [52], and the heterocycles that could have been formed by reaction of the imino group with the anomeric hydroxyl (117-118), have been taken into account and are depicted in Scheme 6. Scheme 6. Acyclic and cyclic structures considered in mutarotational equilibria.
Moreover, for bicyclic structures like 117-120, the two possible orientations of the hydrogen atom at the NH group, i.e., either axial (a) or pseudo-equatorial (e), have been considered as well (Chart 11 and Table 3).The tabulated data also collect the relative energies obtained by computation in the gas phase and using bulk solvation in DMSO, the solvent where NMR spectra are recorded (for optimized structures, see Figure 9).Results obtained with the two hybrid functionals, B3LYP and M06-2X, are quite similar.Both in the gas phase and DMSO, the imine having an anomeric α-configuration is slightly more stable than its β-counterpart, although the difference is, indeed, so small that they can be ranked with identical stability.Both anomers (30 and 93) are interconverted through an acyclic aldehydic form.We estimated the energy of the two conformations adopted by the side chain along with the orientations of the aldehyde group that would lead to each anomer (115 and 116) [52].The pronounced energy difference with respect to the corresponding pyranoid forms, in general ≥7 kcal/mol, explains why the acyclic forms hardly reach detectable concentrations in NMR experiments.It is well known, for example, that although the acyclic form of D-glucose is present in aqueous solutions, more than 99% actually exists as pyranose structures [38,39].Results obtained with the two hybrid functionals, B3LYP and M06-2X, are quite similar.Both in the gas phase and DMSO, the imine having an anomeric α-configuration is slightly more stable than its β-counterpart, although the difference is, indeed, so small that they can be ranked with identical stability.Both anomers (30 and 93) are interconverted through an acyclic aldehydic form.We estimated the energy of the two conformations adopted by the side chain along with the orientations of the aldehyde group that would lead to each anomer (115 and 116) [52].The pronounced energy difference with respect to the corresponding pyranoid forms, in general ≥7 kcal/mol, explains why the acyclic forms hardly reach detectable concentrations in NMR experiments.It is well known, for example, that although the acyclic form of D-glucose is present in aqueous solutions, more than 99% actually exists as pyranose structures [38,39].
ΔGa ≤ 1.9 kcal/mol in DMSO).However, for 119 and 120, the axial disposition becomes significantly stabilized (ΔΔG = ΔGa − ΔGe ≤ 3.2 kcal/mol in DMSO).The small energy difference with respect to 119e (0.89 kcal/mol in DMSO) is surprising, suggesting it is possible that the species could be formed in the reaction mixture.However, as indicated above, the signals characteristic of the oxazolidine ring at ~5 ppm are not observed in the 1 H NMR spectrum, thereby ruling out this speculation.

Anomeric Stabilization of 2-Aminoaldose Derivatives
A well-established principle in conformational analysis is that electronegative anomeric groups preferentially adopt an axial arrangement at the pyranose ring of sugars.This predisposition, contrary to expectations based on steric or solvation factors [53][54][55][56][57][58][59][60], is attributed to the existence of a stereoelectronic effect known as the anomeric effect.Its origin is associated with the hyperconjugation of the electron pairs on oxygen with the anomeric bond, also called the endo-anomeric effect.In turn, the anomeric substituent can generate a similar effect, involving the bonding orbital of the anomeric carbon and the oxygen of the pyranoid ring, which is known as exo-anomeric effect [54].Both effects, Regarding the possible cyclic structures, it is worth noting that those arising from the cyclization of the β-anomer, trans-oxazolidines 117 and 118, are much less stable than the imine structure, ∆∆G DMSO = ∆G oxaz.trans− ∆G imine ≥ 8.6 kcal/mol (in DMSO), probably due to the strain associated with trans-fusion of a six-membered ring to a constrained pentagonal cycle.In stark contrast, for cis-oxazolidines 119 and 120, the steric strain is lower, yet imines represent the most stable tautomers, ∆∆G DMSO = ∆G oxaz.cis− ∆G imine ≥ 0.9 kcal/mol (in DMSO).
Either axial or pseudo-equatorial arrangements of the NH cause little variations in the case of 117 and 118, with the equatorial arrangement being more stable (∆∆G = ∆G e − ∆G a ≤ 1.9 kcal/mol in DMSO).However, for 119 and 120, the axial disposition becomes significantly stabilized (∆∆G = ∆G a − ∆G e ≤ 3.2 kcal/mol in DMSO).The small energy difference with respect to 119e (0.89 kcal/mol in DMSO) is surprising, suggesting it is possible that the species could be formed in the reaction mixture.However, as indicated above, the signals characteristic of the oxazolidine ring at ~5 ppm are not observed in the 1 H NMR spectrum, thereby ruling out this speculation.

