In depth analysis of chiroptical properties of enones derived from abietic acid

Article abstract of DOI:10.1021/acs.joc.7b02911

With the use of inexpensive commercially available abietic acid, a whole series of abietane enones were prepared in high yields. The structures of all the products obtained were determined by comprehensive spectroscopic analysis with particular emphasis on the use of advanced NMR techniques, comparison with previously reported data and, where possible, by single crystal X-ray diffraction. However, in cases where X-ray crystallography was not applicable or compounds tested were unstable, a final stereochemical assignment could be inferred only by electronic circular dichroism (ECD) supported by vibrational circular dichroism to increase credibility. To reveal the relationship between structure and chiroptical properties, we used combined experimental and theoretical analysis of geometries, structural parameters, and chiroptical properties of all enones synthesized. A thorough analysis of their conformational flexibility by examining the effect of solvent and temperature on the ECD spectra was also used to achieve desired objectives. As a result, the impact of substituents adjacent to the enone chromophore on the conformation was determined by demonstrating that even slight changes in the position of hydroxyl and isopropyl groups attached to carbon C13 may substantially affect ECD curves' pattern, leading in some cases to Cotton effects sign reversal.

Full text of DOI:10.1021/acs.joc.7b02911

Subscriber access provided by UNIV OF NEW ENGLAND ARMIDALE
Article
In-Depth Analysis of Chiroptical Properties
of Enones Derived from Abietic Acid
Marek Masnyk, Aleksandra Butkiewicz, Marcin Górecki, Roman
Luboradzki, Piotr Paluch, Marek J. Potrzebowski, and Jadwiga Frelek
J. Org. Chem., Just Accepted Manuscript • DOI: 10.1021/acs.joc.7b02911 • Publication Date (Web): 01 Mar 2018
Downloaded from http://pubs.acs.org on March 2, 2018
Just Accepted
“Just Accepted” manuscripts have been peer-reviewed and accepted for publication. They are posted
online prior to technical editing, formatting for publication and author proofing. The American Chemical
Society provides “Just Accepted” as a service to the research community to expedite the dissemination
of scientific material as soon as possible after acceptance. “Just Accepted” manuscripts appear in
full in PDF format accompanied by an HTML abstract. “Just Accepted” manuscripts have been fully
peer reviewed, but should not be considered the official version of record. They are citable by the
Digital Object Identifier (DOI®). “Just Accepted” is an optional service offered to authors. Therefore,
the “Just Accepted” Web site may not include all articles that will be published in the journal. After
a manuscript is technically edited and formatted, it will be removed from the “Just Accepted” Web
site and published as an ASAP article. Note that technical editing may introduce minor changes
to the manuscript text and/or graphics which could affect content, and all legal disclaimers and
ethical guidelines that apply to the journal pertain. ACS cannot be held responsible for errors or
consequences arising from the use of information contained in these “Just Accepted” manuscripts.
The Journal of Organic Chemistry is published by the American Chemical Society.
1155 Sixteenth Street N.W., Washington, DC 20036
Published by American Chemical Society. Copyright © American Chemical Society.
However, no copyright claim is made to original U.S. Government works, or works
produced by employees of any Commonwealth realm Crown government in the course
of their duties.

Page 1 of 41
The Journal of Organic Chemistry
1
2
3
4
5
6
In-Depth Analysis of Chiroptical Properties of Enones Derived from
Abietic Acid
7
8
9
Marek Masnyk,*† Aleksandra Butkiewicz,† Marcin Górecki,† Roman Luboradzki,‡ Piotr
Paluch,§ Marek J. Potrzebowski,§ and Jadwiga Frelek,*†
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
† Institute of Organic Chemistry, Polish Academy of Sciences, Kasprzaka 44/52, 01-224
Warsaw, Poland;
‡ Institute of Physical Chemistry, Polish Academy of Sciences, Kasprzaka 44/52, 01-224
Warsaw, Poland;
§ Centre of Molecular and Macromolecular Studies, Polish Academy of Sciences,
Sienkiewicza 112, 90-363 Lodz, Poland
marek.masnyk@icho.edu.pl; jadwiga.frelek@icho.edu.pl;
Abstract
With the use of inexpensive commercially available abietic acid, a whole series of abietane
enones have been prepared in high yields. The structures of all the products obtained were
determined by comprehensive spectroscopic analysis with particular emphasis on the use of
advanced NMR techniques, comparison with previously reported data, and where possible by
single crystal Xꢀray diffraction. However, in cases where Xꢀray crystallography was not
applicable or compounds tested were unstable, a final stereochemical assignment could be
only inferred by electronic circular dichroism (ECD) supported by vibrational circular
dichroism (VCD) to increase credibility. To reveal the relationship between structure and
chiroptical properties we used combined experimental and theoretical analysis of geometries,
ACS Paragon Plus Environment

The Journal of Organic Chemistry
Page 2 of 41
1
2
3
4
5
6
7
8
9
structural parameters and chiroptical properties of all enones synthesized. A thorough analysis
of their conformational flexibility by examining the effect of solvent and temperature on the
ECD spectra was also used to achieve desired objectives. As a result, the impact of
substituents adjacent to the enone chromophore on the conformation was determined by
demonstrating that even slight changes in the position of hydroxyl and isopropyl groups
attached to carbon C13 may substantially affect ECD curves’ pattern leading in some cases to
Cotton effects’ sign reversal.
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
INTRODUCTION
It is widely recognized that biological activity of molecules is highly driven by their threeꢀ
dimensional structure. An example of this is the strong dependence of physiological or
pharmacological properties on the chiral recognition by chiral receptors, interacting
predominantly with molecules or phases of the correct spatial arrangement.1 Therefore, access
to methods appropriate for the reliable absolute configuration (AC) assignment and detailed
structural analysis is of paramount importance. Here, chiroptical methods, including in
particular circular dichroism (CD), not only meet but also successfully fulfil such a
requirement. The recent progress in ab initio methods and computer sciences additionally
makes the application of quantum chemical calculations of molecular and chiroptical
properties enforceable.2 Consequently, the reliability of the assignment of the AC, as a result
of combined experimentalꢀtheoretical analysis of the CD results, is unquestionably multiplied,
thus leading to their increasingly frequent and efficient use.3 Nevertheless, examples can be
found that lead to ambiguous results even when using many different chiroptical techniques
combined with quantumꢀchemical calculations. One such exemplary group of compounds are
α,βꢀenones that have been investigated extensively over the past decades, both experimentally
and theoretically, in terms of electronic circular dichroism (ECD) spectroscopy.4 The interest
in enones was undoubtedly related to the fact that this chromophore is one of the most popular
ACS Paragon Plus Environment

Page 3 of 41
The Journal of Organic Chemistry
1
2
3
4
5
6
7
8
9
structural motifs found in numerous natural products exhibiting, in most cases, essential
biological activity. Steroids, terpenoids, and alkaloids can be listed as examples of such
biologically relevant compounds with enone units in their skeletons.5 Also, their defined
absorption in the UVꢀVis region together with a stiffening of the molecular skeleton, thus
reducing the number of conformers contributing to the ECD spectra, resulted in their selection
as suitable model systems for chiroptical studies. As a result of intensive chiroptical
investigations, a number of sector and helicity rules correlating signs and magnitudes of
observed Cotton effects (CEs) in the ECD spectra with threeꢀdimensional structure of α,βꢀ
enone molecules have been developed over the years.4c In most cases, the torsional angle of
the enone chromophore Cβ=Cα–C=O, defined as ω, determines the sign of the CE associated
with the nꢀπ* transition occurring at around 320ꢀ350 nm.4a, 4c A satisfactory correlation
between ECD and stereochemistry might also be found for the πꢀπ* type transitions of α,βꢀ
enones.
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
Gawroński, however, showed the significant effect of the enone dissymmetry and
substituents present in the proximity of the chromophore on the shape of the spectra in this
spectral region.4a, 4c His finding leads to the conclusion that the above factors may have a
dramatic impact on the UV and ECD spectra of α,βꢀunsaturated ketones forcing, in some
cases, the reformulation or even change of the working rules.3d As demonstrated in numerous
works exploring the threeꢀdimensionality of enones via chiroptical properties remains a
nontrivial task and may become a complicated issue in cyclic α,βꢀenones.3d, 4d, 6 We have been
additionally motivated to continue research in this area, by our latest results demonstrating
that in some cases the sign of the nꢀπ* ECD signals in enones could not be satisfactorily
described by a minimum structure model.3d Therefore, further detailed studies on this topic
are justified, if not necessary, as they can lead to the clarification of ambiguities and doubts
still present in the field. These studies should further aid the thorough exploration of the
ACS Paragon Plus Environment

