Monosaccharides as Potential Chiral Probes for ... - ACS Publications

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Monosaccharides as Potential Chiral Probes for the Determination of the Absolute Configuration of Secondary Alcohols Tomasz Laskowski,* Katarzyna Szwarc, Paweł Szczeblewski, Paweł Sowiński, Edward Borowski, and Jan Pawlak Department of Pharmaceutical Technology and Biochemistry, Faculty of Chemistry, Gdańsk University of Technology, Gabriela Narutowicza Street 11/12, 80-233 Gdańsk, Poland S Supporting Information *

ABSTRACT: Herein, a new method for the elucidation of the absolute configuration of chiral secondary alcohols is proposed. This method is an alternative for a widely used approach reported by Mosher and Dale and similar methods that are based on the 1H NMR shift (δ) changes of protons that are attached to the substituents of the oxymethine carbon atom. The presented method is not based on tracking the chemical shift changes and utilizes stereochemically defined monosaccharides as chiral probes. A secondary alcohol is glycosylated, and the resulting glycoside is subjected to NMR studies. The observation of dipolar couplings between the protons of the monosaccharide moiety and the protons of the secondary alcohol moiety via the NOESY/ROESY spectra enables the determination of the absolute configuration of the oxymethine carbon atom.

T

groups are prone to steric compression. Therefore, anomalies are often observed in the predicted ΔδRS trends.11,13 Herein, we propose a new method for the elucidation of the absolute configuration of chiral secondary alcohols that is not based on tracking chemical shift changes. The idea of this method originated from model NMR studies of the antibiotic amphotericin B (AmB) derivative.15 The interpretation of the ROESY spectrum of the AmB derivative revealed the presence of ROE associations between the protons of the stereochemically defined mycosamine (3-amino-3,6-dideoxy-D-mannose) moiety, β-glycosicidally linked to the macrolactone unit, and the protons of the aglycone moiety. This observation, which is presented in Figure 1, provided the background for the definition of the absolute configuration of many other mycosamine-containing polyene macrolides.16−21 Our previous publication on the application of monosaccharides as internal probes for the determination of the absolute configuration of 2-butanol22 outlines the general approach with the essential concept being the observation of dipolar couplings between the protons of the aglycone unit and the respective protons of the monosaccharide moiety via the NOESY/ROESY spectra (Figure 2). The theoretical foundation of the new method is that the glycosidic linkages of synthesized glycosides adopt one dominant stable conformation in most cases. The restrained conformational freedom of the glycosidic linkage is required for the observation of the probe−aglycone diagnostic NOEs/ ROEs. This requirement has been previously met using several glycosides of 2-butanol.22 Because 2-butanol is one of the

he definition of the absolute configuration of secondary alcohols is of interest for many researchers due to the presence of this functional group in natural and synthetic compounds. Remarkable progress in the elucidation of the absolute configuration of this class of compounds is associated with the application of NMR spectroscopy as the primary analytic tool. In 1973, Mosher and Dale proposed a method to define the absolute configuration of asymmetric secondary alcohols.1 This method and other similar methods involve the interpretation of the 1H NMR shift (δ) changes of the protons of the substituents on the oxymethine carbon atom that are induced by the anisotropic effects of a chiral derivatizing agent (CDA).2−6 However, the tentative assumption of the conformation of the CDA moiety may lead to an erroneous assignment of the absolute configuration.7,8 For example, the absolute configuration of a pyrrolo[2,1-b]quinazoline-vasicinone alkaloid determined via its α-methoxy-α-trifluoromethylphenylacetyl (MTPA) derivative was found to be erroneous.9 Moreover, in some cases, the data provided by tracking the Δδ values are incoherent; therefore, the determination of the absolute configuration of a secondary alcohol is problematic and sometimes even impossible. For cytotoxic marine cembranolides, the 1H and 19F ΔδRS data are contradictory and indicate different absolute configurations10 due to the steric compression of the MTPA derivative. The potential influence of the steric compression of the CDA derivatives on the assignments of the absolute configurations of secondary alcohols has been analyzed in a series of studies.7,10−14 The MTPA-based methods are reliable for compounds with equatorial hydroxy groups, but compounds with axial hydroxy © 2016 American Chemical Society and American Society of Pharmacognosy

Received: May 23, 2016 Published: October 26, 2016 2797

DOI: 10.1021/acs.jnatprod.6b00471 J. Nat. Prod. 2016, 79, 2797−2804

Journal of Natural Products

Article

mannose, and L-rhamnose were chosen as chiral probes, and the respective NOEs appeared in only the spectra of the αepimers of the synthesized glycosides. Therefore, for studies of the secondary alcohols, a D-mannose derivative, 2,3,4,6-tetra-Obenzyl-D-mannose, was selected, as this derivative is commercially available and tends to form an α-glycosidic linkage during the glycosylation reaction.23

