Stereochemical assignment of four diastereoisomers of a maculalactone derivative by computational NMR calculations

Accepted Manuscript Stereochemical assignment of four diastereoisomers of a maculalactone derivative by computational NMR calculations Daniel Previdi, Viviani Nardini, Mayla Eduarda Rosa, Vinicius Palaretti, Gil Valdo José da Silva, Paulo Marcos Donate PII:

S0022-2860(18)31259-6

DOI:

https://doi.org/10.1016/j.molstruc.2018.10.064

Reference:

MOLSTR 25793

To appear in:

Journal of Molecular Structure

Received Date: 3 August 2018 Revised Date:

12 October 2018

Accepted Date: 19 October 2018

Please cite this article as: D. Previdi, V. Nardini, M.E. Rosa, V. Palaretti, G.V. José da Silva, P.M. Donate, Stereochemical assignment of four diastereoisomers of a maculalactone derivative by computational NMR calculations, Journal of Molecular Structure (2018), doi: https://doi.org/10.1016/ j.molstruc.2018.10.064. This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.

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Stereochemical assignment of four diastereoisomers of a maculalactone derivative by

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computational NMR calculations

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Authors: Daniel Previdi,* Viviani Nardini, Mayla Eduarda Rosa, Vinicius Palaretti, Gil

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Valdo José da Silva and Paulo Marcos Donate

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Departamento de Química, Faculdade de Filosofia, Ciências e Letras de Ribeirão Preto,

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Universidade de São Paulo, Avenida Bandeirantes, 3900, CEP 14040-901, Ribeirão Preto,

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SP, Brazil.

*Corresponding author. E-mail address: [email protected] (D. Previdi).

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Abstract:

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Naturally occurring γ-butyrolactones and their synthetic analogues display a wide

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range of bioactivities. Here, the multicomponent reaction of dimethyl 2-benzyl-3-

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methylenesuccinate with bromobenzene and benzaldehyde catalyzed by cobalt(II) bromide

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afforded a maculalactone derivative with three stereogenic centers. This reaction presented

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moderate diastereoselectivity and yielded different proportions of all the four possible

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diastereoisomers. The anti:anti (majority), anti:syn, syn:anti, and syn:syn diastereoisomers

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were isolated and characterized by 1D and 2D NMR experiments. Because the stereochemical

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assignment of all the diastereoisomers by Nuclear Overhauser Effect Difference (NOEDiff)

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experiments was not definitive, the 1H and

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theoretical calculations with the density functional theory at the B3LYP/6-31G(d) level. The

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relative configurations of all the four diastereoisomers were assigned by using the CP3

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parameter to compare the experimental and the calculated data and by determining the CP3

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probability, which provided high level of confidence.

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C NMR chemical shifts were predicted by

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Keywords: γ-butyrolactone, maculalactone, assignment, NMR, CP3.

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1. Introduction

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1.1. γ-Butyrolactones The γ-butyrolactone ring exists in many natural products and synthetic analogues that

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display diverse biological activities, such as antitumor,[1,2] antibacterial,[3] antiviral,[4] and

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antiprotozoal[5] action, among other effects.[6–12] This skeleton occurs in numerous natural

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products like mono-, di-, or tri-substituted monocyclic lactones, or it can be part of more

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complex frameworks (Figure 1).[13] Maculalactones, which bear a benzylated γ-

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butyrolactone core, are natural products that have been isolated from the marine

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cyanobacterium Kyrtuthrix maculans.[14–20]

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Figure 1. Examples of natural γ-butyrolactones and some biological activities.

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Recently, Le Floch and co-workers developed a multicomponent reaction involving

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dimethyl itaconate, an aryl halide, and a carbonyl compound catalyzed by cobalt(II) bromide

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to synthesize paraconic acid analogues.[21,22] Our research group has employed microwave

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irradiation to optimize this reaction [23,24] and used it to obtain maculalactone

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derivatives.[25]

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The single-step synthesis of maculalactone derivatives through this multicomponent

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reaction affords γ-butyrolactones with three aromatic substituents and three stereogenic

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centers (Scheme 1). All the reactions are moderately diastereoselective, and their products

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consist of mixtures of all the four possible diastereoisomers as racemic mixtures. In almost all

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cases, the major diastereoisomer is purified by crystallization and has the anti:anti relative 2

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configuration, as determined by NOEDiff NMR experiments.[25] To increase our level of

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confidence in the stereochemical assignment, we isolated the four diastereoisomers, each one

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as a racemic mixture, by preparative HPLC and analyzed them by NMR. Because the full

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characterization of these compounds was not so clear-cut, we decided to calculate the 13C and

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1

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diastereoisomers.

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H NMR chemical shifts directly in order to assign the relative configurations of all the four

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Scheme 1. Syntheses of maculalactone derivatives.