The Journal of Organic Chemistry
Page 4 of 41
1
2
3
4
5
6
7
8
9
structureꢀproperty relationships and find out how even subtle structural changes modulate
them. This work is undertaken exactly for this purpose, assuming that the cisꢀenone
chromophore in the selected new structures should contain substituents in its environment to
allow a methodical comparative analysis. With new model compounds in hand, their
chiroptical properties will be carefully analyzed for compliance with the aboveꢀspecified
objectives. The resulting access to more experimental data should enable clarification of the
existing controversies or at least reduce their numbers. It could also play a significant role in
creating and/or developing new theories. In doubtful cases, which cannot be excluded,
vibrational circular dichroism (VCD) will be used to support and verify the ECD results and
thus achieve strengthening of the conclusions drawn. As VCD is the extension of the
electronic CD into infrared/nearꢀinfrared absorption region of the spectrum, i.e., where
vibrational transitions occur within the ground electronic state of the molecule, therefore it
provides new information about structural features of chiral molecules. Consequently, an
approach based on two independent dichroic methods leads to the reinforcement of
stereochemical argumentation.
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
When looking for model compounds, our attention turned to abietane diterpenes. The
abietaneꢀtype diterpenoids often exhibit outstanding biological activity which is why they
were and are used in therapy, including folk medicine.7,8 Their unique architecture and
structural diversity, places them as a good starting point for the synthesis of a variety of cisꢀ
enones. As a substrate for the planned synthesis, we have chosen abietic acid 1 (Chart 1): a
natural product which is a major component of resin acids abundantly occurring in conifer
oleoresin, used commercially in paints, varnishes, and lacquers. The benefit of using abietic
acid as a substrate in synthesis is its low price. What is more, it was also effectively used for
the synthesis of a wide variety of complex molecules, including other terpenes.9 The review
of relevant literature has demonstrated the ability to obtain appropriate cisꢀenones through a
ACS Paragon Plus Environment

Page 5 of 41
The Journal of Organic Chemistry
1
2
3
4
5
6
7
8
9
simple synthesis consisting of a few steps at most.10 Therefore, taking into account both the
economic as well as the synthetic point of view, we recognized abietic acid as a convenient
starting material for the preparation of a whole range of compounds with suitable
conformationally diverse molecular structures. The prepared and analyzed enones are
presented in Chart 1.
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
Chart 1. Enones under consideration.
This article is organized as follows: first, we describe the synthesis of individual enones
and their structure determination based on the comprehensive spectroscopic analysis. Then,
we analyze CD spectra of the enones with the aim to clarify the impact of structural changes,
solvent applied, and measurement temperature on dichroic properties. Finally, we summarize
the results and findings achieved.
RESULTS AND DISCUSSION
Synthesis of model compounds
Syntheses of all enones discussed herein were accomplished using commercial abietic acid
1 (Scheme 1) freshly purified by crystallization of its isoamylamine salt. The synthetic
strategy for preparation of cisoid enones was based on the observations, reported in the
literature, that the particular double bonds within a conjugated diene system in the abietic acid
are susceptible to selective oxidations in which the second double bond remains unchanged.11
ACS Paragon Plus Environment

The Journal of Organic Chemistry
Page 6 of 41
1
2
3
For our purposes, we adopted cisꢀhydroxylation of the C13ꢀC14 double bond which enabled
4
5
preparation of 7ꢀenꢀ14ꢀone systems9b and iodineꢀwater oxidation suitable for preparation of
6
7
8
8(14)ꢀenꢀ7ꢀone derivatives 11b
.
9
The first target enone 2 containing 7ꢀenꢀ14ꢀone fragment was prepared by slightly
modifying procedure known from literature (Scheme 1).9b Abietic acid 1 was esterified with
diazomethane and the methyl ester 10 was subjected to selective cis hydroxylation with a
catalytic amount of osmium tetroxide and Nꢀmethylmorpholine Nꢀoxide as the oxidant in tꢀ
butyl alcohol/water solution. The resulted diol 11 was then oxidized to hydroxyenone 2 in
mild conditions with benzeneselenic anhydride prepared in situ from diphenyl diselenide and
tertꢀbutyl hydroperoxide.
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
Scheme 1. Synthesis of enone 2.
The 13S,14S absolute configuration of diol 11, and thus the approach of osmium tetroxide
from the βꢀside in the cisꢀhydroxylation reaction, was confirmed by comparing the literature
data of the specific rotation and VCD results.11a, 12 Similarly, the 13S stereochemistry in
previously described hydroxyenone 2 has been corroborated by comparing our and literature
spectroscopic data.11a
Through the preparation of enone 2, we gained easy access to the next two enones 3 and 4.
Simple reduction of the hydroxyl group in 2 with zinc dust in acidic medium afforded
expected epimeric enones 3 and 4 (Scheme 2). The best result, 65% overall yield, was
obtained when hydroxyenone 2 was heated at 50 ºC for a prolonged time in a methanolic
solution of ammonium chloride. Reaction yields of the reductions with zinc carried out in
boiling methanolic solution of ammonium chloride, boiling methanolic solution of acetic acid
ACS Paragon Plus Environment

Page 7 of 41
The Journal of Organic Chemistry
1
2
3
4
5
6
7
8
9
or in acetic acid alone at 50 ºC were not satisfactory. It is noteworthy that enone 4 with an
axial isopropyl group is vulnerable to acidic isomerization and in the presence of traces of
acid readily converts into isomer 3 with an equatorial isopropyl group. We observed a
significant conversion degree of 4 into 3 afterwards, when the NMR sample of 4 was left in a
deuterated chloroform solution for a couple of hours. In additional experiments under
controlled conditions, 3 and 4 were treated separately with pꢀtoluenesulfonyl acid in
dichloromethane solutions at room temperature for 26 hrs. In both reactions the same
mixtures containing 78% of 3 and 22% of 4 were obtained.
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
Scheme 2. Synthesis of enones 3 and 4.
The composition and stereochemistry of 3 was established by Xꢀray analysis. The ORTEP
diagrams of 3 together with those of the remaining enones for which receiving crystalline
structures were possible are presented in Figure S1 (see Supporting Information for ORTEP
plots). For compound 4, however, we were not able to grow crystals of sufficient
crystallographic quality, and exhaustive NMR investigations had to be conducted to prove the
structure. First of all, we had to ensure that whole abietane skeleton in 4 remained unchanged
after reduction. Such changes could not be ruled out due to the reaction conditions (acidic
medium, prolonged reaction time), which could favor a double bond migration or skeletal
rearrangements. Comparison of 2D INADEQUATE spectra of 3 and 4 showed that none of
the phenomena mentioned above occurred and both of the compounds have the same carbon
atoms alignment. As can be seen in Figure 1, in both samples C7 and C8 carbon atoms are
present in the "olefinic region" of 13C chemical shifts (140ꢀ150 ppm) while carbons C12 and
ACS Paragon Plus Environment