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RESULTS AND DISCUSSION Preparation. For ursolic acid, the glycosylation reaction was preceded by synthesizing an ursolic acid methyl ester via reaction with CH2N2. For quinine, a quinine salt with ptoluenesulfonic acid (PTSA) was formed first. The formation of the glycosidic linkage in each case was carried out in two steps using the Schmidt method based on O-glycosyl trichloroacetimidates.24 The 2,3,4,6-tetra-O-benzyl-D-mannose was transformed into 2,3,4,6-tetra-O-benzyl-D-mannopyranosyl trichloroacetimidate via reaction with CCl3CN in the presence of a catalytic amount of NaH in CH2Cl2 as the solvent. Next, the probe was subjected to reactions with methyl ursolate, (+)-menthol, and the quinine salt in the presence of a catalytic amount of PTSA in CH2Cl2. The resulting glycosides were isolated using standard chromatographic methods to yield a mixture of α- and β-epimers of methyl 3-(2,3,4,6-tetra-Obenzyl-D-mannopyranosyloxy)ursolate (designated as 1A and 1B, respectively) in a 10:1 molar ratio, a mixture of α- and βepimers of (+)-1-(2,3,4,6-tetra-O-benzyl-Dmannopyranosyloxy)menthol (designated as 2A and 2B, respectively) in a 2:3 molar ratio, and a mixture of α- and βepimers of 9-(2,3,4,6-tetra-O-benzyl-D-mannopyranosyloxy)quinine (designated as 3A and 3B, respectively) in a 5:1 molar ratio. Compound 1A, the mixture of 2A and 2B, and compound 3A were subjected to NMR studies in benzene-d6. All of the connectivities within each isolated spin system of the resulting glycosides were traced in a straightforward manner using standard DQF-COSY, TOCSY, HSQC, HMBC, and NOESY/ ROESY experiments. In each case, the analysis of the vicinal coupling constants and dipolar couplings of the 2,3,4,6-tetra-Obenzyl-D-mannose moiety confirmed the 4C1 conformation of the D-mannopyranosyl ring. Methyl 3-(2,3,4,6-Tetra-O-benzyl-α-Dmannopyranosyloxy)ursolate (1A). For 1A, the analysis of the vicinal coupling constants and dipolar couplings of the aglycone unit permitted the determination of the (3S*, 5R*, 8R*, 9R*, 10R*, 14S*, 17S*, 18S*, 19S*, 20R*) relative configuration. The probe−aglycone NOEs H1′/H3 and H1′/

Figure 1. Diagnostic ROEs (depicted as bidirectional arrows) between the protons of the mycosamine moiety and the aglycone unit of the AmB derivative, which enabled determination of the absolute configuration of C-19.

Figure 2. Determination of the absolute configuration of the oxymethine carbon atom of a secondary alcohol via dipolar couplings (depicted as bidirectional arrows) between the protons of the aglycone unit and the protons of the monosaccharidic 2,3,4,6-tetra-O-benzyl-Dmannose chiral probe.

simplest chiral secondary alcohols, a glycoside containing more bulky L1 and L2 substituents (Figure 2) will also meet this conformational condition. In this study, the application of the method was verified for more complex, naturally occurring chiral secondary alcohols, and the terpenoids (+)-menthol and ursolic acid and the alkaloid quinine were selected as model compounds. Studies of 2-butanol also indicated that diagnostic dipolar couplings between the protons of the aglycone unit and the respective protons of the monosaccharide moiety occur only when the glycoside contains a glycosidic linkage with a required configuration.22 Previously, 2,3,4,6-tetra-O-acetyl-D-glucose, D-

Figure 3. Determination of the (3S) absolute configuration of 1A. The diagnostic NOEs are depicted as bidirectional arrows. 2798



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Table 1. NMR Data for 1A with Probe−Aglycone Diagnostic NOEs Listed in Bold 1

1

H NMR Data for 1A

position 1ax 1eq 2ax

δ (ppm) 0.58 0.63 1.29

2eq

1.54

3

3.35

5 6ax 6eq 7ax 7eq 9 11a 11b 12 15a 15b 16a 16b 18 19 20

0.70 1.30 1.48 1.28 1.43 1.45 1.85 1.86 5.41 1.01 1.10 1.85 1.98 2.48 1.37 0.91

21a 21b 22a 22b 23 24 25 26 27 29

1.22 1.33 1.67 1.75 1.16 0.88 0.84 0.89 1.13 1.00

JH,H (Hz) Aglycone Unit 13.0 (1eq), (2ax),a 3.1 (2eq) 13.0 (1ax), (2ax),a (2eq)a (1ax),a(1eq),a 13.0 (2eq), 13.0 (3) 3.1 (1ax),(1eq),a 13.0 (2ax), 3.6 (3) 13.0 (2ax), 3.6 (2eq) 12.0 (6ax), 1.5 (6eq) 12.0 (5), (6eq),a(7ax),a (7eq) 1.5 (5),(6ax),a (7ax),a(7eq)a (6ax),a (6eq),a(7eq)a (6ax),a (6eq),a(7ax)a 12.0 (11a),(11b)a 12.0 (9), 3.9 (12) (9),a 3.9 (12) 3.9 (11a), 3.9 (11b) (15b),a (16a),a(16b)a (15a),a(16a),a(16b)a (15a),a(15b),a(16b)a (15a),a (15b),a (16a)a 11.3 (19), 2.0 (22a) 11.3 (18), 11.3 (20), 6.7 (29) 11.3 (19), (21a),a (21b),a 6.2 (30) (20),a (21b),a (22a),a (22b)a (20),a (21a),a (22a),a (22b)a 2.0 (18), (21a),a (21b),a (22a)a (21a), (21b), (22b)

6.7 (19)

H NMR Data for 1A

NOE to proton

position

δ (ppm)

30 0.90 OMe 3.42 Probe Moiety 1′ 5.22 2′ 3.86 3′ 4.16 4′ 4.36 5′ 4.27 6′a 3.85 6′b 3.91

1eq, 3 1ax, 3 23, 25 3, 1′ 1ax, 1eq, 2eq, 5, 24, 1′ 3, 7eq, 24 7eq 7eq

position 5, 6ax, 6eq, 27 27 12, 25, 26 12, 25, 26 11a, 11b, 18, 29 15b, 16a, 16b 15a 15a, 16b 15a, 16a, 19 12, 19, 29 16b, 18

Aglycone Unit 1, CH2 2, CH2 3, CH 4, C 5, CH 6, CH2 7, CH2 8, C 9, CH 10, C 11, CH2 12, CH 13, C 14, C 15, CH2 16, CH2 17, C 18, CH