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1.2. NMR chemical shift calculations and comparison parameters Aiming at the stereochemical assignment of molecules, computational calculations have been increasingly applied to predict NMR properties, including 1H and

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chemical shifts and coupling constants.[26–30] This procedure has been employed to

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characterize several natural products and synthetic compounds.[31–38]

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C NMR

In 2002, Barone and co-workers used this technique in their benchmark studies based

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on easy-to-calculate statistical correlation parameters, such as R2, the mean absolute error

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(MAE), and the corrected mean absolute error (CMAE), to estimate confidence levels for the

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assignments.[39,40] Recently, more refined procedures have been developed to compare

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experimental and calculated NMR chemical shifts and to achieve higher levels of confidence

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than those expected from simple statistical parameters.[41] Three main methods have been

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designed: CP3,[42] DP4,[43,44] and ANN-PRA.[45,46]

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In 2009, Smith and Goodman developed three different comparison parameters,

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designated CP1, CP2, and CP3. They validated these parameters with a set of 28 pairs of

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diastereoisomers, and CP3 gave the best results.[42] CP3, calculated according to Equation 1,

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provided the best match between two experimental spectra and the calculated chemical shifts

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of two diastereoisomers. This parameter is based on the observation that, for similar atoms,

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systematic errors in NMR chemical shift calculations can be removed by comparing the 3

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differences between two calculated chemical shifts and two experimental data. Cancelation of

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the systematic errors indicates that these differences are calculated more accurately than the

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chemical shifts themselves. CP3 has been applied in the stereochemical assignments of

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various types of natural and synthetic compounds.[47–56]

CP3 =

∑ (∆  , ∆  )  ∑ ∆  where

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Equation 1

 ⁄ ∆  , ∆   = ∆  ∆  if ∆  ⁄∆  > 1

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∆  , ∆   = ∆  ∆  otherwise 85

CP3 was developed to solve the problem of attributing two sets of experimental data

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(spectra X and Y) to two possible diastereomeric structures (structures x and y). As a

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consequence, two assignments could be made: X = x and Y = y or X = y and Y = x.

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The differences between the NMR chemical shifts calculated for similar atoms from

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diastereomeric structures (∆δcalc, x – y) are compared to the differences between the

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experimental NMR chemical shifts (∆δexp, X – Y), and CP3 is obtained according to Equation

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1. A positive CP3 value indicates that the assignment is probably right (i.e. X = x and Y = y);

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CP3 equal to one means perfect agreement between the experimental and calculated NMR

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chemical shifts; and a negative CP3 value indicates that the assignment is probably wrong.

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CP3 can be calculated for 1H and 13C NMR separately. The CP3 arithmetic mean for 1H and

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C NMR, called “All Data”, usually affords better results for the assignments. Smith and Goodman (2009) also developed Equation 2 to calculate the probability that

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the assignment is correct. Although CP3 is easy to compute “by hand”, its probability requires

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knowledge of normal distribution descriptors (expectation value and standard deviation) and

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statistical computer programs. The authors provided an applet to compute both CP3 and its

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probability.[62]

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102 (A |R and R  ) =

(A )(R |A )(R  |A ) (A )(R |A )(R  |A ) + (A )(R |A )(R  |A )

Equation 2

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To obtain Equation 2, the authors used conditional probability elements and the Bayes

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theorem. They assumed that the CP3 values for correct and wrong assignments are normally 4

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distributed, independent variables, and that expectation values and standard deviations can be

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approximated on the basis of the values obtained for the original reference.[42] In Equation 2, P(A1|R1 and R2) is the probability that a given assignment is correct.

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Figure 2 illustrates the probability elements in Equation 2. Two assignments (A1 and A2) are

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possible. In the absence of any prediction about which assignment is right, P(A1) = P(A2) =

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0.5. Therefore, two possibilities (R1 and R2) exist: that A1 is right, and consequently A2 is

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wrong (R1), or that A2 is right, so A1 is wrong (R2). To calculate these probabilities, statistics

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computer programs can be employed to compute the area above normal distribution curves.

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The programs use the expectation values and standard deviations for CP3 for right and wrong

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assignments.

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Figure 2. Interpretation of probability elements in Equation 2.

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In this study, we describe the assignment of the relative configurations of all the four

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possible diastereoisomers of maculalactone derivative 4 (Scheme 2) by using a combination

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of NMR experiments, DFT/GIAO chemical shift calculations, and CP3 computation.

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2. Experimental section

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2.1. General

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γ-Butyrolactone 4 was prepared as described in our previous work [25]. A CEM

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Discover® microwave reactor operating at maximum potency of 150 W was used. The

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melting points of the synthesized compounds were obtained on a Bristolscope–871035

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melting point apparatus and are uncorrected. IR spectra were acquired on a Shimadzu IR 5

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Prestige-21 spectrometer equipped with a germanium crystal; the thin solid film method and

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the ATR (Attenuated Total Reflection) technique were employed. NMR experiments (1H, 13C,

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NOEDiff, COSY, HMQC, and HMBC) were run on a Bruker DPX-300 spectrometer with

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CDCl3 as solvent. The Chemical shifts are reported relative to TMS (0.00 ppm for 1H) and

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CDCl3 (77.0 ppm for

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conducted on a HPLC Shimadzu-LC-6AD with a preparative column Phenomenex® – Luna

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C18 (250 x 21.2 mm, particle size: 5 µm, pore size: 100 Å, UV-Visible detector: 210 and 240

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nm, loop: 1000 µL) and isocratic elution with acetonitrile (HPLC grade) and water (Milli-Q)

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at a flow rate of 9.0 mL/min; the sample was diluted in acetonitrile at a concentration around

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100 mg/mL for injection. Gas chromatography analyses were carried out on a GC-FID –

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Shimadzu GC-2010 Plus with a Restek Rtx-5® column (film thickness: 0.25 µm, length: 30

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m, and internal diameter: 0.25 mm) and nitrogen as carrier gas (linear velocity: 36.8 cm/s).

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The heat program was 80 oC for 2 min, which was followed by a rise to 320 oC at 30 oC/min,

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and 320 oC for 10 min. Electron impact (70 eV) mass spectra were registered on a Shimadzu

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GC-MS-QP2010Plus under the same conditions describe above, except that the carrier gas

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was helium.

