Samholides, Swinholide-Related Metabolites from a Marine Cyanobacterium cf. Phormidium sp.

Article abstract of DOI:10.1021/acs.joc.8b00028

Cancer cell cytotoxicity was used to guide the isolation of nine new swinholide-related compounds, named samholides A-I (1-9), from an American Samoan marine cyanobacterium cf. Phormidium sp. Their structures were determined by extensive analysis of 1D and 2D NMR spectroscopic data. The new compounds share an unusual 20-demethyl 44-membered lactone ring composed of two monomers, and they demonstrate structural diversity arising from geometric isomerization of double bonds, sugar units with unique glyceryl moieties and varied methylation patterns. All of the new samholides were potently active against the H-460 human lung cancer cell line with IC₅₀ values ranging from 170 to 910 nM. The isolation of these new swinholide-related compounds from a marine cyanobacterium reinvigorates questions concerning the evolution and biosynthetic origin of these natural products.

Full text of DOI:10.1021/acs.joc.8b00028

Subscriber access provided by UNIV OF DURHAM
Featured Article
The Samholides, Swinholide Related Metabolites
from a Marine Cyanobacterium cf. Phormidium sp.
Yiwen Tao, Pinglin Li, Daojing Zhang, Evgenia Glukhov, Lena
Gerwick, Chen Zhang, Thomas F Murray, and William H Gerwick
J. Org. Chem., Just Accepted Manuscript • Publication Date (Web): 19 Feb 2018
Downloaded from http://pubs.acs.org on February 20, 2018
Just Accepted
“Just Accepted” manuscripts have been peer-reviewed and accepted for publication. They are posted
online prior to technical editing, formatting for publication and author proofing. The American Chemical
Society provides “Just Accepted” as a service to the research community to expedite the dissemination
of scientific material as soon as possible after acceptance. “Just Accepted” manuscripts appear in
full in PDF format accompanied by an HTML abstract. “Just Accepted” manuscripts have been fully
peer reviewed, but should not be considered the official version of record. They are citable by the
Digital Object Identifier (DOI®). “Just Accepted” is an optional service offered to authors. Therefore,
the “Just Accepted” Web site may not include all articles that will be published in the journal. After
a manuscript is technically edited and formatted, it will be removed from the “Just Accepted” Web
site and published as an ASAP article. Note that technical editing may introduce minor changes
to the manuscript text and/or graphics which could affect content, and all legal disclaimers and
ethical guidelines that apply to the journal pertain. ACS cannot be held responsible for errors or
consequences arising from the use of information contained in these “Just Accepted” manuscripts.
The Journal of Organic Chemistry is published by the American Chemical Society.
1155 Sixteenth Street N.W., Washington, DC 20036
Published by American Chemical Society. Copyright © American Chemical Society.
However, no copyright claim is made to original U.S. Government works, or works
produced by employees of any Commonwealth realm Crown government in the course
of their duties.

Page 1 of 29
The Journal of Organic Chemistry
1
2
3
4
5
The Samholides, Swinholide Related Metabolites from a Marine Cyanobacterium cf.
Phormidium sp.
6
7
8
9
Yiwen Tao,†,⊥,1 Pinglin Li,‡,§,⊥,1 Daojing Zhang,⊥,⸡ Evgenia Glukhov,⊥ Lena Gerwick,⊥ Chen
*
Zhang,⊥ Thomas F. Murray,║ William H. Gerwick⊥∇
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
† Key Laboratory of Molecular Target & Clinical Pharmacology, School of Pharmaceutical
Sciences and the Fifth Affiliated Hospital, Guangzhou Medical University, Guangzhou 511436,
People’s Republic of China
‡Key Laboratory of Marine Drugs, Chinese Ministry of Education, School of Medicine and
Pharmacy, Ocean University of China, Qingdao 266003, People’s Republic of China
§Laboratory for Marine Drugs and Bioproducts of Qingdao National Laboratory for Marine
Science and Technology, Qingdao 266235, People’s Republic of China
⊥
Center for Marine Biotechnology and Biomedicine, Scripps Institution of Oceanography,
University of California San Diego, La Jolla, California 92093, United States
⸡
State Key Laboratory of Bioreactor Engineering, East China University of Science &
Technology, Shanghai 200237, People’s Republic of China
║Department of Pharmacology, Creighton University School of Medicine, Omaha, Nebraska
68178, United States
∇
Skaggs School of Pharmacy and Pharmaceutical Sciences, University of California San Diego,
La Jolla, California 92093, United States
1 These authors contributed equally to this paper.
*Corresponding author. Tel: (858) 534-0578; fax: (858) 534-0576
E-mail address: wgerwick@ucsd.edu
Express Mail Mailing address:
Scripps Institution of Oceanography, University of California San Diego
Center for Marine Biotechnology and Biomedicine,
8615 Kennel Way, Sverdrup Hall, Room 3232
La Jolla, California 92037
ACS Paragon Plus Environment

The Journal of Organic Chemistry
Page 2 of 29
1
2
3
4
5
6
Graphical Abstract
7
8
9
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
MS2-based Molecular Network
Samholide A
Cf. Phormidium sp.
Abstract
Cancer cell cytotoxicity was used to guide the isolation of nine new swinholide-related compounds,
named samholides A-I (1-9), from an American Samoan marine cyanobacterium cf. Phormidium
sp. Their structures were determined by extensive analysis of 1D and 2D NMR spectroscopic data.
The new compounds share an unusual 20-demethyl 44-membered lactone ring comprised of two
monomers, and they demonstrate structural diversity arising from geometric isomerization of
double bonds, sugar units with unique glyceryl moieties and varied methylation patterns. All of
the new samholides were potently active against the H-460 human lung cancer cell line with IC50
values ranging from 170 nM to 910 nM. The isolation of these new swinholide-related compounds
from a marine cyanobacterium reinvigorates questions concerning the evolution and biosynthetic
origin of these natural products.
ACS Paragon Plus Environment