22b

21a 2ax, 24, 3′, 5′ 3, 5, 23 2ax, 11a, 11b 11a, 11b 7eq, 9 12, 18

JH,H (Hz)

NOE to proton

6.2 (20)

1.5 1.5 2.6 9.3 9.3 2.0 4.2

(2′) (1′), (2′), (3′), (4′), (5′), (5′),

2.6 (3′) 9.3 (4′) 9.3 (5′) 2.0 (6′a), 4.2 (6′b) 14.5 (6′b) 14.5 (6′a) 13 C NMR Data for 1A

δ (ppm) 38.3 21.9 81.8 38.3 55.8 18.4 33.2 39.9 47.8 36.9 23.5 125.6 138.8 42.1 28.2 24.7 48.2 53.0

2eq, 3, 2′ 1′, 3′ 23, 2′, 5′ 23, 6′a, 6′b, 3′ 5′ 5′

position 19, CH 20, CH 21, CH2 22, CH2 23, CH3 24, CH3 25, CH3 26, CH3 27, CH3 28, CO 29, CH3 30, CH3 Probe Moiety 1′ 2′ 3′ 4′ 5′ 6′

δ (ppm) 39.4 38.9 30.6 36.9 28.9 17.0 15.3 17.0 23.7 177.15 17.0 21.0 94.6 70.7 81.1 70.6 74.0 70.7

a

These coupling constants could not be measured. Signal pattern remains partially unclear due to severe signal overlap and higher order effects.

H2eq confirmed the partial inhibition of the rotation around the glycosidic linkage of 1A. The NOESY spectrum revealed two more dipolar couplings between the protons of the aglycone moiety and the protons of the chiral probe, i.e., H3′/ H23 and H5′/H23. Because the probe was stereochemically defined, these NOEs unambiguously indicated the (3S) absolute configuration (Figure 3). Therefore, the absolute configuration of the stereogenic centers of the aglycone moiety was determined to be (3S, 5R, 8R, 9R, 10R, 14S, 17S, 18S, 19S, 20R), which corresponds to that of ursolic acid.25 The assignments along with the dipolar couplings of 1A are listed in Table 1. (+)-1-(2,3,4,6-Tetra-O-Benzyl-α-D-mannopyranosyloxy)menthol (2A) and (+)-1-(2,3,4,6-Tetra-O-benzyl-β- D mannopyranosyloxy)menthol (2B). For 2A and 2B, the analysis of the vicinal coupling constants and the dipolar couplings of the aglycone moiety confirmed that the substituents of the cyclohexane ring in 2A and 2B are all in equatorial positions, which permitted definition of the (1S*, 2R*, 5S*) relative configuration. The probe−aglycone NOEs H1′/H1 and H1′/H6eq indicated the partial inhibition of rotation around the glycosidic

linkage of 2A. In addition, for 2A, two more NOEs between the protons of the aglycone unit and the protons of the chiral probe were observed, i.e., H5′/H7 and H5′/H9. These NOEs enabled the determination of the (1S) absolute configuration (Figure 4). Therefore, the absolute configuration of the stereogenic centers of the aglycone moiety was determined to be (1S, 2R, 5S), which corresponds to that of (+)-menthol.26,27

Figure 4. Determination of the (1S) absolute configuration of 2A. The diagnostic NOEs are depicted as bidirectional arrows. 2799



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Interestingly, more NOE contacts between the protons of the aglycone moiety and the protons of the chiral probe were observed for 2B. The H1′/H1, H1′/H6eq, H1′/H7, and H1′/ H8 dipolar couplings confirmed the restrained conformational freedom of the glycosidic linkage of 2B. However, the H2′/H7 and H2′/H8 NOEs indicated the (1S) absolute configuration (Figure 5). Therefore, the absolute configuration of the stereogenic centers of the aglycone unit was established as (1S, 2R, 5S), which is in agreement with that of (+)-menthol.


H NMR Data for 2A

position

δ (ppm)

1 2

3.57 1.31

3ax

0.84

3eq 4ax

1.53 0.68

4eq

1.48

5

1.06

6ax 0.73 6eq 1.91 7 2.48 8 0.89 9 0.91 10 0.80 Probe Moiety 1′ 5.22 2′ 3.88 3′ 4.16 4′ 4.39 5′ 4.19

Figure 5. Determination of the (1S) absolute configuration of 2B. The diagnostic NOEs are depicted as bidirectional arrows.

The assignments along with the dipolar couplings of 2A and 2B are listed in Tables 2 and 3, respectively. 9-(2,3,4,6-Tetra-O-benzyl-α- D -mannopyranosyloxy)quinine (3A). The analysis of the vicinal coupling constants and dipolar couplings of the aglycone moiety of 3A permitted definition of the (3R*, 4S*, 8S*, 9R*) relative configuration. The H1′/H9 and H1′/H5q ROEs indicated the partial inhibition of the rotation around the glycosidic linkage of 3A. Moreover, for 3A, only one more and relatively weak probe− aglycone ROE was observed, i.e., H2′/H8. Nevertheless, these data were sufficient to unambiguously indicate the (9R) absolute configuration (Figure 6). Therefore, the absolute configuration of the stereogenic centers of the aglycone unit was determined to be (3R, 4S, 8S, 9R), which is in agreement with that of quinine.28,29 The assignments along with the dipolar couplings of 3A are listed in Table 4. Molecular Modeling. The probe−aglycone dipolar couplings are diagnostic when the glycosidic linkages do not have full conformational freedom. In each case, the conformation of the glycosidic linkages resulting from rotation about the CA−O and O−C1′ bonds can be described by two dihedral angles, φ and ψ (Figure 7). To investigate the conformational properties of the glycosidic linkages, molecular dynamics simulations of 1A, 2A, 2B, and 3A were performed. To validate these calculations, the separation distances between the respective protons during the simulation time were monitored, and these distances correspond to the observed probe−aglycone NOEs/ ROEs. The computed average proton−proton distances are in good agreement with the NOESY/ROESY data (Table 5; also see Supporting Information). The definitions of the dihedral angles for each compound are given in Table 6. For 1A, two major stable conformations of the glycosidic linkage were observed and were designated 1A1 and 1A2 (see Ramachandran plot for 1A in Figure 8). The MD simulations also revealed that for 2A, 2B, and 3A the glycosidic bonds primarily adopt one dominant stable conformation. Therefore, on the basis of the results shown in Figure 8, the glycosidic bonds exhibited limited conformational freedom. Moreover, the number of conformers resulting from the simulations of 1A, 2A,

