An unusual intramolecular trans-amidation

Article abstract of DOI:10.1016/j.tet.2016.01.062

Polyketide biosynthesis engages a series of well-timed biosynthetic operations to generate elaborate natural products from simple building blocks. Mimicry of these processes has offered practical means for total synthesis and provided a foundation for rea

Full text of DOI:10.1016/j.tet.2016.01.062

Tetrahedron xxx (2016) 1e4
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An unusual intramolecular trans-amidation
,
,
,c,d,
*
Heriberto Rivera, Jr. a y, Sachin Dhar a y, James J. La Clair a, Shiou-Chuan Tsai b
,
,
Michael D. Burkart a
*
a Department of Chemistry and Biochemistry, University of California, San Diego, 9500 Gilman Drive, La Jolla, CA 92093-0358, United States
b Department of Molecular Biology and Biochemistry, University of California, Irvine, CA 92697, United States
c Department of Chemistry, University of California, Irvine, CA 92697, United States
d Department of Pharmaceutical Sciences, University of California, Irvine, CA 92697, United States
a r t i c l e i n f o
a b s t r a c t
Article history:
Polyketide biosynthesis engages a series of well-timed biosynthetic operations to generate elaborate
natural products from simple building blocks. Mimicry of these processes has offered practical means for
total synthesis and provided a foundation for reaction discovery. We now report an unusual intra-
molecular trans-amidation reaction discovered while preparing stabilized probes for the study of acti-
norhodin biosynthesis. This rapid cyclization event offers insight into the natural cyclization process
inherent to the biosynthesis of type II polyketide antibiotics.
Received 25 August 2015
Received in revised form 27 January 2016
Accepted 30 January 2016
Available online xxx
Keywords:
trans-Amidation
Polyketide
Ó 2016 Elsevier Ltd. All rights reserved.
Cyclization
Intramolecular reactions
Reaction mechanisms
1. Introduction
The mechanisms guiding polyketide biosynthesis have served as
inspiration for both total synthesis1 and reaction development
efforts.2 Within recent years, the fusion between synthetic organic
chemistryand structural biology has provided a rich forum to further
explore the mechanisms that guide each of the discrete operations
during a biosynthetic process.3 The significance of these advances
has most recently allowed chemoenzymatic methods to address the
total synthesis of complex natural products such as spinosyn A.4
One of the key complications encountered when studying in-
termediate formation within natural product biosynthesis arises
from compound instability. This is particularly problematic in type
II polyketide biosynthesis, as the formation of the acyl carrier
protein (ACP) tethered polyketones such as 1a (Fig. 1), are highly
unstable.5
Fig. 1. Exemplary ketide acyl-carrier protein (ACP) tethered intermediate 1a along
with the corresponding stabilized atom replacement probe 1 and probe-loaded ACP
1b. The black bar denotes the 40-phosphopantetheinyl arm.
catalyze specific cyclization events (Fig. 2). Interested in further
understanding these processes at the structural level, we turned to
the preparation of ‘atom replacement probes’.6
Through these studies, we learned that one could effectively
prepare pantetheinylated mimetics of polyketones by the selective
replacement of specific carbonyls with heteroatoms. As depicted in
1 (Fig. 1), we prepared probes bearing both thioethers and iso-
xazoles to represent key carbonyl units within the polyketone 1a.6
Similarly, we also developed partially-cyclized intermediates of 2a,
as shown in 2 (Fig. 2).
Similar problems with instability also arise after the cyclization
begins, as intermediates such as 2a are prone to uncontrolled aldol-
type cyclizations, if not regulated. Our current hypothesis supports
the concept that the ACP serves to both protect unstable cargo (1a,
Fig. 1) as well as guide these species to the enzymes, which will
* Corresponding authors. E-mail addresses: sctsai@uci.edu (S.-C. Tsai), mbur-
kart@ucsd.edu (M.D. Burkart).
y
These authors contributed equally to the work.
http://dx.doi.org/10.1016/j.tet.2016.01.062
0040-4020/Ó 2016 Elsevier Ltd. All rights reserved.
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2
H. Rivera Jr. et al. / Tetrahedron xxx (2016) 1e4
corresponding naturally-loaded 1a and 2a, respectively.6 Overall,
we were able to synthesize stable mimetics of intermediates at two
stages of the actinorhodin biosynthetic process (Fig. 3).