Anomeric Stabilization of 2-Aminoaldose Derivatives
A well-established principle in conformational analysis is that electronegative anomeric groups preferentially adopt an axial arrangement at the pyranose ring of sugars.This predisposition, contrary to expectations based on steric or solvation factors [53][54][55][56][57][58][59][60], is attributed to the existence of a stereoelectronic effect known as the anomeric effect.Its origin is associated with the hyperconjugation of the electron pairs on oxygen with the anomeric bond, also called the endo-anomeric effect.In turn, the anomeric substituent can generate a similar effect, involving the bonding orbital of the anomeric carbon and the oxygen of the pyranoid ring, which is known as exo-anomeric effect [54].Both effects, together with neighboring gauche effects [61][62][63], are mainly responsible for the conformational arrangements of sugar derivatives and their reactivity [64].In the absence of other factors, the exo-anomeric effect constitutes the most dominant interaction, even in the α-anomer.
The anomeric effect in carbohydrates is a complex, often puzzling, issue, although it can be interpreted by a combination of steric, resonance, hyperconjugation, inductive, hydrogen bonding, electrostatic, and solvation effects.The extent of such effects depends on the model and level of computation chosen [65].It is believed that both steric and electronic interactions make contributions to the conformational preference, as any decomposition of such interactions is more or less arbitrary [66].Some authors suggest that the steric interaction (or eventually a given electrostatic interaction) dominates the anomeric effect [67] and found further computational evidence to disprove the hyperconjugation explanation [68,69].A cautionary corollary is that no single factor accounts for the axial preference of a substituent, while different and correlated interactions should be involved [70].Moreover, the hyperconjugation model involving the electron transfer from the ring heteroatom to an excited state of an axial bond is a minor contributor to the anomeric effect.However, the effects exerted by substituents on the anomeric effect in positions other than the anomeric carbon have been scarcely studied.In any case, experimental data show that the most influential substituents are those located at the position adjacent to the anomeric center.
Anomeric stabilization in tetrahydropyranose sugars (E an ), defined as the non-steric stabilization of the axial conformer, can be quantified by correcting the axial preference of a substituent, ∆G o an , with the steric effects favoring an equatorial arrangement, ∆G o steric (Equation ( 1)): where ∆G o an is the observed free energy change for the balance between the axial and equatorial disposition, i.e., α-anomer ⇌ β-anomer equilibrium (Equation ( 2)): ∆G o steric can be estimated through non-anomeric model compounds, with the A x values of cyclohexane usually employed to this end (Equation ( 3)): Thus, the A OH value for the hydroxyl group in aqueous solution is 1.25 kca/mol [=0.002 × 298 × ln(89/11)] and corresponds to an 89% predominance of cyclohexanol with the OH group placed in equatorial disposition [71,72].When one varies the substituents at non-anomeric positions, a quantitative relationship for the anomeric hydroxyl group can be expressed by Equation ( 4): A parameter capable of quantifying the magnitude of the RAE in imines (∆G o rae ) could be determined as the difference between the stabilization due exclusively to the anomeric effect (∆G o ae ) minus the anomeric stabilization in imines (∆G o imine ).If we take the anomeric effect as the value of E an shown by 121 in DMSO-d 6 , 0.6 ln[(47.1)/(52.9)]+ 1.25 = 1.32 kcal/mol, Equation ( 5) is obtained: The values of Ax in tetrahydropyrans