6′a 6′b

3.86 3.96

position Aglycone Unit 1, CH 2, CH 3, CH2 4, CH2 5, CH 6, CH2 7, CH 8, CH3

JH,H (Hz)

NOE to proton

Aglycone Unit 10.3 (2), 10.5 (6ax), 2.3 (6eq) 10.3 (1), 1.5 (7), 11.0 (3ax), 1.8 (3eq) 11.0 (2), 11.5 (3eq), 10.6 (4ax), (4eq)a 1.8 (2), 11.5 (3ax), (4ax),a (4eq)a 10.6 (3ax), (3eq),a 12.5 (4eq), 10.7 (5) (3ax),a (3eq),a 12.5 (4ax), (5),a 2.0 (6eq) 10.7 (4ax), (4eq),a (6ax),a 2.5 (6eq), 6.1 (10) 10.5 (1), (5),a 12.2 (6eq) 2.3 (1), 2.0 (4eq), 2.5 (5), 12.2 (6ax) 1.5 (2), 6.7 (8), 6.7 (9) 6.7 (7) 6.7 (7) 6.1 (5) 1.6 1.6 1.9 9.5 9.4

(2′) (1′), (2′), (3′), (4′),

1.9 9.5 9.4 1.9

(3′) (4′) (5′) (6′a), 2.6 (6′b)

1.9 (5′), 12.0 (6′b) 2.6 (5′), 12.0 (6′a) 13 C NMR Data for 2A δ (ppm) 75.1 48.4 23.2 34.3 31.2 39.7 25.5 15.7

position 9, CH3 10, CH3 Probe Moiety 1′ 2′ 3′ 4′ 5′ 6′

3ax, 5, 6eq, 1′ 3eq, 6ax, 7, 9 1, 3eq, 4eq, 5 2, 3ax, 4eq, 8, 9 4eq, 6ax 3ax, 3eq, 4ax, 5 1, 3ax, 4eq, 6eq, 10 2, 4ax, 6eq, 10 1, 5, 6ax, 1′ 2, 8, 9, 5′ 3eq, 7 2, 3eq, 7, 5′ 5, 6ax 2′, 5′, 1, 6eq 1′, 3′, 4′ 2′, 4′, 5′ 2′, 3′, 5′ 1′, 3′, 4′, 6′a, 6′b, 7, 9 5′, 6′b 5′, 6′a δ (ppm) 21.0 22.2 94.6 76.5 81.3 75.7 73.6 70.2

a


2B, and 3A most likely depends on the steric hindrance around the oxymethine carbon atom of the aglycone moiety. 2,3,4,6-Tetra-O-benzyl-D-mannose can be successfully used as a chiral probe for determining the absolute configuration of naturally occurring secondary alcohols. Moreover, both the αand β-epimers of the resulting glycosides were useful in the stereochemical studies. This method provides quick and unambiguous results based on clear theoretical foundations. The creation of a probe−aglycone linkage is not complicated, and product isolation is straightforward. Only one set of NMR spectra of the resulting derivative(s) is required, and no reference spectrum of the underivatized secondary alcohol is required for comparison. In addition, for 2A and 2B, the α- and β-epimers of the resulting glycoside do not need to be separated for further stereochemical NMR-based studies. Because many complex chiral secondary alcohols require a 2800



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Table 3. NMR Data for 2B with Probe−Aglycone Diagnostic NOEs Listed in Bold 1

H NMR Data for 2B

position

δ (ppm)

1 2 3ax 3eq 4ax 4eq

3.35 1.35 0.85 1.52 0.77 1.54

5

1.29

6ax 1.28 6eq 2.60 7 2.31 8 0.80 9 0.96 10 0.93 Probe Moiety 1′ 4.42

JH,H (Hz)

NOE to proton

Aglycone Unit 10.3 (2), 10.4 (6ax), 2.8 (6eq) 10.3 (1), 3.4 (7), 11.2 (3ax), 2.5 (3eq) 11.2 (2), 11.6 (3eq), (4ax),a (4eq)a 2.5 (2), 11.6 (3ax), (4ax),a (4eq)a (3ax),a (3eq),a 12.5 (4eq), 10.4 (5) (3ax),a (3eq),a 12.5 (4ax), (5),a 2.2 (6eq) 10.4 (4ax), (4eq),a (6ax),a (6eq),a 6.3 (10) 10.4 (1), (5),a (6eq)a 2.8 (1), 2.2 (4eq), (5),a (6ax)a 3.4 (2), 7.1 (8), 7.6 (9) 7.1 (7) 7.6 (7) 6.3 (5) 1.7 (2′)

2′ 3′ 4′ 5′

3.93 3.41 4.08 3.55

1.7 (1′), 2.1 (3′) 2.1 (2′), 10.1 (4′) 10.1 (3′), 10.1 (5′) 10.1 (4′), 3.0 (6′a), 2.3 (6′b)

