Synthesis and Stereochemical Assignment of Conioidine A: DNA- And HSA-Binding Studies of the Four Diastereomers

Article abstract of DOI:10.1021/acs.jnatprod.0c00871

Conioidine A (1), isolated in 1993 with unknown relative and absolute configuration, was suggested to be a DNA-binding compound by an indirect technique. Four stereoisomers of conioidine A have been synthesized from d- and l-proline, and the natural product has been identified as possessing (4R,6R) absolute configuration. Binding of the conioidine diastereomers to calf thymus DNA (CT DNA) and human serum albumin (HSA) has been investigated by fluorescence spectroscopy and isothermal titration calorimetry (ITC). All stereoisomers display at least an order of magnitude weaker binding to DNA than the control compound netropsin; however, a strong association with HSA was observed for the (4R,6S) stereoisomer.

Full text of DOI:10.1021/acs.jnatprod.0c00871

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Article
Synthesis and Stereochemical Assignment of Conioidine A: DNA-
and HSA-Binding Studies of the Four Diastereomers
Ryan Shaktah, Laura Vardanyan, Elroma David, Alexis Aleman, Dupre Orr, Lawrence A. Shaktah,
Daniel Tamae, and Thomas Minehan*
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ABSTRACT: Conioidine A (1), isolated in 1993 with unknown relative and absolute
configuration, was suggested to be a DNA-binding compound by an indirect technique.
Four stereoisomers of conioidine A have been synthesized from D- and L-proline, and
the natural product has been identified as possessing (4R,6R) absolute configuration.
Binding of the conioidine diastereomers to calf thymus DNA (CT DNA) and human
serum albumin (HSA) has been investigated by fluorescence spectroscopy and
isothermal titration calorimetry (ITC). All stereoisomers display at least an order of
magnitude weaker binding to DNA than the control compound netropsin; however, a
strong association with HSA was observed for the (4R,6S) stereoisomer.
onioidines A and B are pyrrolidine natural products
approximately micromolar affinity. Unlike most naturally
occurring substances that interact with DNA,2 the conioidines
lack aromatic ring systems that are typically important
structural elements for tight binding to nucleic acids. It has
been recently demonstrated that leinamycin, a natural product
that lacks a polycyclic aromatic unit, binds DNA by
intercalation of a (Z,E)-penta-2,4-dienone moiety in the base
pair stack.3 Furthermore, intercalation of hydrophobic amino
acid side chains between base pairs is an important binding
element in numerous DNA−protein complexes.4 To elucidate
the absolute configuration of the conioidines and evaluate their
DNA-binding profile, we undertook the synthesis of all four
diastereomers of conioidine A.
C
isolated from the Texas plant Chamaesaracha conioides by
Chan and co-workers in 1993.1 The gross structure of each
compound was established by means of 1D and 2D NMR
spectroscopy; however, the relative and absolute configurations
of both structures were not elucidated (Figure 1). Intriguingly,
cytotoxicity assays in the presence and absence of exogenous
DNA suggested that both compounds bind DNA with
RESULTS AND DISCUSSION
■
The syntheses commenced from L-proline or D-proline by
reduction with LiAlH4 in THF according to the procedure of
Pericas5 to provide the corresponding prolinols (4S)-3 and
(4R)-3 (Scheme 1). Protection of the pyrrolidine nitrogen
atom as the tert-butyl carbamate followed by tosylation of the
primary hydroxy group and cyano group displacement gave
rise to nitriles (4S)-4 and (4R)-4 in 60% and 75% yields,
Received: August 4, 2020
Figure 1. Four possible diastereomers of conioidines A and B.
© XXXX American Chemical Society and
American Society of Pharmacognosy
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Scheme 1. Synthesis of the Four Diastereomers of Conioidine A
respectively.6 Hydrolysis of the cyano groups under basic
conditions provided the intermediate carboxylic acids, which
were immediately converted to the corresponding Weinreb
amides (4R)-5 and (4S)-5 in 81% and 62% yields, respectively.
Treatment of amides 5 with CH3MgBr in THF rapidly
afforded methyl ketones (4S)-6 (68%) and (4R)-6 (55%).
Davies7a has shown that stereoselective reduction of ketones 6
may be performed either with LiAlH(OtBu)3 in THF at 0 °C
or with Zn(BH4)2 in THF at 0 °C. Reduction of (4S)-6 and
(4R)-6 with Zn(BH4)2 afforded the alcohols (4S,6S)-7 (63%)
and (4R,6R)-7 (58%) (dr = 3:1; both separable from the
(4S,6R) and (4R,6S) diastereomers, respectively, by silica gel
chromatography).7b In contrast, reduction of ketones (4S)-6
and (4R)-6 with LiAlH(Ot-Bu)3 gave rise to alcohols (4S,6R)-
8 (91%) and (4R,6S)-8 (83%) with >20:1 diastereoselection in
each case. Cleavage of the Boc group of (4S,6S)-7, (4R,6R)-7,
(4S,6R)-8, and (4R,6S)-8 with 1:1 TFA/CH2Cl2 and reaction
of the corresponding amino alcohol intermediates with
decanoyl chloride in the presence of Et3N and CH2Cl2 for 8
h at room temperature furnished the corresponding
intermediate amides, which were immediately exposed to 3
equiv of 2,3-dimethylacryloyl chloride in the presence of Et3N
(6 equiv) and DMAP (3 equiv) in CH2Cl2 at room
temperature overnight to afford the four conioidine A
diastereomers (4S,6S)-1, (4R,6R)-1, (4S,6R)-1, and (4R,6S)-
1 in 43%, 29%, 51%, and 56% overall yields from 7 or 8. On
the basis of comparison of the H and 13C NMR and specific
rotation data for all four diastereomers with the values
recorded for the natural product, we conclude that natural
conioidine A possesses the (4R,6R) absolute configuration (see
Supporting Information).