C). High Performance Liquid Chromatography separations were

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146 2.2. Synthetic procedure

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Compound 4 was synthesized as described previously (see Scheme 2).[25] A 25-mL

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round-bottom flask under argon atmosphere was loaded with recently purified acetonitrile (5

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mL),[63] dimethyl 2-benzyl-3-methylenesuccinate (3.1 g, 12.5 mmol), benzaldehyde (0.25

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mL, 2.5 mmol), bromobenzene (0.42 mL, 4 mmol), and zinc dust (0.8 g, 12 mmol). This

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mixture was briefly stirred at room temperature. Next, cobalt(II) bromide (0.13 g, 0.6 mmol),

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trifluoroacetic acid (30 µL), and 1,2-dibromoethane (50 µL) were successively added to the

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previous mixture, which was then irradiated in a CEM Discovery® focused microwave oven

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at 60 °C and 150 W for 20 min. After that, the reaction mixture was filtered through Celite®,

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which was washed several times with ethyl acetate. The organic fractions were combined and

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concentrated under reduced pressure. The crude reaction product was purified by flash

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column chromatography through silica gel (gradient elution with n-hexane/ethyl acetate from

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9:1 to 6:4 (v/v)), to afford 0.8890 g of a diastereomeric mixture of maculalactone derivative 4

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in 89% yield at a diastereomeric ratio (d.r., determined by GC-FID) of 73:14:8:5. The

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diastereomeric mixture was separated by preparative HPLC. The relative configurations of all

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the four diastereoisomers were determined by using a combination of NMR experiments,

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DFT/GIAO chemical shift calculations, and CP3 computation.

164 Methyl 3,4-dibenzyl-5-oxo-2-phenyltetrahydrofuran-3-carboxylate (4a, anti:anti):

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White solid, mp: 137–138 oC. 1H NMR (300 MHz, CDCl3), δ (ppm): 7.50 to 6.30 (m, 15H),

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5.37 (s, 1H), 3.50 (s, 3H), 3.35 (dd, 1H, J = 14.9 Hz, J = 6.7 Hz), 3.23 (d, 1H, J = 15.4 Hz),

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3.17 (d, 1H, J = 15.4 Hz), 3.03 (dd, 1H, J = 6.7 Hz, J = 5.0 Hz), 2.53 (dd, 1H, J = 14.9 Hz, J =

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5.0 Hz).

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134.8 (C), 131.3 (2 CH), 129.5 (2 CH), 129.1 (CH), 129.0 (2 CH), 128.8 (2 CH), 128.6 (2

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CH), 127.7 (CH), 126.9 (CH), 126.0 (2 CH), 81.2 (CH), 61.7 (C), 52.0 (CH3), 47.7 (CH), 36.3

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(CH2), 33.0 (CH2). IR (thin solid film, cm-1): 3063 (Csp2–H), 2950 (Csp3–H), 1771 and 1723

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(C═O), 1218 and 1175 (C–O), 753 and 700 (═C─H). EI-MS (70 eV), m/z (relative

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intensity, %): [M•+] 400 (<5), [M•+ – C7H6O] 294 (<5), [M•+ – C7H6O – C7H7•] 203 (100),

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[M•+ – C7H6O – C7H7• – C4H4O2 – CO] 91 (72), [M•+ – C9H8O2] 252 (50), [M•+ – C9H8O2 –

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MeOH] 220 (33).

C NMR (75 MHz, CDCl3), δ (ppm): 176.1 (C), 170.9 (C), 139.0 (C), 135.1 (C),

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Methyl 3,4-dibenzyl-5-oxo-2-phenyltetrahydrofuran-3-carboxylate (4b, anti:syn):

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White solid, mp: 172–173 oC. 1H NMR (300 MHz, CDCl3), δ (ppm): 7.35 to 6.88 (m, 15H),

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5.77 (s, 1H), 3.73 (s, 3H), 3.20 (dd, 1H, J = 7.5 Hz, J = 4.7 Hz), 3.10 (dd, 1H, J = 14.4 Hz, J =

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7.5 Hz), 2.83 (d, 1H, J = 14.5 Hz), 2.57 (dd, 1H, J = 14.4 Hz, J = 4.7 Hz), 2.48 (d, 1H,

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J = 14.5 Hz). 13C NMR (75 MHz, CDCl3), δ (ppm): 176.4 (C), 172.6 (C), 138.2 (C), 135.9

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(C), 134.9 (C), 130.4 (2 CH), 129.2 (2 CH), 128.9 (CH), 128.6 (2 CH), 128.5 (2 CH), 128.4

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(2 CH), 127.1 (CH), 127.0 (2 CH), 126.8 (CH), 83.9 (CH), 58.3 (C), 52.4 (CH3), 48.5 (CH),

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39.5 (CH2), 33.5 (CH2). IR (thin solid film, cm-1): 3059 (Csp2–H), 2985 (Csp3–H), 1780 and

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1734 (C═O), 1216 and 1124 (C–O), 756 and 699 (═C─H). EI-MS (70 eV), m/z (relative

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intensity, %): [M•+] 400 (<5), [M•+ – C7H6O] 294 (<5), [M•+ – C7H6O – C7H7•] 203 (100),

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[M•+ – C7H6O – C7H7• – C4H4O2 – CO] 91 (95), [M•+ – C9H8O2] 252 (92), [M•+ – C9H8O2 –

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MeOH] 220 (63).