6′a 6′b

3.77 3.82

3.0 (5′), 11.6 (6′b) 2.3 (5′), 11.6 (6′a) 13 C NMR Data for 2B

position Aglycone Unit 1, CH 2, CH 3, CH2 4, CH2 5, CH 6, CH2 7, CH 8, CH3

δ (ppm) 81.8 49.0 23.4 23.7 29.7 43.9 26.0 16.5

position 9, CH3 10, CH3 Probe Moiety 1′ 2′ 3′ 4′ 5′ 6′

3ax, 5, 6eq, 1′ 3eq, 6ax, 7, 9 1, 3eq, 4eq, 5 2, 3ax, 4eq, 8, 9 4eq, 6ax 3ax, 3eq, 4ax, 5 1, 3ax, 4eq, 6eq, 10 2, 4ax, 6eq 1, 5, 6ax, 1′ 2, 8, 9, 1′, 2′ 3eq, 7, 1′, 2′ 2, 3eq, 7 5

Figure 6. Determination of the (9R) absolute configuration of 3A. The diagnostic ROEs are depicted as bidirectional arrows.

This method has its limitations. The creation of a glycosidic linkage may not always be as efficient as expected. For sterically hindered higher secondary alcohols, the reaction is quite slow and requires some optimization similar to that required for quinine. The usefulness of the β-epimers of the resulting glycosides depends on the steric hindrance around the oxymethine carbon atom of the aglycone moiety. β-Glycosides of less sterically hindered asymmetric secondary alcohols (i.e., 2-butanol) do not meet the requirement of restrained conformational freedom of the glycosidic linkages; therefore, the probe−aglycone diagnostic dipolar couplings are not observed in their NOESY/ROESY spectra.22 For both α- and β-epimers, the main requirement for the successful elucidation of the absolute configuration is the proper and stable orientation of the monosaccharide moiety in the resulting glycoside, which can be inferred based on the presence of dipolar coupling between the anomeric proton of the probe and the oxymethine proton of the studied secondary alcohol (H1′/ HA, Figure 7). Lack of this NOE/ROE excludes the valid stereochemical studies because it cannot be determined if the glycosidic linkage has unlimited conformational freedom or if the orientation of the probe is different than expected. However, the presence of the dipolar coupling and at least one more NOE/ROE between the nonanomeric proton of the probe and the proton(s) of the L1/L2 substituent (Figure 2) will always indicate the correct absolute configuration of a secondary alcohol. In addition, the L1/L2 substituents must contain protons within reasonable distances from the oxymethine carbon atom, which is the greatest limitation of this approach. The new method requires further studies. Therefore, more monosaccharides should be tested as probes, and other secondary alcohols should be used as test compounds. The applicability of this approach for the elucidation of the absolute configuration of diols and polyols should also be investigated. Although the Mosher approach has been extended to the assignment of the absolute configuration of tertiary alcohols,30 such an extension of this method may be problematic and would require further study.

2′, 3′, 5′, 1, 6eq, 7, 8 1′, 3′, 4′, 7, 8 1′, 2′, 4′, 5′ 2′, 3′, 5′ 1′, 3′, 4′, 6′a, 6′b 5′, 6′b 5′, 6′a δ (ppm) 21.5 22.6 103.6 75.3 83.1 75.4 76.4 70.3

a


full set of 2D NMR experiments for 1H and 13C assignments, this method is not more complicated or time-consuming than Mosher’s method and other similar methods. The menthol example also confirmed that in some cases this approach may be considerably less complicated than the ΔδRS-based methods, where the thermodynamic parameters (i.e., polarity of the NMR solvent and experimental temperature) must be taken into account to force the high population of the diagnostic conformers. In the ΔδRS-based methods, regardless of the CDA used, both resulting diastereoisomeric derivatives should adopt dominant and mutually corresponding conformations, which is required for reliable shielding/deshielding effects and correct absolute configuration assignments.5 Previous studies have demonstrated that some reagents that have been extensively used for the determination of the absolute configuration of secondary alcohols (e.g., (−)-menthol) did not produce unambiguous results.5

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EXPERIMENTAL SECTION

General Experimental Procedures. Ursolic acid, (+)-menthol, quinine, 2,3,4,6-tetra-O-benzyl-D-mannose, and CCl3CN were obtained from Sigma-Aldrich (Poznań, Poland). The NMR spectra of 1A, 2801



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H NMR Data for 3A

position

δ (ppm)

Aglycone Unit 2a 2.50 2b 2.81 3 1.67 4

1.14

5a

0.89

5b

1.59

6a 6b 7a 7b 8

2.59 4.19 0.71 1.71 3.19

9 6.47 10 4.73 11a/ 4.50 11b 2q 8.77 3q 7.58 5q 7.42 7q 7.19 8q 8.10 OMe 3.90 Probe Moiety 1′ 5.39 2′ 3.87 3′ 4.20 4′ 4.17 5′ 4.33 6a′/6b′ 3.77

JH,H (Hz)

ROE to proton

13.5 (2b), 6.7 (3) 13.5 (2a), 10.7 (3) 6.7 (2a), 10.7 (2b), 2.5 (4), 4.7 (10) 2.5 (3), 4.5 (5a), 4.3 (5b), 4.6 (7a), 4.7 (7b) 4.5 (4), 11.0 (5b), 11.7 (6a), 3.5 (6b) 4.3 (4), 11.0 (5a), 3.0 (6a), 11.1 (6b) 11.7 (5a), 3.0 (5b), 11.5 (6b) 3.5 (5a), 11.1 (5b), 11.5 (6a) 4.6 (4), 10.9 (7b), 10.1 (8) 4.7 (4), 10.9 (7a), 4.0 (8) 10.1 (7a), 4.0 (7b), 4.7 (9)

