Stereoselective Synthesis of 1-Tuberculosinyl Adenosine; A Virulence Factor of Mycobacterium tuberculosis

Article abstract of DOI:10.1021/acs.joc.6b01332

Despite its status as one of the world's most prevalent and deadly bacterial pathogens, Mycobacterium tuberculosis (Mtb) infection is not routinely diagnosed by rapid and highly reliable tests. A program to discover Mtb-specific biomarkers recently identified two natural compounds, 1-tuberculosinyl adenosine (1-TbAd) and N⁶-tuberculosinyl adenosine (N⁶-TbAd). Based on their association with virulence, the lack of similar compounds in nature, the presence of multiple stereocenters, and the need for abundant products to develop diagnostic tests, synthesis of these compounds was considered to be of high value but challenging. Here, a multigram-scale stereoselective synthesis of 1-TbAd and N⁶-TbAd is described. As a key-step, a chiral auxiliary-mediated Diels-Alder cycloaddition was developed, introducing the three stereocenters with a high exo endo ratio (10:1) and excellent enantioselectivity (>98% ee). This constitutes the first entry into the stereoselective synthesis of diterpenes with the halimane skeleton. Computational studies explain the observed stereochemical outcome.

Full text of DOI:10.1021/acs.joc.6b01332

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Article
Stereoselective Synthesis of 1-Tuberculosinyl Adenosine;
a virulence factor of Mycobacterium tuberculosis
Jeffrey Buter, Dorus Heijnen, Ieng Chim Wan, F. Matthias Bickelhaupt,
David C. Young, Edwin Otten, D Branch Moody, and Adriaan J. Minnaard
J. Org. Chem., Just Accepted Manuscript • DOI: 10.1021/acs.joc.6b01332 • Publication Date (Web): 11 Jul 2016
Downloaded from http://pubs.acs.org on July 13, 2016
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Stereoselective Synthesis of 1-Tuberculosinyl Adenosine; a virulence factor of
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Mycobacterium tuberculosis
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Jeffrey Buter,† Dorus Heijnen,†,ꢀ Ieng Chim Wan,†,ꢀ F. Matthias Bickelhaupt,‡ David C. Young,# Edwin
Otten,† D. Branch Moody,# and Adriaan J. Minnaard,†,*
†Stratingh Institute for Chemistry, University of Groningen, Nijenborgh 7, 9747 AG, Groningen, The Netherlands.
‡Department of Theoretical Chemistry and Amsterdam Center for Multiscale Modeling (ACMM), Vrije Universiteit
Amsterdam, De Boelelaan 1083, 1081 HV Amsterdam, The Netherlands.
# Division of Rheumatology, Immunology and Allergy, Brigham and Women’s Hospital, Harvard Medical School
Smith Building Room 538, 1 Jimmy Fund Way, Boston, MA 02115, USA.
Abstract: Despite its status as one of the world’s most prevalent and deadly bacterial pathogens, M. tuberculosis
(Mtb) infection is not routinely diagnosed by rapid and highly reliable tests. A program to discover Mtbꢁspecific
biomarkers recently identified two natural compounds, 1ꢁtuberculosinyl adenosine (1ꢁTbAd) and N6ꢁtuberculosinyl
adenosine (N6ꢁTbAd). Based on their association with virulence, the lack of similar compounds in nature, the
presence of multiple stereocenters and the need for abundant products to develop diagnostic tests, synthesis of these
compounds was considered to be of high value but challenging. Here, a multiꢁgram scale stereoselective synthesis of
1ꢁTbAd and N6ꢁTbAd is described. As a keyꢁstep, a chiral auxiliaryꢁmediated DielsꢁAlder cycloaddition was
developed, introducing the three stereocenters with a high exoꢁendo ratio (10 : 1) and excellent enantioselectivity
(>98% ee). This constitutes the first entry into the stereoselective synthesis of diterpenes with the halimane skeleton.
Computational studies explain the observed stereochemical outcome.
INTRODUCTION
Tuberculosis is a bacterial infectious disease caused by Mycobacterium tuberculosis (Mtb). Mtb is estimated to have
infected 1.4 billion humans and is responsible for a mortality rate exceeding 1.5 million worldwide deaths annually.1
The persistence of Mtb in human hosts can be attributed to at least two key factors. First, intracellular survival of the
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bacterium is granted by successful infection of the endosomal network of phagocytes.2 In these phagocytes, Mtb
arrests phagosomal acidification, which in turn inhibits pHꢁdependent killing mechanisms and other aspects of
phagosome maturation. In its intracellular state, Mtb is protected from immunoglobulin and cellular immune
responses during a decades long infection process. Secondly, its unusually hydrophobic, multiꢁlayered cell wall
functions as an additional source of protection against drugs and immune response.3 In particular the layers outside
the cytosolic membrane are chemically diverse and quite different from lipids found in other cellular organisms. One
catalog organized by Lipid Maps criteria, defines more than 100 lipid subfamilies in Mtb, with more than 90 percent
of these species expressed by actinobacteria and not by human cells or unrelated bacteria.4 Therefore, lipids can
serve as specific chemical markers of mycobacteria.
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M. bovis strains known as Bacillus CalmetteꢁGuerin (BCG) are genetically related to Mtb but are much less virulent
and have been used as attenuated vaccinates in more than 1 billion humans worldwide.1 We reasoned that a
comparative lipidomics screen to identify lipids present in Mtb but absent in BCG, might identify virulence factors
and Mtbꢁspecific molecules useful for specific detection of this pathogen.5 By screening through more than 8,000
compounds, this approach identified 1ꢁtuberculosinyl adenosine (1ꢁTbAd, Figure 1) as previously unknown lipid,
with no known substantially similar molecules in nature.6 Follow up studies identified the pseudoꢁisomer N6ꢁ
tuberculosinyl adenosine (N6ꢁTbAd, Figure 1), which was postulated to arise from an in vivo Dimroth rearrangement
of 1ꢁTbAd.7
Figure 1. Tuberculosinyl adenosines from Mycobacterium tuberculosis.
Very recently, two closely related compounds, tuberculosinylꢁ2’ꢁdeoxyadenosine (“2’ꢁdeoxy 1ꢁTbAd”) and
tuberculosinyl 2’ꢁOꢁacetyladenosine (2’ꢁacetyl TbAd, Figure 1), were discovered.8 The structures of these analogues
were tentatively assigned based on mass spectrometry, confirming the existence of TbAdꢁlike molecules in
mycobacteria.
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In a screen of bacteria or fungi, 1ꢁTbAd and N6ꢁTbAd were only found in Mtb and not in 13 BCG vaccine strains,
environmental bacteria or nonꢁmycobacterial lung pathogens.7 These facts, along with their high rate of biosynthesis
and ready shedding from the cell wall make 1ꢁTbAd and N6ꢁTbAd attractive candidates for development as chemical
markers for tuberculosis infection.9 Supporting this concept, 1ꢁTbAd and N6ꢁTbAd are produced in vivo in infected
mice and are readily detected ex vivo in wholeꢁlung homogenates and serum,7 as well as human serum,10 using a oneꢁ
step RPꢁHPLC/MS method.
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Further, TbAd biosynthesis requires the virulenceꢁassociated enzymes Rv3377c and Rv3378c, which were shown to
be essential for phagosome maturation arrest.11 Therefore, 1ꢁTbAd is expected to be involved in phagosomal survival
of Mtb. Collectively, these studies provide two strong rationales, based on virulence and diagnostics, for a high yield
stereoselective synthesis of 1ꢁTbAd and N6ꢁTbad as reference factors and targets of ELISA technology. Here we
report the first enantioꢁ and diastereoselective total synthesis of 1ꢁTbAd, together with its 13Cꢁlabeled congener, and
N6ꢁTbAd.
RESULTS AND DISCUSSION
In 2010, the groups of Sorensen12 and Snider13 independently reported a synthesis of tuberculosinol.14 Both routes
relied on a DielsꢁAlder reaction between 6,6ꢁdimethylꢁ1ꢁvinylcyclohexene 115 (Scheme 1) and a tigloyl based
dieneophile leading to the racemic core. This DielsꢁAlder reaction is as such very productive, as it forms the three
stereocenters, of which one is quaternary, in the ring. The reaction, however, is also highly demanding as neither the
diene nor the dienophile is very reactive, and the cycloaddition has to occur with exo selectivity. Whereas tiglic
aldehyde generally gives a high endo selectivity (99 : 1),16 it is known from work by Danishefsky et al. that a bulky
tiglic acid derivative leads to an increased exo selectivity.17 In the total synthesis of tuberculosinol by Sorensen et al.,
the DielsꢁAlder reaction was performed with the relatively small ethyl tiglate providing a modest diastereoselectivity
of 2 : 1 in favor of the exo diastereomer.12 Increase of the steric bulk by using Nꢁtigloyloxazolidinone in the Snider
synthesis led to an exo : endo selectivity of ~10:1.13
Although this (diastereoselective) DielsꢁAlder cycloaddition has been reported several times, an enantioselective
version has not been developed.18 This, however, would provide a first and direct entry into the halimane family of
diterpenes in an enantiopure fashion, and in particular to the members of the TbAdꢁcluster. Extensive
experimentation led to the conclusion that most asymmetric catalytic methods, and also the use of several chiral
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auxiliaries, were met with failure.19 Careful inspection of the assumed reaction coordinate, however, led to the
hypothesis that an Evansꢁtype chiral auxiliary could serve as an efficient reagent for chiral induction, as the
substituent might shield one face of the dienophile. Snider et al. had shown that the parent (achiral) oxazolidinone, in
combination with the strong Lewis acid Me2AlCl, produced a reactive dienophile.13 To our delight, upon reaction of
diene 1 with (S)ꢁisopropylꢁNꢁtigloyloxazolidinone 220 and 2.2 eq of Me2AlCl as the Lewis acid,21 the desired exo
isomer 3 was formed in 59% yield and a diastereoselectivity of 10 : 1 (Scheme 1). Determination of the asymmetric
induction was achieved by removal of the chiral auxiliary with lithium ethanethiolate followed by reduction of the
resulting thioester 4 to the corresponding alcohol 5, in 91% over the two steps. Chiral HPLC analysis of 5 showed an
excellent enantioselectivity for the exo isomer exceeding 98% ee. The minor diastereoisomer (endo) exhibited an ee
of ~60%.