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Methyl 3,4-dibenzyl-5-oxo-2-phenyltetrahydrofuran-3-carboxylate (4c, syn:anti):

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White solid, mp: 86–89 oC. 1H NMR (300 MHz, CDCl3), δ (ppm): 7.37 to 7.08 (m, 15H),

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5.43 (s, 1H), 3.66 (dd, 1H, J = 7.4 Hz, J = 6.2 Hz), 3.43 (d, 1H, J = 15.2 Hz), 3.35 (d, 1H, J =

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15.2 Hz), 3.19 (s, 3H), 2.99 (dd, 1H, J = 14.0 Hz, J = 7.4 Hz), 2.94 (dd, 1H, J = 14. Hz, J = 7

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6.2 Hz).

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135.1 (C), 129.8 (2 CH), 129.3 (2 CH), 129.0 (CH), 128.8 (2 CH), 128.6 (2 CH), 128.5 (2

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CH), 127.3 (CH), 126.9 (CH), 125.9 (2 CH), 83.6 (CH), 59.5 (C), 52.0 (CH3), 47. (CH), 37.2

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(CH2), 31.9 (CH2). IR (thin solid film, cm-1): 3060 (Csp2–H), 2951 (Csp3–H), 1780 and 1706

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(C═O), 1219 and 1158 (C–O), 745 and 695 (═C─H). EI-MS (70 eV), m/z (relative

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intensity, %): [M•+] 400 (<5), [M•+ – C7H6O] 294 (<5), [M•+ – C7H6O – C7H7•] 203 (100),

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[M•+ – C7H6O – C7H7• – C4H4O2 – CO] 91 (79), [M•+ – C9H8O2] 252 (68), [M•+ – C9H8O2 –

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MeOH] 220 (50).

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C NMR (75 MHz, CDCl3), δ (ppm): 176.2 (C), 171.5 (C), 138.3 (C), 136.1 (C),

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Methyl 3,4-dibenzyl-5-oxo-2-phenyltetrahydrofuran-3-carboxylate (4d, syn:syn):

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White solid, mp: 98–101oC. 1H NMR (300 MHz, CDCl3), δ (ppm): 7.40 to 6.80 (m, 15H),

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5.71 (s, 1H), 3.71 (s, 3H), 3.62 (dd, 1H, J = 8.2 Hz, J = 4.4 Hz), 3.01 (d, 1H, J = 15.0 Hz),

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2.97 (dd, 1H, J = 14.2 Hz, J = 8.2 Hz), 2.92 (d, 1H, J = 15.0 Hz), 2.74 (dd, 1H, J = 14.2 Hz,

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J = 4.4 Hz).

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(C), 134.1 (C), 130.0 (2 CH), 129.1 (2 CH), 128.8 (CH), 128.6 (2 CH), 128.4 (2 CH), 128.3 (2

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CH), 127.1 (2 CH), 126.8 (CH), 126.6 (CH), 83.7 (CH), 59.1 (C), 52.6 (CH3), 52.5 (CH), 34.4

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(CH2), 32.2 (CH2). IR (thin solid film, cm-1): 3061 (Csp2–H), 2943 (Csp3–H), 1778 and 1730

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(C═O), 1224 and 1169 (C–O), 744 and 696 (═C─H). EI-MS (70 eV), m/z (relative

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intensity, %): [M•+] 400 (<5), [M•+ – C7H6O] 294 (<5), [M•+ – C7H6O – C7H7•] 203 (100),

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[M•+ – C7H6O – C7H7• – C4H4O2 – CO] 91 (63), [M•+ – C9H8O2] 252 (52), [M•+ – C9H8O2 –

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MeOH] 220 (33).

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2.3. Computational studies

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Conformational searches and geometry optimizations followed practically the same

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conditions and parameters as the conditions and parameters used in our previous

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works.[64,65] Conformational searches were performed with the PCModel program version

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7.0 [66] by using the GMMX routine with the parameters depicted in Table 1.

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Table 1. Parameters used during conformational searches with the PCModel program. Parameter

Value

Force field

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First cycle: 1.5 kcal/mol

Energy window

Second cycle: 1.0 kcal/mol

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Maximum conformation minimized

100,000

Boltzmann temperature

300 K

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The geometry of each conformer in the energy window of the conformational search

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was optimized with ORCA version 3.0.1 [67] in vacuum, at the B3LYP-D3(BJ)/def2-TZVP(-

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f) level [68–73], in addition to the grid4 keyword and the RIJCOSX approximation.[74]

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Dispersion interaction (D.I.) between atoms, which represents the energy of the van der Waals

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forces, was taken into account by following the approach described by Grimme and

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coworkers.[75–77]

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After the geometry was optimized, conformers amounting to 90% of the Boltzmann

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distribution were selected. The number of conformers obtained for the four diastereoisomers

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are shown in Table 2.

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Isotropic magnetic shielding (IMS) was calculated with the GIAO method at the

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B3LYP/6-31G(d) level by using Gaussian03.[78] The calculated chemical shifts were

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obtained as the difference between the IMS of a particular 1H or 13C in a conformer and the

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IMS of 1H or 13C in TMS, calculated by the same method.

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Finally, CP3 was determined according to Equation 1 for all the possible assignment

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combinations. Probabilities were obtained by using Equation 2 as well as the expectation

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values and standard deviations from the original reference listed in Table 3.[42]

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Table 2. Number of conformers obtained during the conformational search with molecular

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mechanics and after optimization geometry by DFT. Calculated diastereoisomer

Conformational search

4a anti:anti

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4b anti:syn

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4c syn:anti

50

4d syn:syn

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Selected conformers after optimization*

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* Number of conformers amounting to 90% of the population according to Boltzmann

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distribution.