Page 3 of 29
The Journal of Organic Chemistry
1
2
3
4
Introduction
5
6
7
8
Cyanobacteria (blue-green alga), are a monophyletic bacterial phylum containing more
than 60 genera with more than 400 species.1 As a group, they are extraordinarily rich in structurally
diverse and biologically active natural products.2 Almost 500 new compounds have been isolated
from cyanobacteria,3 with peptides, polyketides and hybrids thereof being the major
representatives. Several of these natural products have inspired the development of new
pharmaceutical agents, and 17 cyanobacterial-derived or inspired agents are in phase I or II drug
development (in 2018) and are part of the global marine pharmaceutical clinical pipeline.4
9
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
The swinholides represent a family of macrolide natural products having a unique dimeric
44-membered or larger lactone ring. The first example was swinholide A, originally isolated by
Kashman and Carmeli from the marine sponge Theonella swinhoei in 1985.5 However, prior to
this discovery, swinholide-type natural products had also been isolated from cultured
cyanobacteria. Moore et al.6 reported in 1977 a novel monomeric macrolide, tolytoxin, from the
terrestrial cyanobacterium Tolypothrix conglutinata var. colorata collected from Fanning Island.
Tolytoxin showed potent cytotoxic and fungicidal properties, and ultimately was found to possess
a structure closely related to a monomeric half of swinholide A. In 1986 the same research group
reported a series of tolytoxin-related macrolides, scytophycins A-E, from the cultured terrestrial
cyanobacterium Scytonema pseudohofmanni.7 The scytophycins were also structurally related to
the swinholides, and possessed potent cytotoxicity as well as broad-spectrum antifungal activity.
In 2005, our laboratory reported two new glycosylated swinholides, ankaraholides A and B, from
a Madagascar cyanobacterium of the genus Geitlerinema sp., together with swinholide A from a
Fijian cyanobacterium Symploca cf. sp.8
To date, nearly 60 closely related swinholide-type compounds have been isolated from
various species of mollusks, sea hares, nudibranchs, red and brown algae, causing the true
biosynthetic origin of this class of natural products to be confusing and ambiguous.2,3,9-11 In some
cases, it is relatively clear that these metabolites are produced by associated or preyed upon
microorganisms (e.g. in the cases of macroalgae or mollusks, respectively). In the case of the
sponge Theonella spp., because large numbers of cyanobacteria are present in the sponge tissue, it
was hypothesized that the swinholides are cyanobacterial secondary metabolites.9 However,
Bewley et al. found that swinholide A was associated with a heterotrophic eubacterial fraction
ACS Paragon Plus Environment

The Journal of Organic Chemistry
Page 4 of 29
1
2
3
4
5
6
7
8
9
rather than the separated sponge and cyanobacterial cells.10 Subsequently, Piel et al. sequenced the
biosynthetic gene cluster for misakinolide A from a metagenomic sample of Theonella sp. along
with its complement of symbionts.11 Isolation of single filaments of the proposed bacterial source
of the misakinolide cluster, Entotheonella serta, followed by multiple displacement amplification
(MDA) and sequencing, confirmed that it was the source organism. Matrix-assisted laser
desorption ionization (MALDI) imaging as well as catalyzed reporter deposition-fluorescence in
situ hybridization (CARD-FISH) analysis provided additional support for this deduction. However,
the same work provided highly analogous biosynthetic gene clusters from two cultured
cyanobacteria, Scytonema sp. PCC 10023 and Planktothrix paucivesiculata PCC 9631, which
produce scytophycin/tolytoxin and luminaolide, respectively. The gene sequencing studies of Piel
et al. clearly revealed that two distinct phyla of microorganisms have the capacity to make this
class of metabolite, and likely reflects a complex set of vertical and horizontal evolutionary
events.11 Recently, a study of the antifungal constituents of two freshwater cyanobacteria, Nostoc
sp. UHCC 0450 and Anabaena sp. strain UHCC 0451, revealed swinholide and scytophycin-type
natural products, respectively.12 Draft genomes of these two organisms revealed the presence of
trans-AT biosynthetic gene clusters responsible for production of these molecules, and they were
remarkably similar in sequence and architecture to those described by Piel for misakinolide A,
scytophycin and luminaolide. A phylogenetic analysis supported their origination through
Horizontal Gene Transfer events.
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
As a class, the swinholides possess a number of significant bioactivities. Tolytoxin was
found to inhibit the growth of a wide array of fungi with MIC values of 0.25-8 nM. It also showed
strong cytotoxicity towards a variety of mammalian cells at similar IC50 values.13 Scytophycins A
and B displayed antiproliferative activity to the KB human nasopharyngeal carcinoma cell line
with an IC50 of 1.2 nM. Other swinholide-related compounds have been found to have similar
cytotoxicities with IC50 values in the low nanomolar to picomolar range.14 The molecular target of
the swinholides in mammalian cells has been determined to be actin, and at least some of these
molecules exhibit nanomolar-potency binding characteristics at the same site of F-actin and G-
actin. These potent biological features have thus inspired in-depth investigations of structurally
diversified swinholide analogues.15
ACS Paragon Plus Environment