The Journal of Organic Chemistry
Page 8 of 41
1
2
3
4
5
C13 are located in the aliphatic part of spectra. Therefore, the only possibility is that enones 3
and 4 are stereoisomers which differ solely in configuration at C13.
6
7
8
9
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
13
Figure 1. Aliphatic part of Cꢀ13C 2D INADEQUATE spectra of 3 (A) and 4 (B) optimized
1
13
13
1
13 13
13 13
for J C C = 33 Hz. Full Cꢀ C 2D INADEQUATE spectra of 3 and 4 optimized for J
=
C C
1
50 Hz are collected in the Supporting Information part together with the complete set of H
NMR, 13C NMR, DEPT, 2D 1Hꢀ13C HSQC spectra of these enones.
Having proven structures 3 and 4, we must note that signals of HꢀC15 protons in 1H NMR
and Hꢀ13C HMBC spectra are present at 2.71 ppm and 1.97ꢀ2.06 ppm (overlapped with Hꢀ
1
C13), respectively. Such a difference equal to 0.7 ppm for very similar compounds seems to
be unusual. However, the methine proton HꢀC15 in 3 belongs to an equatorial isopropyl group
and in some conformations may lie in the plane of the carbonyl group (deshielding zone of
C=O) and therefore its signal is strongly shifted downfield. In the case of 4 an axial
orientation of the isopropyl group prevents planar alignment of the methine proton HꢀC15
together with the carbonyl group, and therefore the proton signal appears in the typical for
such protons region of the spectrum.
Preparation of conjugated enones with 8(14)ꢀenꢀ7ꢀone system is presented in Scheme 3.
The abietic acid methyl ester 10 was oxidized to β,γꢀunsaturated ketone 12 in reaction with
iodine and aqueous potassium bicarbonate according to the literature procedure.11a The
stereochemistry of the C8 carbon atom in 12 has been determined using single crystal Xꢀray
ACS Paragon Plus Environment

Page 9 of 41
The Journal of Organic Chemistry
1
2
3
4
5
6
7
8
9
diffraction analysis (Figure SI1). We expected that the double bond in 12 would easily
migrate in the presence of a base creating a conjugated enone 5. In fact, when we kept the
solution of 12 in THF with triethylamine for 24 hrs at room temperature, the double bond
migration indeed occurred. However, the migration was instantly followed by an air oxidation
at the allylic position to form two epimeric hydroperoxides 6 and 7 (Scheme 3). The same
result was obtained when the reaction was carried out under argon atmosphere in a strictly
deoxygenated mixture of THF and triethylamine. We conclude that the air oxidation of the
allylic position in 8(14)ꢀenꢀ7ꢀone derivative 5 must be fast and undergoes spontaneously at
the workup stage. Therefore, preparation of a pure compound 5 seems to be difficult, if
possible at all, in standard laboratory work requiring column chromatography purification.
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
Scheme 3. Synthesis of enones 5-7.
A sample of a small amount of enone 5 for ECD/NMR measurements was obtained by
isomerization of 12 in deoxygenated toluene in the presence of a catalytic amount of sodium
tꢀbutoxide followed by the separation of 5 from an unchanged 12 with the help of preparative
TLC. The material washed out from the spot assigned to the conjugated enone consisted of
1
enone 5 and freshly formed hydroperoxides 6 and 7 in a ratio 5:5:1 as it was revealed by H
NMR spectrum (Figure 2). It must be mentioned that enone 5 differs significantly in polarity
from hydroperoxides 6 and 7 in respect to TLC chromatography (Rf = 0.52, 0.37 and 0.29
respectively, 30% ethyl acetate in hexane). Hence, it can be excluded that the enone 5 washed
out from the silicagel was contaminated with 6 and 7 due to the imperfect isolation of
particular spots. Detection of the moderately high concentration of hydroperoxides 6 and 7
ACS Paragon Plus Environment

The Journal of Organic Chemistry
Page 10 of 41
1
2
3
besides enone 5 in the same fraction confirms an exceptional susceptibility of the latter to
4
5
oxidation.
6
7
8
9
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
Figure 2. Fragment of 1H NMR spectrum of the enones 5, 6 and 7 mixture covering the range
of vinyl protons and hydroperoxide groups.
Given the extreme sensitivity of enone 5 to even trace amounts of oxygen in the reaction
medium, determination of the stereochemistry at its C13 carbon atom was only possible by
employing the online HPLCꢀECD technique.13 This method, in fact, allows recording ECD
spectra during the HPLC separation without the need to isolate and purify the components of
the mixture tested. By using the silica gel Kromasil® Si100, 5 ꢁm, 25x0.4 cm mm column
and the mixture of MTBE and hexane (isocratic 1:9 mobile phase for 10 min following
injection, then run linear gradient to 2:8 in 10 min, then constant 2:8, v/v) we were able to
separate all three enones (Figure 3A). The retention time of the first chiral compound, i.e.,
enone 5 (red peak, the first negative in ECD chromatogram), was 13 min. The next two
epimeric hydroperoxides 6 and 7 (blue peak and green peak, respectively) with retention
times 27 and 43 min were also negative. The use of stop flow mode enabled recording the
ECD spectrum of enone 5 in the entire diagnostic range, i.e., 220ꢀ450 nm. Comparison of the
computed spectra for 13R and 13S epimers of 5 calculated after geometry optimization at the
B3LYP/TZVP/PCM level of theory and the Boltzmann averaging of all populated conformers
ACS Paragon Plus Environment

Page 11 of 41
The Journal of Organic Chemistry
1
2
3
4
5
6
7
8
9
pointed to the 13S AC (Figure 3B). To prevent drawing erroneous conclusions and increase
the level of confidence of the stereochemical assignment, we performed QC calculations at
different levels of theory. Theoretical predictions resulting from these calculations lead to the
same result (Figure S2), and thus the 13S AC of enone 5 can be regarded as established with a
high degree of certainty. The details of the calculations are presented in the experimental
section as well as in the following part discussing the results of circular dichroism.
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
Figure 3. A: The UV (top) and ECD (bottom) chromatogram of 5-7 mixture on Kromasil®
(Si100, 5 ꢀm) column, eluent 10ꢀ20 % MTBE in hexane, flow 1 mL/min, UV detection at 250
nm, ECD detection at 330 nm; B: onꢀline recorded ECD spectrum of enone 5 (red line) with
the computed at B3LYP/TZVP/PCM(CH3CN) level of theory spectra of its 13R (blue line)
and 13S (green line) epimers.
Interestingly, different results were obtained when the isomerization reaction of 12 was
carried out with a catalytic amount of sodium tertꢀbutoxide in anhydrous toluene in an air
atmosphere. In this case, the allylic oxidation also took place, but three new allylic alcohols 8,
9, and 13 were the products, not the hydroperoxides obtained before (Scheme 4). Alcohols 8,
9, and 13 have been obtained in the yield 26:11:34, respectively.
Scheme 4. Synthesis of enones 8, 9, 13.
ACS Paragon Plus Environment