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Table 3. Expectation values and standard deviations for CP3. 1

Assignment

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C

All data

Right

0.478 ± 0.305

0.547 ± 0.253

0.512 ± 0.209

Wrong

-0.786 ± 0.835

-0.487 ± 0.533

-0.637 ± 0.499

247 3. Results and discussion

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We conducted the cobalt(II) bromide-catalyzed multicomponent reaction of dimethyl

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2-benzyl-3-methylenesuccinate (1) with bromobenzene (2) and benzaldehyde under

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microwave irradiation, to obtain maculalactone derivative 4 as a mixture of four

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diastereoisomers as racemic mixtures at a diastereomeric ratio of 73:14:8:5, determined by

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GC-FID analysis, and relative configurations anti:anti, anti:syn, syn:anti, and syn:syn.[25]

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We purified the diastereoisomers by preparative HPLC. The NMR spectral data (1H,

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DEPT-135, COSY, HMQC, HMBC, and NOEDiff) obtained for the four fractions did not

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allow a clear-cut assignment of each spectrum corresponding to structures 4a-4d. Therefore,

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we had to work out which spectrum referred to a given diastereoisomer (see Figures 3-4 and

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supporting information for all the spectral data).

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C,

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O MeO

+

O

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ACN Zn, CoBr2

O

Br

OMe

+

H

2

O

TFA, DBE MW, 150W 20 min

O

MeO2C

3

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4 89% (73:14:8:5)

11 12

10

13

9

24 23

22

O

2 1 O 3 4 14 15 20 16 19 17 18

MeO2C

4a anti:anti

O

O

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5 6 MeO2C 25 26 21

O

MeO2C

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4b anti:syn

O

4c syn:anti

MeO2C

O O

4d syn:syn

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Scheme 2. Relative configurations of all the possible diastereoisomers of compound 4.*

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*Diastereoisomers 4a-d were obtained as racemic mixtures and the stereochemistry showed in

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the structures of these compounds are the relative configurations between then.

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Figure 3. 1H NMR (300 MHz, CDCl3) spectra of the diastereoisomers of compound 4. 11

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Figure 4. 13C NMR (75 MHz, CDCl3) spectra of the diastereoisomers of compound 4.

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The diastereoselectivity of this multicomponent reaction is probably due to the

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mechanism of reaction and epimerization equilibrium. Scheme 3 presents a proposed

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mechanism for this multicomponent reaction. The reaction starts with an organometallic

273

compound originating from bromobenzene (2). The organometallic compound then reacts

274

with itaconate derivative 1, to produce enolate 5. In turn, this enolate reacts with

275

benzaldehyde (3) through cyclic transition state 6, to yield intermediate 7. The latter

276

intermediate is in equilibrium with intermediate 8, thanks to exchange of the carboxylate

277

group chelating the metal. Finally, a lactonization process gives maculalactone derivative

278

4.[21]

EP

AC C

279

TE D

270

12

TE D

M AN U

SC

RI PT

ACCEPTED MANUSCRIPT

Scheme 3. Mechanism proposed for the multicomponent reaction to obtain maculalactone

282

derivative 4.

283

EP

280 281

The aldol reaction, during which cyclic transition state 6 acquires a chair

285

conformation, probably underlies the diastereoselectivity at carbons 3 and 4.[79,80] Enolate 5

286

can have stereochemistry E/Z (5a and 5b in Scheme 4). Under the reaction conditions (i.e.,

287

temperature = 60 oC), the aldol reaction product can be determined by thermodynamic

288

control.[81] As shown in Scheme 4, enolate E (5a) has transition state 6a with the large

289

substituent R1 in the equatorial position and furnishes intermediate 7a, which has relative

290

configuration anti between groups R2 and R3. The opposite is observed for enolate Z (5b): it

291

has transition state 6b with the large substituent R1 in the axial position and furnishes

292

intermediate 7b, which has relative configuration syn between groups R2 and R3. Saturated

293

six-membered rings are known to exist in the chair/boat conformation. Because the largest

294

substituent is known to be more stable in the equatorial position of the chair conformation,

295

relative configuration anti is probably favored at carbons 3 and 4.

AC C

284

13

ACCEPTED MANUSCRIPT 296 O

M

R1 Ph

R2

MeO2C Ph

Ph

Ph

5a enolate E O

Ph Ph

Ph

297 298

Ph

CO2Me

5b enolate Z

R3 M

4 O R2

7a 3,4-anti

R H Ph 3

O

OMe H O M O Ph

MeO 5 Ph R2 Ph

CO2Me

6b

3

Ph 2 CO2Me 1

Ph

6a

M

H Ph

RI PT

MeO

MeO 5

SC

MeO

O

OMe H M O O Ph R3

3

4 O

2

M

CH2O2Me 1

7b 3,4-syn

Scheme 4. Origin of diastereoselectivity at carbons 3 and 4 of intermediate 7.