are greater than those obtained for cyclohexanes.Accordingly, the calculated anomeric effects (as E an ) in Tables 4-7 are approximate values.The steric interactions in the axial disposition of the substituent are more intense because the C-O bond in tetrahydropyran is shorter than in cyclohexane.Equation (6) extrapolates approximately the values of A X (for cyclohexane ring) to the corresponding value in a tetrahydropyran ring (A X THP ) [73]:    The A OH THP value for the hydroxyl group in tetrahydropyran is 1.93, and the corresponding values of E an would increase by 0.68 (=1.93 − 1.25) kcal/mol (Equation ( 7)): E an THP = −RTlnK an + A OH THP = −0.6 lnK an + 1.93 (7) All the imines studied through this work show a broad preference for an equatorial arrangement of the anomeric hydroxyl, which clearly deviates from the expected anomeric effect.This behavior has its origin in the total or partial inhibition of the exo-anomeric effect in the α-anomer, which stems from the H-bonding between the imine nitrogen and the anomeric hydroxyl.In line with the above equations, all data from the studied equilibria are gathered in Tables 4-7.
Special attention has been paid to imines derived from 2 with benzaldehydes bearing strong EWG (8, 9, 12, 13).As already mentioned, since the lone pair on the nitrogen lies in the nodal plane of the arylimino system, the electronic effects exerted by the substituents at the aromatic nucleus can only be inductive.EWGs could decrease the basicity of the nitrogen atom, thus weakening the intramolecular H-bond with the anomeric hydroxyl and, as a result, the reverse anomeric effect.EDGs (10,11) should exhibit the opposite trend.However, no appreciable variations ascribed to the electronic effect of the substituents could be observed, and the extent of the RAE (∆G o rae ) remains above 1 kcal/mol.Data collected in Table 5 show that the behavior of imines derived from 2-amino-2-deoxy-D-glycero-L-glucoheptopyranose with substituted benzaldehydes (37-41) is essentially identical to those derived from D-glucosamine.Therefore, a similar RAE can be invoked (122) (Chart 13) with comparable ∆G o rae values.Data in Table 6 show that separation of the tetrahydropyran ring from the aromatic moiety by the ethylene bridge in imines 45-49 has no appreciable effect on the magnitude of the RAE, either.Special attention has been paid to imines derived from 2 with benzaldehydes bearing strong EWG (8, 9, 12, 13).As already mentioned, since the lone pair on the nitrogen lies in the nodal plane of the arylimino system, the electronic effects exerted by the substituents at the aromatic nucleus can only be inductive.EWGs could decrease the basicity of the nitrogen atom, thus weakening the intramolecular H-bond with the anomeric hydroxyl and, as a result, the reverse anomeric effect.EDGs (10,11) should exhibit the opposite trend.However, no appreciable variations ascribed to the electronic effect of the substituents could be observed, and the extent of the RAE (ΔG o rae) remains above 1 kcal/mol.Data collected in Table 5 show that the behavior of imines derived from 2-amino-2-deoxy-Dglycero-L-gluco-heptopyranose with substituted benzaldehydes (37-41) is essentially identical to those derived from D-glucosamine.Therefore, a similar RAE can be invoked (122) (Chart 12) with comparable ΔG o rae values.Data in Table 6 show that separation of the tetrahydropyran ring from the aromatic moiety by the ethylene bridge in imines 45-49 has no appreciable effect on the magnitude of the RAE, either.Also, imines 50-58 with fused aromatic rings exhibit the same RAE as those previously mentioned and in similar extent (Table 7).One can conclude with confidence that this effect is neither significantly influenced by the volume of the aromatic substituent nor by its hydrophobicity.