The Journal of Organic Chemistry
Page 12 of 41
1
2
3
4
5
6
7
8
9
Stereochemical assignment of hydroperoxides 6, 7 and alcohols 8, 9 and 13, was
accomplished with the assistance of single crystal Xꢀray analysis. Hydroperoxide 6 and
alcohols 9 and 13 directly provided sufficient crystals for Xꢀray measurements (see Figure
SI1). Reduction of the hydroperoxide 7 with triphenylphosphine in methylene chloride
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
1
afforded material identical in H NMR and 13C NMR spectra with the crystalline alcohol 9
revealing an equatorial position of the hydroperoxide group in 7. An analogous reduction of
crystalline axial hydroperoxide 6 provided material identical with alcohol 8 proving an axial
orientation of its hydroxyl group (Scheme 5, Figure SI1). Thus, based on the single crystal Xꢀ
ray results, the AC at the C13 carbon atom in hydroperoxide 6 and alcohol 9 was directly
assigned to be 13S, and 13R respectively, whereas the one at C8 carbon atom in
β,γꢀ
unsaturated keto alcohol 13 to be 8S. Comparison of the NMR spectra of the respective
reduction products led to an indirect assignment of AC in hydroperoxide 7 as 13R and in
alcohol 8 as 13S. An independent confirmation of this assignment was provided by the full
compliance of experimental and simulated ECD spectra of the corresponding alcohols and
hydroperoxides, as shown in the next section, Table 1 and Supporting Information part.
Scheme 5. Reduction of hydroperoxides 6 and 7.
HO
HOO
HOO
OH
Ph3P
Ph3P
CH2Cl2, RT
CH2Cl2, RT
O
O
O
O
CO2CH3
CO2CH3
CO2CH3
CO2CH3
8
6
7
9
Structures 8 and 9 have been already noted in the literature. The reported physical data for
these compounds, however, were not consistent with the data recorded by us. In 1983 Cilko et
ACS Paragon Plus Environment

Page 13 of 41
The Journal of Organic Chemistry
1
2
3
4
5
6
7
8
9
al.14 described alcohol 8 as a solid of mp 189ꢀ190 ºC and 1H NMR (CDCl3, in his article Cilko
reported only diagnostic signals) δ = 0.70 (s, CH3), 0.88 (d, J = 7 Hz, isopropyl CH3), 0.90 (d,
J = 7 Hz, isopropyl CH3), 1.20 (s, CH3), 6.75 (s, vinylic H). Corresponding values observed in
1
our laboratory for 8 stand as follows: mp not detected (oil), H NMR (CDCl3) δ = 0.85 (s,
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
CH3), 0.94 (d, J = 6.9 Hz, isopropyl CH3), 0.98 (d, J = 6.9 Hz, isopropyl CH3), 1.24 (s, CH3),
6.74 (s, vinylic H).
In 2013 Amato et al.15 described structure 9 as one of the products of abietic acid
oxidation with hydrogen peroxide in the presence of methyltrioxorhenium. The presented
1
NMR data, however, were in strong contradiction with our results. Surprisingly, all listed H
NMR and 13C NMR signals perfectly agreed with our spectra of 13. In our opinion, therefore,
it is highly plausible that the structure claimed before as 9 was in fact structurally tantamount
to the unconjugated hydroxyenone 13.
Oxidations of enones with oxygen in neutral and basic media are wellꢀrecognized
processes in which hydroperoxides, epoxides, and allylic alcohols are typically formed.16 Due
to the erratic character of such oxidations affording plenty of products and difficulties in the
prediction of the major product, these reactions attracted only marginal attention in organic
synthesis. Recently, some successful attempts were undertaken to improve air allylic
hydroxylations of enones by carrying out the reactions in the presence of catalytic amounts of
metal oxides.16 In our case, finding that air oxidation of the nonꢀconjugated enone 12 can be
easily and efficiently directed toward alcohols or hydroperoxides depending on the base used
in the reaction was unexpected. A complete explanation of this phenomenon requires further
experimental work which is in progress in our laboratory.
2.2. Structure – Chiroptical Properties Relationship
For the evaluation of the structure – circular dichroic activity relationship, we took into
account abietane
α,
β
ꢀenones 2-9 of formulas presented in Chart 1 provided in the introductory
ACS Paragon Plus Environment

The Journal of Organic Chemistry
Page 14 of 41
1
2
3
4
5
6
7
8
9
section. Their experimental UVꢀVis and ECD data recorded in acetonitrile solution are
collected in Table 1. The only exception in this respect is the enone 5 whose ECD spectrum
was recorded in the stop flow mode during the HPLC separation with on-line ECD detection
in a mixture of hexane : MTBE 9:1, as already mentioned before. Its UVꢀVis spectrum was
simultaneously measured in the same solvent mixture, and in the Table it is given in arbitrary
units (a.u.) while ECD in mdeg.
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
Compounds 12 and 13 obtained during our synthesis exemplify β,γꢀunsaturated ketones.
Therefore, their chiroptical data as, for example, the location of ECD bands in their ECD
spectra differ significantly from the other compounds analyzed. A blue shift of CE associated
with the nꢀπ* transition, i.e., 280 nm in enones 12 and 13 vs 350 nm in remaining enones,
clearly indicates the absence of conjugation in the first two. Change of orientation of the
substituent at the C8 carbon atom, i.e., 8βꢀH in 12 vs. 8αꢀOH in 13 without reversal of the AC
associated with the formal inversion of chirality according to the CIP sequence rule, does not
significantly affect the shape of ECD spectra. Signs of individual CEs are consistent, and their
positions on the spectra are very similar. The AC assignment was obtained by the single
crystal Xꢀray analysis enabling determination 8S AC in both these compounds (Figure SI1).
Also, calculated ECD spectra agreed well with the observed ECD curves in sign and pattern,
as can be seen in Figure S3 (for calculation details see below and experimental section).
Table 1. UVꢀVis and ECD data of enones 2-9 and 12-13 recorded in acetonitrile. UV and
ECD values are given in M−1cm−1as
ε (λmax/nm) and ꢁε (λmax/nm), respectively. UVꢀVis and
ECD data for enone 5 (for clarity marked in gray) were registered in the stop flow mode
during HPLC separation in a mixture of hexane:ethyl acetate 9:1 and are given in a.u.
(arbitrary units) and mdeg, respectively.
UV [ε (λmax)]
6580 (252.0)
ECD [ꢁε (λmax)]
Enone
73 (329.0)
36 (333.0)
68 (333.0)
0.004 (322.0)
44 (326.0)
50 (325.0)
82 (323.5)
+4.78 (249.5)
+4.15 (193.5)
−0.03 (302.0)
+0.72 (263.0)
+0.08 (341.0)
+0.45 (337.0)
−0.90 (326.0)
−4.80 (345.0)
−0.77 (346.0)
−0.81 (339.0)
−0.64 (348.5)
2
3
4
5
6
7
8
6740 (248.5)
6970 (244.0)
1.429 (243.5)
8430 (242.5)
7510 (245.0)
8760 (244.5)
+1.33 (198.5)
−1.03 (220.4) ((
−1.95 (220.0)
−1.08 (231.6)
−3.15 (241.5) +0.02 (273.2)
+23.0 (250.0)
+5.57 (241.0)
+2.00 (280.0)
+5.96 (205.0)
+5.15 (191.0)
+7.57 (208.5)
+4.24 (204.5)
−6.01 (252.5)
+4.15 (239.0)
ACS Paragon Plus Environment