1
To assess the strengths of binding of the four diastereomers
of conioidine A to calf thymus (CT) DNA, the competitive
ethidium displacement technique was employed to obtain C50
values (the concentration of ligand required to achieve a 50%
decrease in the fluorescence of ethidium bromide) and
apparent association constants (Kapp).8 Titration of CT DNA
(10 μM) and ethidium bromide (10 μM) with (4R,6R)-
conioidine A resulted in a negligible effect on ethidium
fluorescence intensity (measured at 590 nm) over the 0.01−50
μM ligand concentration range (pH = 6.81, Tris-HCl buffer);
indeed, 93% of the initial ethidium fluorescence was measured
at 50 μM of (4R,6R)-1 (Figure 2a), and extrapolation of the
data gave a C50 value of 480 μM (estimated Kapp = 1.8 × 105
M−1 bp−1). Similar results were obtained in ethidium
displacement studies employing (4S,6S)-1, (4R,6S)-1, and
(4S,6R)-1 (C50 = 470, 340, and 380 μM, respectively). Under
otherwise identical experimental conditions, the positive
control netropsin produced a significant decrease in ethidium
emission fluorescence intensity with a directly measurable C50
value of 10 2.5 μM (Kapp = (5.1 0.3) × 106 M−1 bp−1; see
Figure 2b); in contrast, the negative control dextrose showed
B
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Figure 2. (a) Displacement of ethidium bromide from CT DNA by (4R,6R)-1. (b) Plot of % relative fluorescence vs ligand concentration for
(4R,6R)-1, (4S,6S)-1, (4R,6S)-1, (4S,6R)-1, and control compound netropsin binding to CT DNA with displacement of ethidium bromide.
minimal displacement of ethidium from CT DNA with an
extrapolated C50 value of 390 μM. Furthermore, thermal
denaturation studies9 of CT DNA employing UV spectroscopy
showed minimal differences (ΔTM = 0.7 °C; see Supporting
Information) in the helix melting temperature in the presence
or absence of the four diastereomers of 1, whereas the same
experiment performed with netropsin showed strong helix
stabilization (ΔTM = +10.3 °C). These results clearly indicate
that the conioidines interact relatively weakly with duplex CT
DNA.
In light of these data, we explored the possibility that this
family of natural products may form complexes with proteins.
Numerous hydrophobic organic molecules are known to
associate with the serum albumins, and binding to these
proteins typically influences the apparent solubility, distribu-
tion, metabolism, and efficacy of a wide range of drugs.10,19
Titration of the abundant plasma protein human serum
albumin11 (HSA; N form at pH 6.81, Tris-HCl buffer; 10
μM) with (4R,6R)-1 over the 0.5−10 μM12 range showed a
significant quenching of the Trp 214 fluorescence emission at
338 nm (λex = 280 nm; KSV = (7.2 0.4) × 104 M−1, Figure
3a,b).13,14 Analogous experiments with the (4S,6S)-1, (4R,6S)-
1, and (4S,6R)-1 stereoisomers revealed that compound
(4R,6S)-1 bound tightest to HSA (KSV = (1.3 0.1) × 105
M−1), while both (4S,6S)-1 and (4S,6R)-1 had similar HSA
binding affinities to (4R,6R)-1 and the positive control
compound virstatin (KSV = (3.0
0.8) × 104 M−1; see
Supporting Information and Table 1).
ITC titration15 of HSA (57 μM) with (4R,6R)-1 (100 μM
solution, 25 °C, pH = 6.81, Tris-HCl buffer, Figure 3c) gave a
binding constant Kb of (6.4 0.2) × 104 M−1, along with an
enthalpy of binding, ΔH, of −1.9
0.3 kcal/mol and an
entropy of binding, ΔS, of 15.5 0.7 cal/mol·K. ITC titration
of HSA with (4R,6S)-1 gave a binding constant Kb of (1.6
0.3) × 105 M−1, along with an enthalpy of binding, ΔH, of
−1.6 0.1 kcal/mol and an entropy of binding, ΔS, of 18.2
0.8 cal/mol·K. The close agreement of KSV and Kb values
obtained from fluorescence and ITC experiments, respectively,
again confirms that compound (4R,6S)-1 is the tightest binder
of HSA. It should be noted that for all diastereomers except
(4S,6R)-1 the magnitude of the entropy of binding (−TΔS
term) exceeds the magnitude of the enthalpy of binding (ΔH),
suggesting that the hydrophobic effect may play an important
role in the association of these compounds with HSA.26
Competition binding experiments employing warfarin (site I
marker, Kb = 2.8 × 105 M−1)21 or ibuprofen (site II marker, Kb
= 2.9 × 106 M−1)22 and the diastereomers of 1 are currently
underway to clarify which binding site (Sudlow site I or
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Figure 3. (a) Fluorescence quenching at 339 nm by addition of (4R,6R)-1 to HSA. (b) Stern−Volmer plot for data represented in (a). (c) ITC
trace for the binding of (4R,6R)-1 to HSA.
