Total Synthesis of Scytonemide A Employing Weinreb AM Solid-Phase Resin

Article abstract of DOI:10.1021/acs.jnatprod.7b00912

The human 20S proteasome inhibitor scytonemide A (1), a macrocyclic imine originally isolated from the cyanobacterium Scytonema hofmanni, was synthesized via a biomimetic solid-phase peptide synthesis (SPPS) approach employing the Weinreb AM resin. Utilizing this approach, cyclization of the protected heptapeptide via formation of the imine bond occurred spontaneously upon cleavage from the resin in the presence of a reducing agent and subsequent aqueous workup. The final deprotection step necessary to produce the natural product was accomplished under slightly basic conditions, facilitating cleavage of the silyl ether group while leaving the macrocycle intact. Purification of the synthetic scytonemide A was accomplished via normal-phase flash column chromatography, potentially facilitating larger scale preparation of the compound necessary for future mechanistic and SAR studies. The structure of the target compound was confirmed by NMR spectroscopy, which also shed light on differences in the spectroscopic data obtained for the synthetic and natural scytonemide A samples for some of the amide and alcohol signals in the ¹H NMR spectrum.

Full text of DOI:10.1021/acs.jnatprod.7b00912

Article
Cite This: J. Nat. Prod. XXXX, XXX, XXX−XXX
pubs.acs.org/jnp
Total Synthesis of Scytonemide A Employing Weinreb AM Solid-
Phase Resin
Tyler A. Wilson,†,§ Robert J. Tokarski, II, Peter Sullivan, Robert M. Demoret, Jimmy Orjala,
†,§
‡
,†
†
‡
†
L. Harinantenaina Rakotondraibe, and James R. Fuchs*
†Division of Medicinal Chemistry & Pharmacognosy, College of Pharmacy, The Ohio State University, Columbus, Ohio 43210,
United States
‡
Department of Medicinal Chemistry and Pharmacognosy, College of Pharmacy, University of Illinois at Chicago, Chicago, Illinois
60612, United States
*
S Supporting Information
ABSTRACT: The human 20S proteasome inhibitor scytonemide A (1), a
macrocyclic imine originally isolated from the cyanobacterium Scytonema
hofmanni, was synthesized via a biomimetic solid-phase peptide synthesis
(
SPPS) approach employing the Weinreb AM resin. Utilizing this approach,
cyclization of the protected heptapeptide via formation of the imine bond
occurred spontaneously upon cleavage from the resin in the presence of a
reducing agent and subsequent aqueous workup. The final deprotection step
necessary to produce the natural product was accomplished under slightly
basic conditions, facilitating cleavage of the silyl ether group while leaving the
macrocycle intact. Purification of the synthetic scytonemide A was
accomplished via normal-phase flash column chromatography, potentially
facilitating larger scale preparation of the compound necessary for future mechanistic and SAR studies. The structure of the target
compound was confirmed by NMR spectroscopy, which also shed light on differences in the spectroscopic data obtained for the
1
synthetic and natural scytonemide A samples for some of the amide and alcohol signals in the H NMR spectrum.
1
n 2010, Krunic et al. reported the isolation of scytonemides
required the preparation of the linear amino aldehyde precursor
of 1, which was then cyclized via titration with triethylamine
(TEA) in deuterated methanol. Ultimately, the key cyclization
event effectively exploited the pH-dependent equilibrium
between the cyclic and acyclic forms of these peptides
I
A (1) and B from the cultured freshwater cyanobacterium
Scytonema hofmanni (UTEX 1834). Scytonemide A (1), a novel
cyclic peptide containing an unusual imine linkage, was found
to potently inhibit human 20S proteasome chymotrypsin
1
10
catalytic activity in vitro with an IC50 of 96 nM. Although it
previously demonstrated with the nostocyclopeptides.
has not yet been established whether the imine moiety is
responsible for this activity, this structural motif does make
scytonemide A an interesting and potentially challenging target
for chemical synthesis. In addition to scytonemide A, imine
linkages have only been observed in a small number of naturally
occurring macrocycles, namely, in nostocyclopeptides A1 (2),
2
3
4
A2 (3), M1, and koranimine (4). Although scytonemide A,
, and 3 are fairly homologous with respect to their amino acid
2
sequences, nostocyclopetptide M1 and 4, despite also being
heptapeptides, vary more dramatically.
Unfortunately, in the case of scytonemide A, further
development as a potential proteasome inhibitor has been
hampered by the relatively small quantities that can be
successfully isolated from the cyanobacterium (i.e., 1 mg
In addition to the imine functionality, the cyclic nature of this
natural product also makes it a potentially attractive target from
a drug discovery and development perspective. In general,
macrocyclic peptides have received significant attention
recently due to their large surface areas, limited conformational
flexibility, and promising drug properties, suggesting the ability
1
from 9 L of culture). In addition, despite the efficient
syntheses and subsequent transformations of the imines noted
8
above, the purification of scytonemide A itself was not
5
−8
to better inhibit protein−protein interactions.
As further
9
evidence of this trend, Malins et al. recently published the
synthesis of a series of peptide macrocycles, utilizing the cyclic
imines 1 and 4 as intermediates in the production of more
stable amine adducts with non-native motifs. This synthesis
Special Issue: Special Issue in Honor of Susan Horwitz
Received: October 30, 2017
©
XXXX American Chemical Society and
American Society of Pharmacognosy
A
DOI: 10.1021/acs.jnatprod.7b00912
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Journal of Natural Products
Article
reported as a part of this effort. With this in mind, we were
interested in developing an alternative method for the
preparation of scytonemide A that could produce the quantities
of the natural product necessary for biological testing. In
considering the total synthesis of 1, two primary goals for this
effort were established, namely, the efficient and rapid synthesis
of the natural product and the isolation of the desired
compound without the need for high-pressure liquid
chromatography (HPLC) purification.
RESULTS AND DISCUSSION
■
9
10
Similar to the approaches of Malins and Kopp, solid-phase
peptide synthesis (SPPS) methodology was envisioned to
facilitate rapid access to the heptapeptide precursor of 1. In this
case, however, inspiration for the choice of resin/linker was
drawn from the biosynthesis of nostocyclopeptide A1 (Figure
1
A), proceeding via reductive cleavage from the nonribosomal
11
peptide synthetase (NRPS).
In studies of the mechanism through which this process takes
place, reductive cleavage of thioester-containing peptidyl-CoA
substrates designed to mimic the natural system were shown to
result in direct cyclization (self-assembly) to form the imines as
detected by HPLC-MS in both the presence and absence of the
recombinant NRPS reductase domain and the adjacent
10
peptidyl-carrier protein. This study demonstrated that the
cyclization reaction was not dependent on the enzyme itself. By
analogy, therefore, a reductive cleavage of the heptapeptide
directly from a solid-phase resin (Figure 1B) would also be
expected to result in formation of the desired imine.