2. Results and discussion
As part of this effort, we were interested in exploring the sta-
bility of ketides arising from opening of the isoxazole motif. Our
plan was to use the isoxazole as a tool to mimic 1,3-dicarbonyl
units, and hence restrict access to unwanted spontaneous aldol
reactions. Our goal was to deliver a series of bench stable mimetics
that could be used for structural biological studies. We began by
preparing linear and cyclized mimetics and examining their in-
corporation on actACP by NMR.7 We then examined what would
happen upon opening of the isoxazole. To this end, we were able to
open the isoxazole in simpler intermediates such as 3 providing
imine 4 and diketide 5 (Fig. 4) in a sequential fashion. While not
unexpected, this process was not possible with more complicated
materials such as 1 (Fig. 1), which underwent rapid degradation.
LC-MS monitoring of the cleavage process using probes such as
isoxazole 1 clearly showed the rapid formation of multiple products
immediately after treatment with Mo(CO)6, indicating that even
the imine-type intermediates were highly-reactive. We then turned
to explore if this process was possible on our partially-cyclized
materials, such as probe 2 (Fig. 2).
Fig. 2. Exemplary cyclized intermediate 2a along with its associated atom replacement
probe 2 and probe loaded ACP 2b. The black bar denotes the 40-phosphopantetheinyl
arm.
Using the actinorhodin biosynthetic system as a model (Fig. 3),
we were able to demonstrate that probes 1 and 2 can be loaded
onto their ascribed ACP as given by the conversion of 1 to 1b (Fig. 1)
and 2 to 2b (Fig. 2). As part of this study, we were able to apply
solution-based protein NMR methods to demonstrate that in-
termediates 1b and 2b did indeed provide viable mimetics of their
Fig. 4. Isoxazole cleavage. Ring opening of isoxazole 3 results in enaminone 4, which in
turn is hydrolytically-converted to ketide 5.
2.1. Precursor synthesis
We began by examining materials from our recent studies.7
Beginning with 67 (Scheme 1), we were able to selectively depro-
tect the acetal by treatment in aq AcOH at rt to provide mimetic 7.
Treatment of 7 with fresh Mo(CO)6 in refluxing aq CH3CN8 resulted
in a clean conversion to enaminone 8 in an overall 86% yield from 6.
2.2. Intramolecular trans-amidation of benzyl-protected en-
aminone 8
While stable thermally, exposure of 8 to mild acidic conditions
(aq AcOH in CH3CN) resulted in a crude product that lacked mass
spectral signatures of the desired ketone. NMR monitoring of this
reaction (Fig. 5) indicated the formation of two major materials,
one of which was pantetheinamine 99 (Scheme
1
and
Supplementary Figs. S6eS8). The remaining material was attrib-
uted to 10, as given by the presence of a compound containing two
distinct benzylic groups in the crude NMR spectrum. Mass spectral
analysis returned a formula of C26H23NO4, indicating that 10 con-
tained all of the remaining hydrogen, carbon, nitrogen and oxygen
atoms of 8 (C37H46N4O8) after the elimination of 9 (C11H23N3O4).
Following purification of 10, 1D NMR and 2D NMR analysis
allowed complete assignment of the 1H and 13C spectra (see
Supplementary data). As summarized on the left of Fig. 6, the 13C
data was suggestive of the structure of 1,4-dihydroisoquinolin-
Fig. 3. Proposed biosynthesis of actinorhodin from holo-actACP.6 One of the enol/ketol
tautomerization states has been depicted. In solution, multiple tautomers exist. The
processing of substrates on the actACP has been depicted by a color change of the
sphere representing the ACP from red to violet with states 1a and 2a shown in Figs. 1
and 2, respectively.
3(2H)-one motif bearing an exocyclic
a,b-unsaturated ketone.
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H. Rivera Jr. et al. / Tetrahedron xxx (2016) 1e4
3
Specifically, the data provided strong support for the exocyclic
unsaturated ketone due to the presence of the chemical shift for the
carbonyl at 200.7 ppm, the -carbon at 146.4 ppm, and -carbon at
a,b-
a
b
102.5 ppm. Further evidence for the 1,4-dihydroisoquinolin-3(2H)-
one core was also obtained by the 13C peaks at 36.9 and 168.1 ppm.
These assignments were further supported by HMBC correlations,
as shown at the right of Fig. 6.
3. Conclusion
Ongoing efforts have recently led to the development of stabi-
lized probes for studying intermediate processing within polyke-
tide biosynthesis. While some atom replaced motifs provide
sufficient stability (Fig. 4), others due to their access to rapid
intramolecular cyclizations still lack sufficient stability (Scheme 1).