Table 1. Experimental HSA Binding Data for the Four
Diastereomers of 1
decrease in ellipticity of the negative Cotton effects at 208 and
220 nm characteristic of the α-helical structure of the protein
(Figure 4). These results are consistent with a minor structural
perturbation of HSA upon ligand binding, resulting in a slight
decrease in α-helical content. Highly similar results have been
reported for the binding of caffeine19 and scutellarin20 to HSA.
In summary, the relative and absolute configurations of
conioidine A [(4R,6R)-1] have been determined through
chemical synthesis of the four possible diastereomers and
comparison of their spectroscopic data with those reported for
the natural product. In comparison to the known minor groove
binding agent netropsin, the four diastereomers of conioidine
A are relatively weak DNA-binding compounds; in contrast,
the compounds show good affinity for HSA, with the (4R,6S)
diastereomer having the highest measured binding constant
a
1
Kb (×104 M−1
)
ΔH (kcal/mol)
ΔS (cal/mol·K)
4S,6R
4R,6S
4S,6S
4R,6R
5.9 1.2
16.0 3.0
7.2 1.4
6.4 0.2
−29.3 0.1
−1.6 0.1
−1.8 0.4
−1.9 0.3
−76.5 0.7
18.2 0.8
16.4 1.3
15.5 0.7
Binding data obtained by isothermal titration calorimetry.15
a
Sudlow site 2) on the protein is preferred by the conioidines.16
Molecular docking studies17 between HSA (PDB 1AO6)18 and
(4R,6R)-1 indicate that in the lowest energy binding mode the
ligand is bound to Sudlow site I in close proximity to the Trp
214 residue (see Supporting Information).
Finally, electronic circular dichrosim (ECD) spectra of HSA
recorded in the 200−260 nm region in the presence of
increasing concentrations of (4R,6S)-1 revealed a marginal
((1.6
0.3) × 105 M−1). Preliminary cell viability
investigations utilizing MTT assays23 suggest that (4R,6R)-1
is cytotoxic toward MCF-7 cells and that the presence of
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Figure 4. The 200−260 nm region of the ECD spectra of solutions of HSA (15 μM) in the absence (light blue line) and presence of various
concentrations of (4R,6S)-1: blue line 5.0 μM (4R,6S)-1; green line 10 μM (4R,6S)-1; red line 15 μM (4R,6S)-1; black line 20 μM (4R,6S)-1.
dissolved in CH2Cl2 (11 mL) and cooled to 0 °C. Carbonyl
diimidazole (856 mg, 5.28 mmol) was added, and the solution was
allowed to stir at room temperature for 30 min. The solution was
cooled to 0 °C, Et3N (1.68 mL, 12 mmol) and N-methyl-N-
methoxyamine hydrochloride (582 mg, 6 mmol) were added, and the
solution was stirred at room temperature overnight. The mixture was
diluted with CH2Cl2 (10 mL) and saturated NaHCO3 solution (20
mL), and the phases were separated. The aqueous layer was extracted
with Et2O (2 × 25 mL). The combined organic extract was washed
once with 20 mL of saturated aqueous NaCl, dried over anhydrous
Na2SO4, and concentrated under reduced pressure. The crude
Weinreb amide (∼4 mmol) was dissolved in THF (10 mL) and
cooled to 0 °C. A solution of CH3MgBr (1.5 mL, 4.5 mmol, 3 M in
Et2O) was added dropwise, and the solution was allowed to stir at
room temperature for 4 h. The reaction mixture was quenched with
saturated NH4Cl solution (10 mL), and Et2O (10 mL) was added.
The phases were separated, and the aqueous layer was extracted with
Et2O (2 × 25 mL). The combined organic extract was washed once
with 20 mL of saturated aqueous NaCl, dried over anhydrous
Na2SO4, and concentrated under reduced pressure. Purification of the
residue by flash chromatography (SiO2, 20:1 → 4:1 hexanes/EtOAc)
afforded ketone (4S)-6 (586.8 mg, 2.6 mmol, 55%) and ketone (4R)-
6 (363 mg, 1.6 mmol, 34%). (4S)-6 (as a mixture of carbamate
exogenous CT DNA attenuates cell killing, in line with the
observations of Chan.1 Full details on these studies will be
reported elsewhere in due course.
EXPERIMENTAL SECTION
■
General Experimental Procedures. All reagents and solvents
were purchased and used without further purification. Distilled water
was used in all of the experiments. Organic extracts were dried over
Na2SO4, filtered, and concentrated using a rotary evaporator at
aspirator pressure (20−30 mm Hg). Chromatography refers to flash
chromatography and was carried out on SiO2 (silica gel 60, 230−400
mesh). All glassware used in the reactions described below were
flame-dried under vacuum and then flushed with argon gas at room
temperature prior to the addition of reagents and solvents. 1H and 13
C
NMR spectra were measured in CDCl3 at 400 and 100 MHz,
respectively, using Me4Si as internal standard. Chemical shifts are
reported in ppm downfield (δ) from Me4Si. UV spectra were recorded
on a diode array spectrophotometer over the 190−500 nm range with
subtraction of a solvent blank. Fluorescence spectra were recorded
over the 520−670 nm range for ethidium bromide displacement
experiments (the maximum emission wavelength was 590 nm when
the excitation wavelength was 520 nm; ex slit (nm) = 10.0; em slit
(nm) = 10.0; scan speed (nm/min) = 200) and over the 320−410 nm
range for HSA binding experiments (the maximum emission
wavelength was 338 nm when the excitation wavelength was 280
nm; ex slit (nm) = 10.0; em slit (nm) = 10.0; scan speed (nm/min) =
200). ITC thermograms were recorded for titrations performed at 25
°C under the following conditions: DP = 6, 307 rpm, 5 μL injections,
10 s duration with 130 s, 2 s filter, initial delay of 60 s, total of 45
injections.