M AN U

299 300

After conversion of intermediate 7 to 8 and subsequent lactonization to maculalactone

301

derivative 4 (Scheme 3), an epimerization process may occur. This process is catalyzed by

302

bases like methoxide, which is generated during the lactonization reaction and is favored by

303

excess organometallic species (due to excess halide).[82,83]

Epimerization possibly accounts for the diastereoselectivity at carbons 2 and 3

305

(Scheme 5). Diastereoisomer 4c, which possesses relative configuration syn at carbons 2 and

306

3, can be converted to diastereoisomer 4a, which has relative configuration anti at carbons 2

307

and 3 and also a minor steric hindrance at the aromatic substituents on carbons 2 and 3. The

308

same epimerization equilibrium can occur with diastereoisomers 4d and 4b, as shown in

309

Scheme 5.

AC C

EP

TE D

304

14

310 311

M AN U

SC

RI PT

ACCEPTED MANUSCRIPT

Scheme 5. Epimerization equilibrium between the diastereoisomers of compound 4.

312

In accordance with the considerations above, we expected that the major

314

diastereoisomer of maculalactone derivative 4 would be diastereoisomer 4a (anti:anti),

315

whereas the minor diastereoisomer of maculalactone derivative 4 would be diastereoisomer

316

4d (syn:syn). The second major diastereoisomer should be diastereoisomer 4b (anti:syn),

317

followed by diastereoisomer 4c (syn:anti). Therefore, we proposed that spectra A, B, C, and D

318

should correspond to diastereoisomers 4a, 4b, 4c, and 4d, respectively.

TE D

313

To determine the relative configurations of all the four diastereoisomers of compound

320

4 isolated by preparative HPLC, we first obtained the NOEDiff NMR spectra. On the basis of

321

these NMR experiments, we deduced that the major diastereoisomer obtained during the

322

multicomponent

323

diastereoisomer possesses syn:syn relative configuration. The relative configurations of the

324

other two diastereoisomers are not clear-cut. See supporting information for the NOEDiff

325

NMR spectra and more details.

AC C

EP

319

reaction

has

anti:anti

relative

configuration,

whilst

the

minor

326

To conclude these assignments and to increase our confidence level about the relative

327

configurations of the four diastereoisomers obtained during the multicomponent reaction, we

328

decided to compare the experimental and calculated 1H and 13C NMR data by using the CP3

329

parameter.[42] Simpler statistical parameters like R2, MAE, and CMAE cannot distinguish

330

between the four diastereoisomers. See supporting information for more details. 15

ACCEPTED MANUSCRIPT We calculated the 1H and

331

13

C NMR chemical shifts according to the procedure

described in the experimental section. Tables 4 and 5 summarize the experimental and

333

calculated 1H and 13C NMR chemical shifts, respectively. These tables only present the atoms

334

that we used to calculate CP3. We did not employ aromatic hydrogens because their signals

335

are heavily overlapped. We removed carbons 10-12, 16-18, and 23-25 because we were not

336

able to attribute them experimentally on the basis of 2D NMR experiments.

337

Table 4. Experimental and calculated 1H NMR chemical shifts used to calculate CP3.

A

B

C

D

4

5.37

5.77

5.43

5.71

6

3.50

3.73

3.19

3.71

7A

3.35

3.10

2.99

20A

3.23

2.83

20B

3.17

2 7B

Calculated diastereoisomer 4a

4b

4c

4d

6.21

6.45

6.21

6.34

4.29

4.10

3.32

4.48

2.97

4.30

4.02

4.26

3.91

3.43

3.01

3.87

3.32

4.71

3.49

2.48

3.35

2.92

3.79

3.12

3.51

3.48

3.03

3.20

3.66

3.62

3.60

4.16

4.64

4.63

2.53

2.57

2.94

2.74

3.59

4.00

3.55

3.71

TE D EP AC C

339

Experimental spectrum

SC

H

M AN U

338

RI PT

332

16

ACCEPTED MANUSCRIPT Table 5. Experimental and calculated 13C NMR chemical shifts used to calculate CP3. C

Calculated diastereoisomer

B

C

D

4a

4b

4c

4d

7

33.0

33.5

31.9

32.2

35.8

36.0

33.4

33.4

20

36.3

39.5

37.2

34.4

38.0

40.8

40.1

37.1

2

47.7

48.5

47.8

52.5

48.7

49.4

51.0

54.7

6

52.0

52.4

52,0

52.6

51.7

51.1

51.3

52.1

3

61.7

58.3

59.5

59.1

63.0

58.8

59.4

61.4

4

81.2

83.9

83.6

83.7

81.3

82.7

79.7

83.3

15

126.0

127.0

125.9

127.1

19

126.0

127.0

125.9

127.1

9

129.5

129.2

129.3

129.1

13

129.5

129.2

129.3

22

131.4

130.4

26

131.4

21

SC

RI PT

A

120.7

118.7

120.5

120.9

120.8

118.7

120.9

123.5

123.2

122.9

124.2

129.1

123.8

123.4

122.9

122.1

129.8

130,0

126.0

125.3

125.5

124.2

130.4

129.8

130,0

124.6

125.2

125.8

126.4

134.8

135.9

136.1

136.1

129.9

129.7

131.8

128.3

14

135.1

134.9

135.1

134.1

129.8

130.7

130.8

132.2

8

139.0

138.2

138.3

139.6

134.2

130.9

132.6

132.7

5

170.9

172.6

171.5

172.7

163.5

165.2

163.7

164.4

1

176.1

176.4

176.2

175.3

165.8

166.6

166.0

165.2

M AN U

120.6

EP

341

Experimental spectrum

TE D

340

CP3 was developed to compare two sets of experimental data to two possible

343

diastereomeric structures, so just two assignments should be made. In our case, we had four

344

NMR spectral datasets to compare to four possible diastereomeric structures, so the number of

345

possible assignments was much larger—actually, 144 assignments were possible.