With this in mind, the Weinreb aminomethyl (AM) resin was
chosen for the synthesis since reduction of the N-methoxy-N-
alkylamide linkage in the presence of a reducing agent has been
shown to provide the corresponding aldehyde through the
1
2,13
unique reactivity of the Weinreb amide motif.
Since the
1
4
introduction of the Weinreb amide resin, it has been
recognized as a useful tool for the preparation of amino and
15−18
peptidyl aldehydes.
For instance, fellutamide B, another
peptidyl aldehyde with proteasome inhibitory activity, was
19
synthesized using a Weinreb amide resin approach. As
opposed to these applications, cleavage of the scytonemide A
heptapeptide from this resin was expected to generate the
desired peptidyl aldehyde precursor that would subsequently
undergo intramolecular condensation with the free N-terminal
amine to provide the desired cyclic imine.
Figure 1. (A) Biosynthesis of 2 via reductive cleavage of peptide from
Ncp nonribosomal peptide synthetase (NRPS). (B) “Biomimetic”
formation of 1 via cleavage of peptide from Weinreb resin.
In practice, the initial route taken to synthesize 1 employing
the Weinreb resin is illustrated in Scheme 1. The on-resin
heptapeptide 5a was assembled following standard SPPS
protocols. During this process, the Ser, D-Gln, and Tyr
residues were introduced with the relatively stable tert-butyl and
trityl protecting groups due to the potentially harsh basic and
reductive conditions of the subsequent cleavage step and to
provide increased lipophilicity for the extraction and
purification of the cyclized product upon reduction.
of the product mixture using mass spectrometry indicated the
direct formation of the cyclic imine 6a, which could be purified
using normal-phase silica gel column chromatography. The
1
9
formation of the imine was further supported by the presence
1
of a doublet (J = 6.9 Hz) at δ 6.94 in the H NMR spectrum.
H
This doublet showed a correlation with the carbon at δ 165.7
C
in an HSQC experiment (Supporting Information, page S5),
correlating with the data reported for the imine signal of
1
Once assembled, the terminal Fmoc group of heptapeptide
a was removed with piperidine. The N-deprotected resin-
scytonemide A itself. Surprisingly, analysis of the crude
1
5
reaction mixture by H NMR showed no diagnostic signal for
bound amino peptide was then treated with excess lithium
aluminum hydride (LAH) to liberate the desired peptidyl
aldehyde from the resin. This type of reduction has also been
reported by Payne and co-workers in their total synthesis of
fellutamide B. The selective reduction of the Weinreb amide
in these transformations is particularly remarkable due to both
the heterogeneous nature of the reducing agent and the
presence of other potentially reactive carbonyl groups. Analysis
the presence of the aldehyde, suggesting that the condensation
reaction appears to be favored under conditions in which the
aldehyde is generated in the presence of the basic N-terminal
amine. The facile nature of this ring closure may be explained
by drawing comparisons between compound 6 and the closely
related structures of the nostocyclopeptides. The cyclization of
the nostocyclopeptides has been suggested to be aided by the
1
9
20
“elaborated prefolding” of the acyclic aldehyde precursor of 2,
B
DOI: 10.1021/acs.jnatprod.7b00912
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Journal of Natural Products
Article
Scheme 1. Total Synthesis of 1 Using Weinreb AM Resin and Acid-Labile Protecting Groups
21
allowing it to adopt a “product-like” conformation in solution,
synthesis or its cleavage from the resin, as the efficiencies of
the subsequent SPPS coupling steps were not affected and the
cyclic imine 6b could still be obtained through the reduction of
5b without undesired reduction of the unprotected amide.
Removal of the remaining tert-butyl protecting groups at this
stage, however, remained problematic. Under mild acidic
conditions using trifluoroethanol (TFE), the tert-butyl groups
were unaffected. Employing stronger conditions (TFA) once
again resulted in undesired hydrolysis of the imine. Based on
the difficulty in removal of these tert-butyl groups after the
imine was formed, a global deprotection step was carried out on
the resin to remove these groups to provide 5c. Treatment of
this material with LAH resulted in a complex mixture of highly
polar products. Although 1 could be detected by TLC and MS
analyses, purification of the desired compound from the
mixture proved to be difficult without the use of HPLC.
Therefore, a new strategy was required in order to effectively
produce scytonemide A. It was obvious from the results
obtained thus far that the cyclization event could be successfully
accomplished with the aid of protecting groups, but the groups
employed would need to be revised in order to maintain the
desired levels of lipophilicity to aid in chromatographic
separation of this product and would also need to be cleaved
under nonacidic conditions in order to keep the imine moiety
intact. Silyl protecting groups ideally fit these criteria due to
their strong lipophilic properties, relative stability to the
coupling steps, and cleavage under mild, nonacidic conditions
with tetrabutylammonium fluoride (TBAF). For this reason,
the tert-butyldimethylsilyl (TBS)-protected ethers were tar-
geted for both the Ser and Tyr residues.
bringing the aldehyde and amine functionality into close
proximity and facilitating the imine self-assembly.
10
Interestingly, the yield of the product obtained could be
improved by repeating the reduction step two additional times
using the same resin, cleanly providing quantities of the desired
imine upon each treatment. Purification of these combined
fractions via column chromatography provided an overall yield
of 40% based on the theoretical loading of the resin (0.441
mmol/g). The successful resubjection of the resin to obtain
additional macrocyclic product suggests that the steric
environment of the resin hinders nucleophilic attack of the
Weinreb amide motif to some degree in the presence of the
attached heptapeptide.
With compound 6a in hand, at this stage all that remained in
order to complete the synthesis of 1 was the global
deprotection of the acid-labile side chain protecting groups.
Unfortunately, but not unexpectedly, treatment of 6a with
trifluoroacetic acid (TFA) resulted in hydrolysis of the imine
and the formation of a complex mixture of cyclic and acyclic
products as observed by MS analysis. Subjection of this mixture
to solid Na CO in MeOH successfully regenerated the imine,
2
3
although small amounts of byproducts (possibly epimers) with
similar retention times and identical molecular weights were
also observed in the HPLC trace for the product mixture.
HPLC purification was able to successfully provide 1.1 mg of 1
from a 7 mg sample, but the low yield led us to reconsider the
synthetic route, specifically with regard to the protecting groups
employed on the Tyr , D-Gln , and Ser residues.
1
3
5
Initial attempts to address the requirements for the
protecting groups for the LAH reduction step focused on the
sequential removal of the trityl and then tert-butyl groups.