Page 5 of 29
The Journal of Organic Chemistry
1
2
3
4
5
6
7
8
9
In the current study, nine new swinholide-type compounds, termed samholides A-I (1-9,
Figure 1), were obtained from a field collection in American Samoa of the cyanobacterium cf.
Phormidium sp. using a bioassay-guided isolation approach in combination with the MS2-based
molecular networking dereplication tool.16 Their structures were determined by extensive analysis
of 1D and 2D NMR spectra, and they share a unique 20-demethyl (compared with swinholide A)
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
1 R =R =a, 2E4E2'E4'E
31
1
2
2 R =R =a, 2Z4E2'E4'E
O
OH
1
2
11
3 R =R =b, 2E4E2'E4'E
1
2
23
25
29
27
OMe OH OH
OMe
4
5
6
7
8
9
R =R =b, 2Z4E2'E4'E
1
2
R =a, R =b, 2E4E2'E4'E
1
2
9
21
13
17
19
15
O
O
1'
O
R =a, R =b, 2Z4E2'E4'E
1 2
R =a, R =b, 2E4E2'Z4'E
1 2
7
R
1
R =b, R =c, 2E4E2'E4'E
3'
1
2
5
R =R =H, 2E4E2'E4'E
1
2
3
5'
MeO
OMe
34
O
1
39
R
OH
2
O
7'
38
O
37
a= 36
O
9'
13' O
19'
17'
15'
OH
O 32
21'
O
OH OH OMe
25'
MeO
27'
MeO
23'
11'
OMe
HO
OMe
OH
29'
OH
O
31'
b=
OH
c=
O
O
O
O
Figure 1. Structures of compounds 1-9.
44-membered lactone ring that is comprised of two monomers. Structural variations among these
new compounds result from differences in the methylation and esterification patterns decorating
the sugars, as well as the double bond geometries. Cytotoxicity was evaluated using the H-460
human lung carcinoma cell line and showed that all nine were potently active with IC50 values
ranging from 170 to 910 nM. The variations in structure and attendant biological activities provide
some initial structure-activity relationships (SAR), and reveal that the sugar units are important for
high potency. Finally, their isolation from a marine cyanobacterium provides further evidence for
ACS Paragon Plus Environment

The Journal of Organic Chemistry
Page 6 of 29
1
2
3
4
5
6
cyanobacteria being a frequent and plentiful source of these actin-binding dimeric metabolites of
the swinholide family.
7
8
9
Results and Discussion
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
Tufts of a marine cyanobacterium, morphologically identified as cf. Phormidium sp., were
collected at approximately 2 m water depth near American Samoa in July 2014. The preserved
collection was repetitively extracted (2:1 CH2Cl2/MeOH) and fractionated using normal-phase
vacuum liquid chromatography (VLC) to obtain nine fractions (Fr. A-I). Fractions and the crude
extract were screened for cytotoxicity to H-460 human lung cancer cells at 1 and 10 μg/mL.17
While several fractions showed cytotoxicity at the higher concentration, only the two most polar
fractions (H, I) were active at 1 μg/mL. By contrast, the extract and all fractions were inactive in
a neuromodulatory assay that evaluated the ability of these materials to modulate intracellular
calcium mobilization.18 LC-MS/MS molecular networking techniques revealed a distinct cluster
of 19 nodes in fractions H and I (Figure 2 and S104 in the Supporting Information), none of which
matched any GNPS MS2 library standards.16 Reversed-phase HPLC of these two fractions yielded
nine new compounds, given the names samholides A-I (1-9, Figure 1) to reflect their geographical
origins, and these were collectively responsible for the potent cytotoxicity of these fractions.
ACS Paragon Plus Environment

Page 7 of 29
The Journal of Organic Chemistry
1
2
3
4
5
6
7
8
9
Figure 2. MS/MS-based molecular network of the samholides.16 The cosine level was adjusted
to 0.7. Nodes display measured masses of the molecular ions. The yellow nodes are samholide
analogues that were isolated and defined in the current study whereas the green nodes are
potential samholide analogues not yet described due to the small amounts that were present. The
size of the node is reflective of the relative amount of the indicated compounds.
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
Samholide A (1) gave a sodiated molecular ion peak by HRESIMS spectrum at m/z
1880.0657 [M+Na]+ for a molecular formula of C96H160O34Na+ (calcd 1880.0683). However, the
13C NMR (Table 1) of compound 1 showed only 48 distinct carbon signals, suggesting that 1
possessed a homodimeric structure. The 1H NMR of samholide A displayed four doublet methyl
resonances, each of which was comprised of 6 protons for two symmetrical methyl groups, at δH
0.83 (d, J = 7.02 Hz), 0.91 (d, J = 6.93 Hz), 0.98 (d, J = 6.74 Hz), 1.20 (d, J = 6.18 Hz) as well as
two overlapping symmetrical olefinic methyl signals at δH 1.80 (s). A cluster of oxygenated
methines and methoxy groups were located between δH 3.10 and δH 5.00, and 10 olefinic protons
appearing as 5 distinct signals were located between δH 5.67 to δH 7.61. Three olefinic signals, each
composed of two isochronous protons, were deshielded to shifts of δH 5.78 (m), 6.34 (dd, J = 9.44,
3.17 Hz) and 7.61 (d, J = 15.54 Hz), indicating the presence of two trisubstituted, conjugated
dienones. 13C NMR analysis of compound 1 showed four ester carbonyls with two each at δC 170.7
and 170.64; the latter two reinforced the above assignments in that they were conjugated to two
symmetrical dienes with carbon resonances at δC 153.1, 142.9, 123.3 and 113.5. There were also
two symmetrical isolated double bonds at δC 133.9 and 129.7.
ACS Paragon Plus Environment