(2S)-tert-Butyl 2-(2-Oxopropyl)pyrrolidine-1-carboxylate [(4S)-6]
and (2R)-tert-Butyl 2-(2-Oxopropyl)pyrrolidine-1-carboxylate [(4R)-
6]. Nitrile (4S)-424 or (4R)-45 (1.0 g, 4.7 mmol) was dissolved in
MeOH (4.7 mL) and 3 N NaOH (4.7 mL, 14.1 mmol) and heated to
100 °C for one hour, at which time a homogeneous solution was
obtained. The solution was cooled to room temperature and
concentrated in vacuo to remove MeOH. Et2O (10 mL) was added,
and the solution was acidified with 3 N HCl (4.5 mL). The phases
were separated, and the aqueous layer was extracted with Et2O (2 ×
25 mL). The combined organic extracts were once washed with 20
mL of saturated aqueous NaCl, dried over anhydrous Na2SO4, and
concentrated under reduced pressure. The crude acid (4.7 mmol) was
1
rotamers): H NMR (400 MHz, CDCl3) δ 4.16 (m, 1H), 3.32 (m,
2H), 3.15, 2.95 (dd, J = 15.3, 15.6 Hz, 1H), 2.44 (m, 1H), 2.17 (s,
3H), 2.08 (m, 1H), 1.84 (m, 2H), 1.67 (m, 1H), 1.47 (s, 9H); 13C
NMR (100 MHz, CDCl3) δ 207.5, 154.3, 79.5, 53.4, 47.8, 46.4, 31.5,
30.5, 28.4, 23.5; HRMS (ESI) calculated for C12H21NNaO3 250.1419,
found 250.1376 (M + Na)+; [α]25D = −36.5 (c 0.02, CH2Cl2). These
data are in full accord with those reported for (4S)-6.25 (4R)-6 (as a
mixture of carbamate rotamers): 1H NMR (400 MHz, CDCl3) δ 4.09
(m, 1H), 3.29 (t, J = 5.9 Hz, 2H), 2.96 (d, J = 16.0 Hz, 1H), 2.37 (dd,
J = 9.6, 16.0 Hz, 1H), 2.11 (s, 3H), 2.01 (m, 1H), 1.79 (m, 2H), 1.60
(m, 1H), 1.42 (s, 9H); 13C NMR (100 MHz, CDCl3) δ 207.3, 154.2,
79.3, 53.4, 48.2, 46.3, 31.1, 30.4, 28.5, 23.2; HRMS (ESI) calculated
for C12H21NNaO3 250.1419, found 250.1400 (M + Na)+; [α]25
=
D
+18.6 (c 0.05, CHCl3). These data are in full accord with those
reported for (4R)-6.7
(2S)-tert-Butyl 2-[(2S)-2-Hydroxypropyl]Pyrrolidine-1-carboxy-
late [(4S,6S)-7] and (2R)-tert-Butyl 2-[(2R)-2-Hydroxypropyl]-
Pyrrolidine-1-carboxylate [(4R,6R)-7]. Ketone (4S)-6 or (4R)-6
(120 mg, 0.53 mmol) was dissolved in THF (1 mL), and the mixture
was cooled to 0 °C; Zn(BH4)2 (1 mL of a 4 M solution, 4 mmol) was
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added, and the mixture was stirred at 0 °C for 6 h. The mixture was
quenched by addition of a saturated solution of Rochelle’s salt (5 mL)
and stirred at room temperature for 2 h. After dilution of the mixture
with EtOAc (10 mL), the phases were separated and the aqueous
layer was extracted with Et2O (2 × 25 mL). The combined organic
extract was washed once with 20 mL of saturated aqueous NaCl, dried
over anhydrous Na2SO4, and concentrated under reduced pressure.
Purification of the residue by flash chromatography (SiO2, 10:1 → 3:1
hexanes/EtOAc) afforded alcohol (4S,6S)-7 (76.4 mg, 0.33 mmol,
63%) or (4R,6R)-7 (70.3 mg, 0.31 mmol, 58%). (4S,6S)-7 (as a
mixture of carbamate rotamers): 1H NMR (400 MHz, CDCl3) δ 5.13
(br s, 1H), 4.15 (m, 1H), 3.71 (m, 1H), 3.29 (t, J = 5.8 Hz, 2H),
1.96−1.82 (m, 3H), 1.58 (m, 1H), 1.44 (s, 9H), 1.17 (d, J = 6.2 Hz,
3H); 13C NMR (100 MHz, CDCl3) δ 156.5, 79.8, 63.8, 53.7, 46.5,
45.5, 31.1, 28.4, 23.5, 22.5; HRMS (ESI) calculated for
mixture was allowed to warm to room temperature and stir overnight.