During these studies, it was shown that the trityl protecting
The TBS-protected Fmoc-Tyr-OH required for the synthesis
of the heptapeptide was prepared according to the procedure of
22
Taleski et al. This same procedure was also modified to
group on the D-Gln residue was not necessary for peptide
produce the corresponding TBS-protected Fmoc-Ser-OH
3
C
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Journal of Natural Products
Scheme 2). For the preparation of this compound,
Article
(
however, indicated formation of the monosilylated imine
product 12a as opposed to the bis-protected product. In an
effort to determine which silyl group had been lost during the
course of this reaction, further spectroscopic data were needed.
commercially available Fmoc-Ser(tBu)-OH (7) was first
Scheme 2. Synthesis of Fmoc-Ser(TBS)-OH via Application
22
1
13
of the Protocol of Taleski et al.
A comparison of the H NMR and C NMR spectroscopic
data of silylated peptide 12a with the isolated 1 indicated little
to no difference in the Ser 3a (Δ = 0.00 ppm) and 3b (Δ
5
δH
δH
=
0.01 ppm) proton shifts and 3a/b carbon (Δ = 0.01 ppm)
δC
shifts. In contrast, clear differences in the chemical shifts
between the tyrosine 5/9 (Δ = 0.11 ppm) and 6/8 (Δ
=
=
δH
δH
0
4
.09 ppm) protons and 5/9 (Δ = 0.26 ppm) and 6/8 (Δ
δC
δC
.54 ppm) carbons were observed, suggesting that the silyl
protecting group had been removed from the Ser residue. A
5
selective 1D NOESY experiment also revealed a through-space
correlation between the methyl protons of the TBS group and
the protons on Tyr positions 6/8 (Figure S2). In addition, an
1
HMBC experiment revealed that an exchangeable proton,
2
observed as a triplet around 5.55 ppm, was found to have J and
3
J correlations with Ser -C2 and Ser -C3, indicating that this
converted into the allyl ester 8. The tert-butyl ether was then
deprotected with TFA, and the primary alcohol was reprotected
as the TBS ether 9. Removal of the allyl group from 9 was
affected with Pd(PPh ) to form the desired amino acid 10,
which was then used for preparation of the heptapeptide.
With the TBS-protected tyrosine and serine residues in hand,
the heptapeptide was once again assembled utilizing these
amino acids to produce 11a (Scheme 3), applying the same
methodology used to produce 5. Subjection of 11a to LAH was
expected to yield the bis-silylated macrocycle. Once again, the
reduction proceeded smoothly, and an imine macrocycle was
obtained as the major product. Analysis by mass spectrometry,
5
5
was the Ser5 alcohol proton (Figure S8). Overall, these
spectroscopic data confirmed the structure of the monosilylated
imine 12a, verifying the presence of the TBS group on the Tyr1
residue. The loss of the protecting group during the course of
this reaction is interesting because TBS ethers are typically
compatible with reductive reaction conditions. There have been
reports, however, of the loss of TBS groups in the presence of
LAH when other heteroatoms are proximal to the silyl
3
4
23−26
ether.
Mechanistically, de Vries and co-workers suggested
that this type of deprotection proceeds through an intra-
molecular hydride delivery from an aluminum species
coordinated to the nearby heteroatom group through either a
deprotonation or reduction event. In the present case, it is
conceivable that the silyl group removal is affected by
coordination of the aluminum species to the N atom of the
Ser residue, resulting in a seven-membered transition state for
delivery of the hydride.
23
Scheme 3. Revised Protecting Group Strategy Using Silyl-
Protected Ser and Tyr for Completion of the Total Synthesis
of 1
In an effort to improve the stability of the silyl protecting
group on the Ser residue, the more stable and sterically
encumbered triisopropylsilyl (TIPS) ether (13) was also
prepared and introduced into the heptapeptide. In this case,
treatment with LAH resulted in the production of two products
in an approximate ratio of 2.4:1. The major product in this case
was still found to be imine 12a, corresponding to removal of
the TIPS group. In addition, however, the bis-silylated product
12b could be isolated in this case, reflecting the attenuated
reactivity of the TIPS group toward reductive deprotection as
compared to the TBS moiety. Because 12b was obtained in
lower yields, however, imine 12a obtained from reduction of
11a was taken on to compound 1.
To complete the synthesis of scytonemide A, therefore, silyl
ether 12a was subjected to standard silyl deprotection
conditions using TBAF. These slightly basic reaction conditions
cleanly provided the deprotected material in 51% yield without
affecting ring opening or epimerization, based on the presence
1
of a single imine signal in the H NMR. Importantly, this
product could be cleanly isolated as a white solid using normal-
phase silica gel column chromatography and did not require
1
13
1
HPLC purification. The H and C NMR data for the natural
scytonemide A and the synthetic sample are shown in Table 1.
13
Although the resonances observed in the C NMR spectrum
1
agree with the reported values, the H NMR spectra of the
product obtained did not completely match the reported
isolation spectra. This was quite evident when the spectra were
D
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Journal of Natural Products
Article
Table 1. Comparison of H and 13C NMR Data of Naturally Occurring Scytonemide A with Synthesized Scytonemide A in
1
a
DMSO-d6
b
natural scytonemide A
δC, type
synthetic 1
δH, mult. (J in Hz)
position
δH, mult. (J in Hz)
δC, type
Tyr1
1
2
172.93, C
74.0, CH
40.1, CH2
172.39, C
73.5, CH
3.53, dd (11.3, 2.8)
3.54, dd (11.2, 2.4)
3.05, dd (13.4, 2.4)
2.48, dd (13.4, 11.2)
3
3
4
5
6
7
a
3.04, dd (13.5, 2.4)
39.45, CH2
b
2.48, ddd (13.5, 13.5, 2.4)
127.6, C
131.4, CH
115.3, CH
156.4, C
127.32, C
130.91, CH
114.78, CH
155.8, C
/9
/8
6.86, d (8.5)
6.61, d (8.5)
6.86, d (8.5)
6.61, d (8.5)
OH
1
9.15, s
Gly2
171.28, C
40.6, CH2
170.84, C
2
a
4.32, m
39.88, CH2
4.32, dd (16.8,10.3)
3.42, dd (16.8, 2.8)
7.75, dd (10.3, 2.8)
2
b
3.42, m
NH
1
7.78, dd (10.0, 2.6)
D-Gln3
172.58, C
53.8, CH
25.7, CH2
172.14, C
53.52, CH
25.23, CH2
2
4.33
4.31, dt (6.8, 6.5)
1.85, m
3
3
4
4
5
a
1.85, m
1.76, m
2.11
b
a
1.76, m
31.8, CH2
173.68, C
31.26, CH2
173.20, C
2.11, ddd (15.5, 10.3, 5.6)