Page 15 of 41
The Journal of Organic Chemistry
1
2
3
4
5
7190 (247.0)
762 (234.0)
2580 (220.5)
55 (326.0)
113 (286.0)
105 (281.5)
+7.37 (204.0)
+4.43 (216.5)
+2.52 (222.5)
−2.51 (255.0)
−1.01 (335.0)
−1.29 (289.0)
−4.55 (288.0)
9
+9.5 (188.5)
+11.7 (185.0)
12
13
6
7
8
9
The UVꢀVis spectra of
arising at around 330 and 240 nm with an intensity characteristic for the corresponding nꢀπ*
and * electronic transitions, respectively. As can be seen in Table 1, in their ECD spectra
α,βꢀunsaturated ketones 2-9 display two wellꢀresolved bands
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
πꢀπ
there are up to four bands visible within the spectral range of 190ꢀ360 nm. The ECD spectra
of hydroperoxides 6 and 7 are very similar in sign and pattern to those of their hydroxyl
counterparts 8 and 9 (Figure S4) thus demonstrating an equivalent effect of both these
substituents on the spectra. This is a consequence of a comparable electronic system in both
hydroperoxide and hydroxyl groups. Due to the small differences in the spectra, further
discussion of these compounds will be considered together as hydroxyl derivatives 8 and 9.
Regarding the spectra pattern and CEs signs, the ECD spectra of
α,βꢀenones 4, and 6-9
show similar profiles with a negative nꢀπ* CE of comparable intensities of about ꢁε ≈ 1. Two
remaining enones. i.e., 2 and 3, exhibit a positive nꢀπ* CE of very low and low intensity,
respectively. In their majority, the enones studied are homohelical as the enone torsion angle
ω
and the ene torsion angle
τ defined as C/H−C=C−C(=O), where C/H is syn to the
C
−
C(=O) bond,17 are of the same sign (Table 2). Also worth mentioning is the small size of
the
τ angle in solution, thus mining only a minimal twist of the enone double bond and
therefore practically not affecting the ECD spectra.
Table 2. The enone torsion angle
signs of experimental nꢀ * Cotton effects measured in acetonitrile compared with the same
CE signs simulated at different levels of theory. The ene torsion angle is defined as
C/H C=C C(=O), where C/H(R) is syn to the C C(=O) bond, for the enones of interest given
ω defined as C=CꢀC=(O), the ene torsion angle τ, and the
π
τ
−
−
−
for lowest optimized energy conformations and for crystallographic samples where available.
nꢀπ*CE sign
Exp. Theory
Enone Level of theory / Xꢀray data
ω
τ
B3LYP/TZVP/PCM(CH3CN)
Xꢀray
2
3
−11.4
+26.6
+16.6
−0.2
+0.5
+1.6
−/+
+
+
+
B3LYP/augꢀccꢀpVDZ/PCM(CH3CN)
ACS Paragon Plus Environment

The Journal of Organic Chemistry
Page 16 of 41
1
2
3
4
5
6
7
8
9
CAMꢀB3LYP/augꢀccꢀpVDZ/PCM(CH3CN)
+18.0
+17.1
+18.3
+18.8
+28.5
+28.8
+27.0
+27.3
−14.8
+1.4
+1.5
+1.3
+
+
+
+
−
−
B3LYP/TZVP/PCM(CH3CN)
CAMꢀB3LYP/TZVP/PCM(CH3CN)
Xꢀray
−4.1
+1.9
+1.5
+2.0
+1.6
4
B3LYP/augꢀccꢀpVDZ/PCM(CH3CN)
CAMꢀB3LYP/augꢀccꢀpVDZ/PCM(CH3CN)
B3LYP/TZVP/PCM(CH3CN)
CAMꢀB3LYP/TZVP/PCM(CH3CN)
B3LYP/augꢀccꢀpVDZ/PCM(CH3CN)
−
−
−
−
−
−
−
−
−
−
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
−0.4
5
6
CAMꢀB3LYP/augꢀccꢀpVDZ/PCM(CH3CN)
B3LYP/TZVP/PCM(CH3CN)
−14.9
−15.2
−15.7
−0.3
−0.7
−0.5
−
−
−
−
CAMꢀB3LYP/TZVP/PCM(CH3CN)
Xꢀray
−13.7
−13.1
−17.3
−10.5
−8.0
+7.1
−1.4
+0.3
B3LYP/augꢀccꢀpVDZ/PCM(CH3CN)
B3LYP/augꢀccꢀpVDZ/PCM(CH3CN)
B3LYP/augꢀccꢀpVDZ/PCM(CH3CN)
Xꢀray
−
−
−
−
−
−
7
8
−2.2
−3.0
+0.5
9
B3LYP/augꢀccꢀpVDZ/PCM(CH3CN)
−18.2
−
−
In this context, the intrinsic helicity of the enone chromophore expressed by angle
ω
should remain a decisive factor for the sign of nꢀ * CE by assuming that the external
π
dissymmetric perturbations have only a limited impact on the optical activity of enones as
required by the Kirk rule.4b At first glance, this seems to be true as the investigated enones
exhibit a positive/negative CE in the nꢀ
twist of the enone system. The only exceptions in this regard are enones 2 and 4 having
opposite signs of angle and nꢀ * CE. Moreover, the data from Table 2 demonstrate that the
second principle of Kirk’s rule, concerning opposing signs of nꢀ * and * CEs, is not
π* transition spectral range for a positive/negative
ω
π
π
πꢀπ
fulfilled for 2, 4, 7, and 9. Even taking into account the bisignate character of nꢀπ* CE of
enone 2, it can be stated that Kirk's rule does not cover all enones investigated. Therefore, to
achive a more inꢀdepth insight into incompatibilities mentioned above and to be able to draw
more definite conclusions we have decided to expand our dichroic experiments on solvent
dependency studies and variable temperature measurements in both polar and nonꢀpolar
solvents.
ACS Paragon Plus Environment

Page 17 of 41
The Journal of Organic Chemistry
1
2
3
4
5
6
7
8
9
For all the enones, quantum chemical calculations were implemented to support
experimental data. At the outset, the conformational analysis was done with CONFLEX 718
and ComputeVOA v. 1.019 programs using MMFF94s20 and MMFF9420ꢀ21 force fields,
respectively (for details see the experimental section). For enone 2, two identical sets of five
conformers in an energy window of 5 kcal/mol resulted in both force fields. For further
analysis, contributions to the overall spectra of all conformers received were taken into
account. The comparison of experimental and Boltzmann averaged ECD spectra showed good
agreement with the isomer of 13S absolute configuration (Figure 4). To a similar conclusion
led the analysis of VCD spectra based on the same five conformers. This way, i.e., on the
grounds of two independent chiroptical methods, the AC of hydroxyenone 2 has been
confirmed as consistent with the literature data.9b
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
Figure 4. The experimental and simulated ECD (left) and VCD (middle) spectra of
hydroxyenone 2 averaged with the Boltzmann distribution at B3LYP/TZVP/PCM(CH3CN)
level of theory (insert shows the nꢀ
top: Differences in Gibbs energy (
π
* range of ECD spectra recorded in acetonitrile); Right
ꢁ
G), population (Pop.), and enone torsion angle values (ω)
for individual conformers of hydroxyenone 2; Right bottom: overlay of conformers 2a and 2c
showing structural differences within the cyclohexanone ring and substituents on carbon C13
ACS Paragon Plus Environment

The Journal of Organic Chemistry
Page 18 of 41
1
2
3
4
5
in two of the three lowest energy conformers. For remaining conformers see Supporting
Information section.
6
7
8
The ECD spectra of enones 3 and 4 were further analyzed to confirm the previously
9
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
assigned AC at the C13 carbon atom. As it turned out, these spectra differ in the sign of the nꢀ
π* CE thus indicating a different AC as expected. Comparison of the experimental and
computed at the B3LYP/augꢀccꢀpVDZ level of theory spectra, considering the Boltzmann
averaging from all populated above 1% conformers in combination with previous data from
Xꢀray for 3 and NMR for 4, confirmed AC to be 13R and 13S respectively (Figure 5).
Nonetheless, the experimental and simulated spectra show a lack of satisfactory compliance,
particularly pronounced for the enone 3 in the πꢀπ* range. Moreover, in both of these enones,
the computed spectra do not accurately reflect the positive CE present at around 260 nm with
varying intensity for 3 and 4. The solvent change also does not affect the shape of the ECD
curves of enone 3. In the case of enone 4, however, this small positive CE at ~260 nm
observed in hexane, acetonitrile, and chloroform disappears in polar solvents such as
methanol and trifluoroethanol (Figure 5).
Figure 5. Comparison of computed at the B3LYP/augꢀccꢀpVDZ/PCM(CH3CN) level of
theory UV/Vis and ECD spectra of enones 3 (A) and 4 (B) obtained as a populationꢀweighted
ACS Paragon Plus Environment