Here, we report on a rather rare example of an intramolecular
trans-amidation reaction. As illustrated in Scheme 1, the conversion
of 8 to 10 occurred within 4 h at 23 ꢀC under mildly acidic condi-
tions. This suggests a strong driving force intrinsically built within
the cyclic framework within atom-replacement probe 8. While an
uncommon event, the trans-amide formation observed herein
(Scheme 1) not only demonstrates a unique reactivity but also
provides a further means to synthetically-divert polyketide bio-
synthetic methods to enable access to new motifs, such as 10. This
finding suggests another alternative pathway that, while not
accessed by the synthase, outlines the inherent non-enzyme cata-
lyzed reactivity within the highly-reactive intermediates in poly-
ketide biosynthesis. Mining these reactions not only expands the
molecular diversity available through biosynthetic processes but
also offers access to new structural motifs, a key step in expanding
the utility of natural products. Overall, this study supports the
importance of understanding polyketide biosynthesis not only by
offering a means to comprehend and optimize natural product
biosynthesis, but also by providing a platform for the advance of
biosynthetically-derived natural product-like scaffolds.10
Scheme 1. Advance of cyclic mimetic 6. Samples of 8 can be prepared two steps and in
excellent yield from 6. While stable to heat, compound 8 readily undergoes intra-
molecular trans-amidation resulting the formation of adduct 10 at rt under mildly
acidic conditions.
4. Experimental section
4.1. General
Unless otherwise noted, all reagents and chemical compounds
were purchased from Alfa Aesar, Strem Chemicals, SigmaeAldrich,
Cambridge Isotopes or TCI and used without further purification.
Flash chromatography was carried out on 40e63 mesh Geduran
Silica Gel 60 (EMD Millipore). Thin layer chromatography (TLC) was
conducted on 250 mm Silica Gel 60 F254 glass plates (EMD Milli-
pore). NMR spectra were recorded on a Mercury Plus 400 MHz
(Varian), a ECA 500 MHz (Jeol), a DMX 400 MHz (Bruker), a DMX
500 MHz (Bruker) or a VX 500 MHz equipped with XSens cold
probe (Varian) spectrometer. FID files were processed using Mes-
tRenova version 10.0.1 (MestreLab Research). Mass spectrometric
analyses were conducted on the following instruments: a LCQ Deca
(ThermoFinnigan), MAT900XL (ThermoFinnigan), LTQ Orbitrap XL
(ThermoScientific), or a LCT Premier (Waters) mass spectrometer.
Reactions were conducted under Ar atmosphere in a round bottom
flask or vial capped with a rubber septa and were stirred using
a Teflon coated stir bar. All mixtures are provided as v:v ratios.
Fig. 5. 1H NMR time course analysis depicting the conversion of 8 (blue circles) to 10
(red spheres) in 2:2:1 CD3OOD:D2O:CD3OD. An expansion of the region between 5.60
and 6.35 ppm has been provided. Full spectral data has been provided in
Supplementary Figs. S6eS8.
4.2. Experimental procedures and data of synthetic
intermediates
4.2.1. Isoxazole 7. PMB-protected isoxazole
6
(54.0 mg,
0.0683 mmol) was dissolved in 1 mL of 4:1 AcOH:H2O. The solution
was stirred for 4 h, after which starting material was no longer
present by TLC analysis using 1:9 MeOH/CH2Cl2. Solvent was
azeotropically removed by rotary evaporation with addition of
Fig. 6. NMR data confirming the assignment of 10. (left) Select 13C NMR assignments
and (right) select 1H, 13C HMBC correlations.
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4
H. Rivera Jr. et al. / Tetrahedron xxx (2016) 1e4
toluene (3ꢁ5 mL). Flash chromatography (CH2Cl2 to 1:9
MeOH:CH2Cl2) provided 43.6 mg (95%) of isoxazole 7; TLC Rf¼0.5
(silica gel, MeOH:CH2Cl2 1:9); 1H NMR (500 MHz CDC13) 7.45e7.26
(m, 10H), 6.64 (d, J¼2.3 Hz, 1H), 6.63 (d, J¼2.3 Hz, 1H), 6.14 (d,
J¼1.0 Hz, 1H), 5.08 (s, 2H), 5.06 (s, 2H), 4.64 (br s, 1H), 3.88 (s, 1H),
3.64 (dddd, J¼3.7, 6.9, 6.9, 13.8 Hz, 2H), 3.54e3.35 (m, 5H), 3.16
(ddd, J¼2.6, 5.8, 14.1 Hz, 2H), 3.13 (ddd, J¼3.3, 8.2, 14.0 Hz, 2H), 2.47
(d, J¼1.0 Hz, 3H), 2.35 (m, 2H), 1.02 (s, 3H), 0.88 (s, 3H); 13C NMR
course of 24 h by 1H NMR analysis on a VX 500 MHz equipped with
XSens cold probe (Varian) spectrometer.