1.99, ddd (15.5, 10.3, 5.6)
b
1.99
NH
8.55, d (6.3)
7.33, br s
8.54, d (6.5)
7.31, br s
NH2
6
.78, br s
6.77, br s
Ile4
1
2
3
170.15, C
58.0, CH
35.7, CH
16.4, CH3
24.1, CH2
12.5, CH3
169.75, C
57.51, CH
35.22, CH
15.95, CH3
23.61, CH2
11.96, CH3
4.17, dd (9.2, 3.9)
2.06, m
4.18, dd (9.1, 3.9)
2.06, m
3
4
5
-Me
0.86, d (7.0)
1.28, m
0.86, d (7.0)
1.28, m
0.83, m
0.84, t (7.4)
8.17, d (9.1)
NH
1
8.17, d (9.3)
Ser5
Val6
Leu7
172.56, C
53.0, CH
62.8, CH2
172.06, C
52.58, CH
62.22, CH2
2
4.93, m
4.90, td (9.5, 6.5)
3.89, ddd (9.9, 9.5, 5.8)
3.62, ddd (9.9, 6.5, 5.8)
5.55, t (5.8)
3
a
3.87, dd (10.0, 10.0)
3.60, dd (9.8, 6.0)
3
b
OH
NH
1
7.36, d (9.0)
7.37, d (9.5)
170.31, C
59.7, CH
29.0, CH
17.4, CH3
19.7, CH3
169.77, C
59.18, CH
28.44, CH
16.97, CH3
19.18, CH3
2
4.01, dd (7.7, 4.2)
2.22, m
4.01, dd (7.6, 4.2)
2.24, sept d (7.0, 4.2)
0.89, d (7.0)
3
3
-Me
0.90 d (7.0)
0.93, d (7.0)
8.40, br s
6.80, d (1.8)
4.37, m
4
0.93, d (7.0)
NH
1
8.16, d (7.6)
166.0, CH
49.6, CH
40.6, CH2
165.60, CH
49.11, CH
39.80, CH2
6.81, d (1.8)
2
4.37, dddd (10.6, 9.3, 4.7, 1.8)
1.05, ddd, (13.6, 10.6, 5.3)
0.81, m
3
3
4
4
5
a
1.05
b
0.81
24.8, CH
23.4, CH3
21.7, CH3
1.29, m
24.26, CH
22.82, CH3
21.16, CH3
1.29, m
-Me
0.80, d (6.7)
0.72, d (6.5)
7.90, d (9.4)
0.80, d (6.7)
0.72, d (6.5)
NH
7.78, d (9.3)
a
1
13
1
13
b
Frequency: natural 900 MHz for H, 226 MHz for C, synthetic 700 MHz for H, 176 MHz for C. J. Nat. Prod. 2010, 73, 1927−1932.
overlaid (Figure S10), as a few of the previously reported
signals in the aromatic/amide region were seen to shift
differently and two “new” resonances were observed at 5.55
and 9.15 ppm. Although differences were noted in comparison
to the isolated material, the obtained spectrum of the synthetic
On the basis of the similarities in the 13C spectra and the alkyl
1
region of the H spectra reported for all three samples, we were
relatively confident that the synthetic material was in fact
scytonemide A. In order to explain the observed differences and
to unambiguously confirm the identity of the synthesized
9
material was identical to the spectrum reported by Malins et al.
E
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Figure 2. Expanded view of HSQC spectrum of 1 showing the cross-peak corresponding to the diagnostic imine moiety.
Figure 3. 1D TOCSY experiment, with irradiation of Ser OH (blue), overlaid with a spectrum of 1 (red).
product, however, more extensive NMR experiments were
undertaken.
amide and alcohol protons (Figure S1) and confirmed the
carbon framework of scytonemide A.
In an effort to examine and assign the protons in the
aromatic/amide region, an HSQC experiment was used to
confirm the presence of the macrocyclic imine and identify
which protons lacked a carbon correlation, suggesting that
these were “exchangeable” heteroatom protons (Figure 2 and
page S28 in the Supporting Information). Based on this
experiment, the imine and tyrosine protons could be easily
identified, and the fact that only one macrocyclic imine signal
existed in this sample was confirmed. The remaining protons
were likely amide −NH or possibly alcohol −OH protons. This
was further supported by the disappearance of these signals, as
well as the signal at 5.55 ppm, in deuterated methanol
At this stage, the “new” signals at 5.55 and 9.15 ppm
remained to be assigned. As all of the amides were accounted
for, it seemed likely that these two protons corresponded to the
Ser and Tyr −OH signals. Neither of these resonances had
been reported in the spectrum of the isolated material, although
1
very broad signals are visible in these regions in the H NMR
spectrum provided in the Supporting Information of the
isolation paper. A triplet (5.58 (t, J = 5.4 Hz, 1H) similar to the
splitting of the signal at 5.55 ppm of scytonemide A also
1
appeared in the H NMR spectrum of compound 12a, but the
singlet at 9.15 was not present in that spectrum. Based on the
assignment of the silyl group on the Tyr residue of 12a and the
observed splitting pattern, this suggested that the triplet was the
Ser −OH proton. This was confirmed by a selective TOCSY
experiment in which the triplet was irradiated, providing
correlations with the Ser protons at 3.62, 3.89, and 4.90 ppm
(Supporting Information, page S29). Further comparison of the
silylated precursor 12a and the final product, along with
HMBC (Figures S3−S8) and selective TOCSY spectra (Figure
S9) for these compounds, facilitated identification of individual
F
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Journal of Natural Products
Figure 3, Table 1). The remaining singlet at 9.15 ppm is likely
Article
(
5 mL), CH
was then applied to ensure proper coupling.
2
Cl
2
(2 × 5 mL), and DMF (2 × 5 mL). The Chloranil test
the phenol signal of the Tyr residue. Despite the fact that no
correlations were observed for this proton in the HMBC and
TOCSY experiments, analogy for this assignment can be drawn
Loading Efficiency. Loading efficiency was determined in
accordance with the procedure reported by Malins et al., employing
9
2
7
1
the extinction coefficient reported by Eissler et al. The Fmoc-Leu-
loaded Weinreb resin was shaken in a solution of piperidine/DMF (3
mL, 1:9 v/v) for 5 min. The solvent was filtered off, and the procedure
was then repeated for 10 min. The filtrates were then combined, and
an aliquot (150 μL) was diluted 20-fold to 3 mL. A 300 μL amount of
this solution was diluted 10-fold to 3 mL and placed in a quartz
cuvette to measure UV absorbance of the piperidine-fulvene adduct (λ
from the reported spectrum of scytonemide B, which
possesses a similar tyrosine −OH signal reported as a singlet
at 9.24 ppm.
Altogether, the spectroscopic data reported herein confirm
the structural assignment of the synthetic material as
scytonemide A. A complete NMR assignment of scytonemide
A (1) is summarized in Table 1. In accordance with our plans
from the outset of the project, purification of the desired
product was successfully achieved using normal-phase silica gel
flash column chromatography. The overall synthetic strategy is
expected to facilitate the preparation of the quantities of the
natural product necessary for further biological evaluation of its
proteasome inhibitory activity and subsequent preparation of
structural analogues.