Theoretical Analysis of Anomer Stability
Theoretical calculations have also been conducted to assess the relative stability of both anomers through the whole structural range of the imines synthesized here.Since aldehydes 23, 26 and 27 do not have a plane of symmetry, their imines can adopt two different conformational dispositions for the imine group, which have been calculated as well.Results collected in Table 8 have been obtained with the M06-2X functional and using def2-TZVP as basis set.The def2-TZVP base indicates a greater stability of the β-anomer.The corresponding optimized geometries are shown in Figures 10 and 11. Figure 10.Optimized calculated structures of some representative α-anomers at the M06-2X/def2-TZVP.Figure 10.Optimized calculated structures of some representative α-anomers at the M06-2X/def2-TZVP.

OR PEER REVIEW 19 of 44
In all α-anomers, the anomeric hydroxyl is oriented towards the electron pair of the nitrogen, generating an intramolecular hydrogen bond.The β-anomers cannot form it, and this hydroxyl points to the endocyclic oxygen.The results of a natural bonding orbital (NBO) [74] analysis, carried out for α-and βanomers of some representative imines and involving the heteroatoms attached to the anomeric carbon and C-2, are shown in Tables S28 and S29 (numbering is shown in Figure 12).The energy difference (∆G) calculated in DMSO (SMD method [51]), the solvent in which the anomeric equilibria have been evaluated, has allowed us to determine the expected proportion of the β-anomer at 298 K according to Equation (8) (Table 8, last column).The calculated equilibrium percentages of β-anomers vary from 63 to 85%, similarly to those determined experimentally.
The results of a natural bonding orbital (NBO) [74] analysis, carried out for αand β-anomers of some representative imines and involving the heteroatoms attached to the anomeric carbon and C-2, are shown in Tables S28 and S29 (numbering is shown in Figure 12).The results of a natural bonding orbital (NBO) [74] analysis, carried out for α-and βanomers of some representative imines and involving the heteroatoms attached to the anomeric carbon and C-2, are shown in Tables S28 and S29 (numbering is shown in Figure 12).All imines show similar stabilizing interactions for both anomers, regardless of the absence or presence of solvent.The stereoelectronic interactions of the lone pair on the nitrogen atom with the proton at C-2, n→σ*C2-H (~6-7 kcal/mol) and the iminic CH, n→σ*=CH (~12-13 kcal/mol), contribute to the perpendicular disposition of the imino group with respect to the pyranose plane, as deduced by NOE experiments.
The lone pairs on the endocyclic oxygen show delocalization with the antiparallel neighboring C-C and C-H bonds, with values for the nO10→σ*C-C and nO10→σ*C-H interactions of ~6-8 kcal/mol.In the α-anomer, the interaction responsible for the anomeric effect, nO10→σ*C1-O35, amounts to ~13.5 kcal/mol.The electron pairs on the anomeric hydroxyl oxygen show similar effects, highlighting an exo-anomeric effect in the β-anomer, nO35→σ*C1-O10, of ~15-17 kcal/mol.This effect is absent in the α-anomer due to hydrogen bonding between the anomeric hydroxyl and the imine nitrogen.
A more realistic calculation considers explicit solvent molecules, specifically water, around the imine molecule (Table 9).There are five water molecules that interact directly with the hydroxyl groups through intermolecular hydrogen bonds and form the first solvation shell, with the exception of α-anomers, whose anomeric hydroxyl is involved in the All imines show similar stabilizing interactions for both anomers, regardless of the absence or presence of solvent.The stereoelectronic interactions of the lone pair on the nitrogen atom with the proton at C-2, n→σ* C2-H (~6-7 kcal/mol) and the iminic CH, n→σ* =CH (~12-13 kcal/mol), contribute to the perpendicular disposition of the imino group with respect to the pyranose plane, as deduced by NOE experiments.
The lone pairs on the endocyclic oxygen show delocalization with the antiparallel neighboring C-C and C-H bonds, with values for the n O10 →σ* C-C and n O10 →σ* C-H interactions of ~6-8 kcal/mol.In the α-anomer, the interaction responsible for the anomeric effect, n O10 →σ* C1-O35 , amounts to ~13.5 kcal/mol.The electron pairs on the anomeric hydroxyl oxygen show similar effects, highlighting an exo-anomeric effect in the β-anomer, n O35 →σ* C1-O10 , of ~15-17 kcal/mol.This effect is absent in the α-anomer due to hydrogen bonding between the anomeric hydroxyl and the imine nitrogen.
A more realistic calculation considers explicit solvent molecules, specifically water, around the imine molecule (Table 9).There are five water molecules that interact directly with the hydroxyl groups through intermolecular hydrogen bonds and form the first solvation shell, with the exception of α-anomers, whose anomeric hydroxyl is involved in the H-bond with the iminic nitrogen atom.The proportion of the β-anomer in DMSO, deduced from the relative stability between both anomers, is ~82%-99%, an estimation almost coincidental with the experimental values determined in that solvent.This percentage increases as the solvent's dielectric constant increases, thus predicting a complete preponderance of the β-anomer in water.

Intramolecular Hydrogen Bond Strength
Based on calculated geometrical data, the strength of this intramolecular H-bonding could be approximately estimated by a well-known empirical relationship (Equation ( 9)) [75], where d D. ..A represents the calculated distance between donor (O) and acceptor (N) atoms involved in H-bonding (Table 10).

D-H•
Similar results are obtained at the M06-2X/6-311G(d,p) and def2-TZVP levels of theory, being approximately 6.5-7 kcal/mol, both in the gas phase and in the presence of solvent molecules (DMSO).Such energy values point to a moderate strength of the OH . . .N bond in α-anomers (Table 10, last column).

Inhibition of the Reverse Anomeric Effect
When an intramolecular hydrogen bond cannot be formed in the α-anomer, then the RAE is totally or partially eliminated.This occurs when the Schiff base adopts an enamine structure.Thus, for example, when enamine 123 [17] is allowed to remain in DMSO-d 6 solution, equilibration with its β-anomer (124), which is the minor species (β-form: 15.3%), could be established after more than 2 months (Scheme 7).In this case, the intramolecular H-bonding is established with the carbonyl group of the enamine fragment, thereby inhibiting the bonding to the anomeric hydroxyl, and the exo-anomeric effect is totally or partially restored.