Page 19 of 41
The Journal of Organic Chemistry
1
2
3
4
5
6
7
8
sum of the calculated spectra of individual conformers with experimental spectra recorded in
CH3CN. Insert in ECD part B shows a comparison of experimental and computed ECD
spectra of the enone 4 in the range of an nꢀπ* transition indicating lack of positive fragment in
9
the calculation; Panel C: ECD spectra of 3 (top) and 4 (bottom) measured in various solvents
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
(TFE = trifluoroethanol).
Having certainty about maintaining of entire carbon skeleton, we decided to check the
reliability of our ECD calculations by ensuring that nonꢀsatisfactory accuracy between
experiment and theory does not result from the use of an inadequate level of theory.
Therefore, for computational purposes, we applied various combinations of hybrid functionals
(CAMꢀB3LYP,22 B3LYP23) and basis sets (TZVP,24 augꢀccꢀpVDZ25) preceded by a thorough
conformational search run with force fields MMFF94s20 implemented to Conflex18 and
MMFF26 to Spartan.27 This step was followed by geometry reꢀoptimization of all energy
minima found by DFT with regard to the solvent effect of acetonitrile by applying continuum
solvation model (PCM).28 Yet, improving the experiment compliance with theory could not
be achieved. In contrast to the UVꢀVis spectra which are well reproducible irrespective of the
level of theory, in ECD spectra even the sign of nꢀπ* CE becomes inverted depending on the
level used, as can be seen in Figure 6A. Regardless of this, the calculations have shown that
the sign of the CE at ~320 nm is sensitive to subtle structural factors, such as, the
conformation of the isopropyl group at C13 which significantly affects the value of the
rotational strength. Such an impact is particularly pronounced in the case of enone 3. On the
other hand, the lack of good agreement between experiment and computation is not too
surprising given the small intensities of the considered bands.29
ACS Paragon Plus Environment

The Journal of Organic Chemistry
Page 20 of 41
1
2
3
4
5
6
7
8
9
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
Figure 6. Comparison of experimental and computed UVꢀVis (top) and ECD (bottom) spectra
at various levels of theory of enones 3 (A) and 4 (B); Comparison of computed IR (top) and
VCD (bottom) spectra of enones 3 (C) and 4 (D) obtained as a populationꢀweighted sum of
the calculated spectra of individual conformers with experimental spectra.
Given the inconsistencies between ECD experiment and theory, we decided to apply a
quantitative approach to compare experimental and calculated spectra of enones 3 and 4, and
ipso facto obtain an answer to the question of how trustworthy assignment based on ECD is.
To accomplish this, we took advantage of the similarity analysis implemented to the SpecDis
1.70 software.30 For clarity and brevity, we show here only results based on spectra calculated
at the B3LYP/TZVP/PCM(CH3CN) level of theory. Table 3 illustrates the similarity factors
(SF) obtained using relevant calculated and experimental data for the best numerical level of
matching between them. Moreover, for each comparison, the UV shift correction factors,
band broadening and enantiomeric similarity index (ESI) are shown.
Table 3. A numerical analysis of the comparison between experimental and computed spectra
for 3 and 4 using the SpecDis.30 Note: the experimental and calculated ECD/UVꢀVis spectra
at B3LYP/TZVP/PCM(CH3CN) level of theory were fit in the range from 195 nm to 400 nm
by using UV shift correction ranging from −30 to +30 nm, and bandwidth values from 0.1 to
0.5 eV. Abbreviations used: SF = similarity factor index; ESI = enantiomeric similarity index
(the highest value found using criteria mentioned above) defined as the difference between SF
of a compound and SF of its enantiomer.
Enone
UV shift
(nm)
Bandwidth
(eV)
SF
SF for
enantiomer
ESI
ꢀ5
0.43
0.853
0.115
0.738
3
ACS Paragon Plus Environment

Page 21 of 41
The Journal of Organic Chemistry
1
2
3
ꢀ6
0.34
0.923
0.031
0.892
4
4
5
6
7
From Table 3 it becomes clear that for 4 the ESI value of ∼89% is large enough to suggest
8
9
high credibility of 13S configurational assignment. On the other hand, ESI of epimer 3
amounting ~74%, so being 17% lower than that of 4, can nevertheless be considered as
indicative of 13R absolute configuration. However, to obtain a reliable assignment of the
absolute configuration of enones 3 and 4 on the CD basis, we used VCD again. The
agreement between experimental and simulated VCD spectra obtained for the same set of
conformers as for ECD support predetermined AC (Figure 6CꢀD). The resulting reliability of
the assignment was entirely satisfactory as the confidence level algorithm for comparing VCD
experimental and theoretical spectra19a, 31 was 100% for 4 in the 1500ꢀ850 cm−1 range. For the
1800ꢀ850 cm−1 range this algorithm was a little lower and amounted to 96%. In fact, most of
the bands in experimental VCD spectra are matched by calculations in sign but some of them
not perfectly in magnitude. This inaccuracy may be related to the flexibility of the molecules
within the isopropyl and ester groups. Consequently, the 13S AC for 4, confirmed by two
independent chiroptical methods supported by NMR data can be considered as certain.
Our DFT calculations show that unlike the lowestꢀenergy ECD band, the band at around
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
260 nm is not pure as, in addition to enone πꢀπ* transition, it consists of various types of
electronic excitations from the ester group at C4 and substituents on the C13 carbon atom (for
details see Supporting Information section).
Polar substituents have a significant influence on the spectra through their conformational
mobility and intraꢀ or intermolecular interaction. This is particularly evident for enone 2,
whose spectra, depending on the polarity of the solvent, are almost perfect mirror images in
the range of nꢀπ* transition (Figure 7). Cotton effect corresponding to this transition is for
nonpolar solvents such as hexane or chloroform bisignate with +/− sign sequence on going
ACS Paragon Plus Environment

The Journal of Organic Chemistry
Page 22 of 41
1
2
3
4
5
6
7
8
9
towards lower energies. In polar solvents, however, the signs’ sequence turns to the opposite
which ultimately results in spectra of almost mirror image character for hexane and methanol
(Figure 7). Thus, we are dealing here with a system that exhibits chiralityꢀinversion effect
dependent upon the polarity of the solvent used. It should also be noted that these calculations
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
do not reproduce the bisignate features of the nꢀπ* band.
Figure 7. A: Experimental ECD spectra of the enone 2 presented in the range of nꢀπ*
transition in various solvents together with the spectrum simulated at
B3LYP/TZVP/PCM(CH3CN) level of theory; B: ECD spectra of enone 2 recorded in MI13
(methylcyclohexane/isopentane, 1:3, v/v); C: ECD spectra of enone 2 recorded in EPA
(Et2O/isopentane/EtOH, 5:5:2, v/v). The symbol ꢂε denotes the difference of the molar
decadic absorption coefficients of left and right circularly polarized light, and λ – the
wavelength. The lower temperature curves have been corrected for solvent contraction.
Such a strong solvent effect on ECD spectra was also confirmed by variable temperature
measurements. In a nonpolar solvent (MI13: methylcyclohexane/isopentane, 1:3, v/v) already
at −40 °C there is a change of bisignate nꢀπ* CE sign sequence from +/− to +/+ (viewed in the
direction of decreasing energy) with maxima occurring at 312 and 355 nm. Further lowering
temperature to −80 °C causes the formation of one positive ECD band with a maximum at
349 nm with a wellꢀdeveloped vibrational fine structure characteristic for enone carbonyls.
This shape remains unchanged with an additional decrease in temperature to −180 °C (Figure
7). In a polar solvent (EPA: Et2O/isopentane/EtOH, 5:5:2, v/v) and temperatures ranging from
+25 °C to −120 °C, nꢀπ* CE remains bisignate (−/+ sign sequence in descending energy
terms) with a gradual disappearance of the very weak in intensity positive part which finally
ACS Paragon Plus Environment