−1
−1
=
289.8 nm, ε = 6089 M cm ) for quantification of the Leu residue
c
loaded onto the resin. The theoretical maximum yields of the isolated
peptides are based on the numerical value obtained from this resin
loading, as opposed to the theoretical loading reported by the
manufacturer.
Fmoc Deprotection. The resin was then shaken in a solution of
piperidine/DMF (3 mL, 1:9 v/v) for 5 min and then repeated for 10
min. The resin was filtered and washed with DMF (2 × 5 mL),
CH Cl (2 × 5 mL), and DMF (2 × 5 mL). A Chloranil test was
2
2
applied to ensure deprotection had occurred. The Fmoc deprotection
process was repeated after each amino acid coupling.
EXPERIMENTAL SECTION
■
Amino Acid Coupling. To the resin (1.0 equiv) was added a
solution of amino acid (3.0 equiv), HATU (2.9 equiv), and DIPEA
(3.5 equiv) in DMF (0.2 M relative to amino acid), and the mixture
was agitated for 15 min. The solvent was then filtered off, and the
procedure was then repeated for 15 min. The resin was filtered and
washed with DMF (2 × 5 mL), CH Cl (2 × 5 mL), and DMF (2 × 5
General Experimental Procedures. Melting points were
recorded using a Thomas-Hoover melting point capillary apparatus.
Optical rotation was measured on an Anton Paar MCP 150
polarimeter. UV absorption spectra were measured on a Hitachi
U2910 spectrophotometer. Infrared (IR) absorption spectra were
recorded on a Thermo Nicolet 6700 FT-IR spectrometer. NMR
2
2
mL). The Chloranil test was applied to ensure coupling had occurred.
28
spectra were recorded at 300 K using a Bruker AV 300 MHz, AVIII
Chloranil Test. After each Fmoc deprotection and amino acid
coupling step, the Chloranil test for free amines was used to ensure
that the Fmoc group had been removed or the proper amino acid had
been coupled. Solutions of acetaldehyde/DMF (2% v/v) and
chloranil/DMF (2% m/v) were prepared. After filtration of the
resin, a glass capillary tube was used to remove a small amount of the
beads. The capillary tube was then placed into an Eppendorf tube. The
acetaldehyde/DMF was then added dropwise (100 μL), followed by
the chloranil/DMF (100 μL). After chloranil/DMF addition, the
Eppendorf tubes were slightly shaken to ensure mixing with the resin,
then allowed to stand for 1 min. The resin was visually checked for
color development: deep blue colored beads indicated free amine
(complete Fmoc deprotection), while colorless/clear beads indicated
no free amine (complete coupling of the amino acid).
General Procedure for Cleavage from Weinreb AM Resin.
The on-resin heptapeptide (1.0 equiv) was swollen in dry THF (0.05
M resin) and cooled to 0 °C. LiAlH4 (11.0 equiv) was added
portionwise, and the mixture was allowed to stir for 2.25 h. The
mixture was again cooled to 0 °C and diluted with EtOAc (5 mL). The
mixture was then quenched with saturated Rochelle’s salt solution (5
mL) and allowed to stir for 15 min to ensure quenching. The mixture
was then filtered using a fritted filter to remove any solid particulates.
The resulting filtrate was extracted three times using EtOAc. The
combined EtOAc fractions were concentrated in vacuo to yield the
desired cyclic imine.
1
4
00 MHz, or Ascend 700 MHz NMR. H NMR chemical shifts are
reported in parts per million (ppm) and are referenced to the solvent
13
residual signals: CDCl (δ = 7.26 ppm) and DMSO-d (δ = 2.50).
C
3
6
NMR chemical shifts are reported in ppm and are referenced to the
solvent residual signal signals: CDCl (δ = 77.16 ppm) and DMSO-d6
3
(δ = 39.52). Electrospray ionization mass spectra (ESIMS) were
recorded on a Thermo LTQ Orbitrap mass spectrometer. Thin-layer
chromatography was performed on Sorbtech UV254 aluminum backed
sheets. The compounds were visualized by UV light at 254 nm or by
staining with KMnO . Flash column chromatography was carried out
4
using Sorbtech 40−63 μm silica gel with solvents as described. Ratios
of solvent systems used for flash chromatography are expressed in v/v
as specified. LC-MS was performed using Waters 2795 HPLC with a
Waters 2996 photodiode array detector operating at 200−800 nm, and
compounds were loaded using MeCN or MeOH. An Ace Excel 3 C18-
PFP column (150 × 4.6 mm, 1.7 μm particle size) was used with a
−1
flow rate of 1 mL·min . Compounds were loaded in MeCN or
MeOH. Separations involved a mobile phase of H O (solvent A) and
2
MeOH (solvent B) using a linear gradient of 5−95% solvent B over 25
min. All reactions were performed at room temperature unless
otherwise stated. Commercially available chemicals were used as
purchased. Ice/water was used as the temperature bath to achieve 0
°
C. Dry tetrahydrofuran (THF) and CH Cl were obtained from an
Innovative Technology PureSolv system.