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Scheme 1. Asymmetric total synthesis of the tuberculosinyl adenosines, 1ꢁTbAd and N6ꢁTbAd.
Remarkably, and despite the interest in tuberculosinol and related compounds, the absolute stereochemistry of
tuberculosinol had not been unequivocally established. It was tentatively assigned22 by comparing the sign of the
optical rotation of tuberculosinol with that of neopolypodatetraene A, which has the same bicyclic core structure, of
known absolute configuration.23 This is not fully convincing since small differences in structure can lead to a
different sign of optical rotation. In addition, tuberculosinol is formed by the geranylgeranyl pyrophosphate cyclase
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Rv3377c whereas neopolypodatetraene A is formed by a squalene cyclase. We therefore sought for a definite
confirmation of the absolute stereochemistry.
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DielsꢁAlder cycloadduct 3 turned out to be crystalline and its Xꢁray structure (see SI) established the absolute
stereochemistry of tuberculosinol, based on the known stereocenter in the chiral auxiliary. The optical rotation of 5
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([α]D = + 27.0 (c = 0.2, CHCl3)) matched with that of the Sorensen laboratory ([α]D = +28.3 (c = 0.2, CHCl3)),12
obtained by chiral SFC (supercritical fluid chromatography) of the racemate. Since alcohol 5 is obtained from 3, and
the precursor of the naturally occurring enantiomer of tuberculosinol, the absolute stereochemistry corresponds to
that in Scheme 1 (see SI for the Xꢀray structure).
The sequence from DielsꢁAlder adduct 3 to alcohol 5 was chosen to circumvent moderate yields (40ꢁ50%) in the
direct removal of the chiral auxiliary with LiAlH4. NaBH4 was completely unreactive, presumably due to the
significant steric crowding around the carbonyl functionality. This inaccessibility was observed once more upon
attempts to reduce thioester 4 to aldehyde 6 with either DIBALꢁH or a Fukuyama reduction (Pd/C, Et3SiH).24
With alcohol 5 in hands, the route was pursued along the lines of the Sorensen12 and Snider13 syntheses. A Leyꢁ
Griffith oxidation was performed on alcohol 5 to form aldehyde 6 which subsequently was condensed with acetone
to furnish enone 7, in 70% yield over the two steps. The enone was then efficiently reduced by Wilkinson’s catalyst
in combination with triethylsilane in 90% yield. Installation of the double bond was achieved by a Hornerꢁ
WadsworthꢁEmmons olefination with triethyl phosphonoacetate to produce the desired enoate 9 in a 9 : 1 E/Z
mixture. This mixture was reduced with DIBALꢁH, which after purification afforded pure, naturally occurring,
tuberculosinol in 82% yield over the two steps.
To complete the synthesis of the tuberculosinyl adenosines, tuberculosinol was converted into tuberculosinyl
chloride 10, using a CoreyꢁKim chlorination, in near quantitative yield.25 10 turned out to be unstable on silica, but
purification was achieved by straightforward precipitation of the residual NCS and succinimide by the addition of
pentane. A thorough study of the alkylation of adenosine with 10 led to a procedure involving NaI in DMF (0.5 M in
10).26 This produced 1ꢁTbAd in 76% yield after purification. The compound was identical in all aspects to the natural
isolate.
In addition to the synthesis of naturally occurring 1ꢁTbAd, we also used this alkylation procedure to construct the
derivatives (Z)ꢁ1ꢁTbAd, 2’ꢁdeoxy 1ꢁTbAd8 and 13Cꢁlabelled 1ꢁTbAd (see supporting information). The latter
compound, made from adenosine fully 13Cꢁlabelled in the ribose, was prepared to facilitate the development of 1ꢁ
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TbAd as a biomarker for tuberculosis. Isotopeꢁlabelled biomarkers are important as standards for accurate
quantification with HPLCꢁMS.
In order to verify its chemical structure as proposed by Lau et al.,8 2’ꢁdeoxy 1ꢁTbAd was produced by reacting
tuberculosinyl chloride with 2’ꢁdeoxy adenosine. CID/MSꢁanalysis of synthetic 2’ꢁdeoxy 1ꢁTbAd produced MS
spectra closely matching that of the proposed natural product. This, and the fact that 2’ꢁdeoxy adenosine is an
abundant nucleoside, provides strong evidence that 2’ꢁdeoxy 1ꢁTbAd is a Mycobacterium tuberculosis produced
natural product.
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The finishing touch of our synthesis was the construction of N6ꢁTbAd. It is known that 1ꢁalkyl adenosines rearrange
to N6ꢁalkyl adenosines under nucleophilic conditions via a Dimroth rearrangement.27 Treatment of 1ꢁTbAd with 60%
aqueous Me2NH brought about this rearrangement to N6ꢁTbAd quantitatively. This result concluded our total
synthesis of the naturally occurring tuberculosinyl adenosines and directly confirmed the existence of a plausible,
nonꢁenzymatic basis for generation of the N6ꢁproduct from 1ꢁTbAd, as hypothesized previously.7
The excellent stereoinduction observed in the DielsꢁAlder reaction deserves particular attention. In 2010 the Snider
laboratory used the achiral tigloylꢁbased oxazolidinone, and stated “The unsubstituted oxazolidinone was chosen to
minimize steric interactions between the αꢀmethyl group and the oxazolidinone and because asymmetric induction
seemed unlikely in an exo DielsꢀAlder reaction in which a chiral oxazolidinone would be far away from the diene
(1).” 13
This statement was based on the mechanistic model proposed by Danishefsky (Figure 2A), explaining exoꢁselectivity
in the DielsꢁAlder reaction with diene 1.17 A steric clash between the diene’s geminal dimethyl group and the bulky
dienophile disfavors the endo approach, hence providing the exoꢁDielsꢁAlder adduct selectively. This model,
however, only addresses the dienophile in its sꢁtrans conformation. Although this model predicts the observed exoꢁ
selectivity, it ignores a possible contribution of the sꢁcis conformation of the dienophile.28
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Danishefsky's 1994 Model
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O
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Ar
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H3C
H3C
H3C
VS
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Endo approach
Exo approach
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Disfavored
Favored
s-trans Diels-Alder cycloaddition
B
Me
Me
Me Al
Me Al
H3C
H3C
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iPr
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iPr
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H
H3C
H3C
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iPr
Exo Re-face approach
Exo Si-face approach
Disfavored
Endo approach
Disfavored
Possible
s-cis Diels-Alder cycloaddition
C
Me
Me
Al
H3C
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O
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iPr
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iPr
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H3C
Me
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Al
Al
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iPr
H3C
H3C
H3C
H3C
Endo approach
Exo Re-face approach
Exo Si-face approach
Disfavored
Disfavored
Possible
Figure 2. Models for stereoinduction in the DielsꢁAlder cycloaddition between diene 1 and dienophile 2.
In a combined experimental and computational study by Houk, Gouverneur et al., it was shown that Evans chiral
auxiliaryꢁbased dienophiles provide efficient chiral induction and endo or exoꢁselectivity depending on the
substitution at the αꢁposition of the dienophile and the diene.29 The reaction takes place from the sꢁcis conformation
of the dienophile as expected as the diene is not substituted at the αꢁposition, like in the current tigloylꢁbased
dieneophile.
In Figure 2B, a model for the [4+2] cycloaddition between diene 1 and dienophile 2, in its sꢀtrans conformation, is
presented. As in Danishefsky’s model17 the endo approach is disfavored for reasons previously mentioned. The exo
approach on the Siꢁface of the dienophile seems disfavored as the isopropyl group on the chiral oxazolidinone
clashes with the vinyl substituent of the diene. The Reꢁface approach on the other hand seems plausible as no
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obvious steric interactions shield this face from reaction. However, this facial approach would provide the Dielsꢁ
Alder adduct 3 with a stereochemistry opposite to that observed experimentally.
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Considering the sꢀcis conformer of dienophile 2 (Figure 2C), the endo approach is again disfavored, this time
because of a clash between the dimethylaluminum function and the geminal dimethyl. The Reꢁface appears
unapproachable due to a steric interaction between the isopropyl group and the geminal dimethyl in diene 1. A Siꢁ
face approach does not result in such steric clash and provides DielsꢁAlder adduct 3 with the experimentally obtained
stereochemistry. This model therefore seems to be a better picture of the course of the reaction, despite the
expectation that the sꢀcis conformation of the dienophile is unfavorable compared to the sꢀtrans conformation.
Therefore, we studied the reaction through DFT calculations.
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Calculations were carried out using the ADF program suite30 at the BP8631/TZ2P level of theory. The geometries of
all stationary points were optimized in the gas phase and verified to be proper local minima or transition states
through vibrational analyses. The gasꢁphase harmonic frequencies were used for the enthalpic and entropic
corrections to the free energies at 233.15 K (ꢁ40 °C).
First, it was determined whether the sꢀcis conformer is energetically feasible by calculating the sꢀtrans/sꢀcis
equilibrium for Me2Alꢁcomplexed dienophile 2. We found the sꢀtrans conformer is favored over the sꢁcis conformer
by 0.2 kcal/mol on the Gibbs free energy surface at 233.15 K, providing a ratio of 6 : 4 respectively at ꢁ40oC (Figure
3). With a rotational barrier of ꢀG‡ = 3.2 kcal/mol, however, the conformers are readily interconvertible and, due to
the CurtinꢁHammet principle, the observed product can be reached via the sꢁcis conformer.32
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Figure 3. DFT BP86/TZ2P calculated reaction coordinate for the DielsꢁAlder cycloaddition between diene 1 and dienophile 2.