The Journal of Organic Chemistry
Page 8 of 29
1
2
3
4
5
Table 1. NMR data for compound 1 in CDCl3 at 600 MHz (1H)a and 125 MHz (13C)b, respectively.
13C
1H
COSY
HMBC
HSQC-TOCSY
ROESY
6
7
8
9
1/1'
2,2'
3,3'
4/4'
170.6
113.5
153.1
133.9
12.1
5.78 (overlapped)
7.6 d (15.5)
3/3'
2/2'
3/3', 1/1'
2/2', 4/4', 4/4'-Me, 5/5'
2/2', 3/3'
2/2', 3/3'
4/4'-Me
2/2', 5/5', 15/15', 38/38'-OH, 39/39'-OH
4,4'-Me
1.74 s
5/5'
3,4,5
4/4'-Me, 5/5'
2/2', 6/6’, 8/8'
3/3’, 7/7', 39/39'-OH
5,5'
142.9
6.34 dd (9.44, 3.17)
6/6', 4/4'-Me
4/4'-Me, 3/3', 6/6', 7/7'
4/4'-Me, 5/5', 6/6', 7/7', 8/8', 9/9'
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
2.50 d (12.76)
2.37 m
4.10 m
2.33 m
1.53 m
4.21 d (11.81)
5.68 d (10.26)
5.77 m
2.08 d (17.58)
1.96 m
4/4'-Me, 8/8’, 9/9', 10/10’
6,6'
7,7'
8,8'
33.5
79.0
39.9
5/5', 7/7'
6/6', 8/8'
7/7', 9/9'
4/4', 5/5', 7/7'
4/4'-Me, 5/5', 6/6', 7/7', 8/8', 9/9'
5/5', 6/6', 7/7', 8/8', 9/9'
5/5', 6/6', 32/32'
6/6', 7/7', 9/9', 10/10'
5/5', 32/32'
4/4'-Me, 13/13’
5/5', 6/6', 7/7', 8/8', 9/9'
9,9'
10,10'
11,11'
68.7
129.7
123.3
8/8', 10/10'
9/9', 11/11', 12/12'
10/10', 12/12'
8/8', 10/10', 11/11', 13/13'
9/9', 12/12'
9/9', 12/12', 13/13'
5/5', 6/6', 7/7', 8/8', 9/9', 10/10', 11/11', 12/12'
8/8', 9/9', 10/10', 11/11', 12/12', 13/13', 14/14'
8/8', 9/9', 10/10', 11/11', 12/12', 13/13', 14/14'
6a/6a', 14/14’
8/8'
12,12'
13,13'
14,14'
31.5
63.4
36.4
11/11', 13/13'
12/12', 14/14'a
13/13', 15/15'
10/10', 11/11', 13/13', 14/14'
7/7', 10/10', 11/11', 12/12', 13/13', 14/14'
12/12', 13/13', 14/14', 15/15'
8a/8a'
3.69 m
1.86 m
1.64 m
4.07 m
1.59 m
0.83 d (6.85)
3.84 t (9.5)
1.76 m
3.95 m
1.94 m
13/13', 15/15', 16/16'
12/12', 13/13', 14/14', 15/15'
15,15'
16,16'
16,16'-Me 9.3
17,17'
18,18'
19,19'
75.0
41.2
14/14'
16/16'-Me, 15/15'
16/16'
14/14', 15/15'-OMe, 16/16'-Me, 16/16', 17/17'
16/16'-Me, 18/18'
15,16,17
15/15', 16/16'-Me, 16/16', 18/18', 19/19'
17/17', 19/19'
12/12', 13/13', 14/14', 15/15'
6a/6a', 5/5', 17/17'-OH
13/13', 14/14', 15/15', 16/16', 16,16'-Me
16,16'-Me, 16/16', 17/17', 18/18', 19/19'
16/16'-Me, 18/18', 17/17', 19/19'
17/17', 18/18', 19/19', 20/20'
73.5
41.4
69.6
18/18'
16/16'-Me
17/17'-OH
18/18', 19/19', 20/20', 21/21'
20,20'
42.1
21/21',
20/20', 21/21', 19/19'
1.52 m
23/23', 23/23'-OH, 39/39'-OH, 38/38'-OH
21/21', 22/22'-Me, 24/24'-Me
21,21'
22,22'
22,22'-Me 9.7
23,23'
24,24'
24,24'-Me 17.2
25,25'
26,26'
27,27'
28,28'
29,29'
30,30'
31,31'
70.3
40.5
5.84 d (11.37)
20/20'
1'/1', 19/19', 23/23', 20/20', 22/22', 22/22'-Me
19/19', 20/20'
1.65 m
22/22'-Me, 23/23'
21,22,23
21/21', 22/22', 24/24'-Me, 25/25'
23/23' , 26/26'
23,24,25
22,22'-Me, 22/22', 23/23', 24/24',25/25', 26/26'
22,22'-Me, 22/22', 23/23'
22/22'-Me, 22/22', 23/23'
22/22', 23/23', 24/24'-Me, 24/24', 25/25', 26/26'
24,24'-Me, 24/24', 25/25', 26/26', 27/27'
24,24'-Me, 24/24', 25/25', 26/26', 27/27'
0.90 d (6.85)
3.18 d (9.01)
1.65 m
0.95 d (6.67)
1.22 m
1.80 m
1.22 m
3.94 m
1.77 m
1.55 m
3.50 m
1.94 m
1.15 m
22/22'
22/22', 24/24'
75.8
33.4
24/24'
23.2
29.3
71.5
35.1
73.5
38.9
64.7
26/26',
25/25', 27/27',
29/29', 31/31'
29/29', 30/30'
28/28', 31/31'
29/29'
24/24'-Me, 25/25', 26/26', 27/27'
24/24'-Me, 25/25', 26/26', 28/28', 29/29', 30/30'
27/27', 28/28', 29/29', 31/31', 30/30'
28/28', 29/29', 30/30', 31/31', 31/31'-Me,
28/28', 29/29', 30/30', 31/31', 31/31'-Me
3.64 m
31/31'-Me
31/31'
27/27', 29/29', 31/31'-Me
30,31
7/7', 33/33', 36/36'
32/32', 34/34', 37/37'
33/33', 34/34'-OMe, 35/35'
28/28', 29/29', 30/30', 31/31', 31/31'-Me
28/28', 29/29', 30/30', 31/31', 31,31'-Me
32/32', 33/33', 34/34', 35/35', 36/36'
32/32', 33/33', 34/34', 35/35', 36/36'
32/32', 33/33', 34/34', 35/35', 36/36'
32/32', 33/33', 34/34', 35/35', 36/36'
31,31'-Me 21.9
1.16 d (6.19)
4.78 d (6.09)
4.76 dd (8.21, 6.29)
3.25 t (7.50)
3.33 m
32,32'
33,33'
34,34'
35,35'
102.1
73.5
82.4
79.3
5/5', 7/7', 34/34'
34/34'-OMe, 35/35'
32/32'
34/34'
33/33'
33/33'
ACS Paragon Plus Environment

