Application of the DP4 Probability Method to Flexible Cyclic Peptides with Multiple Independent Stereocenters: The True Structure of Cyclocinamide A

Article abstract of DOI:10.1021/acs.orglett.8b01756

A DP4 protocol has been successfully utilized to establish the true structure of the natural product cyclocinamide A, a flexible cyclic peptide with four isolated stereocenters. Benchmarking the necessary level of theory required to successfully predict t

Full text of DOI:10.1021/acs.orglett.8b01756

Letter
pubs.acs.org/OrgLett
Cite This: Org. Lett. XXXX, XXX, XXX−XXX
Application of the DP4 Probability Method to Flexible Cyclic
Peptides with Multiple Independent Stereocenters: The True
Structure of Cyclocinamide A
‡
‡
Jason K. Cooper,† Kelin Li, Jeffrey Aube, David A. Coppage,§ and Joseph P. Konopelski*,§
́
†Joint Center for Artificial Photosynthesis and Chemical Science Division, Lawrence Berkeley National Laboratory, Berkeley,
California 94720, United States
‡Division of Chemical Biology and Medicinal Chemistry, UNC Eshelman School of Pharmacy, University of North Carolina at
Chapel Hill, Chapel Hill, North Carolina 27599, United States
§Department of Chemistry and Biochemistry, University of California, Santa Cruz, California 95064, United States
S
* Supporting Information
ABSTRACT: A DP4 protocol has been successfully utilized to establish the
true structure of the natural product cyclocinamide A, a flexible cyclic peptide
with four isolated stereocenters. Benchmarking the necessary level of theory
required to successfully predict the NMR spectra of three previously
synthesized isomers of cyclocinamide A led to the prediction of the natural
stereochemistry as 4S, 7R, 11R, 14S, which has been confirmed by total
synthesis.
n 1997, Crews and co-workers described the isolation,
Istructure, and biological activity (solid tumor activity in a
disk diffusion assay) of the cyclic peptide cyclocinamide A
(CC-A).1 This initial work, together with a second
publication,2 proposed the all-S stereoisomer (1a, Figure
1A) for this natural product constructed of α- and β-amino
acids. Initial synthetic work, which did not benefit from the
stereochemical claims made in the second CC-A publication,
produced the 4R, 7S, 11R, 14S (1b)3 and 4R, 7S, 11S, 14S
(1c)4 isomers. However, neither was found to be the natural
product. Ireland and co-workers isolated a similar compound
that they dubbed cyclocinamide B (CC-B, 2), which was
claimed to differ from its predecessor in two ways: the
addition of a second chlorine atom at C36 and defined
stereochemistry of 4S, 7R, 11S, 14R.5
One of us (UCSC) has prepared 1a,6 the 4S, 7S, 11R, 14S
isomer 1d,6 2,7 and ent-1b7 (i.e., the compound with the core
of 2 and the glycine-pyrrole side chain of compounds 1).
Figure 1. (A) Cyclocinamide structures 1 and 2, stereochemistry
and shorthand designation; (B) truncated structure of CC-A 3 for
computational studies.
None of these compounds corresponded to either natural
1
product as determined by comparison of the H and 13C
NMR data. However, it was determined that CC-A and CC-
B were either enantiomers or identical with respect to their
stereochemistry by comparison of their respective NMR
data.7
Herein, we report the assignment of the stereochemistry of
cyclocinamide A as 1e (4S, 7R, 11R, 14S) through the
Received: June 5, 2018
extension of the DP4 analysis to this 14-membered,
© XXXX American Chemical Society
A
DOI: 10.1021/acs.orglett.8b01756
Org. Lett. XXXX, XXX, XXX−XXX