2b, 3, 8, 10, 11 2a, 3, 6a 2a, 2b, 5a, 6a, 10, 11

4.7 (8), 2.3 (3q) 4.7 (3), 13.8 (11a/11b) 13.8 (10)

Aglycone Unit 2, CH2 3, CH 4, CH 5, CH2 6, CH2 7, CH2 8, CH 9, CH 10, CH 11, CH2 2q 3q 4q

4, 5a, 6b

calculated probe−aglycone proton/proton average distances [nm]

3q 7b, 8, 9, 2q 8, 9, OMe, 1′ 8q, OMe 7q 5q, 7q

1.7 1.7 2.2 9.4 9.8 3.9

(2′) (1′), (2′), (3′), (4′), (5′)

2′, 1′, 2′, 2′, 1′, 5′

(3′) (4′) (5′) (6a′/6b′)

1A

2b, 3, 5a, 6b 5b, 6a, 9 4, 7b, 8, 10, 11 4, 7a, 9, 3q 2a, 7a, 9, 10, 11, 3q, 5q, 2′ 6b, 7b, 8, 3q, 5q, 1′ 2a, 3, 4, 7a, 8, 11 2a, 3, 4, 7a, 8, 10

(3q) (9), 5.9 (2q) (7q) (5q), 7.4 (8q) (7q)

13

position

3, 5b, 6a

Table 5. Probe−Aglycone Diagnostic NOEs/ROEs and the Average Proton−Proton Distances Corresponding to These Signals, Resulting from MD Simulations of 1A, 2A, 2B, and 3A

4, 5b, 7a, 7b, 10, 11

5.9 2.3 2.8 2.8 7.4

2.2 9.4 9.8 3.9

Figure 7. General definition of the φ and ψ dihedral angles.

5′, 3′, 4′, 3′, 3′,

54.3 37.0 26.5 23.9 43.6 17.7 60.1 66.5 137.1 115.6 146.7 118.5 144.6

position 4qa 5q 6q 7q 8q 8qa OMe Probe Moiety 1′ 2′ 3′ 4′ 5′ 6′

2B

3A

H1′/H3: 0.2406

H1′/H1: 0.3123

H1′/H2eq: 0.2259 H3′/H23: 0.4803 H5′/H23: 0.3515

H1′/H6eq: 0.2140 H5′/H7: 0.2961 H5′/H9: 0.5349

H1′/H1: 0.2233 H1′/H6eq: 0.2685 H1′/H7: 0.3846

H1′/H5q: 0.3379 H1′/H9: 0.2166

H1′/H8: 0.4652 H2′/H7: 0.3523 H2′/H8: 0.4816

H2′/H8: 0.4419

The progress of the reaction was monitored by TLC (Si60, Merck, Darmstadt, Germany) using an n-hexane/EtOAc solvent system (5:1, v/v) (product Rf = 0.46). After 1 h, the mixture was passed through a thin layer of silica gel to remove the remaining NaH. The solvent was evaporated under reduced pressure to yield 252 mg of 2,3,4,6-tetra-Obenzyl-D-mannopyranosyl trichloroacetimidate (referred to as “trichloroacetimidate”). Synthesis of Methyl 3-(2,3,4,6-Tetra-O-benzyl-α- D mannopyranosyloxy)ursolate (1A). Generation of Methyl Ursolate. Ursolic acid (50 mg) was dissolved in CH2Cl2 (1 mL), and the mixture was cooled in an ice bath. Then, CH2N2 in Et2O was added dropwise. The reaction was performed at room temperature with constant stirring, and the progress was monitored by TLC using an nhexane/EtOAc solvent system (5:1, v/v) (product Rf = 0.28). The solvent was evaporated under reduced pressure, and the crude reaction mixture was dissolved in 1 mL of n-hexane/EtOAc (10:1, v/v). The product was isolated by flash chromatography on Merck Kieselgel 60 high-purity-grade silica gel (0.063−0.200 mm) using an n-hexane/ EtOAc solvent system (10:1, v/v). A methyl ursolate yield of 38 mg was obtained. Glycosylation Reaction. The trichloroacetimidate (59 mg) was dissolved in CH2Cl2 (1 mL); then, methyl ursolate (38 mg) was added. The reaction was initiated by the addition of a catalytic amount of p-toluenesulfonic acid. The reaction progress was monitored by TLC (Si60, Merck, Darmstadt, Germany) using an n-hexane/EtOAc solvent system (5:1, v/v) (1A Rf = 0.53). After 48 h, the mixture was passed through a thin layer of basic Al2O3 to remove the remaining PTSA. The solvent was evaporated under reduced pressure, and the crude reaction mixture was dissolved in 1 mL of n-hexane/EtOAc (15:1, v/v). The 1A product was isolated by flash chromatography on Merck Kieselgel 60 high-purity-grade silica gel (0.063−0.200 mm) using an n-hexane/EtOAc solvent system (15:1, v/v). A yield of 8.7 mg of 1A as a colorless oil was obtained. Synthesis of (+)-1-(2,3,4,6-Tetra-O-benzyl-α-Dmannopyranosyloxy)menthol (2A) and (+)-1-(2,3,4,6-Tetra-Obenzyl-β-D-mannopyranosyloxy)menthol (2B). The trichloroacetimidate (84 mg) was dissolved in CH2Cl2 (1 mL); then, (+)-menthol (20 mg) was added. The reaction was initiated by addition of a catalytic amount of PTSA. The reaction progress was monitored by TLC (Si60, Merck, Darmstadt, Germany) using an n-hexane/EtOAc solvent system (5:1, v/v) (2A and 2B Rf = 0.56). After 24 h, the mixture was passed through a thin layer of basic Al2O3 to remove the remaining PTSA. The solvent was evaporated under reduced pressure,