For these studies, the sꢀcis exo Siꢁface approach was chosen as the reference point. IRC analysis were performed on both
transition states on the sꢀcis pathway (blue lines) and are connected with solid lines. The dashed lines connect the structures in
which IRC was not performed, but was found to exist on the potential energy surface. (Relative electronic energies are given in
kcal/mol, and relative Gibbs free energies are given in kcal/mol in parentheses). The geometry of diene 1, aluminumꢁcomplexed
sꢀcis conformer of the dienophile (2a) aluminumꢁcomplexed sꢀtrans conformer of the dienophile (2b), and the transition state
between 2a and 2b corresponding to the rotation along the C(2)ꢁC(3) bond (2c) were optimized separately. The energies of the
three states 1+2a, 1+2b and 1+2c are the sum of the corresponding diene and aluminumꢁcomplexed dienophile fragments (See
Supporting Information).
The energy profile for the entire reaction pathway was calculated next. On this basis, and according to the models in
Figure 2, the endo approach for both the sꢁtrans and sꢀcis conformers can be ruled out. Attack of the sꢁtrans
conformer from the exo Siꢁface, and attack of the sꢁcis conformer from the exo Reꢀface are prohibited too as
modeling showed significant steric clash for initial bond formation (see Supporting Information). The two models in
which calculations were carried out, were the Reꢁface approach and the Siꢀface approach for the sꢀtrans and sꢀcis
conformers respectively (both exo attack).
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The reaction of the sꢀtrans conformer (Figure 3, red pathway) has a considerably higher activation energy (ꢀG‡ =
19.4 kcal/mol) for the initial and enantioꢁdetermining step, compared to the reaction of the sꢀcis conformer (blue
path, ꢀG‡ = 15.5 kcal/mol). This convincingly explains the reaction outcome.
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The DielsꢁAlder reaction was found to proceed stepwise rather than concerted. After the first bond formation, the
cationic intermediate is in an energy well with a relative energy of 12.4 kcal/mol, before proceeding to form the
second bond with a saddle point at 14.8 kcal/mol. From this point the reaction is thermodynamically downhill to
form the Me2Al complexed DielsꢁAlder adduct 3 with a relative ꢀG = ꢁ1.3 kcal/mol.
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The differences in activation energies between the two pathways can be rationalized using the activation strain
model.33 In this model, the electronic activation energy (∆E‡) is the sum of strain energy (∆E‡strain), which is
associated with the deformation of the starting materials to the geometry they acquire in the TS, and the interaction
energy (∆E‡int), which is associated with the favorable electronic interactions between the deformed starting
materials, i.e., ∆E‡ = ∆E‡strain + ∆E‡
int
As outlined in Table 1, the difference in ∆E‡ between TS 12a and TS 12b is mainly due to strain, not due to orbital
interaction. Upon inspection on the geometry of the dienophile at the transition state, the dihedral angle O(1)ꢁC(2)ꢁ
C(3)ꢁC(4) is deviating 17.2⁰ and 18.2⁰ from planarity for TS 12a and TS 12b respectively, while the corresponding
dihedral angle of the sꢀcis and sꢀtrans conformers are deviating 29.8⁰ and 43.0⁰ from planarity respectively.
Transition states ꢀE‡
ꢀE‡
ꢀE‡
ꢀH‡
4.2
8.4
2.2
5.8
TꢀS‡
ꢁ11.4
ꢁ11.3
ꢁ12.6
ꢁ13.9
ꢀG‡
15.6
19.7
14.8
19.7
strain
int
3.4
7.7
ꢁ0.2
3.9
16.5
23.4
79.1
75.6
ꢁ13.1
ꢁ15.7
ꢁ79.3
ꢁ71.6
TS 12a
TS 12b
TS 14a
TS 14b
Table 1. Activation strain analysis (in kcal/mol) for the four transition states shown in Figure 3.
The larger deviation from planarity, of the dihedral angle O(1)ꢁC(2)ꢁC(3)ꢁC(4), in the sꢀtrans conformer might be
attributed to a steric interaction between the hydrogen atom at the stereocenter of the oxazolidinone scaffold, and that
of the hydrogen atom at C(4). The distance between these two hydrogen atoms is 2.07 Å for the sꢀtrans conformer,
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while the closest distance between the hydrogen atom at the oxazolidinone and the hydrogens on the αꢁmethyl group
in the sꢀcis conformer is 2.17 Å. Since both distances are less than the sum of the van der Waals radii of two
hydrogen atoms (2.4 Å), the steric interactions between the hydrogen atoms in both cases are nonꢁnegligible, and the
steric interaction in the sꢀcis conformer is indeed less severe than that in the sꢀtrans conformer. The sꢀcis conformer
can thus retain a relatively planar geometry without experiencing severe steric interactions. The larger difference of
the dihedral angle between the ground state and the transition state of the sꢀtrans conformer compared to that of the
sꢀcis conformer could explain the difference in activation strain.
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While ∆E‡ plays a larger role in determining the ∆E‡ of the second transition state, this difference is argued to be
int
less important due to the fact that enantioselectivity is determined at the first transition state.
This computational study shines light on the stereochemical course of DielsꢁAlder reactions with tigloyl (i.e., α,βꢁ
disubstituted) chiral oxazolidinones. Although the CurtinꢁHammet principle in DielsꢁAlder reactions has been
described before,28 in the use of tigloyl based dienophiles this concept is novel.
CONCLUSIONS
In summary, 1ꢁTbAd and N6ꢁTbAd have been prepared as pure stereoisomers in 10 and 11 steps respectively, starting
from diene 1. Installation of the three chiral centers in tuberculosinol was efficiently achieved (exo:endo = 10:1,
>98% ee for the exo isomer) employing a chiral auxiliaryꢁaided DielsꢁAlder reaction. The synthesis has been scaled
to produce 2.4 g of 1ꢁTbAd in 21% overall yield. In addition, the family members N6ꢁTbAd and 2’ꢁdeoxy 1ꢁTbAd
have been prepared, confirming the structure of the latter. The compounds are currently studied as biomarkers for
tuberculosis, which is assisted by the availability of 13Cꢁlabeled 1ꢁTbAd.
The stereoselectivity observed in the DielsꢁAlder reaction was also investigated in detail. Inꢀsilico studies showed the
cycloaddition to proceed via the thermodynamically less stable sꢀcis conformer of the dienophile because this
conformer needs less deformation in the transition state which results in a lower reaction barrier.
This asymmetric and exoꢁselective DielsꢁAlder reaction sets the stage for the synthesis of other diterpenes bearing
the halimane skeleton as present in tuberculosinol (i.e. akaterpin,34 siphonodictyal D,35 neopolypodatetraene A,23
cacospongionolide F,36 11R*ꢁacetoxyhalimaꢁ5,13Eꢁdienꢁ15ꢁoic acid,37 and mamanuthaquinone38).
EXPERIMENTAL SECTION
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6,6-dimethyl-1-vinylcyclohex-1-ene (1):15
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To a solution of cyclohexanone (9.91 ml, 96 mmol) and iodomethane (12.5 ml, 201 mmol, 2.05 eq) in dry THF (500
mL), cooled to ꢁ20 °C, was added dropwise, using a dropping funnel, a solution of KOtBu (22.6 g, 201 mmol, 2.05
eq) in dry THF (125 mL). Upon addition, a white precipitate formed and the reaction turned yellow. After addition
the mixture was stirred for 60 min at this temperature after which GC/MS indicated complete conversion of the
cyclohexanone. The reaction mixture was quenched with an aqueous saturated NH4Cl solution (200 mL). Quenching
resulted in significant, but not complete, disappearance of the white precipitate, which was completely removed by
filtration of the quenched mixture over a glass filter. The phases were separated and the aqueous phase was extracted
twice with ether. The combined organic layers were dried over Na2SO4, filtered, and concentrated under reduced
pressure. Flash column chromatography of the resulting oil using pentane:ether (4:1) afforded 2,2ꢁ
dimethylcyclohexanone (18.3 g, 145 mmol, 49% yield) ~90% pure.
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Note: The reaction was performed 3x on this scale. The extractions were performed on the combined reaction
mixtures. The yield represents that of the combined reactions. In order to obtain the desired 90% purity, the
individual column fractions were analyzed with GC/MS.
To a stirred solution of vinylmagnesium bromide (174 mL, 174 mmol, 1.2 eq) at 0 °C was added dropwise a solution
of 2,2ꢁdimethylcyclohexanone (18.3 g, 145 mmol) in dry THF (50 mL) over 30 min. The reaction was allowed to
warm up to room temperature and stirred for 2 h. The reaction mixture was carefully quenched with an aqueous
saturated NH4Cl solution (75 mL). After phase separation the aqueous layer was extracted with ether (3x25 mL). The
organic layers were combined and dried over Na2SO4, filtered and concentrated under reduced pressure. The
resulting yellowish oil (27.3 g, >99% yield) was used in the subsequent dehydration reaction.
The crude alcohol was dissolved in benzene (300 mL). To the stirred solution, anhydrous CuSO4 (50 g, 313 mmol,
2.2 eq) was added. The suspension was refluxed under DeanꢁStark conditions overnight. The reaction mixture was
cooled to rt and thereafter filtered over Celite® and flushed with pentane. The filtrate was concentrated under
reduced pressure and the residual oil was subjected to flash column chromatography employing pentane as the
eluent. Pure 6,6ꢁdimethylꢁ1ꢁvinylcyclohexꢁ1ꢁene 1 (10.5 g, 77 mmol, 53% yield) was obtained as a colorless oil.