Organic Letters
Letter
conformationally flexible, cyclic peptide with four unrelated
stereocenters. In addition, we present the total synthesis of
this isomer, thus verifying the DP4 prediction.
Table 1. Computational Data and DP4 Predictions
minimum energy
conformations
The failure to find correspondence between the claimed
and true structures of natural products by synthetic means
has been the experience of many other researchers8 and has
led us to a new strategy. The prediction of 1H and 13C
chemical shifts by purely computational means has advanced
greatly in recent years.9 In addition, the classic problem of
natural product identification, that of having one set of
experimental NMR data that could be assigned to one of
several stereochemically possible structures, has been ex-
plored by Smith and Goodman10 with the development of
the DP4 application. Use of this procedure results in the
assignment of a probability of identity between a given
molecular
DP4 protocol
a
b
c
isomer stereochemistry mechanics
Gaussian
predictions (%)
1a
all-S
105
39
64
102
121
71
13
8
16
19
15
8
0.0
26.3
0.0
0.3
73.2
0.2
ent-1b
1c
7R, 14R
4R
1d
11R
1e
1f
7R, 11R
7R
1g
1h
11R, 14R
14R
61
50
9
7
0.0
0.0
a
Number of low energy conformations from molecular mechanics
b
calculations. Number of low energy (<4 kcal/mol−1) conformations
1
experimental set of H and/or 13C NMR spectra and each of
c
from single-point energy calculations. Probabilities using DP4 applet
from comparison of cyclocinamide A (CC-A) experimental NMR
data with computational NMR data from all isomers.
the corresponding computationally derived data sets for the
stereoisomers.11
Implementation of this methodology to the present
problem, however, was not without its challenges. The
stereogenic centers in CC-A are isolated from one another;
no useful NOE data beyond the nearest neighbor relation-
ships were obtained, and the four amide bonds effectively
isolate each spin system.1 Furthermore, there was no
detectable interaction between the glycine-pyrrole fragment
at C11, the tryptophan residue at C7, and the asparagine side
chain at C14. In addition, the flexibility of CC-A would
require the incorporation of a number of low-energy
conformations that contribute to the final calculated spectra.
Structure 3 was employed in the calculations. Such an
approach was deemed reasonable due to the lack of
interactions between side chains on the macrocycle (vide
supra). In addition, we had previously argued that evaluation
of the chemical shift data rested most reasonably on the ring
sp3 carbons and hydrogens, where all the stereochemistry
resides, and not on the more conformationally mobile side
chains.
Table 1, the number of low energy conformations ranged
from 7 for the 14R structure to 19 for 1d (11R). These
1
structures were then used to calculate the H and 13C NMR
shielding constants with the mPW1PW91/6-311+G(3df,2p)
level of theory. To transform the shielding constants to
chemical shift data, N-methylacetamide in DMSO was
employed as the reference, calculated at the same level of
theory.9,15,16 The Gibbs free energy of each conformer was
used to assess its contribution to the Boltzmann distribution
of structures contributing to the final NMR shielding
constants. From these results, 92−99% identity was obtained
from the DP4 analysis for each of the three structures; only
the core sp3 centers (1H and 13C spectra) were used in the
comparison (Supporting Information, Tables S1 and S2).17
With a confirmed computational method in place, the final
DP4 analysis was performed on all eight possible diaster-
eomers of CC-A. Table 1 gives the final results, which
identified the 7R, 11R isomer 1e as the most probable
stereochemical match to the natural product. The only other
isomer with an appreciable, but distinctly lower, probability of
identity with CC-A was ent-1b, which the previous synthetic
work had shown was not the desired product.
Based on this analysis, we prepared isomer 1e using an
analogous route to that employed in the production of 2 and
ent-1b (Scheme 1). Thus, methyl R-5-bromotryptophan 4
was coupled with 5 (prepared as previously reported6) to
afford dipeptide 6, which itself was subjected to TFA
deprotection and coupled with commercially available (S)-
Fmoc-Asn(Tr)-OH 7 to provide the 4S, 7S, 11R-tripeptide 8
(CC-A numbering). Additional deprotection and coupling,
this time with (R)-10, provided tetrapeptide 11 in a
moderate 49% yield from 8, but with 40% of recovered 9,
which could be recycled. Carrying out this reaction for an
extended time did not lead to improved yields. The
conversion of 11 to seco-acid 13 required deprotection of
the Fmoc amine and carboxylic acid saponification. The latter
reaction proved sensitive, with the best results being obtained
from carefully monitored treatment with a base at 0 °C for 3
h; extended exposure of 13 to the base at room temperature
led to extensive decomposition. Cyclization was accomplished
by treating 13 with DEPBT, affording compound 14 in 71%
yield. Reductive deprotection of 14 removed both the Boc
and the terminal amide trityl group, setting up amine
coupling with carboxylic acid 156 to give hexapeptide 16 in
1
The work initially focused on computing the H and 13C
chemical shifts of the three isomers most recently prepared:
1a (all-S), 1d (11R), and ent-1b (7R, 14R). The computa-
tional protocols would be continually refined until the DP4
application could assign a high probability of identity upon
comparison of a given experimental set of 1H and 13C
chemical shifts with the corresponding calculated spectra for
that isomer when challenged with the calculated spectra of all
three isomers. Once this benchmarking task was complete,
the same methodology would be applied to all possible
stereoisomers of cyclocinamide A for comparison with the
experimental shifts of the natural product.12
Table 1 gives an overview of the initial conformational
search results for each isomer using molecular mechanics
calculations (Spartan 1013). However, the initial levels of
theory and basis sets employed within Gaussian 0914 used to
refine the energy levels and provide the calculated spectral
data did not produce adequate results. Specifically, we were
unable to obtain correspondence of the experimental vs
calculated values for the 7R, 14R isomer with the DP4
method from our initial calculations. Successful implementa-
tion was finally achieved by an additional geometry
optimization from the conformations with energies <4 kcal
mol−1 from the minimum using B3LYP/6-311+G(d,2p),
which also included a frequency calculation on the final
structures to obtain the Gibbs free energies. As shown in
B
DOI: 10.1021/acs.orglett.8b01756
Org. Lett. XXXX, XXX, XXX−XXX