Inhibition of the Reverse Anomeric Effect
When an intramolecular hydrogen bond cannot be formed in the α-anomer, then the RAE is totally or partially eliminated.This occurs when the Schiff base adopts an enamine structure.Thus, for example, when enamine 123 [17] is allowed to remain in DMSO-d6 solution, equilibration with its β-anomer (124), which is the minor species (β-form: 15.3%), could be established after more than 2 months (Scheme 7).In this case, the intramolecular H-bonding is established with the carbonyl group of the enamine fragment, thereby inhibiting the bonding to the anomeric hydroxyl, and the exo-anomeric effect is totally or partially restored.Other examples are illustrated by compounds 125 [76] and 126 [15,20] (Chart 13, Table 11).Schiff base 127 [77] crystallizes as the α-anomer, but in solution, it equilibrates with the β-anomer, which is the predominant species [13].In this case, the RAE is only partially attenuated.Other examples are illustrated by compounds 125 [76] and 126 [15,20] (Chart 14, Table 11).Schiff base 127 [77] crystallizes as the α-anomer, but in solution, it equilibrates with the β-anomer, which is the predominant species [13].In this case, the RAE is only partially attenuated.Other examples are illustrated by compounds 125 [76] and 126 [15,20] (Chart 13, Table 11).Schiff base 127 [77] crystallizes as the α-anomer, but in solution, it equilibrates with the β-anomer, which is the predominant species [13].In this case, the RAE is only partially attenuated.
Chart 13.Compounds showing attenuated RAE or completely lacking the effect.
Protonation of the nitrogen atom represents the other way to sequester its lone pair.Thus, hydrochloride 128 [77] shows a complete reversal of the anomeric populations with respect to 127 and has an Ean coincidental with that of 123 and 125.The free energy variation for protonated 128 (or deprotonated 127) is 1.4 kcal/mol, with the exo-anomeric effect playing a dominant role.In addition, a strong intramolecular hydrogen bond is probably generated between the NH + and the axial anomeric hydroxyl (α-anomer) (129), which restores the exo-anomeric effect (Scheme 8).The conformational rigidity of the imine group makes difficult the formation of this H-bonding in the β-anomer.Protonation of the nitrogen atom represents the other way to sequester its lone pair.Thus, hydrochloride 128 [77] shows a complete reversal of the anomeric populations with respect to 127 and has an E an coincidental with that of 123 and 125.The free energy variation for protonated 128 (or deprotonated 127) is 1.4 kcal/mol, with the exo-anomeric effect playing a dominant role.In addition, a strong intramolecular hydrogen bond is probably generated between the NH + and the axial anomeric hydroxyl (α-anomer) (129), which restores the exo-anomeric effect (Scheme 8).The conformational rigidity of the imine group makes difficult the formation of this H-bonding in the β-anomer.It is worth pointing out that this type of intramolecular bond formed by protonation has also been described for 2-aminocyclohexanol derivatives and is so powerful that it can invert the chair conformation of the cyclohexane ring (130), even though the substituents adopt axial dispositions (131) (Scheme 9) [78][79][80][81][82][83][84][85].
In the case of D-mannosamine (132) in which the amino group is arranged axially in a 4 C1 conformation of the pyranose ring, there is no possibility to remove the exo-anomeric effect.Accordingly, it should adopt a 1 C4 chair conformation that would place all the substituents in axial dispositions, thus making an impossible structure.In fact, no Schiff bases of this aminosugar with an imine structure have been described, although two enamines It is worth pointing out that this type of intramolecular bond formed by protonation has also been described for 2-aminocyclohexanol derivatives and is so powerful that it can invert the chair conformation of the cyclohexane ring (130), even though the substituents adopt axial dispositions (131) (Scheme 9) [78][79][80][81][82][83][84][85].It is worth pointing out that this type of intramolecular bond formed by protonation has also been described for 2-aminocyclohexanol derivatives and is so powerful that it can invert the chair conformation of the cyclohexane ring (130), even though the substituents adopt axial dispositions (131) (Scheme 9) [78][79][80][81][82][83][84][85].
In the case of D-mannosamine (132) in which the amino group is arranged axially in a 4 C1 conformation of the pyranose ring, there is no possibility to remove the exo-anomeric effect.Accordingly, it should adopt a 1 C4 chair conformation that would place all the substituents in axial dispositions, thus making an impossible structure.In fact, no Schiff bases of this aminosugar with an imine structure have been described, although two enamines Scheme 9. Ring inversion in aminocyclohexanols through protonation.
In the case of D-mannosamine (132) in which the amino group is arranged axially in a 4 C 1 conformation of the pyranose ring, there is no possibility to remove the exoanomeric effect.Accordingly, it should adopt a 1 C 4 chair conformation that would place all the substituents in axial dispositions, thus making an impossible structure.In fact, no Schiff bases of this aminosugar with an imine structure have been described, although two enamines have been reported (Chart 15), namely 133 [86] and 134 [87][88][89].In such cases, the existence of the RAE can hardly be detected.As a result, the RAE does not affect 2-aminoaldoses for which the imino group adopts an axial arrangement in the more stable conformer.Scheme 9. Ring inversion in aminocyclohexanols through protonation.