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1H NMR (400 MHz, CDCl3) δ 6.32 (dd, J = 17.2, 10.9 Hz, 1H), 5.78 (t, J = 3.9 Hz, 1H), 5.28 (d, J = 17.2 Hz, 1H),
4.92 (d, J = 10.9 Hz, 1H), 2.05 (q, J = 5.7 Hz, 2H), 1.61 (dt, J = 12.0, 5.9 Hz, 2H), 1.53 – 1.46 (m, 2H), 1.07 (s, 6H).
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13C NMR (101 MHz, CDCl3) δ 144.7, 137.2, 123.2, 112.8, 39.6, 33.3, 28.5, 26.3, 19.3.
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Note: Diene 1 is rather volatile and therefore concentration, after column chromatography, was performed very
carefully. As a result the NMR spectra of diene 1 contain benzene and traces of pentane.
(S,E)-4-isopropyl-3-(2-methylbut-2-enoyl)oxazolidin-2-one (2):20
To vigorously stirred thionyl chloride (27.2 ml, 375 mmol, 1.5 eq) in a 250 mL 3ꢁneck round bottom flask (open
flask!) was added portionwise (E)ꢁ2ꢁmethylbutꢁ2ꢁenoic acid (25 g, 250 mmol). Each portion was added after
significant gas evolution (HCl, SO2) ceased. After addition of all the acid the reaction mixture (open flask!) was
heated to 40 °C until no gas evolution was observed. The 3ꢁneck round bottom flask was equipped with a reflux
condenser, and the reaction was refluxed for 1 h. After this time no gas evolution was observed. The excess of
thionyl chloride was removed by concentration under reduced pressure. The product was subsequently distilled in the
rotavapor to afford (E)ꢁ2ꢁmethylbutꢁ2ꢁenoyl chloride E (29.6 g, 250 mmol, quantitative) as a clear liquid.
Note: The reaction is exothermic with vigorous evolution of HCl/SO2 gas.
To a cooled solution, –60 °C, of (S)ꢁ4ꢁisopropyloxazolidinꢁ2ꢁone (25.0 g, 194 mmol) in dry THF (500 mL) was
added dropwise nꢀbutyllithium (133 mL in hexanes, 213 mmol, 1.1 eq) by the aid of a dropping funnel. Upon
addition, the reaction mixture became turbid. After addition the reaction was allowed to warmꢁup to 0 °C and was
stirred at this temperature for 30 min where after the mixture was reꢁcooled to ꢁ60 °C and (E)ꢁ2ꢁmethylbutꢁ2ꢁenoyl
chloride (29.6 g, 250 mmol, 1.3 eq) was added dropwise by the aid of a syringe pump over 20 min. Upon addition
the reaction mixture turned yellow/orange. After addition of the acid chloride the reaction was allowed to warm up to
rt and the reaction became transparent. The reaction was stirred overnight where after it was carefully quenched
using aqueous HCl (1 M, 150 mL). After phase separation the aqueous layer was extracted twice with ether. The
combined organic layers were washed with a saturated aqueous solution of NaHCO3 and dried using Na2SO4, filtered
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and concentrated under reduced pressure. A solid formed which was dissolved in CH2Cl2 (15 mL) and subjected to
flash column chromatography employing pentane:ether (3:2) as the eluent to afford pure (S,E)ꢁ4ꢁisopropylꢁ3ꢁ(2ꢁ
methylbutꢁ2ꢁenoyl)oxazolidinꢁ2ꢁone 2 (25 g, 118 mmol, 61% yield) as a waxy solid.
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1H NMR (400 MHz, CDCl3) δ 6.20 (q, J = 7.0 Hz, 1H), 4.51 (dt, J = 9.0, 4.9 Hz, 1H), 4.31 (t, J = 8.9 Hz, 1H), 4.17
(dd, J = 8.9, 5.5 Hz, 1H), 2.36 (dq, J = 13.8, 6.9 Hz, 1H), 1.90 (s, 3H), 1.80 (d, J = 7.0 Hz, 3H), 0.91 (t, J = 6.8 Hz,
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6H).
13C NMR (101 MHz, CDCl3) δ 171.9, 153.8, 134.7, 131.9, 63.5, 58.4, 28.4, 18.0, 15.1, 14.2, 13.5.
HRMS: (ESI+) Calculated mass [M+H]+ C11H18NO3 = 212.1281, found: 212.1281. Calculated mass [M+Na]+
+
C11H17NO3Na+ = 234.1101 found: 234.1101.
(S)-4-isopropyl-3-((1R,2S,8aR)-1,2,5,5-tetramethyl-1,2,3,5,6,7,8,8a-octahydronaphthalene-1-
carbonyl)oxazolidin-2-one (3):
To a stirred solution of dienophile 2 (9.13 g, 43.2 mmol, 1.1 eq) in dry (CH2)2Cl2 (250 mL) at ꢁ40 °C under N2 was
added Me2AlCl (86.5 mL, 1 M in hexanes, 86.5 mmol, 2.2 eq) over 15 min, by the aid of a dropping funnel. The
yellow reaction mixture was stirred for 20 min and diene 1 (5.35 g, 39.3 mmol) in dry (CH2)2Cl2 (90 mL) was added
dropwise over 15 min by the aid of a dropping funnel. The reaction was then allowed to warm to rt and was stirred
for 36 h at this temperature. GC/MS indicated complete conversion of the diene.
The reaction mixture was cooled to 0 °C and carefully quenched by dropwise addition of aqueous 1 M HCl (50 mL).
After phase separation the aqueous layer was extracted with CH2Cl2 (3 x 20 mL). The combined organic layers were
dried over Na2SO4, filtered and concentrated under reduced pressure. Flash column chromatography with
pentane:ether
(6:1)
provided
(S)ꢁ4ꢁisopropylꢁ3ꢁ((1R,2S,8aR)ꢁ1,2,5,5ꢁtetramethylꢁ1,2,3,5,6,7,8,8aꢁ
octahydronaphthaleneꢁ1ꢁcarbonyl)oxazolidinꢁ2ꢁone 3 as a white crystalline solid (8.0 g, 19.6 mmol, 59% yield,
exo:endo = 10:1).
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Note: the reaction temperature was monitored inside the flask. Lower temperatures leads to crystallization of the
5
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dienophile.
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1H NMR (400 MHz, CDCl3) δ 5.47 (d, J = 5.7 Hz, 1H), 4.54 (d, J = 8.2 Hz, 1H), 4.33 – 4.13 (m, 2H), 3.34 (d, J =
12.8 Hz, 1H), 3.15 (tt, J = 12.5, 6.5 Hz, 1H), 2.39 – 2.30 (m, 1H), 1.95 (dt, J = 17.5, 5.5 Hz, 1H), 1.79 – 1.63 (m,
2H), 1.54 (tt, J = 7.0, 3.1 Hz, 1H), 1.43 – 1.29 (m, 2H), 1.28 – 1.13 (m, 2H), 1.06 (s, 3H), 1.03 (s, 3H), 1.01 (s, 3H),
0.90 (t, J = 6.3 Hz, 6H), 0.78 (d, J = 6.8 Hz, 3H).
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13C NMR (101 MHz, CDCl3) δ 178.0, 153.0, 144.8, 116.1, 62.9, 61.2, 53.5, 40.9, 37.6, 36.4, 31.3, 29.7, 29.5, 28.9,
28.3, 22.2, 18.5, 16.5, 14.5, 12.4.
+
HRMS: (ESI+) Calculated mass [M+H]+ C21H34NO3 = 348.2533, found: 348.2536. Calculated mass [M+Na]+
C21H33NO3Na+ = 370.2353, found: 370.2355.
Melting point: 95ꢁ97 °C
Optical rotation: [α]D23 = +57.4 (c = 0.0135, CHCl3).
(1R,2S,8aR)-S-ethyl 1,2,5,5-tetramethyl-1,2,3,5,6,7,8,8a-octahydronaphthalene-1-carbothioate (4):
To a solution of ethanethiol (8.3 mL, 115 mmol, 5.9 eq) in dry THF (200 mL), cooled to 0 °C, was added dropwise
nꢁbutyllithium (57.6 mL, 92 mmol, 4.7 eq). Upon addition a white precipitate formed. After addition the milk white,
now viscous, suspension was allowed to warm to rt. DielsꢁAlder adduct 3 (8.0 g, 19.6 mmol) in dry THF (50 mL)
was added and the reaction was stirred for 7 h. Full conversion was observed by TLC and GC/MS analysis. The
reaction was diluted with ether and quenched by addition of a saturated aqueous NH4Cl solution. The white
precipitate dissolved. The phases were separated and the aqueous phase was extracted with ether. The combined
organic phases were dried over MgSO4, filtered and concentrated under reduced pressure to afford a yellow oil.
Upon standing, the oxazolidinone chiral auxiliary crystallized. Pentane was then added to the crystallized product
and the resulting suspension was cooled to ꢁ20 °C where after the cold suspension was filtered over a sintered glass
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filter (pore size 3). The residue was rinsed with cold pentane (ꢁ20 °C) to recover the chiral auxiliary as needle shaped
transparent crystals (2.2 g). The filtrate was concentrated under reduced pressure to afford crude (1R,2S,8aR)ꢁSꢁethyl
1,2,5,5ꢁtetramethylꢁ1,2,3,5,6,7,8,8aꢁoctahydronaphthaleneꢁ1ꢁcarbothioate 4 (7.2 g, 25.7 mmol >99% yield) as a
yellowish oil. The product was considered sufficiently pure to be used in the next step.
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Alternatively flash column chromatography can be performed: The oil/crystal mixture was dissolved in a minimum
amount of ether and loaded on the column. Elution using pentane:ether (98:2) as the eluent afforded pure
(1R,2S,8aR)ꢁSꢁethyl 1,2,5,5ꢁtetramethylꢁ1,2,3,5,6,7,8,8aꢁoctahydronaphthaleneꢁ1ꢁcarbothioate 4 (5.06 g, 18.0 mmol,
96% yield) as a yellowish oil. (The 96% yield was obtained for a reaction starting from 6.5 g of DielsꢀAlder adduct
3).