5q, 9 4′, 8 5′ 5′ 4′, 6a′/6b′

C NMR Data for 3A

δ (ppm)

2A

δ (ppm) 126.0 99.9 159.0 122.1 131.8 144.7 56.4 92.6 76.0 80.3 75.6 72.1 70.2

2A, 2B, and 3A were recorded using a Varian Unity 500 Plus spectrometer (500 MHz) with a sample concentration of 10 mg/mL. The chemical shifts are reported in δ (ppm) units using 1H (residual) from benzene-d6 (δ = 7.16 ppm) as the internal standard. Generation of 2,3,4,6-Tetra-O-benzyl-D-mannopyranosyl Trichloroacetimidate. The 2,3,4,6-tetra-O-benzyl-D-mannose (207 mg) was dissolved in CH2Cl2 (2 mL). Then, 180 μL of CCl3CN was added, and the mixture was stirred for 15 min at room temperature. The reaction was initiated by addition of a catalytic amount of NaH. 2802



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Table 6. Definitions of φ and ψ Dihedral Angles for Each Compound φ definition ψ definition

1A

2A

2B

3A

C2−C3−O−C1′ C3−O−C1′−O′

C6−C1−O−C1′ C1−O−C1′−O′

C6−C1−O−C1′ C1−O−C1′−O′

C8−C9−O−C1′ C9−O−C1′−O′

Figure 8. Ramachandran plots obtained from the MD simulations of 1A, 2A, 2B, and 3A. 2D NMR Experiments on 2A and 2B. The 2D 1H NMR spectra were measured in phase-sensitive mode with a spectral width of 3805 Hz. The DQF-COSY spectrum was acquired in a 4032 × 800 matrix with 16 accumulations per increment and processed in a 4 K × 2 K matrix. The TOCSY spectrum was acquired with a mix time of 110 ms in a 2048 × 300 matrix with 16 accumulations per increment in a 2 K × 1 K matrix. The NOESY spectrum was acquired with a mix time of 300 ms in a 2048 × 300 matrix with 16 accumulations per increment in a 2 K × 1 K matrix. The HSQC and HMBC experiments were performed with pulse field gradients. The HSQC spectrum was acquired in phase-sensitive mode. The spectral windows for the 1H and 13C axes were 3805 and 20 111 Hz, respectively. The data were collected in a 1344 × 320 matrix and processed in a 2 K × 1 K matrix. The HMBC spectrum was acquired in absolute value mode. The spectral windows for the 1H and 13C axes were 3805 and 20 113 Hz, respectively. The data were collected in a 1664 × 320 matrix and processed in a 2 K × 1 K matrix. 2D NMR Experiments on 3A. The 2D 1H NMR spectra were measured in phase-sensitive mode with a spectral width of 4692 Hz. The DQF-COSY spectrum was acquired in a 4096 × 600 matrix with 8 accumulations per increment and processed in a 4 K × 2 K matrix. The TOCSY spectrum was acquired with a mix time of 110 ms in a 2048 × 300 matrix with 8 accumulations per increment in a 2 K × 1 K matrix. The ROESY spectrum was acquired with a mix time of 300 ms in a 2048 × 300 matrix with 16 accumulations per increment in a 2 K × 1 K matrix. The HSQC and HMBC experiments were performed with pulse field gradients. The HSQC spectrum was acquired in phasesensitive mode. The spectral windows for the 1H and 13C axes were 4692 and 20 109 Hz, respectively. The data were collected in an 1856 × 400 matrix and processed in a 2 K × 1 K matrix. The HMBC spectrum was acquired in absolute value mode. The spectral windows for the 1H and 13C axes were 4692 and 21 367 Hz, respectively. The

and the crude reaction mixture was dissolved in benzene (1 mL) with the addition of five drops of CH2Cl2. The 2A and 2B mixture was separated by preparative TLC (Si60, Merck, Darmstadt, Germany) using an n-hexane/EtOAc solvent system (5:1, v/v). A yield of 10.7 mg of the 2A and 2B mixture was obtained as a colorless oil. Synthesis of 9-(2,3,4,6-Tetra-O-benzyl-α-D-mannopyranosyloxy)quinine (3A). The trichloroacetimidate (99 mg) was dissolved in CH2Cl2 (1 mL); then, quinine-PTSA salt (47 mg) was added. The reaction was initiated by the addition of a catalytic amount of PTSA. The progress was monitored by TLC (Si60, Merck, Darmstadt, Germany) using a MeOH/H2O/NH3(aq) solvent system (10:1:0.05, v/ v/v) (3A Rf = 0.46). After 48 h, the mixture was passed through a thin layer of basic Al2O3 to remove the remaining PTSA. The solvent was evaporated under reduced pressure, and the crude reaction mixture was dissolved in benzene (1 mL). Product 3A was purified by water extraction, and a yield of 9.3 mg of 3A was obtained as a colorless oil. 2D NMR Experiments on 1A. The 2D 1H NMR spectra were measured in phase-sensitive mode with a spectral width of 4243 Hz. The DQF-COSY spectrum was acquired in a 3712 × 650 matrix with 16 accumulations per increment, which was processed in a 4 K × 2 K matrix. The TOCSY spectrum was acquired with a mix time of 110 ms in a 2048 × 360 matrix with 16 accumulations per increment in a 2 K × 1 K matrix. The NOESY spectrum was acquired with a mix time of 300 ms in a 2048 × 290 matrix with 32 accumulations per increment in a 2 K × 1 K matrix. The HSQC and HMBC experiments were performed with pulse field gradients. The HSQC spectrum was acquired in phase-sensitive mode. The spectral windows for the 1H and 13C axes were 4243 and 17 597 Hz, respectively. The data were collected in an 1856 × 374 matrix and processed in a 2 K × 1 K matrix. The HMBC spectrum was acquired in absolute value mode. The spectral windows for the 1H and 13C axes were 4243 and 18 855 Hz, respectively. The data were collected in an 1856 × 380 matrix and processed in a 2 K × 1 K matrix. 2803