346

AC C

342

We conducted all these assignments and computed CP3 and its probability for the 1H

347

and 13C NMR data together (“All Data”). Table 6 shows these results in a simple manner. A

348

green check indicates that CP3 is positive and its probability is high (> 95%); a yellow check

349

indicates that CP3 is positive and its probability is low, or that CP3 is negative and its

350

probability is high; and a red “X” indicates that CP3 is negative and its probability is low. For

351

more details about Table 6, see supporting information.

352

Table 6 also contains all the possible assignments. For example, the first line shows

353

the assignments attributed to diastereoisomer 4a in spectrum A and to the other 17

ACCEPTED MANUSCRIPT 354

diastereoisomers (diastereoisomers 4b, 4c and 4d) in spectra B, C, and D. This table is

355

reflected over the diagonal, which means that the first line is equivalent to the first column.

356

The last three columns are the sum of the green check, the yellow check, and the red “X”. If we analyze Table 6 over the four lines for a single spectrum; i.e., the first four lines

358

of spectrum A, for example, the line that presents the largest numbers of green and yellow

359

checks indicates that this spectrum corresponds to the diastereoisomer in question. For

360

spectrum A, the first line (diastereoisomer 4a) is the line with the largest numbers of green

361

and yellow checks (total of 9), indicating that the assignment of spectrum A to

362

diastereoisomer 4a is probably right. Another important observation is that when the other

363

spectra are attributed to diastereoisomer 4a (look at lines 2, 3, and 4 and columns 5, 9, and 13

364

for spectra B, C, and D, respectively), all the assignments present a red “X”, indicating that

365

CP3 is negative, and that the probability of the assignment is low. This confirms that the

366

former assignment is right, spectrum A really refers to diastereoisomer 4a. Hence, the best

367

assignments are spectra A, B, C, and D corresponding to diastereoisomers 4a (anti:anti), 4b

368

(anti:syn), 4c (syn:anti), and 4d (syn:syn), respectively.

M AN U

SC

RI PT

357

CP3 could provide “false positives” when the assignment is made for two spectra with

370

two diastereoisomers but just one spectrum corresponds to one of the two diastereoisomers

371

and has been attributed to the right one. For example, spectra A and C are attributed to

372

diastereoisomers 4a and 4b, respectively. This assignment furnishes a positive value for CP3,

373

and the probability that this assignment is right is high (green check). Because “false

374

positives” may happen, we must look over all the possible assignments and find out which are

375

the best.

EP

AC C

376

TE D

369

18

ACCEPTED MANUSCRIPT

377

Table 6. Possible assignments of spectra A, B, C, and D to diastereoisomers 4a, 4b, 4c, and 4d. Spectrum Diastereoisomer

4a

4b

B 4c

4d

4a

4b

C 4c

4d

4a

4b

4c

4d









4a

Sum

4b

4c

4d




















7 0 2 1 1 8 2 2 2 1 5 0 1 1 2 8

2 3 2 1 0 1 0 2 2 1 3 3 1 3 0 1

0 6 5 7 8 0 7 5 5 7 1 6 7 5 7 0

378

4a


4b    A 4c 

4d 
 4a    4b


B 4c
  4d

4a   

4b
     C 4c




4d

 
 4a   
  4b

    D 4c
  
 4d






Green check indicates that CP3 is positive and its probability is high.

379


Yellow check indicates that CP3 value is positive and its probability is low, or that CP3 is negative and its probability is high.

380

 Red “X” indicates that CP3 is negative and its probability is low.

TE D

 









SC

M AN U





  










  
  







   



 
   









  




AC C

EP

‘

Spectrum

381

  




D

RI PT

A

19

ACCEPTED MANUSCRIPT

382

Finally, we detail the assignments that are necessary and enough to conduct the

383

stereochemical assignments of all the four possible diastereoisomers of maculalactone derivative 4.

384

Table 7 gives these assignments and the CP3 values for the 1H and

385

correct assignments (A = 4a, B = 4b, C = 4c, and D = 4d) yield positive values for CP3, whereas

386

wrong assignments furnish negative values for CP3.

13

C NMR data. Presumably

Table 7. CP3 values for the 1H and 13C NMR data. CP3

Assignment

13

H

C

1

H and 13C

0.74

0.65

A = 4b and B = 4a

-0.98

-0.86

A = 4a and C = 4c

0.26

0.03

0.15

A = 4c and C = 4a

-1.27

-0.62

-0.94

A = 4a and D = 4d

0.65

A = 4d and D = 4a

-1.27

B = 4b and C = 4c

0.42

B = 4c and C = 4b

-0.95

B = 4b and D = 4d

0.69

B = 4d and D = 4b

-0.86

0.70

-0.92

0.47

0.56

-0.81

-1.04

0.30

0.36

-0.93

-0.94

0.67

0.68

-1.03

-0.94

0.26

0.66

0.46

-1.52

-0.84

-1.18

TE D

C = 4d and D = 4c

SC

A = 4a and B = 4b

C = 4c and D = 4d

389

1

M AN U

388

RI PT

387

The graphs in Figure 5 correspond to the plots of the CP3 values obtained for the 1H and 13C

391

NMR data for the assignments listed in Table 7. The horizontal lines represent one, two, and three

392

standard deviations from the expected CP3 value when the assignment is correct. For all the

393

assignments that give negative values, these values are below three standard deviations. This means

394

that element P(R2|A2) in Equation 2 will furnish a value that is smaller than 0.05, which is typical of

395

normal distribution. Actually, this value is usually responsible for the high probabilities obtained

396

with CP3. Mathematically speaking, when element P(R2|A2) in Equation 2 has value near zero, the

397

probability that a given assignment is right is close to one or 100%. Indeed, all the assignments

398

shown in Table 7 and presumed right present probabilities of 100.0%, so they are the correct ones.