1H NMR (400 MHz, CDCl3) δ 5.46 (d, J = 5.3 Hz, 1H), 2.88 (q, J = 7.4 Hz, 2H), 2.79 (d, J = 12.9 Hz, 1H), 2.02 –
1.86 (m, 2H), 1.75 (ddt, J = 13.4, 11.5, 3.6 Hz, 1H), 1.53 – 1.46 (m, 3H), 1.44 – 1.36 (m, 1H), 1.25 (t, J = 7.4 Hz,
3H), 1.21 – 1.10 (m, 2H), 1.08 (s, 3H), 1.05 (s, 3H), 0.98 (s, 3H), 0.79 (d, J = 6.5 Hz, 3H).
13C NMR (101 MHz, CDCl3) δ 207.9, 144.9, 116.0, 56.7, 43.5, 40.6, 36.8, 36.0, 31.4, 29.7, 29.4, 28.2, 23.1, 21.7,
15.9, 14.8, 9.6.
HRMS: (ESI+) Calculated mass [M+H]+ C17H29OS+ = 281.1934, found: 281.1932.
Optical rotation: [α]D23 = +8.7 (c = 0.0184, CHCl3).
((1R,2S,8aR)-1,2,5,5-tetramethyl-1,2,3,5,6,7,8,8a-octahydronaphthalen-1-yl)methanol (5):12,13
To a cooled (0 °C) solution of crude thioester 4 (7.2 g, 25.7 mmol) in dry THF (150 mL) was added portionwise
lithium aluminum hydride (4.9 g, 128 mmol, 5 eq). After addition, the reaction was allowed to warmꢁup to rt where
after the reaction was heated to 40 °C and stirred for 2 h. GC/MS and TLC analysis indicated complete conversion of
the starting material. The reaction mixture was cooled to 0 °C, diluted with ether and carefully quenched using a
saturated aqueous Rochelle salt solution. After addition, the quenched reaction mixture was stirred for 30 min. After
phase separation, the aqueous layer was extracted three times with ether where after the combined organic layers
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were washed with brine. The organic phase was then dried over MgSO4, filtered and concentrated under reduced
pressure to yield ((1R,2S,8aR)ꢁ1,2,5,5ꢁtetramethylꢁ1,2,3,5,6,7,8,8aꢁoctahydronaphthalenꢁ1ꢁyl)methanol 5 (6.2 g, 30
mmol, quantitative yield) as yellow oil. The material was deemed pure enough to be used in the next step without
purification.
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Alternatively flash column chromatography can be performed: purification of the obtained yellow oil, using
pentane:ether (9:1) as the eluent, afforded pure ((1R,2S,8aR)ꢁ1,2,5,5ꢁtetramethylꢁ1,2,3,5,6,7,8,8aꢁ
octahydronaphthalenꢁ1ꢁyl)methanol 5 (4.7 g, 21.1 mmol, 91% yield) as a yellowish oil. (The 91% yield reflects that
obtained over the past 2 steps, starting from DielsꢀAlder adduct 3. With 96% isolated yield for the oxazolidinone
cleavage this corresponds to 95% isolated yield for the reduction of thioester 4 to alcohol 5).
Chiral HPLC analysis of alcohol 5 showed an enantiomeric excess exceeding 98% for the exo isomer. Also the endo
isomer proved to be enantiomerically enriched, possessing an enantiomeric excess of ~60%.
1H NMR (400 MHz, CDCl3) δ 5.43 (d, J = 5.2 Hz, 1H), 3.48 (d, J = 11.4 Hz, 1H), 3.39 (d, J = 11.3 Hz, 1H), 2.36 (d,
J = 12.9 Hz, 1H), 1.91 – 1.81 (m, 1H), 1.81 – 1.71 (m, 2H), 1.70 – 1.63 (m, 2H), 1.61 – 1.51 (m, 2H), 1.38 (s, 1H),
1.28 – 1.14 (m, 2H), 1.05 (s, 3H), 1.00 (s, 3H), 0.86 (d, J = 6.5 Hz, 3H), 0.51 (s, 3H).
13C NMR (101 MHz, CDCl3) δ 146.1, 116.1, 65.8, 41.1, 39.3, 38.1, 36.3, 31.9, 31.3, 29.9, 28.9, 27.7, 22.3, 15.1,
11.6.
HRMS: (ESI+) Calculated mass [M+H]+ C15H27O+ = 223.2056, found: 223.2059.
Chiral HPLC analysis on a Chiracel ADꢁH column, nꢁHeptane : iꢁPrOH = 98 : 2, 40 °C, flow = 0.5 mL/min, UV
detection at 190 nm, 210 nm and 254 nm, retention times (min): 15.7 (endo minor), 15.1 (endo major), 17.7 (exo
major), and 21.1 (exo minor, not detected for chiral alcohol 5)
(1R,2S,8aR)-1,2,5,5-tetramethyl-1,2,3,5,6,7,8,8a-octahydronaphthalene-1-carbaldehyde (6): 12,13
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To a solution of alcohol 5 (4.7 g, 21.1 mmol) in dry CH2Cl2 (80 mL) were added TPAP (371 mg, 1.06 mmol, 5
mol%), 4ꢁmethylmorpholineꢁNꢁ4ꢁoxide (or its monohydrate) (3.22 g, 27.5 mmol, 1.3 eq) and 3 Å molecular sieves.
The reaction was stirred at rt for 2 h after which TLC and GC/MS analysis indicated complete conversion of the
starting material. The reaction mixture was diluted using pentane which resulted in precipitation of the TPAP. The
mixture was filtered over Celite® to remove the TPAP. NMO and its reduced analogue were removed by flash
column chromatography using pentane:ether (95:5) as the eluent to afford pure (1R,2S,8aR)ꢁ1,2,5,5ꢁtetramethylꢁ
1,2,3,5,6,7,8,8aꢁoctahydronaphthaleneꢁ1ꢁcarbaldehyde 6 (4.43 g, 20.1 mmol, 95% yield) as a yellowish oil which
crystallized upon standing.
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1H NMR (400 MHz, CDCl3) δ 9.38 (s, 1H), 5.52 – 5.47 (m, 1H), 2.50 (d, J = 13.1 Hz, 1H), 2.00 – 1.91 (m, 1H), 1.90
– 1.80 (m, 1H), 1.80 – 1.69 (m, 1H), 1.57 – 1.49 (m, 2H), 1.45 – 1.37 (m, 1H), 1.36 – 1.28 (m, 1H), 1.27 – 1.14 (m,
2H), 1.08 (s, 3H), 1.04 (s, 3H), 0.80 (s, 3H), 0.78 (d, J = 6.5 Hz, 3H).
13C NMR (101 MHz, CDCl3) δ 207.2, 143.9, 116.6, 52.3, 40.6, 38.2, 36.3, 32.7, 30.5, 29.7, 29.1, 28.7, 21.8, 16.1,
7.5.
HRMS: (ESI+) Calculated mass [M+H]+ C15H25O+ = 221.1900, found: 221.1901.
Melting point: 52ꢁ53 °C
(E)-4-((1S,2S,8aS)-1,2,5,5-tetramethyl-1,2,3,5,6,7,8,8a-octahydronaphthalen-1-yl)but-3-en-2-one (7): 12,13
A solution of NaHMDS (30 mL, 2 M in THF, 30 mmol, 3 eq) diluted to a 1 M solution with dry THF (30 mL) was
cooled to ꢁ78 °C. The orange solution became slightly turbid and viscous, however stirring was assured. Acetone (4.4
ml, 60.3 mmol, 3 eq) was added dropwise to the turbid solution which became clear upon addition. The mixture was
stirred at this temperature for 20 min after which a solution of aldehyde 6 (4.43 g, 20.1 mmol) in dry THF (125 mL)
was added dropwise over 20 min by the aid of a dropping funnel. After addition the reaction was taken out of the
cooling bath and allowed to warmꢁup to rt (by the aid of a water bath at rt). The reaction was stirred for 2 h after
which TLC and GC/MS indicated complete consumption of the aldehyde 6.
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The reaction was diluted with ether and quenched with a saturated aqueous NaHCO3 solution. The phases were
separated and the organic phase was washed with distilled water and brine. The combined aqueous layers were backꢁ
extracted once with ether and the combined organic layers were dried over MgSO4, filtered and concentrated under
reduced pressure. Flash column chromatography employing pentane:ether (95:5) afforded (E)ꢁ4ꢁ((1S,2S,8aS)ꢁ1,2,5,5ꢁ
tetramethylꢁ1,2,3,5,6,7,8,8aꢁoctahydronaphthalenꢁ1ꢁyl)butꢁ3ꢁenꢁ2ꢁone 7 as an amorphous solid (3.4 g, 13.1 mmol,
65% yield)
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Note: The aldol condensation was also performed on a 3.5 g scale which resulted in a slightly higher isolated yield
of 74%. It is very important to monitor the reaction and not let it run overnight. This gives overꢀcondensation and
therefore lowers isolated yields.
1H NMR (400 MHz, CDCl3) δ 6.60 (d, J = 16.2 Hz, 1H), 6.02 (d, J = 16.3 Hz, 1H), 5.49 (d, J = 5.8 Hz, 1H), 2.26 (s,
3H), 2.13 (d, J = 13.1 Hz, 1H), 1.93 (dt, J = 17.8, 4.7 Hz, 1H), 1.80 – 1.68 (m, 1H), 1.62 – 1.34 (m, 5H), 1.17 (dd, J
= 12.6, 5.1 Hz, 1H), 1.07 (s, 3H), 1.00 (s, 3H), 0.96 – 0.89 (m, 1H), 0.78 (s, 3H), 0.72 (d, J = 6.8 Hz, 3H).