Page 9 of 29
The Journal of Organic Chemistry
1
2
3
4
5
6
7
8
9
4.00 dd (14.79,
7.83)
3.30 m
36,36'
62.3
35/35'
32/32', 34/34', 35/35'-OMe
32/32', 33/33', 34/34', 35/35', 36/36'
37/37'
38,38'
170.7
72.4
3/3'
4.06 m
38/38'-OH, 39/39'
38/38', 39/39'-OH
37/37', 39/39'
38/38'
38/38', 39/39'
38/38', 39/39'
3.70 m
3.50 m
3.37 s
3.32 s
3.45 s
3.42 s
4.82 s
5.77 m
4.64 br s
5.03 d (3.00)
4.81 br s
24/24'-Me
39,39'
64.7
15,15'-OMe 57.5
29,29'-OMe 55.4
34,34'-OMe 60.1
35,35'-OMe 58.3
17,17'-OH
19/19'-OH
23,23'-OH
38,38'-OH
39,39'-OH
15/15'
29/29'
34/34'
35/35'
17/17'
15,15'-OMe
29,29'-OMe
34,34'-OMe
35,35'-OMe
16/16'-Me, 17/17', 18/18'
19/19', 18/18'
22/22'-Me, 22/22', 23/23', 31/31'
38/38', 39/39'
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
19/19'
23/23'
38/38'
39/39'
22/22'
37/37', 38/38', 39/39'
21/21', 24/24'-Me, 31/31'
2/2', 3/3', 21/21'
3/3', 5/5', 21/21'
38/38', 39/39'
a
1H and 2D NMR spectra were run on a Bruker Advance III DRX-600 MHz NMR spectrometer.
13C NMR spectra were run on a Varian X-Sens 500 MHz NMR spectrometer (125 MHz).
b
ACS Paragon Plus Environment

The Journal of Organic Chemistry
Page 10 of 29
1
2
3
4
5
6
7
8
1
13
HSQC-TOCSY (Table 1) further enabled assignment of the H and C NMR data to two
symmetrical pentose units with anomeric carbons at δC 102.1 and four oxygenated carbons each at
δC 82.4, 73.2, 79.3 and 62.3. These NMR data were generally indicative of a swinholide-related
polyketide structure possessing a sugar unit, and thus was of a structure class related to that of
ankaraholide A which had been previously isolated from another cyanobacterium, Geitlerinema
sp. collected in Madagascar.8
9
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
The main differences in the NMR data between compound 1 and ankaraholide A were between
C-16 and C-22, and C2 and C5, which nevertheless showed highly similar Δδ-values ranging from
0.8 to 1.4 ppm, relative to the average of 0.3 ppm for the remainder of the comparable carbon
signals (Table S1 in Supporting Information). Considering the variability of the methyl groups at
C-16 and C-20 in the known swinholides,3 these differences between 1 and ankaraholide suggested
that it might possess the uncommon 16-methyl-20-demethyl skeleton, a carbon framework only
previously appearing in the partial monomer structure of swinholide G.18 This speculation was
supported by HMBC analysis that revealed that the long-range correlations from the methyl groups
at C-16 and C-22 formed two distinct and non-overlapping spin networks. Confirmation of this
framework was obtained by COSY, HMBC, and HSQC-TOCSY experiments (Table 1 and Figures
S9-S13 in Supporting Information) which outlined a linear spin system decorated with secondary
methyl and oxymethine groups, and yielded a homodimeric structure possessing a 20,20’-
didemethyl swinholide A skeleton (Figure 3). Three additional carbon signals were observed in 1
compared to ankaraholide A, and included one ester carbonyl group at δC 170.7, one oxygenated
methine at δC 72.4, and one oxygenated methylene at δC 64.7. HMBC and COSY data clarified that
these latter resonances and their associated protons were connected and formed a glyceric acid
moiety. A neutral fragment ion loss of m/z 88 was observed in the MS/MS spectrum of 1,
supporting the nature of this substituent.
ACS Paragon Plus Environment