2 2
General Procedure for Solid-Phase Peptide Synthesis Using
Tyr(tBu) -Gly -D-Gln(trt) -Ile -Ser-(tBu) -Val -Leu Cyclic Imine
1
2
3
4
5
6
7
α
the Weinreb Aminomethyl Resin. Loading. N -Fmoc-N-methoxy-
(6a). Fmoc-Leu-loaded Weinreb AM resin (1000 mg, substitution =
0.331 mmol) was sequentially coupled with Fmoc-Val-OH, Fmoc-
Ser(tBu)-OH, Fmoc-Ile-OH, Fmoc-D-Gln(trt)-OH, Fmoc-Gly-OH,
and Fmoc-Tyr(tBu)-OH according to the SPPS protocol. Fmoc
deprotection was performed as stated to produce the free amine on the
β-alanine AM resin (Weinreb AM resin) (1.0 equiv, substitution =
0
.4−0.8 mmol/g) was swollen in CH Cl (5 mL) for 20 min. The
2
2
resin was then soaked in a solution of piperidine/dimethylformamide
(
DMF) (4.86 mL, 1:9 v/v) and agitated for 5 min. The solvent was
then filtered off, and the procedure was then repeated for 10 min. The
Tyr residue. Cleavage from the resin as detailed yielded the crude
1
resin was then filtered with DMF (2 × 5 mL), CH Cl (2 × 5 mL),
product. The crude residue was then purified via flash chromatography
2
2
and DMF (2 × 5 mL). A Chloranil test was then applied. After a
positive Chloranil test (colored beads), the resin was soaked in a
solution of Fmoc-Leu-OH (3.0 equiv), 2-(1H-7-azabenzotriazol-1-yl)-
(eluent: 44:40:10:5:1 EtOAc/hexanes/CH Cl /MeOH/TEA) to yield
2
2
the desired cyclic imine 6a as an off-white powder (146.9 mg, 40%
−1 1
yield): mp 138−140 °C; IR (neat) 3305, 2967, 1655, 1529 cm ; H
1
,1,3,3-tetramethyluronium hexafluorophosphate (HATU) (2.9
NMR (400 MHz, CDCl ) δ 7.98 (d, J = 10.1 Hz, 1H), 7.93 (d, J = 5.1
3
equiv), and N,N-diisopropylethylamine (DIPEA) (3.5 equiv) in
DMF (0.2 M relative to Fmoc-Leu-OH). The beads were then
agitated for 1 h. The solvent was then filtered off, and the procedure
was then repeated for 1 h. The resin was then filtered with DMF (2 ×
Hz, 1H), 7.44 (dd, J = 9.6, 3.6 Hz, 1H), 7.36−7.15 (m, 15H), 7.06 (d,
J = 6.3 Hz, 1H), 6.94 (d, J = 1.9 Hz, 1H), 6.91 (d, J = 8.5 Hz, 2H),
6.86−6.80 (m, 3H), 6.69 (d, J = 9.5 Hz, 1H), 6.44 (d, J = 9.2 Hz, 1H),
4.64 (tdd, J = 10.6, 4.23, 1.8 Hz, 1H), 4.50 (dd, J = 10.1, 3.2 Hz, 1H),
G
DOI: 10.1021/acs.jnatprod.7b00912
J. Nat. Prod. XXXX, XXX, XXX−XXX

Journal of Natural Products
Article
4
9
3
3
1
1
0
.47−4.41 (m, 1H), 4.33 (dd, J = 9.2, 3.3 Hz, 1H), 4.26 (dd, J = 16.4,
.6 Hz, 1H), 4.15 (p, J = 4.99 Hz, 1H), 3.88 (dd, J = 10.2, 4.8 Hz, 1H),
.77 (dd, J = 11.2, 2.6 Hz, 1H), 3.48−3.38 (m, 2H), 3.27 (dd, J = 16.4,
.6 Hz, 1H), 2.79−2.61 (m, 2H), 2.56−2.46 (m, 1H), 2.41−2.30 (m,
H), 2.30−2.18 (m, 2H), 2.05−1.92 (m, 1H), 1.51−1.39 (m, 1H),
.34 (s, 9H), 1.30 (s, 9H), 1.33−1.28 (m, 1H), 1.22−1.11 (m, 1H),
.97 (d, J = 6.9 Hz, 3H), 1.02−0.93 (m, 2H), 0.93−0.85 (m, 9H), 0.83
OH, Fmoc-Ile-OH, Fmoc-D-Gln-OH, Fmoc-Gly-OH, and Fmoc-
Tyr(TBS)-OH according to the SPPS protocol. Fmoc deprotection
was performed as stated to produce the free amine on the Tyr residue.
1
Cleavage from the resin as detailed yielded the crude product. The
product was then purified via flash chromatography (eluent:
60:10:5:2.5 v/v EtOAc/MeCN/MeOH/H O) to yield the desired
2
cyclic imine 12a as an off-white powder (67.7 mg, 24% yield): IR
(
d, J = 6.6 Hz, 6H); 13C NMR (100 MHz, CDCl ) δ 173.31, 172.68,
(neat) 3296, 2958, 2929, 2857, 2539, 1647, 1541, 1510, 1258, 838
3
−
1 1
1
1
5
2
72.66, 171.30, 171.01, 170.94, 170.88, 165.7, 154.0, 144.3, 132.5,
30.1, 128.7, 128.3, 127.4, 124.1, 78.4, 76.2, 74.5, 71.0, 61.8, 59.3, 57.7,
4.4, 53.6, 50.2, 41.4, 40.7, 40.4, 36.6, 33.1, 29.0, 28.8, 28.0, 25.0, 24.2,
cm ; H NMR (400 MHz, DMSO-d ) δ 8.54 (d, J = 6.4 Hz, 1H),
6
8.24−8.11 (m, 2H), 7.86−7.71 (m, 2H), 7.38 (d, J = 9.7 Hz, 1H), 7.31
(s, 1H), 6.97 (d, J = 8.5 Hz, 2H), 6.79 (d, J = 1.7 Hz, 1H), 6.77 (s,
1H), 6.70 (d, J = 8.4 Hz, 2H), 5.58 (t, J = 5.4 Hz, 1H), 4.92 (td, J =
9.9, 6.1 Hz, 1H), 4.42−4.27 (m, 3H), 4.19 (dd, J = 9.2, 3.9 Hz, 1H),
4.02 (dd, J = 7.6, 4.2 Hz, 1H), 3.89 (td, J = 9.7, 6.7 Hz, 1H), 3.79−3.60
(m, 1H), 3.58 (dd, J = 11.2, 2.5 Hz, 1H), 3.43 (dd, J = 16.7, 2.5 Hz,
1H), 3.11 (dd, J = 13.2, 2.3 Hz, 1H), 2.60−2.55 (m, 1H), 2.31−2.19
(m, 1H), 2.18−1.95 (m, 3H), 1.81−1.71 (m, 2H), 1.45−1.26 (m, 3H),
1.11−0.98 (m, 1H), 0.94 (s, 9H), 0.92 (d, J = 6.3 Hz, 3H), 0.90−0.84
(m, 7H), 0.83 (s, 3H), 0.81 (d, J = 6.7 Hz, 3H), 0.73 (d, J = 6.5 Hz,
+
3.2, 21.2, 19.8, 16.7, 16.5, 12.0; HRESIMS m/z 1121.6438 [M + Na]
(calcd for C H N O Na, 1121.6410).
63 86 8 9
(
S)-Allyl 2-((((9H-fluoren-9-yl)methoxy)carbonyl)amino)-3-(tert-
butoxy)propanoate (8). To a cooled solution of Fmoc-L-Ser(tBu)
4.00 g, 10.43 mmol) in DMF (9.5 mL) at 0 °C was added dropwise
DIPEA (3.64 mL, 20.9 mmol) followed by allyl bromide (1.81 mL,
09 mmol). The mixture was allowed to warm to room temperature
(
2
and stirred for 16 h. The reaction was then diluted with EtOAc (25
mL). The solution was washed with H O (3 × 25 mL), dried
3H), 0.16 (s, J = 3.0 Hz, 6H); 13C NMR (101 MHz, DMSO-d ) δ
2
6
(
Na SO ), and concentrated in vacuo. The resulting solid was purified
173.23, 172.32, 172.13, 172.06, 170.82, 169.82, 169.76, 165.69, 153.52,
131.17, 130.13, 119.32, 73.29, 62.23, 59.21, 57.52, 53.33, 52.52, 49.21,
(39.90, 39.72, 39.41 HSQC), 35.23, 31.26, 28.45, 25.75, 25.52, 25.25,
24.29, 23.62, 23.03, 20.92, 19.18, 16.99, 15.95, 11.95, −4.47, −4.48;
2
4
by column chromatography (eluent: 90:10 hexanes/EtOAc) to afford
the desired allyl ester 8 (3.75 g, 85%) as a white powder: IR (neat)
3
−1 1
443, 3338, 2973, 1726, 1508, 1198, 1103, 1085, 759, 740 cm ; H
+
NMR (400 MHz, CDCl ) δ 7.80 (d, J = 7.5 Hz, 2H), 7.62 (t, J = 7.0
HRESIMS m/z 881.4924 [M + Na] (calcd for C H N O SiNa,
3
42 70
8
9
Hz, 2H), 7.43 (t, J = 7.4 Hz, 2H), 7.35 (t, J = 7.4 Hz, 2H), 5.95 (ddd, J
881.4927).