13C NMR (101 MHz, CDCl3) δ 198.7, 158.2, 144.7, 129.6, 116.5, 43.7, 43.0, 40.8, 36.7, 36.2, 31.1, 29.7, 29.3, 28.3,
27.4, 22.0, 16.4, 10.3.
HRMS: (ESI+) Calculated mass [M+H]+ C18H29O+ = 261.2213 found: 261.2215. Calculated mass [M+Na]+
C18H28ONa+ = 283.2032, found: 283.2034.
Melting point: 70ꢁ72 °C
4-((1R,2S,8aS)-1,2,5,5-tetramethyl-1,2,3,5,6,7,8,8a-octahydronaphthalen-1-yl)butan-2-one (8):12,13
To a solution of α,βꢁunsaturated ketone 7 (3.4 g, 13.1 mmol) in dry (CH2)2Cl2 (150 mL) were added Wilkinson's
catalyst (909 mg, 0.98 mmol, 7.5 mol%) and triethylsilane (10.4 mL, 65.5 mmol, 5 eq). The reaction mixture was
heated to reflux and stirred for 90 minutes after which TLC indicated complete conversion of the starting material.
The reaction was cooled to rt where after it was quenched with an aqueous solution of HCl (6 M, 100 mL). The
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mixture was stirred for 30 minutes after which the phases were separated and the organic layer was washed with
water, followed by a saturated aqueous solution of NaHCO3 and brine. The organic layer was dried over MgSO4,
filtered and concentrated under reduced pressure. Flash column chromatography employing pentane:ether (95:5)
afforded pure 4ꢁ((1R,2S,8aS)ꢁ1,2,5,5ꢁtetramethylꢁ1,2,3,5,6,7,8,8aꢁoctahydronaphthalenꢁ1ꢁyl)butanꢁ2ꢁone 8 (3.1 g,
11.7 mmol, 90% yield) as a slightly yellow oil.
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1H NMR (400 MHz, CDCl3) δ 5.41 (s, 1H), 2.37 – 2.28 (m, 2H), 2.14 (s, 3H), 1.99 (d, J = 12.5 Hz, 1H), 1.88 – 1.60
(m, 4H), 1.60 – 1.32 (m, 6H), 1.17 (td, J = 13.0, 4.4 Hz, 1H), 1.04 (s, 3H), 0.98 (s, 3H), 0.78 (d, J = 6.8 Hz, 3H),
0.63 (s, 3H).
13C NMR (101 MHz, CDCl3) δ 209.6, 145.9, 116.3, 41.0, 40.1, 37.8, 36.7, 36.2, 33.6, 31.6, 30.2, 30.1, 29.8, 29.0,
27.6, 22.3, 16.0, 15.1.
ethyl
(9):13
3-methyl-5-((1R,2S,8aS)-1,2,5,5-tetramethyl-1,2,3,5,6,7,8,8a-octahydronaphthalen-1-yl)pent-2-enoate
ethyl 2ꢁ(diethoxyphosphoryl)acetate (6.96 mL, 35.1 mmol, 3 eq) in dry THF (50 mL) was added dropwise to a
suspension of sodium hydride (1.4 g, 60% in oil, 35.1 mmol, 3 eq) in dry THF (50 mL) at 0 °C by the aid of a
dropping funnel. The suspension turned into a clear solution and was stirred for 20 min after which ketone 8 (3.1 g,
11.7 mmol) in dry THF (100 mL) was added dropwise over 10 minutes by the aid of a dropping funnel. The cooling
bath was removed and the mixture was allowed to warm to rt. The reaction vessel was then sealed under N2 and
placed in an oil bath at 80 °C and was allowed to stir for 6 h. TLC and GC/MS indicated full conversion of the
starting material.
The reaction was cooled to rt and quenched with saturated aqueous NH4Cl. The reaction mixture was diluted with
ether and the phases were separated. The aqueous phases was extracted with ether were after the combined organic
layers were dried over MgSO4, filtered and concentrated under reduced pressure to afford a yellow oil.
Flash column chromatography employing pentane:ether (95:5) afforded crude ethyl 3ꢁmethylꢁ5ꢁ((1R,2S,8aS)ꢁ1,2,5,5ꢁ
tetramethylꢁ1,2,3,5,6,7,8,8aꢁoctahydronaphthalenꢁ1ꢁyl)pentꢁ2ꢁenoate 9 (4.0 g, 12.0 mmol, 103%) as an E/Z mixture
of ~9:1.
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Note: The sodium hydride was preꢀwashed three times with pentane to remove the oil.
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1H NMR (400 MHz, CDCl3) δ 5.67 (s, 1H), 5.45 – 5.40 (m, 1H), 4.13 (q, J = 7.1 Hz, 2H), 2.17 (s, 3H), 2.13 (d, J =
13.1 Hz, 1H), 2.03 (dt, J = 10.1, 4.4 Hz, 2H), 1.90 – 1.67 (m, 4H), 1.65 – 1.44 (m, 5H), 1.44 – 1.34 (m, 2H), 1.26 (d,
J = 6.9 Hz, 2H), 1.22 – 1.14 (m, 1H), 1.05 (s, 3H), 0.99 (s, 3H), 0.81 (d, J = 6.7 Hz, 3H), 0.63 (s, 3H).
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13C NMR (101 MHz, CDCl3) δ 166.9, 161.3, 146.0, 116.3, 115.3, 59.5, 41.0, 40.0, 37.2, 36.2, 34.7, 34.6, 33.5, 31.7,
29.9, 29.1, 27.6, 22.3, 19.2, 16.3, 15.2, 14.5.
(E)-3-methyl-5-((1R,2S,8aS)-1,2,5,5-tetramethyl-1,2,3,5,6,7,8,8a-octahydronaphthalen-1-yl)pent-2-en-1-ol
(Tuberculosinol):12,13
To a solution of crude enoate 9 (4.0 g, 12.0 mmol) in dry CH2Cl2 (75 mL) cooled to ꢁ78 °C was added dropwise
DIBALꢁH (36.0 mL, 1.0 M in CH2Cl2, 36.0 mmol, 3 eq) over 15 min by the aid of a dropping funnel. The cooling
bath was removed and the reaction was allowed to slowly warm up to rt. TLC and GC/MS indicated complete
conversion of the starting material after 1 h of reaction time.
The reaction mixture was carefully quenched using MeOH (10 mL) where after saturated aqueous Rochelle salt
solution (75 mL) was added at 0 °C. The mixture was stirred for 30 min while warming up to rt where after the
phases were separated. The aqueous phase was extracted three times with CH2Cl2 and the combined organic layers
were dried over MgSO4, filtered and concentrated under reduced pressure to afford a yellowish oil. Flash column
chromatography was executed using pentane:ether (4:1) as the eluent, affording pure (E)ꢁ3ꢁmethylꢁ5ꢁ((1R,2S,8aS)ꢁ
1,2,5,5ꢁtetramethylꢁ1,2,3,5,6,7,8,8aꢁoctahydronaphthalenꢁ1ꢁyl)pentꢁ2ꢁenꢁ1ꢁol (2.33 g, 8.0 mmol, 68% yield) as a
colorless oil.
A mixture of (E)ꢁtuberculosinol and (Z)ꢁtuberculosinol was also obtained (0.9 g). This was subjected to an additional
chromatographic purification (pentane:ether = 5:1), affording 460 mg of (E)ꢁtuberculosinol, giving a combined yield
of 82%.
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Also (Z)ꢁtuberculosinol (0.4 g, 1.4 mmol, 11%) was isolated as a colorless oil.
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Analytical data for (E)ꢁtuberculosinol:
1H NMR (400 MHz, CDCl3) δ 5.43 – 5.33 (m, 2H), 4.08 (d, J = 6.8 Hz, 2H), 2.57 (s, 1H), 2.13 (d, J = 12.6 Hz, 1H),
1.92 – 1.85 (m, 2H), 1.81 – 1.67 (m, 3H), 1.65 (s, 3H), 1.59 – 1.28 (m, 6H), 1.28 – 1.10 (m, 1H), 1.02 (s, 3H), 0.97
(s, 3H), 0.78 (d, J = 6.8 Hz, 3H), 0.59 (s, 3H).
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13C NMR (101 MHz, CDCl3) δ 146., 140.1, 123.1, 116.1, 59.1, 40.9, 39.8, 36.9, 36.1, 35.0 33.4, 32.8, 31.7, 29.8,
29.0, 27.4, 22.3, 16.5, 16.2, 15.1.
HRMS: (ESI+) Calculated mass [M+Na+]+ C20H34ONa+ = 313.2502, found: 313.2500
Optical rotation: [α]D23 = +40.1 (c = 0.024, EtOH) and [α]D23 = +39.5 (c = 0.013, CHCl3).
Analytical data for (Z)ꢁtuberculosinol:
1H NMR (400 MHz, CDCl3) δ 5.46 – 5.42 (m, 1H), 5.39 (t, J = 6.9 Hz, 1H), 4.13 (d, J = 7.0 Hz, 2H), 2.21 (d, J =
13.2 Hz, 1H), 1.99 (ddp, J = 17.4, 12.6, 5.3 Hz, 3H), 1.91 – 1.79 (m, 1H), 1.76 (s, 3H), 1.67 – 1.47 (m, 4H), 1.47 –
1.15 (m, 6H), 1.07 (s, 3H), 1.02 (s, 3H), 0.85 (d, J = 6.7 Hz, 3H), 0.62 (s, 3H).
13C NMR (101 MHz, CDCl3) δ 145.8, 139.9, 123.9, 116.0, 58.7, 40.9, 39.7, 37.0, 36.0, 35.3, 33.2, 31.6, 29.7, 29.0,
27.5, 25.4, 23.6, 22.2, 16.0, 15.1.