Organic Letters
Letter
Scheme 1. Synthesis of 7R, 11R-Cyclocinamide A
66% yield (2 steps). Finally, TBDPS removal by treatment
that the DP4 protocol can be effectively utilized to guide
synthetic target identification on flexible molecules, provided
the necessary level of theory for the problem can be
ascertained.19
with TBAF gave the target alkaloid 1e in 44% yield.
1
The H and 13C NMR spectra of 1e were compared to the
values originally reported by Crews1 (Table S3). Figures S1
and S2 provide a graphical comparison of the ring sp3
carbons and hydrogens of all four of the recently prepared
CC-A isomers (1a, ent-1b, 1d, and 1e) to the natural
material. Table S4 provides the corresponding 13C data in
tabular form color-coded for the magnitude of chemical shift
variation from that of the natural material; the same
comparison of natural CC-B with synthetic 2 is also provided
in this table. The close correspondence of the 7R, 11R isomer
spectral data with corresponding natural product data
strongly support the assignment of compound 1e as
cyclocinamide A. The most notable difference in the
synthetic sample is the doublet appearing at δ 6.00 ppm,
which we assign to the hydroxyl OH group (a likely
candidate for exchange in the natural sample).18 Otherwise,
the largest disparity between our synthetic sample and the
originally isolated compound is the magnitude of the
ASSOCIATED CONTENT
■
S
* Supporting Information
The Supporting Information is available free of charge on the
ACS Publications website at DOI: 10.1021/acs.or-
glett.8b01756.
Details and tables of computational data, comparison of
NMR data between final synthetic compound and
natural product, full experimental, and NMR spectra
(PDF)
AUTHOR INFORMATION
■
Corresponding Author
*E-mail: joek@ucsc.edu.
ORCID
dextrorotatory specific rotation: [α]21 +102.5 for the
D
synthetic material vs [α]21 +29 for the original isolated
D
sample (c 0.1, MeOH for both).
Jason K. Cooper: 0000-0002-7953-4229
In conclusion, we have succeeded in establishing the
absolute stereochemistry and structure of the marine natural
product cyclocinamide A. In addition, we have demonstrated
́
Jeffrey Aube: 0000-0003-1049-5767
Joseph P. Konopelski: 0000-0002-7910-8596
C
DOI: 10.1021/acs.orglett.8b01756
Org. Lett. XXXX, XXX, XXX−XXX

Organic Letters
Letter
́
́
G.; Lindeberg, G.; Karlen, A.; Hallberg, M.; Erdelyi, M.; Hallberg, A.
J. Med. Chem. 2010, 53, 8059−8071.
Notes
The authors declare no competing financial interest.
ACKNOWLEDGMENTS
■
Partial support for the synthetic chemistry in the form of
start-up funding awarded from the University of North
Carolina at Chapel Hill to J.A. is gratefully acknowledged. We
also acknowledge the UNC Department of Chemistry Mass
Spectrometry Core Laboratory for HR-MS analysis, and Dr.
Sarah M. Scarry and Dr. Greg Young (UNC) for assistance
with NMR. The work of D.A.S. was supported by grants from
the NIH (R01 CA47135 and R25 GM104552). One of us
(J.P.K.) would like to offer his sincere thanks to his UC
colleagues, Prof. D. Tantillo and Dr. M. Lodewyk (UC
Davis), for their many conversations regarding the computa-
tional work, and Prof. P. Crews (UCSC) for many fruitful
discussions on the subject of this paper and for sharing the
underwater image of Psammocinia aff. bulbosa used in the
graphical abstract.
REFERENCES
■
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D
DOI: 10.1021/acs.orglett.8b01756
Org. Lett. XXXX, XXX, XXX−XXX