In the case of D-mannosamine (132) in which the amino group is arranged axially in a 4 C1 conformation of the pyranose ring, there is no possibility to remove the exo-anomeric effect.Accordingly, it should adopt a 1 C4 chair conformation that would place all the sub stituents in axial dispositions, thus making an impossible structure.In fact, no Schiff bases of this aminosugar with an imine structure have been described, although two enamines have been reported (Chart 14), namely 133 [86] and 134 [87][88][89].In such cases, the existence of the RAE can hardly be detected.As a result, the RAE does not affect 2-aminoaldoses for which the imino group adopts an axial arrangement in the more stable conformer.Furthermore, the RAE could modify the conformational behavior of imines derived from some 2-aminoaldoses.This is portrayed by imines of 2-amino-2-deoxy-pentopyra noses with D-arabino or L-xylo configurations.The most abundant species in the tautomeric equilibrium should be the α-anomer in 1 C4 conformation (135/136), since the RAE would be feasible in the β-anomer (137/138) (Scheme 10).Furthermore, the RAE could modify the conformational behavior of imines derived from some 2-aminoaldoses.This is portrayed by imines of 2-amino-2-deoxy-pentopyranoses with D-arabino or L-xylo configurations.The most abundant species in the tautomeric equilibrium should be the α-anomer in 1 C 4 conformation (135/136), since the RAE would be feasible in the β-anomer (137/138) (Scheme 10).Scheme 9. Ring inversion in aminocyclohexanols through protonation.
In the case of D-mannosamine (132) in which the amino group is arranged axially in a 4 C1 conformation of the pyranose ring, there is no possibility to remove the exo-anomeric effect.Accordingly, it should adopt a 1 C4 chair conformation that would place all the sub stituents in axial dispositions, thus making an impossible structure.In fact, no Schiff bases of this aminosugar with an imine structure have been described, although two enamines have been reported (Chart 14), namely 133 [86] and 134 [87][88][89].In such cases, the existence of the RAE can hardly be detected.As a result, the RAE does not affect 2-aminoaldoses for which the imino group adopts an axial arrangement in the more stable conformer.Furthermore, the RAE could modify the conformational behavior of imines derived from some 2-aminoaldoses.This is portrayed by imines of 2-amino-2-deoxy-pentopyra noses with D-arabino or L-xylo configurations.The most abundant species in the tautomeric equilibrium should be the α-anomer in 1 C4 conformation (135/136), since the RAE would be feasible in the β-anomer (137/138) (Scheme 10).In addition, the RAE may be present in other aminosugar derivatives and even in unprotected aminoaldoses themselves (Table 12).Thus, by replacing the hydroxyl at C-2 of D-glucose (139) by an amino group in D-glucosamine (140), the anomeric balance is shifted towards the equatorial anomer (β).However, protonation in the corresponding hydrochloride (2) reverses the anomeric ratio, making the α-anomer the more abundant species.The same applies to other protonated 2-aminosugars, such as 6 [16] and 142 [90].This behavior is completely parallel to that shown by 127 and 128, suggesting a similar performance of the RAE.Likewise, this trend occurs when replacing the amino group by the N-acetyl group, where the lone pair on the nitrogen atom participates in strong delocalization with the amide carbonyl.Thus, the anomeric ratio is reversed in 141 [91,92] with respect to 140 and becomes equal to that observed in compound 2.
As conclusive statement, one may say that whenever there is a heteroatom with at least one lone pair at C-2, a hydrogen bond could form with the hydroxyl of the α-anomer and inhibit the exo-anomeric effect.Such an inhibition should weaken the hydrogen bonding.Apparently, this happens when the presence of the hydroxyl at C-2 decreases the amount of the α-anomer, as illustrated by comparing 2-deoxy-D-glucose (111) and 2-deoxy-Dgalactose (143) with D-glucose (139) and D-galactose (144), respectively (Chart 16).Overall and despite the intriguing complexity of all factors influencing the anomeric equilibrium, the RAE should invariably be taken into account in further studies.As conclusive statement, one may say that whenever there is a heteroatom with at least one lone pair at C-2, a hydrogen bond could form with the hydroxyl of the α-anomer and inhibit the exo-anomeric effect.Such an inhibition should weaken the hydrogen bonding.Apparently, this happens when the presence of the hydroxyl at C-2 decreases the amount of the α-anomer, as illustrated by comparing 2-deoxy-D-glucose (111) and 2-deoxy-D-galactose (143) with D-glucose (139) and D-galactose (144), respectively (Chart 15).Overall and despite the intriguing complexity of all factors influencing the anomeric equilibrium, the RAE should invariably be taken into account in further studies.