CH2Cl2 (10 mL) and a saturated solution of NaHCO3 were added
(10 mL), and the layers were separated. The aqueous layer was
extracted with EtOAc (2 × 25 mL). The combined organic extract
was washed once with 20 mL of saturated aqueous NaCl, dried over
anhydrous Na2SO4, and concentrated under reduced pressure.
Purification of the residue by flash chromatography (SiO2, 5:1
hexanes/EtOAc → 2:1 hexanes/EtOAc) afforded the four diaster-
eomers (4S,6S)-1, (4R,6R)-1, (4S,6R)-1, and (4R,6S)-1. (4S,6R)-1
1
(101 mg, 0.344 mmol, 51%, as a mixture of amide rotamers): H
NMR (400 MHz, CDCl3) δ 6.89 (m, 1H), 4.99 (m, 1H), 4.20, 3.85
(m, 1H), 3.47−3.40 (m, 2H), 2.31 (t, J = 7.6 Hz, 1H), 2.21 (t, J = 8.1
Hz, 1H), 1.89−1.75 (m, 5H), 1.77 (s, 3H), 1.74 (d, J = 7.0 Hz, 3H),
1.64 (m, 2H), 1.44 (m, 1H), 1.27 (d, J = 6.2 Hz, 3H), 1.27−1.24 (m,
12H), 0.86 (t, J = 6.7 Hz, 3H); 13C NMR (100 MHz, CDCl3) δ
171.8, 167.8, 137.4, 137.1, 128.8, 128.6, 68.5, 68.3, 54.4, 53.8, 46.7,
45.3, 41.8, 39.2, 35.1, 34.3, 31.8, 30.1, 29.6, 29.5, 29.4, 29.3, 29.2,
25.5, 24.8, 23.9, 22.6, 21.8, 20.9, 20.5, 14.4, 14.0, 12.1, 12.0; HRMS
(ESI) calculated for C22H40NO3 366.2990, found 366.2958 (M +
C12H23NNaO3 252.1576, found 252.1555 (M + Na)+; [α]25
=
D
−5.8 (c 0.01, CHCl3). These data are in full accord with those
reported for (4S,6S)-7.7 (4R,6R)-7 (as a mixture of carbamate
rotamers): 1H NMR (400 MHz, CDCl3) 4.16 (m, 1H), 3.72 (m, 1H),
3.30 (m, 2H), 1.99−1.85 (m, 3H), 1.60 (m, 1H), 1.47 (s, 9H), 1.40
(m, 2H), 1.18 (d, J = 6.0 Hz, 3H); 13C NMR (100 MHz, CDCl3) δ
156.5, 79.8, 63.7, 53.8, 46.4, 45.6, 31.1, 28.4, 23.4, 22.6; HRMS (ESI)
calculated for C12H23NNaO3 252.1576, found 252.1478 (M + Na)+;
H)+; [α]25 = −9.9 (c 0.01, MeOH). (4S,6S)-1 (84 mg, 0.28 mmol,
D
43%, as a mixture of amide rotamers): 1H NMR (400 MHz, CDCl3) δ
6.82 (m, 1H), 4.96 (q, J = 6.4 Hz, 1H), 4.08, 3.83 (m, 1H), 3.38 (m,
2H), 2.21 (t, J = 8.3 Hz, 2H), 2.08 (m, 1H), 1.91−1.78 (m, 9H),
1.66−1.58 (m, 3H), 1.31−1.25 (m, 16H), 0.88 (t, J = 6.7 Hz, 3H);
13C NMR (100 MHz, CDCl3) δ 171.6, 167.5, 136.7, 129.0, 69.6, 54.9,
46.6, 39.3, 35.0, 31.8, 29.5, 29.4, 29.2, 24.7, 24.1, 22.6, 20.0, 14.2,
14.0, 11.9; HRMS (ESI) calculated for C22H39NNaO3 388.2828,
[α]25 = +7.5 (c 0.02, CHCl3). These data are in full accord with
D
those reported for (4R,6R)-7.7
(2S)-tert-Butyl 2-[(2R)-2-Hydroxypropyl]pyrrolidine-1-carboxy-
late [(4S,6R)-8] and (2R)-tert-Butyl 2-[(2S)-2-Hydroxypropyl]-
pyrrolidine-1-carboxylate [(4R,6S)-8]. Ketone (4S)-6 or (4R)-6
(320 mg, 1.41 mmol) was dissolved in THF (5 mL), and the mixture
was cooled to −78 °C; LiAlH(OtBu)3 (1.07 g, 4.24 mmol, 3 equiv)
was added, and the mixture was warmed to 0 °C and stirred at this
temperature for 6 h. The mixture was quenched with a few drops of
water and diluted with EtOAc (10 mL). The solution was cooled to 0
°C, filtered through Celite, and concentrated in vacuo. Purification of
the residue by flash chromatography (SiO2, 10:1 → 3:1 hexanes/
EtOAc) afforded alcohol (4S,6R)-8 (293 mg, 1.28 mmol, 91%) or
(4R,6S)-8 (267 mg, 1.17 mmol, 83%). (4S,6R)-8 (as a mixture of
found 388.2838 (M + Na)+; [α]25 = −38.5 (c 0.01, MeOH).