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data were collected in a 2048 × 420 matrix and processed in a 2 K × 1 K matrix. Molecular Modeling. Molecular dynamics (MD) simulations were performed for 1A, 2A, 2B, and 3A that were explicitly solvated in a cubic box with 484, 493, 490, and 490 benzene molecules, respectively. Benzene was chosen as the environment due to its use as the NMR solvent. The force field parameters for 1A, 2A, 2B, 3A, and benzene were obtained from the CHARMM generalized force field .31 All of the energy minimizations and MD simulations were carried out using GROMACS 4.6.5.32 The particle mesh Ewald technique with a cutoff of 1 nm and a grid spacing of approximately 0.1 nm was employed to evaluate the electrostatic forces.33 The van der Waals interactions were calculated using a Lennard-Jones potential with a cutoff of 1 nm. The simulation was conducted at a constant temperature of 300 K and a constant pressure of 1 bar using the weak coupling method34 with relaxation times of 0.1 and 0.5 ps, respectively. All of the covalent bond lengths were constrained using the P-LINCS35 and SETTLE36 algorithms. After an initial thermalization for 2 ns, the four systems were simulated for 100 ns using the leapfrog scheme with a time step of 2 fs.

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(14) Ohtani, I.; Kusumi, T.; Ishitsuka, M. O.; Kakisawa, H. Tetrahedron Lett. 1989, 30, 3147−3150. (15) Sowiński, P.; Pawlak, J.; Gariboldi, P.; Borowski, E. Magn. Reson. Chem. 1992, 30, 275−279. (16) Pawlak, J.; Sowiński, P.; Gariboldi, P.; Borowski, E. J. Antibiot. 1993, 46, 1598−1604. (17) Pawlak, J.; Sowiński, P.; Garboldi, P.; Borowski, E. J. Antibiot. 1995, 48, 1034−1038. (18) Sowiński, P.; Pawlak, J.; Borowski, E. J. Antibiot. 1995, 48, 1288−1291. (19) Płosiński, M.; Laskowski, T.; Sowiński, P.; Pawlak, J. Magn. Reson. Chem. 2012, 50, 818−822. (20) Szwarc, K.; Szczeblewski, P.; Sowiński, P.; Borowski, E.; Pawlak, J. Magn. Reson. Chem. 2015, 53, 479−484. (21) Szwarc, K.; Szczeblewski, P.; Sowiński, P.; Borowski, E.; Pawlak, J. J. Antibiot. 2015, 68, 504−510. (22) Seroka, P.; Płosiński, M.; Czub, J.; Sowiński, P.; Pawlak, J. Magn. Reson. Chem. 2006, 44, 132−138. (23) Ferrier, R. J. Fortschr. Chem. Forsch. 1970, 14, 389−429. (24) Zhu, X.; Schmidt, R. R. Angew. Chem., Int. Ed. 2009, 48, 1900− 1934. (25) Corey, E. J.; Ursprung, J. J. J. Am. Chem. Soc. 1956, 78, 183− 188. (26) Read, J. J. Soc. Chem. Ind., London 1927, 46, 871−876. (27) Sassa, T.; Kenmoku, H.; Sata, M.; Murayama, T.; Kato, N. Biosci., Biotechnol., Biochem. 2003, 67, 475−479. (28) Rabe, P.; Kindler, K. Ber. Dtsch. Chem. Ges. B 1939, 72, 263− 264. (29) Woodward, R. B.; Doering, W. E. J. Am. Chem. Soc. 1945, 67, 860−874. (30) Izumi, S.; Moriyoshi, H.; Hirata, T. Bull. Chem. Soc. Jpn. 1994, 67, 2600−2602. (31) Vanommeslaeghe, K.; Hatcher, E.; Acharya, C.; Kundu, S.; Zhong, S.; Shim, J.; Mackerell, A. D. J. Comput. Chem. 2010, 31, 671− 690. (32) Hess, B.; Kutzner, C.; Van Der Spoel, D.; Lindahl, E. J. Chem. Theory Comput. 2008, 4, 435−447. (33) Darden, T.; York, D.; Pedersen, L. J. Chem. Phys. 1993, 98, 10089−10092. (34) Berendsen, H. J.; Postma, J. P. M.; van Gunsteren, W. F.; DiNola, A. R. H. J.; Haak, J. R. J. Chem. Phys. 1984, 81, 3684−3690. (35) Hess, B. J. Chem. Theory Comput. 2008, 4, 116−122. (36) Miyamoto, S.; Kollman, P. A. J. Comput. Chem. 1992, 13, 952− 962.

ASSOCIATED CONTENT

S Supporting Information *

The Supporting Information is available free of charge on the ACS Publications website at DOI: 10.1021/acs.jnatprod.6b00471. NMR spectra (images) of studied glycosides, MD simulation analysis, and Cartesian coordinates of 1A, 2A, 2B, and 3A (PDF)

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AUTHOR INFORMATION

Corresponding Author

*Tel: +48 58 347 20 79. Fax: +48 58 347 11 44. E-mail: [email protected]. Notes

The authors declare no competing financial interest.

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DEDICATION This work is dedicated to the late Jan Pawlak. REFERENCES

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