(4aS,5R,6S)-5-((E)-5-chloro-3-methylpent-3-en-1-yl)-1,1,5,6-tetramethyl-1,2,3,4,4a,5,6,7-
octahydronaphthalene (10):
To a suspension of 1ꢁchloropyrrolidineꢁ2,5ꢁdione (1.0 g, 7.61 mmol, 1.3 eq) in dry CH2Cl2 (15 mL), cooled to ꢁ20
°C, was added dropwise dimethyl sulfide (0.65 mL, 8.78 mmol, 1.5 eq) in dry CH2Cl2 (5.0 mL). The milk white
suspension was allowed to warm to 0 °C for 15 min after which the temperature was lowered to ꢁ40 °C.
Tuberculosinol (1.7 g, 5.85 mmol) in dry CH2Cl2 (20 mL) was added slowly by the aid of a syringe pump over 15
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min. After addition, the cooling bath was removed and the reaction was allowed to warmꢁup to rt. The reaction was
allowed to stir at this temperature for 2 h after which TLC and GC/MS analysis indicated complete conversion of the
tuberculosinol. The reaction mixture was concentrated under reduced pressure and treated with pentane upon which
succinimide oiled out. The mixture was decanted and filtered and the elute was concentrated under reduced pressure
affording nearly pure (4aS,5R,6S)ꢁ5ꢁ((E)ꢁ5ꢁchloroꢁ3ꢁmethylpentꢁ3ꢁenꢁ1ꢁyl)ꢁ1,1,5,6ꢁtetramethylꢁ1,2,3,4,4a,5,6,7ꢁ
octahydronaphthalene 10 (1.85 g, 6.0 mmol, >99% yield) as a yellow oil contaminated only with DMSO formed
during the reaction.
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Analytical data for (E)ꢁtuberculosinyl chloride:
1H NMR (400 MHz, CDCl3) δ 5.51 – 5.41 (m, 2H), 4.09 (d, J = 7.9 Hz, 2H), 2.16 (d, J = 12.8 Hz, 1H), 1.96 (dt, J =
10.4, 4.6 Hz, 2H), 1.83 (ddd, J = 17.7, 14.5, 3.5 Hz, 2H), 1.75 (s, 3H), 1.72 (s, 1H), 1.63 – 1.25 (m, 7H), 1.20 (td, J =
12.9, 4.8 Hz, 1H), 1.06 (s, 3H), 1.01 (s, 3H), 0.82 (d, J = 6.8 Hz, 3H), 0.63 (s, 3H).
13C NMR (101 MHz, CDCl3) δ 146.1, 144.0, 119.9, 116.3, 41.3, 41.1, 39.9, 37.1, 36.2, 34.8, 33.5, 32.9, 31.8, 29.9,
29.1, 27.6, 22.4, 16.5, 16.3, 15.2.
HRMS: (APCI) Calculated mass [MꢁClꢁ]+ C20H33+ = 273.2582, found: 273.2576
Analytical data for (Z)ꢁtuberculosinyl chloride:
1H NMR (400 MHz, CDCl3) δ 5.46 – 5.42 (m, 1H), 5.39 (t, J = 6.9 Hz, 1H), 4.13 (d, J = 7.0 Hz, 2H), 2.21 (d, J =
13.2 Hz, 1H), 1.99 (ddp, J = 17.4, 12.6, 5.3 Hz, 3H), 1.91 – 1.79 (m, 1H), 1.76 (s, 3H), 1.67 – 1.47 (m, 4H), 1.47 –
1.15 (m, 6H), 1.07 (s, 3H), 1.02 (s, 3H), 0.85 (d, J = 6.7 Hz, 3H), 0.62 (s, 3H).
13C NMR (101 MHz, CDCl3) δ 145.8, 139.9, 123.9, 116.0, 58.7, 40.9, 39.7, 37.1, 36.0, 35.3, 33.2, 31.6, 29.7, 29.0,
27.5, 25.4, 23.6, 22.2, 16.0, 15.1.
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6-amino-9-((2R,3R,4S,5R)-3,4-dihydroxy-5-(hydroxymethyl)tetrahydrofuran-2-yl)-1-((E)-3-methyl-5-
((1R,2S,8aS)-1,2,5,5-tetramethyl-1,2,3,5,6,7,8,8a-octahydronaphthalen-1-yl)pent-2-en-1-yl)-9H-purin-1-ium (1-
TbAd):6
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To a solution (0.5 M) of nearly pure tuberculosinyl chloride 10 (1.8 g, 5.8 mmol) in peptide grade DMF (11.6 mL)
was added sodium iodide (1.05 g, 7.0 mmol, 1.2 eq) and adenosine (1.87 g, 7.0 mmol, 1.2 eq). The suspension was
stirred in the dark at rt overnight, forming a dark turbid solution. The reaction mixture was concentrated under
reduced pressure and subsequently purified using flash column chromatography (15% MeOH in CH2Cl2) affording
1ꢁTbAd (2.4 g, 4.43 mmol, 76% yield)
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The reaction can also be performed in dimethylacetamide as the solvent. Starting from tuberculosinyl chloride 10
(0.4 g, 1.29 mmol), 1ꢁTbAd (560 mg, 1.04 mmol, 79% yield) was obtained.
Note: 1ꢀTbAd proved to be difficult to separate from unreacted adenosine due to tailing of the 1ꢀTbAd. It is
1
recommended to use a long column (30 cm) and analyze individual fractions by HꢀNMR analysis to determine the
presence of free adenosine. Visualization of the adenosine on a TLC plate proved to be difficult.
Analytical data for naturally occurring 1ꢀTbAd:
1H NMR (400 MHz, CD3OD) δ 8.62 (s, 1H), 8.49 (s, 1H), 6.08 (d, J = 5.2 Hz, 1H), 5.51 – 5.42 (m, 2H), 4.91 (d, J =
6.6 Hz, 2H), 4.62 (t, J = 5.1 Hz, 2H), 4.58 (s, 1H), 4.38 – 4.31 (m, 2H), 4.15 (q, J = 3.3 Hz, 1H), 3.87 (dd, J = 12.3,
2.9 Hz, 1H), 3.77 (dd, J = 12.2, 3.4 Hz, 1H), 2.23 (d, J = 12.1 Hz, 2H), 2.12 – 2.04 (m, 2H), 1.89 (s, 3H), 1.87 – 1.82
(m, 1H), 1.84 – 1.71 (m, 2H), 1.65 – 1.47 (m, 5H), 1.47 – 1.36 (m, 3H), 1.21 (ddd, J = 12.7, 5.7 Hz, 1H), 1.06 (s,
3H), 1.01 (s, 3H), 0.85 (d, J = 6.7 Hz, 3H), 0.66 (s, 3H).
13C NMR (101 MHz, CD3OD) δ 152.5, 147.8, 147.6, 147.3, 147.1, 143.7, 121.5, 117.3, 115.9, 90.4, 87.4, 76.4, 71.8,
62.6, 49.4, 42.0, 41.1, 38.1, 36.9, 35.9, 34.5, 33.9, 32.6, 30.3, 29.5, 28.5, 23.2, 17.4, 16.6, 15.5.
HRMS: (ESI+) Calculated mass [M]+ C30H46N5O4+ = 540.3544, found: 540.3542.
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Analytical data for nonꢀnatural (Z)ꢀ1ꢀTbAd constructed from (Z)ꢁtuberculosinyl chloride:
1H NMR (400 MHz, CD3OD) δ 8.56 (2x s, 2H), 8.41 (2x s, 1H), 6.09 – 6.02 (m, 1H), 5.44 (dt, J = 23.8, 6.5 Hz, 2H),
4.88 (d, J = 6.6 Hz, 2H), 4.61 (t, J = 4.6 Hz, 1H), 4.33 (t, J = 4.3 Hz, 1H), 4.14 (d, J = 3.1 Hz, 1H), 3.91 – 3.82 (m,
1H), 3.76 (dd, J = 12.8, 2.5 Hz, 1H), 2.32 – 2.14 (m, 2H), 2.07 (t, J = 8.2 Hz, 1H), 1.88 (s, 3H), 1.85 – 1.71 (m, 3H),
1.65 – 1.47 (m, 5H), 1.43 (dd, J = 14.6, 8.6 Hz, 2H), 1.22 (dd, J = 12.4, 6.2 Hz, 1H), 1.06 (s, 3H), 1.02 (2x s, 3H),
0.86 (2x d, J = 6.8 Hz, 3H), 0.66 (2x s, J = 10.9 Hz, 3H).
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13C NMR (101 MHz, CD3OD) δ 152.7, 152.5, 147.8, 147.6, 147.3, 147.1, 147.0, 143.6, 143.4, 121.6, 121.5, 117.2,
117.2, 116.5, 116.0, 90.4, 87.4, 76.2, 71.7, 62.6, 42.0, 41.9, 41.0, 38.2, 38.0, 36.9, 36.9, 35.8, 34.4, 34.3, 33.8, 32.6,
30.3, 29.5, 28.7, 28.5, 26.8, 23.9, 23.2, 23.1, 17.5, 16.6, 16.6, 15.8, 15.6.
HRMS: (ESI+) Calculated mass [M]+ C30H46N5O4+ = 540.3544, found: 540.3542.
Note: The appearance of additional signals in the NMR spectra of (Z)ꢁ1ꢀTbAd, compared to that of naturally
occurring (E)ꢁ1ꢀTbAd, is attributed to rotamers.