Page 23 of 41
The Journal of Organic Chemistry
1
2
3
in
−
160 °C becomes purely negative. In this solvent, the fine vibrational structure within nꢀπ*
4
5
6
is visible in the entire temperature range, i.e., from +25 to
−180 °C.
7
8
The EPA results indicate more pronounced conformational homogeneity of the rings
9
comprising the enone chromophore evidenced by only slight changes in the shape of the ECD
curve along with the lowering of the temperature. In the nonpolar solvent MI13, in turn,
lowering the temperature eliminates gradually high energy conformers from equilibrium by
shifting it towards more stable conformers. In this solvent, chromophore is stabilized by
intramolecular hydrogen bonding between the hydroxyl group at C13 and the enone carbonyl
equaling to 2.26 Å. The presence of this Hꢀbond with the formation of an additional fiveꢀ
membered ring was independently verified by IR spectroscopy dilution experiments in CCl4
(Figure S5). The creation of this Hꢀbond changes the identity of the chromophore indicating
the presence of different molecular species in polar and nonpolar solvents. Consequently, we
are dealing with various behaviors depending on solvent polarity ultimately resulting in
differences in the spectra shapes. The most exciting conclusion from this variable temperature
measurements is apparent solventꢀ and temperatureꢀmediated chirality inversion, as can be
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
clearly seen when comparing the spectra in MI13 and EPA starting from
°C.
−120 °C up to −180
Remaining enones, in particular 8ꢀ9 in which intramolecular interactions do not exist, do
not exhibit such a spectacular dependency on solvent and temperature. The impact of various
solvents differing in polarity on the ECD spectra of enones 8 and 9 is in line with
expectations. This means that with increasing solvent polarity, the band corresponding to the
nꢀπ* transition is subjected to a hypsochromic shift and the πꢀπ* to a bathochromic one in
accord with the polarity of the solvents, and their intensity does not show any substantial
changes (Figure S6). On the other hand, measurements at variable temperatures, ranging from
+25 °C to
−
180 °C, performed in a polar solvent EPA (Et2O/isopentane/EtOH, 5:5:2, v/v)
ACS Paragon Plus Environment

The Journal of Organic Chemistry
Page 24 of 41
1
2
3
result in only a slight increase in the intensity of the bands and their bathochromic shift of 10
4
5
6
nm in the range of nꢀπ* transition (Figure 8 CꢀD, top). More pronounced changes in intensity
7
8
and a hypsochromic shift of 7 and 9 nm, respectively for 8 and 9, show these enones in a
9
nonpolar solvent MI13 (methylcyclohexane/isopentane, 1:3, v/v) at the same temperature
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
range, i.e., from +25 °C to
−
180 °C (Figure 8 CꢀD, bottom). However, these changes remain
ꢄꢅꢆ
within limits accepted for compounds of relative conformational stability, as the index
I
ꢀꢁꢂꢃ
describing the relative change of ꢁεmax in ascending temperature terms amounts to ca. 48% in
both cases. This value indicates a greater degree of conformational freedom in a nonpolar
solvent, nevertheless still points to a noticeable hindrance to an unlimited mobility.32
Moreover, it indicates the lack of necessity to stabilize the rings containing the enone
chromophore and substituents through hydrogen bonding or other nonꢀcovalent interactions.
Figure 8. ECD spectra in the nꢀπ* range of enones 3 (A), 4 (B), 8 (C), and 9 (D) recorded
top: in EPA (Et2O/isopentane/EtOH, 5:5:2, v/v), and bottom: in MI13
(methylcyclohexane/isopentane, 1:3, v/v); The symbol ꢂε denotes the difference of the molar
decadic absorption coefficients of left and right circularly polarized light, and λ – the
wavelength. The lower temperature curves have been corrected for solvent contraction.
The variable temperature measurements further corroborated of the relative
conformational stability within the skeleton including the chromophoric unit of enones 8 and
9 although, for both of them several conformers in the 10 kcal/mol energy window were
ACS Paragon Plus Environment

Page 25 of 41
The Journal of Organic Chemistry
1
2
3
4
5
6
7
8
9
found. These conformers mainly differ in the conformation of substituents at C4 and C13.
They are essentially rotamers of isopropyl and ester groups as well rotamers of the hydroxyl
at C13 but without a hydrogen bond between the H atom of this OH group and the carbonyl
oxygen as evidenced by the distance between them equal to 4.56 Å in 9.
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
Lowering the measurement temperature of enones 3 and 4 results in much stronger
changes in both the pattern of the ECD curves and the intensity of the individual bands
depending on the polarity of the solvent used (Figure 8 AꢀB). In the EPA solvent, the ECD
curve of enone 3 starts already at −80 °C to become bisignate reaching its final shape at −180
°C with a minimum at 312 nm and ꢁε
= −0.1 followed by a maximum at 350 nm and ꢁε =
+0.32 (Figure 8A, top). This result can be explained by a progressive elimination of highꢀ
energy conformers from the equilibrium along with shifting this equilibrium to the most stable
conformer. Concurrently, the main positive band of 3 occurring in EPA at r.t at 339 nm is
subjected to a bathochromic shift by 11 nm. In this case, such a shift means that the most
stable conformation was frozen out at the lowest temperature or at least has reached a highest
possible population. In the case of enone 4, however, lowering the temperature causes solely a
gradual intensity increase of the nꢀπ* band, in line with the expected shift of equilibrium
towards more stable conformations. On the contrary, in the MI13 case (MI13:
methylcyclohexane/isopentane, 1:3, v/v) a hypsochromic shift in the λmax of the nꢀπ* CEs,
i.e., ꢁλmax, equaling to 12 nm and 27 nm for enones 3 and 4, respectively, occurs with
decreasing temperature (Figure 8AꢀB, bottom). It is accompanied, as expected, by an increase
in intensity along with a decrease in temperature. In this nonpolar solvent, the most significant
changes in the shape of temperatureꢀdependent ECD curves are observed for enone 4. In the
temperature range of +25 to
nm to 313 nm is accompanied by a gradual intensity decrease of the main negative band.
Further lowering of temperature to 160 °C changes the shape of the curve to bisignate with a
−140 °C, the hypsochromic shift of the nꢀπ* transition from 333
−
ACS Paragon Plus Environment