Page 11 of 29
The Journal of Organic Chemistry
1
2
3
4
5
6
7
8
9
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
Figure 3. Key COSY and HMBC correlations of the monomeric structure present in samholide
A (1).
While the spin system of H-32 to H-36 for a pentapyranose sugar was apparent in compound
1, H-32 and H-33 were overlapped and thus introduced a degree of uncertainty. However, this
was resolved by consideration of the HMBC network between these proton and carbon atoms
(Tables 1 and Figure 3). Moreover, this spin connectivity between H-32/H-33/H-34/H-35/H2-36
was directly observed in the COSY spectrum of compound 3 (Table 2). The large vicinal diaxial
coupling constants between H-32, H-33, H-34, H-35 and Ha-36 (all between 6-9 Hz, Table 1) in
1 were indicative of the β-xylopyranoside unit, and this was further confirmed by ROESY
correlations between H-32 and H-34 and Ha-36 (Figure 4 and Figures S14-S15). These xylose
units were attached to C-7/7’ on the basis of reciprocal HMBC correlations between H-7/7’ and
C-32/32’, and H32/32’ and C-7/7’. HMBC correlations between two OMe resonances at δH 3.45
and 3.42 with carbons at δC 82.4 and 79.3 were indicative of two di-O-methyl-xylopyranoside units,
and by consideration of the 13C NMR assignments for this sugar (Table 1), these could be placed
at C-34 and C-35. The glyceric acid moiety was connected through the oxygen atom at C-33 by
HMBC correlation of H-33 with C-37.
ACS Paragon Plus Environment

The Journal of Organic Chemistry
Page 12 of 29
1
2
3
4
Table 2. 1H NMR data for compounds 2-9 in CDCl3 at 600 MHz.
8a
5
6
7
8
2
3
δH/δH’
4
5
6
7
9
δH/δH’
δH
5.79 m
δH’
5.67 m
δH
5.69 m
δH’
5.82 m
δH
δH’
δH
5.68 d
δH’
5.81 d
(15.68
)
7.44 d
(15.54
)
δH
5.80 m
δH’
5.66 d
(12.72
)
6.46 d
(12.69
)
δH
δH’
2,2'
3,3'
5.79 d (15.59)
5.79 d (15.48)
5.80 d (15.6)
5.80 d (15.62)
(12.47)
9
6.51 (d (12.6)
1.86 s
7.50 d
(15.6)
7.53 (d 15.56)
1.82 s
6.46 d
(12.41)
7.39 d
(15.56)
7.57 d
(15.55)
7.52 d
(15.56)
6.52 d
(12.62)
7.47 d
(15.63)
7.55 d (15.6)
7.47 d (15.62)
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
4,4'-
Me
5,5'
1.77 s
1.84 s
1.82 s
1.75 s
1.82 s
1.86 s
1.81 s
1.78 s
1.86 s
1.83 s
1.80 s
5.84 dd (7.06,
6.27)
6.14 dd,
(6.53,
6.40)
2.62 br d
(13.34)
2.28 m
6.31 dd (7.55,
7.37)
5.98 t
(6.33)
6.09 t
(6.23)
6.33 m
6.32 m
5.86 t (7.00)
6.14 t
(6.87)
6.11 t (6.88)
5.97 br
s
6.35 m
6.19 t (7.03)
6,6'
2.55 dd
(14.73, 5.62)
2.38 m
2.59 ddd (2.85,
6.14, 14.47)
2.37 dt (8.86,
14.34)
2.56 m
2.34 m
2.64 m
2.36 m
2.50 d
(13.39)
2.35 m
2.67 d
(12.99)
2.35 m
2.57 ddd
(4.06, 6.48,
13.87)
2.40 dt (7.02,
15.36)
2.69
ddd
(4.38,
6.03,
11.73)
2.28
2.59 m
2.34 m
2.57 m
2.41 dt
(7.38,
2.60 m
2.38 m
2.47 ddd (3.24,
7.14, 14.94)
2.31 dt (7.56,
15.19)
14.56)
dt
(7.45,
15.12)
4.00 m
7,7'
8,8'
4.06 m
4.04 m
4.01 m
4.02 m
3.99 m
3.97 m
4.03 m
3.97 m
4.08 m
4.04 ddd (4.25,
7.90, 11.76)
1.89 m
2.28 m
1.54 m
2.25 ddd (5.02,
9.81, 15.0)
1.63 m
2.10 m
1.64 m
2.29 m
1.67 m
2.10 m
1.55 m
2.10 m
1.67 m
2.19 m
1.54 m
2.05 m
1.64 m
2.26 m
1.62 m
1.52 m
9,9'
4.30 m
5.70 m
4.30 m
4.36 m
5.74 m
4.21 d
(11.37)
5.72 m
4.31 brs
4.29 m
5.69 m
5.80 m
4.39 m
5.78 m
5.69 m
4.28 m
4.38 m
4.29 m
5.74 m
4.34 d (10.74)
5.65 m
10,10'
5.74 m
5.78 m
5.71 m
5.71 m
11,11'
12,12'
5.80 m
2.01 m
5.74 m
1.97 m
5.80 m
1.96 m
5.80 m
5.74 m
1.95 m
5.77 m
1.97 m
5.81 m
1.98 m
2.08 d (17.61)
1.98 m
1.97 m
13,13'
14,14'
3.67 m
3.63 m
3.64 m
3.69 m
3.68 m
3.79 m
3.64 m
3.68 m
1.87 m
1.62 m
4.06 m
1.78 m
1.64 m
4.01 m
1.87 m
1.55 m
3.78 m
1.84 m
1.63 m
4.05 m
1.88 m
1.56 m
1.84 m
1.58 m
3.82 m
1.77 m
1.64 m
4.08 m
1.86 m
1.59 m
3.83 m
15,15'
3.78 ma
1.65 m
3.82
ma
16,16'
16,16'-
Me
1.54 m
1.51 m
0.78 d (6.98)
1.55 m
1.59 m
1.63 m
0.83 d (7.13)
1.66 m
1.68 m
0.81 d (6.0)
0.82 d
(6.0)
0.83 d
(6.54)
0.84 d
(6.32)
3.80 m
0.80 d
(7.14)
3.88 m
0.78 d
(7.02)
0.82 d (6.91)
0.81 d
(7.13)
3.86 m
0.84 d
(7.75)
0.79 d
(6.36)
0.78 d
(6.35)
0.83 d (7.02)
17,17'
3.85 m
3.84 t (9.70)
3.76 m
3.79 m
3.85 m
3.84 m
18,18'
19,19'
20,20'
21,21'
1.76 m
1.63 m
1.70 m
1.41 m
3.88 t, 10.55
1.79 m
1.41 m
1.74 m
1.55 m
3.88 m
1.70 m
1.55 m
3.88 m
1.85 m
1.50 m
1.72 m
1.41 m
3.90 m
1.87 m
1.50 m
3.84 m
3.93 m
3.88, m
5.71 m
3.85 m
5.64 m
3.79 m
3.99 m
3.87 m
5.65 m
3.85 m
5.69 m
1.94 m
1.53 m
1.87 t 12.35
1.52 m
5.56 d (11.09)
1.87 m
1.53 m
1.90 m
1.55 m
5.71 m
1.87 m
1.53 m
1.87 m
1.57 m
1.85 m
1.53 m
5.55 d (11.17)
1.89 m
1.55 m
5.65 m
5.63 d (9.82)
5.66 m
5.60 d
(10.39)
5.63 d
(10.00
)
22,22'
1.64 m
0.87 d (6.81)
1.65 m
1.64 m
0.89 d
1.69 m
1.76 m
1.73 m
1.76 m
0.91 m
1.74 m
22,22'-
Me
0.89 d
(6.86)
0.90 d (6.89)
0.87 d
(8.45)
0.91 d
(6.92)
0.89 d
(7.13)
0.89 d (6.78)
0.88 d
(6.78)
0.86 d (7.03)
0.90 d
(6.95)
0.91 d (6.93)
(7.47)
ACS Paragon Plus Environment