Conclusions
New imines have been synthesized by condensation of D-glucosamine and 2-amino-2-deoxy-D-glycero-L-gluco-heptopyranose with mono-and polycyclic aromatic aldehydes, as well as cinnamylidene aldehydes.They all crystallize as equatorial anomers (β); although in the cases of benzaldehyde, 2-fluorobenzaldehyde, 4-methoxynaphthaldehyde and 2-naphthaldehyde, the corresponding axial anomers (α) could also be isolated.Such imines have been thoroughly characterized by preparing their per-O-acetyl derivatives.In solution, the Schiff bases derived from 2-amino-2-deoxyaldoses and alkyl and arylaldehydes establish an equilibrium between both anomers.The prevalence of the β-anomer is independent of the nature of the alkyl or aryl moiety carried by the imine nitrogen.When imines crystallize rapidly, the α-anomer is usually formed; otherwise, the β-anomer or mixtures of both anomers are obtained.This RAE can judiciously be ascribed to the total or partial inhibition of the exo-anomeric effect in the α-anomer, and results from of an intramolecular hydrogen bond between the anomeric hydroxyl and the nitrogen atom.As Chart 16.Hexose and 2-aminohexose derivatives exhibiting potential RAE.

Conclusions
New imines have been synthesized by condensation of D-glucosamine and 2-amino-2-deoxy-D-glycero-L-gluco-heptopyranose with mono-and polycyclic aromatic aldehydes, as well as cinnamylidene aldehydes.They all crystallize as equatorial anomers (β); although in the cases of benzaldehyde, 2-fluorobenzaldehyde, 4-methoxynaphthaldehyde and 2-naphthaldehyde, the corresponding axial anomers (α) could also be isolated.Such imines have been thoroughly characterized by preparing their per-O-acetyl derivatives.In solution, the Schiff bases derived from 2-amino-2-deoxyaldoses and alkyl and arylaldehydes establish an equilibrium between both anomers.The prevalence of the β-anomer is independent of the nature of the alkyl or aryl moiety carried by the imine nitrogen.When imines crystallize rapidly, the α-anomer is usually formed; otherwise, the β-anomer or mixtures of both anomers are obtained.This RAE can judiciously be ascribed to the total or partial inhibition of the exo-anomeric effect in the α-anomer, and results from of an intramolecular hydrogen bond between the anomeric hydroxyl and the nitrogen atom.As a key structural prerequisite, the iminic group in the pyranose ring should adopt an equatorial arrangement.The effect is reduced and disappears completely when Schiff bases either adopt enamine structures or undergo protonation of the imino group.Also, the protonation of the starting 2-amino-2-deoxyaldopyranoses modifies the anomeric balance, thus suggesting the action of RAE in such compounds.Theoretical calculations show that the formation of hydrogen bonding between the anomeric hydroxyl and the imine nitrogen is responsible for removal of the exo-anomeric effect on the α-anomer of 2-arylimino-2deoxyaldopyranoses, which together with solvent effects (in terms of continuous solvation or discrete solvation) provide sufficient evidence supporting the preferential formation of the β-anomer.

Computational Details
The computational DFT study was initially carried out using the B3LYP [45,46] and the M06-2X [47] hybrid density functionals in conjunction with 6-31G(d,p) and 6-311G(d,p) basis sets [42,43], as implemented in the Gaussian09 package [93].The M06-2X method was chosen on the basis of previous studies showing its accuracy in determining conformational energies associated with non-covalent interactions.To assess the influence of the level of theory on anomer stability, the def2-TZVP valence-triple-ζ basis set [44] was also employed in combination with the M06-2X functional for geometry optimizations.As mentioned, the latter has proven to be reliable enough for estimating structure and binding in other carbohydrate derivatives [48][49][50].In all cases, frequency analyses were carried out to confirm the existence of true stationary points on the potential energy surface.All thermal corrections were calculated at the standard values of 1 atm at 298.15 K. Solvent effects were modeled through density-based, self-consistent reaction field (SCRF) theory of bulk electrostatics, i.e., the solvation model based on density (SMD) [51], as implemented in the Gaussian09 suite of programs.This solvation method accounts for long-range electrostatic polarization (bulk solvent) together with short-range effects due to cavitation, dispersion, and solvent structural effects.
We assessed mutarotational equilibria and solvent effects in 2-iminoaldose derivatives using four approaches: (a) gas-phase, as the absence of solvent allows determining the intrinsic stability of each species; (b) continuum solvation: anomerization was studied in solution with a description of the solvent as a continuum dielectric medium, using specifically the SMD model [51]; (c) microsolvation: calculations were conducted in the gas phase, but one or several water molecules were added to the resulting structures of the stationary points in order to determine the stabilization induced by hydrogen bonding, and (d) microsolvation and continuum solvation, which represents the hybrid between methods (b) and (c).Here, the assembly of the imine with one or several water molecules was studied in a continuum and polarizable dielectric medium.