=
1
22.7, 10.8, 5.6 Hz, 1H), 5.71 (d, J = 9.0 Hz, 1H), 5.38 (dd, J = 17.2,
.2 Hz, 1H), 5.27 (dd, J = 10.5, 0.96 Hz, 1H), 4.70 (dd, J = 5.6, 0.98
Hz, 2H), 4.56 (dt, J = 8.9, 2.9 Hz, 1H), 4.52−4.34 (m, 2H), 4.29 (t, J =
Tyr(TBS) -Gly -D-Gln -Ile -Ser(TIPS) -Val -Leu Cyclic Imine (12b).
1
2
3
4
5
6
7
Fmoc-Leu-loaded Weinreb AM resin (1000 mg, substitution = 0.295
mmol) was sequentially coupled with Fmoc-Val-OH, Fmoc-Ser(TBS)-
OH, Fmoc-Ile-OH, Fmoc-D-Gln-OH, Fmoc-Gly-OH, and Fmoc-
Tyr(TBS)-OH according to the SPPS protocol. Fmoc deprotection
7
1
1
1
4
.2 Hz, 1H), 3.90 (dd, J = 9.0, 2.8 Hz, 1H), 3.65 (dd, J = 9.0, 3.1 Hz,
13
H), 1.24−1.13 (br s, 9H); C NMR (101 MHz, CDCl ) δ 170.36,
56.15, 144.02, 143.84, 141.31, 141.30, 131.72, 127.71, 127.09, 127.07,
25.22, 125.17, 119.98, 118.48, 73.50, 67.21, 65.97, 62.15, 54.73,
3
was performed as stated to produce the free amine on the Tyr residue.
1
Cleavage from the resin as detailed yielded the crude product. The
product was then purified via flash chromatography (eluent:
+
7.18, 27.33; HRESIMS m/z 424.2108 [M + H] (calcd for
+
C H NO 424.2119), 446.1922 [M + Na] (calcd for
70:10:5:2.5 v/v EtOAc/MeCN/MeOH/H O) to yield the desired
2
5
30
5,
2
C H NO Na, 446.1938).
cyclic imine 12b as an off-white powder (19.8 mg, 7% yield): IR (neat)
3305, 2959, 2857, 2478, 1651, 1510, 1462, 1261, 918 cm ; H NMR
25
29
5
−1 1
(
S)-Allyl 2-((((9H-fluoren-9-yl)methoxy)carbonyl)amino)-3-
(
(tertbutyldimethylsilyl)oxy)-propanoate (9). To a solution of
(400 MHz, DMSO-d ) δ 8.44 (d, J = 6.0 Hz, 1H), 8.08 (d, J = 8.8 Hz,
6
TFA/H O (4:1 v/v, 34 mL) was added 8 (3.27 g, 7.72 mmol).
1H), 7.91−7.84 (m, 1H), 7.68 (s, 1H), 7.52 (d, J = 8.7 Hz, 1H), 7.33
(s, 1H), 7.01 (d, J = 1.7 Hz, 1H), 6.91 (d, J = 8.4 Hz, 2H), 6.78 (s, J =
14.8 Hz, 1H), 6.73 (s, 1H), 6.70 (d, J = 8.4 Hz, 2H), 4.90−4.81 (m,
1H), 4.38−4.29 (m, 3H), 4.12 (dd, J = 8.5, 4.0 Hz, 1H), 4.05 (d, J =
6.5 Hz, 1H), 3.83 (dd, J = 13.2, 7.9 Hz, 2H), 3.63 (dd, J = 10.7, 2.6 Hz,
1H), 3.43 (dd, J = 16.5, 5.5 Hz, 1H), 3.15−3.11 (m, 1H), 2.53 (d, J =
2.4 Hz, 1H), 2.22 (ddd, J = 15.5, 11.7. 5.3 Hz, 2H), 2.14 (dd, J = 9.5,
6.3 Hz, 1H) 2.02 (ddd, J = 9.9, 7.9, 4.3 Hz, 3H), 1.84−1.77 (m, 2H),
1.45−1.36 (m, 3H), 1.05 (d, J = 3.1 Hz, 9H), 0.94 (s, 18H), 0.85 (d, J
= 3.7 Hz, 3H), 0.83 (d, J = 4.4 Hz, 6H), 0.78 (d, J = 6.5 Hz, 5H), 0.16
2
After 2 h the mixture was concentrated in vacuo to afford the desired
alcohol intermediate as a yellow solid. This solid was immediately
redissolved into CH Cl2 (38.6 mL), followed by the addition of
2
imidazole (1.68 g, 24.7 mmol). The mixture was then cooled to 0 °C
prior to the addition of TBS-Cl (2.91 g, 19.3 mmol). The reaction was
then allowed to stir for 16 h before quenching with H O. Extraction
2
was performed using CH Cl (3 × 75 mL). The organic fractions were
2
2
then combined, dried (Na SO ), and concentrated in vacuo. The
2
4
resulting residue was purified by column chromatography (eluent
0:10 v/v hexanes/EtOAc) to afford the TBS-protected ether 9 (3.56
g, 95.8%) as a white powder: IR (neat) 3443, 3346, 2952, 2930, 1727,
(s, 6H); 13C NMR (176 MHz, DMSO-d ) δ 173.42, 172.51, 171.58,
9
6
171.51, 170.33, 170.24, 170.07, 166.27, 166.26, 153.61, 130.78, 130.71,
130.22, 119.79, 119.35, 119.30, 73.76, 64.31, 58.27, 53.16, 49.74,
45.61, 40.49, 40.02, 35.34, 35.32, 31.27, 28.75, 25.58 25.55, 24.29,
23.44, 22.85, 21.17, 19.11, 18.07, 17.95, 17.94, 17.88, 17.80, 17.79,
15.63, 11.80, 11.66, 11.40, 11.35, 8.60, −4.49, −4.52; HRESIMS m/z
−
1 1
1507, 1253, 1198, 837, 740 cm ; H NMR (400 MHz, CDCl ) δ 7.80
3
(
d, J = 7.5 Hz, 2H), 7.65 (t, J = 8.0 Hz, 2H,), 7.43 (t, J = 7.5 Hz, 2H),
.34 (t, J = 7.4 Hz, 2H), 5.95 (ddd, J = 22.7, 10.9, 5.8 Hz, 1H), 5.69 (d,
J = 8.6 Hz, 1H), 5.33 (dd, J = 37.0, 13.8 Hz, 2H), 4.70 (d, J = 5.70 Hz,
7
+
2
7
0
H), 4.50 (dt, J = 8.70, 2.60 Hz, 1H), 4.47−4.35 (m, 2H), 4.29 (t, J =
1037.6237 [M + Na] (calcd for C H N O Si Na, 1037.6262).