Page 13 of 29
The Journal of Organic Chemistry
1
2
3
4
5
6
7
8
9
23,23'
3.17 d (9.69)
3.12 d
(9.59)
3.19 m
3.09 m
3.20 m
1.69 m
0.95 d
(7.23)
1.35 m
1.20 m
1.86 m
1.27 m
3.15 d (9.39)
3.12 d
(9.83)
3.12 m
1.66 m
3.20 m
3.14 dd (1.52,
9.66)
1.66 m
24,24'
24,24'-
Me
1.67 m
0.97 d (7.15)
1.68 m
0.93 d (6.73)
1.69 m
1.68 m
0.98 d (6.80)
1.68 m
0.93 m
0.95 d
(7.07)
0.97 d (6.10)
0.96 d
(7.17)
0.96 d
(6.80)
0.97 d (6.18)
0.96 d
(6.3)
0.98 d (6.74)
25,25'
1.25 m
1.34 m
1.17 m
1.83 m
1.26 m
1.37 m
1.25 m
1.86 m
1.25 m
1.39 m
1.35 m
1.24 m
1.86 m
1.22 m
1.36 m
1.21 m
1.84 m
1.26 m
1.37 m
1.23 m
1.87 m
1.24 m
1.24 m
1.87 m
1.25 m
26,26'
1.88 m
1.25 m
27,27'
28,28'
3.99 m
1.82 m
1.59 m
3.53 m
3.98 m
1.79 m
1.59
3.52 ddd 4.4,
9.83, 14.17
1.95 m
3.98 m
1.82 m
1.56 m
3.52 m
3.98 m
1.81 m
1.59 m
3.52 m
3.99 m
1.82 m
1.58 m
3.52 m
3.97 m
1.80 m
1.57 m
3.52 m
3.97 m
1.79 m
1.58 m
3.52 m
3.98 m
1.81 m
1.58 m
3.52 m
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
29,29'
30,30'
31,31'
1.99 m
1.18 m
3.67 m
1.97 m
1.16 m
3.68 m
1.98 m
1.19 m
3.69 m
1.99 m
1.18 m
3.69 m
1.94 m
1.17 m
3.67 m
1.97 m
1.17 m
3.70 m
1.98 m
1.16 m
3.69 m
1.18 m
3.69 dddd 2.88,
6.13, 12.0, 15.0
1.20 d (6.18)
31,31'-
Me
32,32'
1.18 d (6.36)
4.67 d (6.91)
1.17 d
(6.14)
4.74 d
(6.80)
1.20 d (5.9)
1.17 d
(5.90)
4.81 d
(4.98)
4.74 t
6.11
1.19 d
1.19d (6.25)
1.18 d (6.24)
1.20 d
(6.3)
4.43 d
(6.29)
3.37 m
1.21 d (6.6)
1.20 d (6.18)
(6.44)
4.46 d
(5.45)
3.27 m
4.43 d (6.64)
3.28 t 7.15
3.24 m
4.39 m
4.46 d
(6.6)
3.29 m
4.65 d (6.77)
4.39 d
(6.72)
3.52 m
4.71 m
4.47 d
(6.69)
4.53 d
(5.45)
3.36 m
―
―
―
33,33'
34,34'
4.82 (dd, 8.33, 7.02)
4.83 dd 6.85,
8.39
3.35 m
4.83 dd 7.18,
8.13
3.40 m
3.34 m
3.20 m
3.32 m
3.25 m
3.21 m
3.31 m
3.21 m
3.26 m
3.21 m
3.26 m
35,35'
36,36'
3.34 m
3.24 m
3.25 m
3.31 m
3.30 m
3.31 m
3.61 m
3.36 m
―
―
4.05 m
3.28 m
3.98 m
3.16 m
3.99 m
3.21 m
4.01 m
3.25 m
3.99 m
3.25 m
3.99 m
3.23 m
3.99 m
3.18 m
3.96 m
3.31 m
38,38'
39,39'
4.28 m
4.20 brs
4.13 m
3.70 m
4.27 m
3.80 m
4.21 m
3.81 m
―
―
―
―
―
―
―
―
―
―
―
―
―
―
―
―
3.88 m
3.66 m
15,15'-
OMe
29,29'-
OMe
34,34'-
OMe
35,35'-
OMe
3.34 s
3.32 s
3.48 s
3.44 s
3.35 s
3,32 s
3.56 s
3.45 s
3.35 s
3.34 s
3.59 s
3.35 s
3.33 s
3.34 s
3.48 s
3.32 s
3.34 s
3.32 s
3.35 s
3.36 s
3.35 s
3.34 s
3.32 s
3.32 s
3.44 s
3.34 s
3.34 s
3.55 s
3.47 s
3.43 s
3.56 s
3.45 s
3.58 s
3.48 s
3.44 s
3.58 s
3.45s
―
―
3.45 s
3.44 s
3.45
―
a The data at the same atom position could be exchanged.
ACS Paragon Plus Environment