Natural Bond Orbital (NBO) and Steric Analysis
Natural bond orbital analysis was performed with NBO 6.0 [74].Intramolecular interactions of the stabilization energies were obtained using second-order perturbation theory and listed in the SI.For each donor NBO(i) and acceptor NBO(j), the stabilization energy E 2 associated with electron delocalization between donor and acceptor is estimated as where q i is the donor orbital occupancy, ε i , ε j are diagonal elements (orbital energies), and F ij is the off-diagonal NBO Fock matrix element.In the natural bond orbital (NBO) approach, a hydrogen bond is viewed as an interaction between an occupied non-bonded natural orbital n A of the acceptor atom A and the unoccupied antibonding orbital of the DH bond σ DH *.

Synthesis of Schiff Bases
New and reported substances were obtained according to the following general procedures.Method 1: To a solution of 2 or 6 (23.2 mmol) in 1M NaOH (25 mL) was added the appropriate aromatic aldehyde (25.0 mmol), and the mixture was stirred at room temperature.A solid precipitated and was collected by filtration and washed successively with cold water, cold ethanol, and ethyl ether, and dried under vacuum on silica gel.Method 2: Sodium hydrogen carbonate (0.50 g, 6.0 mmol) was added to a solution of 2 or 6 (4.7 mmol) in water (6 mL).To the resulting mixture, a solution of the appropriate aromatic aldehyde (4.7 mmol) in methanol (saturated solution) was added dropwise.The mixture was stirred at room temperature until precipitation, and then left in the refrigerator (~5

Figure 2 .
Figure 2. Preferred conformations in solution for imines derived from 2 and 6.

Figure 2 .
Figure 2. Preferred conformations in solution for imines derived from 2 and 6.

Figure 2 .
Figure 2. Preferred conformations in solution for imines derived from 2 and 6.

Figure 2 .
Figure 2. Preferred conformations in solution for imines derived from 2 and 6.

Chart 11 .
Axial and pseudo-equatorial orientations of the N-H bond in 117 and 118.

Chart 11 .
Axial and pseudo-equatorial orientations of the N-H bond in 117 and 118.

Figure 12 .
Figure 12.Numbering used in the NBO analysis of both anomers of some representative imines.

Figure 12 .
Figure 12.Numbering used in the NBO analysis of both anomers of some representative imines.
a At room temperature.b Just dissolved.

Table 2 .
Relative energy minima found for 56 and 112 a .

Table 2 .
Relative energy minima found for 56 and 112 a .

Table 3 .
Relative energies (kcal/mol) for species involved in mutarotational equilibria of imines 30 and 93 a .
Scheme 6. Acyclic and cyclic structures considered in mutarotational equilibria.

Table 3 .
Relative energies (kcal/mol) for species involved in mutarotational equilibria of imines 30 and 93 a .
Chart 12. Axial and pseudo-equatorial orientations of the N-H bond in 117 and 118.

Table 3 .
Relative energies (kcal/mol) for species involved in mutarotational equilibria of imines 30 and 93 a .
a In DMSO-d 6 .b In %. c Anomeric stabilization referred to cyclohexanol.
a In DMSO-d 6 .b In %. c Anomeric stabilization referred to cyclohexanol.
a In DMSO-d 6 .b In %. c In pyridine.d Anomeric stabilization referred to cyclohexanol.
a In DMSO-d 6 .b In %. c In pyridine.d Anomeric stabilization referred to cyclohexanol.

Table 8 .
Calculated stability of imine anomers derived from 2 a,b .

Table 9 .
Calculated stability of hydrated imine anomers with five molecules of water a,b .

Table 11 .
Anomeric composition (%) at equilibrium for imines and enamines derived from compounds 2 and 6 a .Compounds showing attenuated RAE or completely lacking the effect.

Table 11 .
Anomeric composition (%) at equilibrium for imines and enamines derived from compounds 2 and 6 a .
a In D 2 O. b In DMSO-d 6 .