D
(4R,6S)-1 (110 mg, 0.37 mmol, 56%, as a mixture of amide rotamers):
1H NMR (400 MHz, CDCl3) δ 6.90 (m, 1H), 5.00 (m, 1H), 4.20,
3.85 (m, 1H), 3.47−3.38 (m, 2H), 2.28 (t, J = 7.8 Hz, 1H), 2.25 (t, J
= 7.1 Hz, 2H), 1.91−1.76 (m, 9H), 1.64 (m, 2H), 1.44 (m, 1H),
1.41−1.27 (m, 16H), 0.89 (t, J = 6.6 Hz, 3H); 13C NMR (100 MHz,
CDCl3) δ 171.8, 167.8, 137.3, 128.8, 68.5, 54.4, 53.8, 46.7, 45.3, 41.8,
39.2, 35.1, 34.3, 31.8, 30.1, 29.6, 29.5, 29.4, 29.3, 25.5, 24.8, 23.9,
22.6, 21.8, 20.9, 20.5, 14.4, 14.0, 12.1; HRMS (ESI) calculated for
C22H40NO3 366.2990, found 366.3042 (M + H)+; [α]25 = +8.0 (c
1
D
carbamate rotamers): H NMR (400 MHz, CDCl3) 3.95 (br s, 1H),
0.01, MeOH). (4R,6R)-1 (70 mg, 0.19 mmol, 29%, as a mixture of
amide rotamers): 1H NMR (400 MHz, CDCl3) δ 6.82 (q, J = 4.3 Hz,
1H), 4.96 (q, J = 4.0 Hz, 1H), 4.08 (m, 1H), 3.41−3.35 (m, 2H), 2.21
(t, J = 8.1 Hz, 2H), 2.08 (m, 1H), 1.91−1.80 (m, 6H), 1.78 (d, J = 8.0
Hz, 3H), 1.64 (m, 3H), 1.32−1.30 (m, 13H), 1.31 (d, J = 8.0 Hz,
3H), 0.89 (t, J = 7.0 Hz, 3H); 13C NMR (100 MHz, CDCl3) δ 171.6,
167.5, 136.7, 129.0, 69.6, 54.9, 46.6, 39.3, 35.1, 31.8, 29.5, 29.2, 24.7,
24.1, 22.6, 20.0, 14.3, 14.0, 12.0; HRMS (ESI) calculated for
C22H39NaO3 388.2828, found 388.2832 (M + Na)+; [α]25D = +25.7 (c
0.03, MeOH). These data are in accord with those reported for
conioidine A.1
Competitive Ethidium Bromide Displacement Experiments.
Constant concentrations of CT-DNA (10 μM) and EtBr (10 μM)
(each in Tris-HCl buffer, pH 6.81) were titrated with increasing
concentrations of the diastereomers of 1 (from 1 mM and 100 μM
stock solutions in Tris-HCl buffer, pH 6.81) or netropsin. The
maximum emission wavelength was 490 nm when the excitation
wavelength was 520 nm. Fluorescence titrations were recorded from
520 to 692 nm after an equilibration period of 3 min: ex slit (nm) =
10.0; em slit (nm) = 10.0; scan speed (nm/min) = 200.
Thermal Denaturation Studies. UV thermal denaturation
samples (2 mL) were prepared by mixing CT-DNA in Tris-HCl
buffer (pH 6.81) in 1 cm path length quartz cuvettes. The DNA to
ligand ratio was 40:1. Absorbance readings were taken at 260 nm for
temperatures ranging from 25 to 95 °C. Temperature was increased
gradually with a speed of 1 °C/min with an absorbance reading every
2 °C. First derivative plots were used to determine the TM value.
ECD Studies. Small aliquots (0.6−5.0 μL) of a concentrated
(4R,6S)-1 solution (1 mM) were added to a solution of HSA (15 μM
in pH = 6.81 in Tris-HCl buffer), inverted twice, and incubated for 5
min at 20 °C. The ECD spectra were recorded as an average of three
scans from 220 to 310 nm, and data recorded in 0.1 nm increments.