Acknowledgements
Funding for this project was provided by National Institutes of
Health GM095970 and GM100305. We also thank Dr. Dr. X. Huang
(UC San Diego), Dr. A. Mrse (UC San Diego), Dr. Y. Su (UC San Diego)
and Dr. P. Dennison (UC Irvine) for assistance with collection of
NMR and MS data.
(125 MHz, CDCl3) 174.0, 172.2, 172.1,
*
172.0,
*
169.1, 160.9, 159.6,
158.5, 136.7, 136.3, 136.3, 128.8, 128.7, 128.4, 128.2, 127.8, 127.0,
111.8, 108.7, 104.9, 100.4, 77.9, 71.1, 70.6, 70.4, 42.6, 39.8, 39.7, 39.5,
*
39.3, 39.2,
*
36.1, 35.3, 22.4, 20.4, 12.4; HRMS (m/z): [MþH]þ calcd
Supplementary data
for C37H44N4O8þHþ 673.3232; found 673.3230. Multiple confir-
mations of 7 were observed during NMR analysis with duplicate
NMR signatures indicated above by an asterisk.
Supplementary data associated with this article can be found in
the online version, at http://dx.doi.org/10.1016/j.tet.2016.01.062.
4.2.2. Enaminone 8. Isoxazole 7 (40.0 mg, 0.0595 mmol) was dis-
solved in 2 mL of 3:1 MeCN:H2O in 10 mL Teflon tube (Nalgene Oak
Ridge Centrifuge Tubes). Solid Mo(CO)6 (15.7 mg, 0.060 mmol) was
added and the tube was flushed with Ar, capped, and warmed to
85 ꢀC. TLC analysis using 1:9 MeOH:DCM indicated that con-
sumption of starting material was complete within 6 h. The crude
reaction mixture was transferred to a 50 mL round bottom flask and
evaporated to dryness. Flash chromatography (CH2Cl2 to 1:9
MeOH:CH2Cl2) provided 36.1 mg (90% yield) of enaminone 8;
Rf¼0.45 (silica gel, 1:9 MeOH:CH2Cl2); 1H NMR (500 MHz CDC13)
7.39e7.24 (m, 10H), 6.99 (t, J¼5.9 Hz,1H), 6.75 (t, J¼6.0 Hz, 1H), 6.55
(d, J¼2.3 Hz, 1H), 6.54 (d, J¼2.3 Hz, 1H), 5.08 (s, 1H), 5.05 (d,
J¼2.3 Hz, 1H), 5.02 (s, 2H), 3.79 (br s, 2H), 3.54e3.38 (m, 7H), 3.28
(m, 2H), 3.12 (dddd, J¼5.3, 5.3, 5.3, 10.5 Hz, 2H), 2.30 (ddd, J¼4.3,
7.4, 15.3 Hz, 1H), 2.20 (m, 1H), 2.06 (s, 3H), 0.95 (s, 3H), 0.86 (s, 3H);
13C NMR (125 MHz, CDCl3) 197.5, 174.9, 172.4, 172.1, 160.1, 159.8,
157.0, 136.6, 136.5, 135.0, 128.8, 128.7, 128.4, 128.0, 127.8, 127.0,
120.8, 108.4, 100.2, 98.1, 77.9, 71.0, 70.6, 70.3, 41.0, 40.0, 39.6, 39.3,
36.2, 35.3, 29.7, 21.7, 20.6; HRMS (m/z): [MþH]þ calcd for
References and notes
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37H46N4O8þHþ 675.3388; found 675.3389.
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was dissolved in 2:2:1 AcOH:H2O:MeCN and stirred at rt. TLC
analysis using 1:9 MeOH:CH2Cl2 indicated consumption of starting
material after 4 h. The crude reaction mixture was evaporated to
dryness azeotropically with toluene (3ꢁ5 mL). Flash chromatog-
raphy (CH2Cl2 to 1:5 CH2Cl2:MeOH) provided 8.6 mg (70% yield) of
10; 1H NMR (500 MHz CDC13) 12.69 (br s, 1H), 7.51e7.34 (m, 10H),
6.89 (s, 1H), 6.60 (d, J¼2.4 Hz, 1H), 6.42 (d, J¼2.4 Hz, 1H), 2.01 (s,
3H); 13C NMR (125 MHz, CDCl3) 200.7, 168.1, 161.7, 159.9, 146.4,
138.5, 135.9, 135.5, 128.9, 128.8, 128.6, 128.1, 127.7, 107.9, 106.2,
102.5, 100.2, 71.6, 70.5, 36.9, 31.4; HRMS (m/z): [MþH]þ calcd for
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C
26H23NO4þHþ 414.1700; found 414.1701.
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€
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Please cite this article in press as: Rivera, H.; Jr.et al., Tetrahedron (2016), http://dx.doi.org/10.1016/j.tet.2016.01.062