AC C

EP

390

399

20

M AN U

SC

RI PT

ACCEPTED MANUSCRIPT

400 401

Figure 5. Plots of CP3 values obtained for the 1H and

402

shown in Table 7.

403

13

C NMR data regarding the assignments

As an example, figure 6 illustrates the CP3 parameter values obtained. It was generated by

405

simulating spectra with the chemical shifts obtained from the experimental and calculated 1H NMR

406

data from spectra A and B, and the diastereoisomers 4a and 4b that were used to compute the CP3

407

parameter.

TE D

404

It is possible to note that the differences in between chemical shifts (∆δ) for similar atoms in

409

calculated spectra for diastereoisomers 4a and 4b are better matched with the differences in

410

experimental chemical shifts for the assignments A = 4a and B = 4b, indicating that a positive value

411

for CP3 parameter was expected for this assignment and a negative value for the opposite one. For

412

example, the difference of chemical shifts for atom H4 in calculated spectra of diastereoisomer 4a

413

and 4b is – 0.24 (∆δcalc = 6.21 – 6.45; Table 4) and the difference for chemical shifts of atom H4

414

between experimental spectra A and B is – 0.40 (∆δexp = 5.37 – 5.77; Table 4) which contribute

415

with a positive increment for the CP3 parameter value because both differences of chemical shifts

416

possess the same algebraic sign. On the other hand, the difference of chemical shifts for atom H6 in

417

calculated spectra of diastereoisomer 4a and 4b is 0.19 (∆δcalc = 4.29 – 4.10; Table 4) and the

418

difference for chemical shifts of atom H6 between experimental spectra A and B is – 0.23 (∆δexp =

419

3.50 – 3.73; Table 4) which contribute with a negative increment for the CP3 parameter value. In

420

fact, the only difference between algebraic signs of ∆δcalc and ∆δexp in assignments A = 4a and B =

AC C

EP

408

21

ACCEPTED MANUSCRIPT

421

4b is for atom H6. The overall evaluation of chemical shift differences of corresponding atoms

422

between the two calculated spectra compared to the two experimental ones leads to a positive value

423

of which indicates that the CP3 parameter has a positive value (CP3 = 0.74) and the assignments in

424

question are correct. The opposite assignments, A = 4b and B = 4a, has a negative value for the

425

CP3 parameter value (CP3 = – 0.98) indicating that these assignments are wrong.

TE D

M AN U

SC

RI PT

426

Figure 6. Differences between the chemical shifts for spectra A and B and for diastereoisomers 4a

429

and 4b.

AC C

430

EP

427 428

431

Figure 6 also illustrate how the systematic errors in NMR chemical shift calculations can be

432

removed by comparing the differences between two calculated chemical shifts and two

433

experimental data, indicating that these differences are calculated more accurately than the chemical

434

shifts themselves.

435 436

4. Conclusion

437

Assignment of the relative configurations of the four diastereoisomers of maculalactone

438

derivative 4, initially deduced on the basis of the mechanism proposed for the multicomponent

439

reaction, was confirmed through the use of the CP3 parameter. This parameter provides high level 22

ACCEPTED MANUSCRIPT

440

of confidence as revealed by the calculated CP3 probability. In summary, DFT/GIAO calculations

441

of 1H and 13C NMR chemical shifts can be used in combination with CP3 to compare experimental

442

and calculated NMR data for complete assignment of all the four possible diastereoisomers of a γ-

443

butyrolactone ring with three stereogenic centers.

444 445

Acknowledgments The authors thank Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP,

447

grants 2013/18254-9, 2015/05454-5 and 2016/04896-7), Coordenação de Aperfeiçoamento de

448

Pessoal de Nível Superior - Brasil (CAPES) - Finance Code 001, and Conselho Nacional de

449

Desenvolvimento Científico e Tecnológico (CNPq) for financial support and fellowships.

RI PT

446

AC C

EP

TE D

M AN U

SC

450

23

ACCEPTED MANUSCRIPT

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452

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(–)-cubebin,

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Med.

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ACCEPTED MANUSCRIPT Stereochemical assignment of four diastereoisomers of a maculalactone derivative by computational NMR calculations

Authors: Daniel Previdi*, Viviani Nardini, Mayla Eduarda Rosa, Vinicius Palaretti, Gil

RI PT

Valdo José da Silva and Paulo Marcos Donate

Departamento de Química, Faculdade de Filosofia, Ciências e Letras de Ribeirão Preto, Universidade de São Paulo, Avenida Bandeirantes, 3900, CEP 14040-901, Ribeirão Preto,

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SP, Brazil.

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*Corresponding author. E-mail address: [email protected] (D. Previdi).

Highlights

• NMR chemical shifts calculation and the relative configurations of lactones

TE D

• Stereochemical assignment of diastereoisomers of a tri-benzylated γ-butyrolactone • Determination of the relative configurations of a maculalactone derivative • CP3 parameter applied to distinguish four diastereoisomers

AC C

EP

• Assignment of the relative configurations corroborated a proposed reaction mechanism