3.80 (m, 1H), 3.30 (m, 2H), 1.97 (m, 1H), 1.81 (m, 2H), 1.62 (m,
1H), 1.42 (s, 9H), 1.16 (d, J = 5.0 Hz, 3H); 13C NMR (100 MHz,
CDCl3) 155.4, 79.5, 66.3, 55.5, 46.3, 45.3, 32.1, 28.4, 23.9, 23.7;
HRMS (ESI) calculated for C12H23NNaO3 252.1576, found 252.1597
(M+Na)+; [α]25 = −24.8 (c 0.08, CHCl3). These data are in full
D
accord with those reported for (4S,6R)-8.7 (4R,6S)-8 (as a mixture of
1
carbamate rotamers): H NMR (400 MHz, CDCl3) 3.89 (br s, 1H),
3.65 (m, J = 2.9 Hz, 1H), 3.62 (br s, 1H), 3.24 (m, 2H), 1.91−1.86
(m, 1H), 1.74 (m, 3H), 1.59 (m, 1H), 1.37 (s, 9H), 1.12 (d, J = 6.2
Hz, 3H); 13C NMR (100 MHz, CDCl3) δ 155.2, 79.4, 66.0, 55.2,
46.2, 44.9, 31.7, 28.4, 23.9, 23.7; HRMS (ESI) calculated for
C12H23NNaO3 252.1576, found 252.1579 (M + Na)+; [α]25D = +33.3
(c 0.02, CHCl3). These data are in full accord with those reported for
(4R,6S)-8.7
(4S,6S)-Conioidine A, (4R,6R)-Conioidine A, (4R,6S)-Conioidine
A, and (4S,6R)-Conioidine A. Alcohol (4S,6S)-7, (4R,6R)-7, (4S,6R)-
8, or (4R,6S)-8 (150 mg, 0.66 mmol) was dissolved in 1:1 TFA/
CH2Cl2 (1.5 mL) at room temperature, and the mixture was stirred
for 30 min, at which time it was concentrated in vacuo. The crude
residue was azeotroped with benzene (5 mL) twice, and CH2Cl2 (1.5
mL) was added. Et3N (0.25 mL, 1.6 mmol) was added, and the
mixture was cooled to 0 °C. Decanoyl chloride (0.15 mL, 0.72 mmol)
was added dropwise, and the mixture was warmed to room
temperature and stirred overnight. A solution of saturated NaHCO3
(5 mL) was added, and the layers were separated. The aqueous layer
was extracted with EtOAc (2 × 25 mL). The combined organic
extract was washed once with 20 mL of saturated aqueous NaCl, dried
over anhydrous Na2SO4, and concentrated under reduced pressure.
The crude intermediate alcohol (∼189 mg, 0.67 mmol) was dissolved
in CH2Cl2 (2 mL) and cooled to 0 °C. Pyridine (0.216 mL, 2.68
mmol), DMAP (163 mg, 1.34 mmol), and 2,3-dimethylacryloyl
chloride (158 mg, 1.34 mmol) were added successively, and the
F
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Article
ITC Studies. Ligands 1 (100 μM in pH = 6.81 Tris-HCl buffer)
were titrated against HSA (57 μM in pH = 6.81, Tris-HCl buffer) at
25 °C. ITC thermograms were recorded for titrations under the
following conditions: DP = 6, 307 rpm, 5 μL injections, 10 s duration
with 130 s, 2 s filter, initial delay of 60 s, total of 45 injections.
Molecular Docking Studies. Compound (4R,6R)-1 was
minimized using Spartan’14 for Macintosh. Molecular docking studies
were performed with (4R,6R)-1 and HSA (PDB 1AO6)18 using
AutoDock Vina.17 The search space included both Sudlow sites I and
II.
(2) Tse, W. C.; Boger, D. L. Chem. Biol. 2004, 11, 1607.
(3) Fekry, M. I.; Szekely, J.; Dutta, S.; Breydo, L.; Zang, H.; Gates,
K. S. J. Am. Chem. Soc. 2011, 133, 17641.
(4) Williams, L. D.; Maher, L. J., III Annu. Rev. Biophys. Biomol.
Struct. 2000, 29, 497.
(5) Alza, E.; Cambeiro, X. C.; Jimeno, C.; Pericas, M. A. Org. Lett.
2007, 9, 3717.
(6) (a) Mahato, C. K.; Mukherjee, S.; Kundu, M.; Pramanik, A. J.
Org. Chem. 2019, 84, 1053. (b) Tampio L’Estrade, E.; Edgar, F. G.;
Xiong, M.; Shalgunov, V.; Baerentzen, S. L.; Erlandsson, M.; Ohlsson,
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7344.
(7) (a) Davies, S. G.; Fletcher, A. M.; Roberts, P. M.; Smith, A. D.
Tetrahedron 2009, 49, 10192. (b) We attempted to access (4S,6S)-7
and (4R,6R)-7 from the (4S,6R)-8 and (4R,6S)-8 isomers,
respectively, via the Mitsunobu reaction and ester hydrolysis. This
process gave <20% yields of (4S,6S)-7 and (4R,6R)-7 because of the
production of a cyclic carbamate side product, likely arising from
intramolecular cyclization of the Boc group under Mitsunobu
conditions. All other attempted reductions using AlH4 or BH4
reducing agents gave low diasteroselectivities or favored production
of (4S,6R)-8 and (4R,6S)-8..
ASSOCIATED CONTENT
* Supporting Information
The Supporting Information is available free of charge at
https://pubs.acs.org/doi/10.1021/acs.jnatprod.0c00871.
■
sı
1
Experimental details, characterization data, and H and
13C NMR spectra for all compounds in Scheme 1;
fluorescence, UV, ECD, and ITC binding data for the
four diastereomers of 1 (PDF)
AUTHOR INFORMATION
Corresponding Author
(8) Morgan, A. R.; Lee, J. S.; Pulleyblank, D. E.; Murray, N. L.;
Evans, D. H. Nucleic Acids Res. 1979, 7, 547.
■
(9) (a) Wilson, W. D.; Tanious, F. A.; Fernandez-Saiz, M.; Rigl, C.
T. Methods in Mol. Biol. Drug-DNA Interact. Protoc. 1997, 90, 219. (b)
A 40:1 CT DNA:ligand ratio was utilized for these studies.
(10) Yasseen, Z. J.; El-Ghossain, M. O. J. Biomed. Sci. 2016, 5, 19.
(11) Liu, C.; Liu, Z.; Wang, J. PLoS One 2017, 12, No. e0176208.