51
90
8
9
2
.3 Hz, 1H), 4.14 (dd, J = 10.1, 2.5 Hz, 1H), 3.97−3.86 (m, 1H),
(S)-2-((((9H-Fluoren-9-yl)methoxy)carbonyl)amino)-3-
13
.96−0.85 (br s, 9H), 0.13−0.00 (d, 6H); C NMR (101 MHz,
((triisopropylsilyl)oxy)propanoic Acid (13). Compound 13 was
3
0
1
CDCl ) δ 170.17, 155.96, 144.00, 143.79, 141.32, 141.31, 131.58,
prepared according to the procedure reported by Aguda et al.
H
3
NMR and 13C NMR spectra were in agreement with the previously
reported data.
1
6
4
+
27.72, 127.09, 127.07, 125.22, 125.15, 119.99, 118.81, 67.24, 66.13,
3.69, 56.04, 47.15, 25.75, 18.23, −5.50, −5.56; HRESIMS m/z
+
82.2348 [M + H] (calcd for C H NO Si, 482.2357), 504.2163 [M
Scytonemide A (1). Cyclic imine 12a (25 mg, 0.03 mmol) was
dissolved in MeOH (3 mL). TBAF (1 M in THF, 33 μL, 0.03 mmol)
was then added dropwise. The reaction mixture was allowed to stir for
16 h. Solvent was then removed in vacuo, yielding an off-white
powder. Trituration with hexanes (3 × 5 mL) was then performed to
remove any nonpolar impurities. Crude material was then purified by
column chromatography (eluent 60:10:5:2.5 v/v EtOAc/MeCN/
2
7
36
5
+
Na] (calcd for C H NO SiNa, 504.2177).
27
35
5
(
S)-2-((((9H-Fluoren-9-yl)methoxy)carbonyl)amino)-3-((tert-
butyldimethylsilyl)oxy)propanoic Acid (10). Compound 10 was
29
1
prepared according to the procedure reported by Gagnon et al.
H
NMR and 13C NMR spectra were in agreement with the previously
reported data.
Tyr(TBS) -Gly -D-Gln -Ile -Ser -Val -Leu Cyclic Imine (12a).
Fmoc-Leu-loaded Weinreb AM resin (1000 mg, substitution = 0.295
mmol) was sequentially coupled with Fmoc-Val-OH, Fmoc-Ser(TBS)-
MeOH/H O) to yield the desired product 1 (11.5 mg, 51%) as a
1
2
3
4
5
6
7
2
25
25
white solid: [α]D −14 (c 0.05, MeOH), isolated scytonemide A, [α]
D
1
−15 (c 0.0013, MeOH); IR (KBr) 3613, 3346, 2962, 2365, 1647,
H
DOI: 10.1021/acs.jnatprod.7b00912
J. Nat. Prod. XXXX, XXX, XXX−XXX

Journal of Natural Products
Article
−
1
1
1
543, 1519, 1416, 1236, 1068 cm ; Table 1 for H NMR and 13C
(9) Malins, L. R.; deGruyter, J. N.; Robbins, K. J.; Scola, P. M.;
Eastgate, M. D.; Ghadiri, M. R.; Baran, P. S. J. Am. Chem. Soc. 2017,
139, 5233−5241.
+
NMR data; HRESIMS m/z 767.4024 [M + Na] (calcd for
C H N O Na, 767.4063).
36
56
8
9
̈
(10) Kopp, F.; Mahlert, C.; Grunewald, J.; Marahiel, M. A. J. Am.
ASSOCIATED CONTENT
Supporting Information
The Supporting Information is available free of charge on the
Chem. Soc. 2006, 128, 16478−16479.
■
(
(
11) Becker, J. E.; Moore, R. E.; Moore, B. S. Gene 2004, 325, 35−42.
12) Nahm, S.; Weinreb, S. M. Tetrahedron Lett. 1981, 22, 3815−
*
S
3
818.
ACS Publications website at DOI: 10.1021/acs.jnat-
(13) Mentzel, M.; Hoffmann, H. M. R. J. Prakt. Chem./Chem.-Ztg.
prod.7b00912.
1
997, 339, 517−524.
1
H and 13C NMR spectra for compounds 8, 9, and 12b;
H, C, HSQC, and HMBC NMR spectra for imines 6a
and 12a; and H, C, HSQC, and selected TOCSY
NMR spectra for scytonemide A (1) (PDF)
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AUTHOR INFORMATION
Corresponding Author
Tel: (614) 247-7377. E-mail: fuchs.42@osu.edu.
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*
(
(
ORCID
Drugs 2013, 11, 2382−2697.
(20) Kopp, F. Macrocyclization and Fatty Acid Modification during the
Jimmy Orjala: 0000-0002-4420-2048
L. Harinantenaina Rakotondraibe: 0000-0003-2166-4992
James R. Fuchs: 0000-0002-8743-6389
Author Contributions
Synthesis of Nonribosomal Peptides; Universitat
008.
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sbibliothek Marburg,
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§
T. A. Wilson and R. J. Tokarski contributed equally to the
work presented herein.
Notes
(
5
(
The authors declare no competing financial interest.
Tetrahedron 1990, 46, 1653−1658.
ACKNOWLEDGMENTS
The authors would like to thank the NIH for funding provided
through the 2P01 CA125066 program project [Project 1
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(25) Nicolaou, K. C.; Yang, Z.; Liu, J. J.; Ueno, H.; Nantermet, P. G.;
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L.H.R.), Project 2 (J.O.), and Core B (J.R.F.)], a CBIP
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Fellowship (GM08512) for T.A.W., and a Fellowship (5T32
AT007533) for P.S. We also would like to thank Dr. C.
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DEDICATION
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(30) Aguda, A. H.; Lavallee, V.; Cheng, P.; Bott, T. M.; Meimetis, L.
Dedicated to Dr. Susan Band Horwitz, of Albert Einstein
College of Medicine, Bronx, NY, for her pioneering work on
bioactive natural products.
G.; Law, S.; Nguyen, N. T.; Williams, D. E.; Kaleta, J.; Villanueva, I.;
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