The Journal of Organic Chemistry
Page 26 of 41
1
2
3
minimum at 309 nm and maximum at 348 nm indicating the presence of different conformers
4
5
6
in the equilibrium. An additional decrease of the temperature to
−180 °C shifts the
7
8
9
equilibrium toward the conformer characterized by a positive CE causing the positive part to
become much more pronounced at the expense of the negative one.
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
The usually observed increase of intensity of ECD bands at a lower temperature is on
account of greater conformational homogeneity favoring the most stable conformers and a
shift of some maxima with decreasing temperatures can be attributed to the soluteꢀsolvent
interaction. This interaction, referred to as solvatochromic effect and related to the
dependency of absorption spectra on solvent polarity, causes the observed variations in the
position, intensity, and pattern of the absorption spectra. In consequence, experimentally
established directions and magnitudes of solvatochromic shifts are applicable for the
determination of the nature of electronic transition, and the estimation of dipole moments in
the ground and excited states.33
CONCLUSION
In this paper, the synthesis of a series of abietane enones serving as model compounds for
chiroptical considerations has been described. We have demonstrated that for reliable
stereochemical conclusions to be achieved a combination of at least two different chiroptical
methods, e.g., ECD and VCD, supported by calculations, is necessary. Such an approach
prevents drawing erroneous conclusions and simultaneously increases the confidence level of
the achieved assignment by analysis of the results in respect of electronic transitions.
Furthermore, the inclusion in the calculations of solvent polarity, which affects the electronic
structure of the solute, additionally stresses the importance of the role it plays in the case of
the enones tested. Thus, applying solvents of different polarity or Hꢀbond/nonꢀHꢀbond
competitive, and variableꢀtemperature ECD measurements enabled us to examine their impact
on resulting environment change. Consequently, such an approach based on compatibility
ACS Paragon Plus Environment

Page 27 of 41
The Journal of Organic Chemistry
1
2
3
4
5
6
7
8
9
with the experiment allowed for the circumvention of possible errors caused by the choice of
a functional and/or negligence of solvent effects. At least for enone 2, we were able to
demonstrate a relatively rare phenomenon of chirality inversion resulting in practically perfect
mirror image ECD spectra, depending on both solvent and temperature applied.
As shown in the present study, experimental data supported by theoretical estimations
resulting from the QC calculations offer an improved and reliable analysis of spectroscopic
properties (position of the absorption band, molar extinction coefficient, and band shape) of a
series of abietane enones. The results obtained ipso facto encourage a much more extensive
use of chiroptical spectroscopy due to its effectiveness and credibility. In particular,
application of dichroic methods for the determination of the AC is relevant in cases where
other techniques, including Xꢀray crystallography, fail, are not applicable or give inconclusive
results. For instance, the structure determination of the extremely sensitive to oxidation enone
5 became possible by employing the on-line HPLCꢀCD technique supported by calculations.
Thus, the final finding of this work is to demonstrate the vital role of chiroptical methods in
determining molecular structure.
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
EXPERIMENTAL SECTION
General Techniques
All solvents were dried and distilled before use. All reactions were monitored by thinꢀ
layer chromatography using aluminumꢀbacked silica gel plates 60 F254; visualization was
accomplished with UV light and/or staining with 50% H2SO4. Standard flash chromatography
procedures were followed using silica gel with particle size 40ꢀ63 ꢁm. Melting points were
recorded in a melting point stage and are not corrected. Optical rotation was measured in
CHCl3 solutions at ambient temperature and is quoted in units of 10−1 deg cm2 g–1. FTꢀIR
spectra were recorded on an FTꢀIR spectrophotometer for KBr pellets or films or examined
directly in the solid or liquid state without further preparation with Universal Attenuated Total
ACS Paragon Plus Environment

The Journal of Organic Chemistry
Page 28 of 41
1
2
3
Reflectance (ATR) accessory in an FTꢀIR spectrophotometer. 1H NMR spectra were recorded
4
5
13
at 400, 500 or 600 MHz and C NMR at 125 and 150 MHz using CDCl3 or C6D6 as solvents
6
7
8
9
and TMS as internal standard and are reported as
signal H (7.26 ppm) and CDCl3
δ
values (ppm) relative to residual CHCl3
13
δ
δ
C (77.16 ppm), respectively. Cꢀ13C 2D INADEQUATE
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
spectra were recorded in concentration ca. 200 mg/400 uL C6D6.
Mass spectra (MS) were obtained with magnetic sector mass spectrometer equipped with
an electron impact (EI) ion source and the EBE double focusing geometry mass analyzer at 70
eV. The instrument was controlled and recorded data were processed using MassLynx 4.1
software package. Electrospray ionization (ESI) experiments were performed on a mass
spectrometer equipped with an electrospray ion source and qꢀTOF type mass analyzer under
normal conditions. PFK solution was used as a calibrant for HRMS measurements. The
instrument was controlled and recorded data were processed using MassLynx V4.1 software
package.UV and ECD spectra of abietic enones 2–9 and 12-13 were measured essentially in
acetonitrile. In selected cases, however, they were also measured in chloroform, methanol,
hexane or 2,2,2ꢀtrifluoroethanol to investigate the effect of solvent on the spectrum. Spectra
were recorded between 180–500 nm at room temperature using solutions with concentrations
in the range 4.6 × 10−4 to 2.6 × 10−3 M, in quartz cells with path length 0.02, 0.1, 0.2, 0.5, 1 or
2 cm. All spectra were recorded using 100 nm/min scanning speed, a step size of 0.2 nm, a
bandwidth of 1 nm, a response time of 0.5 sec, and an accumulation of 5 scans. The spectra
were background corrected.
Lowꢀtemperature ECD measurements of 2, 3, 4, 8, and 9 were performed in the
temperature range 298 – 93 K in a solution of MI13 (methylcyclohexane/isopentane, 1:3, by
vol.) and EPA (Et2O/isopentane/EtOH, 5:5:2, by vol.) as solvents. The solutions with
concentrations in the range from 1.8 × 10−3 to 6.7 × 10−3 M were measured between 280
−
450 nm in 1 cm quartz cell. Baseline correction was achieved by subtracting the spectrum of
ACS Paragon Plus Environment

Page 29 of 41
The Journal of Organic Chemistry
1
2
3
4
5
a reference solvent obtained under the same conditions. All spectra were normalized to ꢁε
using volume correction for MI13 and EPA.
6
The VCD and IR spectra of 2-4, 9 and 11 were measured with 4 cm−1 resolution using
CD3CN as a solvent. The FTꢀVCD spectrometer was equipped with dual sources and dual
PEM (Photoelastic Modulator) technology. A solution with concentrations in the range 0.12
M to 0.17 M was measured in a BaF2 cell with a path length of 100 ꢁm (3 and 9) or 102 ꢁm
(2) assembled in a rotating holder (14 scycle–1). The ZnSe PEM of the instrument was
optimized at 1400 cm−1. The spectra were measured from 5 to 12 hr to find the best signalꢀtoꢀ
noise ratio. Baseline correction was achieved by subtracting the spectrum of a solvent
obtained under the same conditions.
7
8
9
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
Single crystal Xꢀray diffraction measurements for 3, 6, 9, 12 and 13 were carried out at 100
K with graphite monochromated Cu Kα radiation (1.54184 Å). All nonꢀhydrogen atoms were
refined as anisotropic while hydrogen atoms were placed in calculated positions and refined in
riding mode. The data reduction was made by using the CrysAlisPRO34 software. The
structures were solved by direct methods and refined on F2 by FullꢀMatrix leastꢀsquares by
using SHELXS97 and SHELXL97.35 Crystallographic data of 3, 6, 9, 12 and 13 reported here
have been deposited with the Cambridge Crystallographic Data Centre (Deposition No.
CCDC 1483005 for 3, 1467004 for 6, 1467005 for 9, 1486042 for 12, and 1467003 for 13).
These data can be obtained free of charge via www.ccdc.cam.ac.uk/data_request/cif.
Computational details
Conformational search for enones under study was done using ComputeVOATM19a
CONFLEX 718 and/or SPARTAN 1427 programs with MMFF94,21 MMFF94s20 and MMFF26
force fields within 10 kcal mol–1 energy windows. Subsequently, all structures were submitted
to the Gaussian 0936 program for optimisation at the DFT level using the
B3LYP/TZVP/PCM(CH3CN), B3LYP/augꢀccꢀpVDZ/PCM(CH3CN), CAMꢀB3LYP/TZVP/
ACS Paragon Plus Environment