(12) Concentrations of 1 greater than 10 μM used in the
fluorescence experiments resulted in the formation of a precipitate,
likely an HSA·1 complex.
(13) Chatterjee, T.; Pal, A.; Dey, S.; Chatterjee, B. K.; Chakrabati, P.
PLoS One 2012, 7, No. e37468.
(14) Na, N.; Zhao, D.-Q.; Li, H.; Jiang, N.; Wen, J.-Y.; Liu, H.-Y.
Molecules 2016, 21, 54.
Thomas Minehan − Department of Chemistry and
Biochemistry, California State University, Northridge,
Northridge, California 91330, United States; orcid.org/
0000-0002-7791-0091; Email: thomas.minehan@csun.edu
Authors
Ryan Shaktah − Department of Chemistry and Biochemistry,
California State University, Northridge, Northridge, California
91330, United States
Laura Vardanyan − Department of Chemistry and Biochemistry,
California State University, Northridge, Northridge, California
91330, United States
Elroma David − Department of Chemistry and Biochemistry,
California State University, Northridge, Northridge, California
91330, United States
Alexis Aleman − Department of Chemistry and Biochemistry,
California State University, Northridge, Northridge, California
91330, United States
Dupre Orr − Department of Chemistry and Biochemistry,
California State University, Northridge, Northridge, California
91330, United States
Lawrence A. Shaktah − Department of Chemistry and
Biochemistry, California State University, Northridge,
Northridge, California 91330, United States
Daniel Tamae − Department of Chemistry and Biochemistry,
California State University, Northridge, Northridge, California
91330, United States
(15) Callies, O.; Hernandez Daranas, A. Nat. Prod. Rep. 2016, 33,
881.
(16) (a) Sudlow, G. D. J. B.; Birkett, D. J.; Wade, D. N. Mol.
Pharmacol. 1976, 12, 1052. (b) We anticipate that the conioidines
bind in Sudlow site I because similar hydrophobic organics containing
long n-alkyl chains such as octanoic, myristic, lauric, and palmitic
acids have all been shown to bind that site: (c) Kawai, A.; Chuang, V.
T. G.; Kouno, Y.; Yamasaki, K.; Miyamoto, S.; Anraku, M.; Otagiri,
M. Biochim. Biophys. Acta, Proteins Proteomics 2017, 1865, 979.
(d) Yang, F.; Bian, C.; Zhu, L.; Zhao, G.; Huang, Z.; Huang, M. J.
Struct. Biol. 2007, 157, 348. (e) Bijelic, A.; Dobrov, A.; Roller, A.;
Rompel, A. Inorg. Chem. 2020, 59, 5243. (f) Qi, J.; Gou, Y.; Zhang, Y.;
Yang, K.; Chen, S.; Liu, L.; Wu, X.; Wang, T.; Zhang, W.; Yang, F. J.
Med. Chem. 2016, 59, 7497. (g) The HSA−conioidine titration
binding studies showed a strong quenching of the fluorescence of Trp
214, which is located in proximity to Sudlow site I. Furthermore, the
binding constant for (4R,6S) conioidine A (Kb = (1.6 0.3) × 105
M−1) more closely resembles the binding constant of the site I marker
warfarin (Kb = 2.8 × 105 M−1) than that of the site II marker
ibuprofen (Kb = 2.9 × 106 M−1).
Complete contact information is available at:
https://pubs.acs.org/10.1021/acs.jnatprod.0c00871
(17) Molecular docking was performed by building ligand (4R,6R)-1
in Spartan’14 for Macintosh (Wavefunction, Inc., Irvine, CA, USA),
minimizing its energy using the Hartree−Fock 3-21G basis set and
transforming the derived PDB files into PDBQT files using AutoDock
Tools; the PDB file 1AO6 (HSA) was also transformed into a
PDBQT file, and then docking was performed with AutoDock Vina:
Trott, O.; Olson, A. J. J. Comput. Chem. 2009, 31, 455.
(18) Sugio, S.; Kashima, A.; Mochizuki, S.; Noda, M.; Kobayashi, K.
Protein Eng., Des. Sel. 1999, 12, 439.
(19) (a) We suggest that the binding of the conioidines to HSA
occurs in a noncovalent fashion. To test the possibility of covalent
reactivity by thiol conjugate addition to the enoate group of the
conioidines, we exposed (4S,6S)-1 to ethanethiol (5 molar equiv) in
Notes
The authors declare no competing financial interest.
ACKNOWLEDGMENTS
We thank the National Science Foundation (CHE-1508070)
for their generous support of our research program.
■
REFERENCES
■
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Journal of Natural Products
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Article
CH2Cl2 for 48 h at 37 °C. No reaction was observed. Furthermore, we
exposed (4S,6S)-1 to ethanethiol (5 molar equiv) and a catalytic
quantity (10 mol %) of the base DBU in CH2Cl2 for 48 h at 37 °C.
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disulfide linkages, we anticipate that major structural changes to the
protein would be required for covalent attachment via cysteine thiol
conjugate addition to the enoate side chain of the natural product.
However, the changes observed during ECD titration of HSA with
(4R,6S)-1 (Figure 4) indicate only a minor structural perturbation of
the protein upon ligand binding, which is consistent with what is
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(4S,6S)-1, and (4R,6R)-1, are all within experimental error, and the
binding constant of (4R,6S)-1 is only a factor of 1.5 times greater than
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viewpoint.
H
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