13C-labelled 1-TbAd:
To a solution (0.5 M) of nearly pure tuberculosinyl chloride (142 mg, 0.459 mmol, 1.25 eq) in peptide grade DMF
(0.92 mL) was added sodium iodide (69 mg, 0.459 mmol, 1.25 eq) and [1’,2’,3’,4’,5’ꢁ13C5] adenosine (100 mg, 0.367
mmol). The suspension was stirred in the dark at rt overnight, forming a dark turbid solution. The reaction mixture
was concentrated under reduced pressure where after purified using flash column chromatography (15% MeOH in
CH2Cl2) affording [1’,2’,3’,4’,5’ꢁ13C5] 1ꢁTbAd (100 mg, 0,18 mmol, 57% yield) as an offꢁwhite solid.
1H NMR (400 MHz, CD3OD) δ 8.56 (s, 1H), 8.42 (s, 1H), 6.26 (s, 1H), 5.84 (s, 1H), 5.52 – 5.40 (m, 2H), 4.58 (s,
1H), 4.51 (s, 1H), 4.42 (s, 1H), 4.33 (s, 1H), 4.14 (s, 1H), 4.04 (d, J = 11.4 Hz, 1H), 3.94 (d, J = 9.7 Hz, 1H), 3.77 –
3.63 (m, 1H), 3.59 (d, J = 12.0 Hz, 1H), 2.22 (d, J = 13.3 Hz, 2H), 2.07 (t, J = 8.3 Hz, 2H), 1.88 (s, 3H), 1.86 – 1.70
(m, 3H), 1.65 – 1.47 (m, 4H), 1.47 – 1.35 (m, 2H), 1.21 (td, J = 12.4, 5.5 Hz, 1H), 1.06 (s, 3H), 1.01 (s, 3H), 0.84 (d,
J = 6.7 Hz, 3H), 0.65 (s, 3H).
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13C NMR (101 MHz, CD3OD) δ 152.6, 147.8, 147.3, 147.1, 147.0, 143.6, 121.6, 117.3, 116.0, 90.4 (dd, J = 42.0, 3.7
Hz), 87.4 (t, J = 41.7, 38.5 Hz), 76.2 (dd, J = 42.0, 37.8 Hz), 71.7 (td, J = 38.1, 3.8 Hz), 62.6 (d, J = 41.7 Hz), 49.9,
42.0, 41.0, 38.0, 36.9, 35.8, 34.5, 33.8, 32.6, 30.3, 29.5, 28.5, 23.1, 17.4, 16.6, 15.5.
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HRMS: (ESI+) Calculated mass [M+H]+ C25(13C)5H46N5O4+ = 545.3712 found: 545.3702.
(2R,3S,4R,5R)-2-(hydroxymethyl)-5-(6-(((E)-3-methyl-5-((1R,2S,8aS)-1,2,5,5-tetramethyl-1,2,3,5,6,7,8,8a-
octahydronaphthalen-1-yl)pent-2-en-1-yl)amino)-9H-purin-9-yl)tetrahydrofuran-3,4-diol (N6-TbAd):7
A solution of 1ꢁTbAd (550 mg, 1.02 mmol) in 60% Me2NH in water (5.5 mL) was stirred for 90 min. NMR analysis
indicated complete conversion of the 1ꢁTbAd. The reaction mixture was concentrated under reduced pressure and
subsequently subjected to flash column chromatography, with 15% MeOH in CH2Cl2, to afford N6ꢁTbAd as a white
solid (550 mg, 1.02 mmol, quantitative yield) as yellowish oil.
Note: The rearrangement could be performed with similar results using Et2NH or iPr2NEt (2 M) in MeOH.
1H NMR (400 MHz, CD3OD) δ 8.25 (s, 1H), 8.23 (s, 1H), 5.95 (d, J = 6.4 Hz, 1H), 5.51 – 5.44 (m, 1H), 5.41 (t, J =
6.5 Hz, 1H), 4.74 (t, J = 5.6 Hz, 1H), 4.35 – 4.29 (m, 1H), 4.20 (s, 1H), 4.17 (s, 1H), 3.89 (dd, J = 12.6, 2.1 Hz, 1H),
3.74 (dd, J = 12.9, 2.3 Hz, 1H), 2.24 (d, J = 15.9 Hz, 1H), 2.07 – 1.93 (m, 3H), 1.91 – 1.82 (m, 2H), 1.80 (s, 3H),
1.65 – 1.49 (m, 4H), 1.49 – 1.36 (m, 3H), 1.21 (td, J = 11.9, 7.3 Hz, 2H), 1.06 (s, 3H), 1.02 (s, 3H), 0.85 (d, J = 6.6
Hz, 3H), 0.65 (s, 3H).
1H NMR (400 MHz, CDCl3) δ 8.19 (s, 1H), 7.81 (s, 1H), 5.86 (bs, 1H), 5.82 (d, J = 7.2 Hz, 1H), 5.44 (d, J = 4.7 Hz,
1H), 5.36 (d, J = 7.3 Hz, 1H), 5.00 (s, 1H), 4.47 (d, J = 4.9 Hz, 1H), 4.33 (s, 1H), 4.19 (bs, 2H), 3.94 (d, J = 12.9 Hz,
1H), 3.76 (d, J = 12.9 Hz, 1H), 3.23 (s, 2H), 2.49 (bs, 2H), 2.16 (d, J = 10.4 Hz, 1H), 2.01 – 1.90 (m, 2H), 1.85 (d, J
= 23.8 Hz, 2H), 1.76 (s, 3H), 1.72 (s, 1H), 1.64 – 1.28 (m, 6H), 1.21 (tt, J = 13.0, 6.2 Hz, 2H), 1.06 (s, 3H), 1.01 (s,
3H), 0.82 (d, J = 6.6 Hz, 3H), 0.63 (s, 3H).
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13C NMR (101 MHz, CDCl3) δ 154.6, 152.4, 147.0, 146.1, 141.7, 140.1, 120.7, 119.0, 116.2, 91.1, 87.6, 73.9, 72.5,
63.2, 41.0, 39.9, 38.9, 37.0, 36.2, 35.0, 33.5, 32.8, 31.7, 29.9, 29.1, 27.5, 22.3, 16.8, 16.3, 15.2.
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HRMS: (ESI+) Calculated mass [M+H]+ C30H46N5O4+ = 540.3544 found: 540.3542.
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6-amino-9-((2R,4S,5R)-4-hydroxy-5-(hydroxymethyl)tetrahydrofuran-2-yl)-1-((E)-3-methyl-5-((1R,2S,8aS)-
1,2,5,5-tetramethyl-1,2,3,5,6,7,8,8a-octahydronaphthalen-1-yl)pent-2-en-1-yl)-9H-purin-1-ium (2’-deoxy 1-
TbAd):
To a solution (0.5 M) of nearly pure tuberculosinyl chloride 10 (300 mg, 0.97 mmol) in peptide grade DMA (2 mL)
was added sodium iodide (175 mg, 1.17 mmol, 1.2 eq) and 2’ꢁdeoxyꢁadenosine monohydrate (314 mg, 1.17 mmol,
1.2 eq). The suspension was stirred in the dark at rt overnight, forming a dark turbid solution. The reaction mixture
was concentrated under reduced pressure and subsequently purified using flash column chromatography (15%
MeOH in CH2Cl2) affording 2’ꢁdeoxy 1ꢁTbAd (190 mg, 0.36 mmol, 37% yield) as a white solid.
Also a slightly impure fraction of 2’ꢁdeoxy 1ꢁTbAd (150 mg, 0.29 mmol, 31%) was obtained. The impurity, based on
NMR analysis, remained unresolved.
1H NMR (400 MHz, CDCl3) δ 8.54 (s, 1H), 8.44 (s, 1H), 6.46 (t, J = 6.5 Hz, 1H), 5.46 (s, 2H), 4.90 (d, J = 6.5 Hz,
2H), 4.57 (s, 1H), 4.09 – 4.01 (m, 1H), 3.77 (qd, J = 12.1, 3.5 Hz, 2H), 2.76 (dt, J = 13.0, 6.3 Hz, 1H), 2.56 – 2.44
(m, 1H), 2.21 (d, J = 12.5 Hz, 2H), 2.11 – 1.99 (m, 2H), 1.89 (s, 3H), 1.86 – 1.70 (m, 3H), 1.54 (d, J = 12.3 Hz, 4H),
1.42 (dd, J = 17.1, 8.8 Hz, 2H), 1.19 (td, J = 12.3, 5.8 Hz, 1H), 1.05 (s, 3H), 1.00 (s, 3H), 0.83 (d, J = 6.6 Hz, 3H),
0.64 (s, 3H).
13C NMR (101 MHz, CDCl3) δ 152.5, 147.7, 147.05, 146.99, 146.95, 143.5, 121.4, 117.2, 116.1, 89.5, 86.3, 72.3,
63.0, 49.9, 42.0, 41.8, 41.0, 38.0, 36.9, 35.8, 34.5, 33.9, 32.6, 30.3, 29.5, 28.5, 23.1, 17.5, 16.6, 15.6.
HRMS: (ESI+) Calculated mass [M]+ C30H46N5O3+ = 524.3595, found: 524.3588.
ASSOCIATED CONTENT
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The associated analytical data (1HꢁNMR, 13CꢁNMR and APT spectra for all compounds, CIDꢁMS of 2’ꢁdeoxy 1ꢁ
TbAd, crystallographic, and computational data) can be found in the supporting information. This material is
available free of charge via the Internet at http://pubs.acs.org.
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AUTHOR INFORMATION
Corresponding Author
* Correspondence can be addressed to Prof. Dr. Adriaan J. Minnaard – a.j.minnaard@rug.nl
Author Contributions
All authors have given approval to the final version of the manuscript. D. Heijnen and I. C. Wan contributed equally.
Notes
The authors hereby declare no competing financial interests.
ACKNOWLEDGMENTS
A.J.M. acknowledges The Netherlands Organization for Scientific Research (NWOꢁCW) for a VICI grant. Mrs
Tiemersma is kindly acknowledged for HRMS analysis. Dr. J. Poater, and Dr. R. W. A. Havenith are acknowledged
for their input regarding the computational chemistry ꢀ.
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