The Journal of Organic Chemistry
Page 14 of 29
1
2
3
4
5
6
7
8
9
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
Figure 4. Key ROESY correlations of the 2,3-di-O-methyl-β-xylopyranoside and glyceryl
moieties in 1 (energy minimized using standard settings for MM2 method in Chem3D 16.0).
All known swinholide-type compounds, irrespective of origin (sponge, cyanobacteria, algae,
nudibranch), possess a highly analogous monomeric carbon skeleton as well as stereo-
configurations at comparable chiral centers (Figure 5); this latter aspect has been confirmed in two
cases by X-ray crystallographic analysis7,9 as well as via total synthesis.19-21 As a result, it has been
proposed that these swinholide-type metabolites are produced by highly similar polyketide
synthase-type biosynthetic gene clusters.11 In the present case, similarities between the 1H and 13C
NMR chemical shifts and coupling constants of the protons at the chiral centers of compound 1
and those of previously reported swinholides, as well as the ankaraholides, strongly suggest that
they have the same configurations at comparable centers (Table 1 and Supporting Information
Tables S1, S2).7,8 With the relative configuration of the sugar units defined as above from coupling
constant analysis, these could be related to the aglycone stereo-configuration by several ROESY
correlations. ROESY correlations of H-32/H-7 (H-32’/H-7’), 39-OH/H-5 (39’-OH/H-5’), 39-
OH/H-3 (39’-OH/H-3’), 38-OH/H-2 (38’-OH/H-2’), 38-OH/H-21’ (38’-OH/H-21), and 38-OH/H-
3 (38’-OH/H-3’), in combination with molecular modeling, revealed that the sugar unit must be of
L configuration (Figure 4). The configuration at C-38 (C-38’) was not discernable from these data,
but given that
ACS Paragon Plus Environment

Page 15 of 29
The Journal of Organic Chemistry
1
2
3
4
5
6
7
8
9
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
ACS Paragon Plus Environment

The Journal of Organic Chemistry
Page 16 of 29
1
2
3
4
5
Figure 5. The monomeric structures and origins of the known swinholide-type compounds. The
numbers in brackets refer to carbon position of dimerization.
6
7
8
9
all occurrences of glyceric acid in 26 cyanobacterial natural products are 2R, we predict samholide
A to possess 38R (38’R) stereochemistry as well. Indeed, the glyceric acid unit configuration was
rigorously established as D by chiral HPLC analysis of the acid hydrolysate of compound 5 in
comparison with authentic standards. The geometry of the C-2-C-3 and C-4-C-5 double bonds
were determined to both be E, the former by a characteristic 15.6 Hz J value between H-2 and H-
3, and the latter from ROESY correlations between H-2 and the C-4 methyl group, H-3 and H-5
and H2-6 and the C-4 methyl group (Figure 4). Thus, both the constitutive and stereo-structure of
samholide A (1) was established.
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
The molecular formula of samholide B (2) was identical to that of compound 1 by HRESIMS,
1
13
and it had a very similar profile of H and C NMR shifts as well (Table 2 and 3). The main
1
difference in the H NMR of 2 was a different pattern of olefinic protons, including one that
integrated for a single proton and was quite distinct from those in samholide A [δH 6.51 (d, J =
12.6 Hz)]. Moreover, the C-4 and C-4’ vinyl methyl groups were present as two distinct signals,
and a number of other resonances appeared “twinned”. These data, along with a second proton
ascribed to H-2’ that had the same large trans-type coupling as present in 1 (JH2’/H3’ = 15.6 Hz),
along with ROESY correlations between H-2/H-3, H3/H-5, H-2’/4’-Me, and H-3’/H-5’, indicated
that compound 2 was the C-2 double bond isomer of 1 with an overall heterodimeric structure.
Table 3. 13C NMR data for compounds 2-9 in CDCl3 at 125 MHz (δH/δH’ in ppm).
8c
2
3
4
5
6
7
9
1/1'
2/2'
167.9/170.1 170.3 167.4/169.0
116.3/114.3 114.2 116.7/115.8
148.6/152.2 152.4 148.5/150.5
170.5/169.9 167.6/169.5 169.5/167.4
170.3
169.6
113.5/114.4 116.5/115.3 114.5/116.2 114.3/113.9 114.5
153.1/152.1 148.7/151.1 153.9/148.3 152.2/152.1 152
3/3'
4/4'
134.1
134.6
12.4
133.6
133.7/134.5
12.1/12.4
134.7
134.1
134.4
12.5/12.4
141/134
33.6/33.3
76.0/76.3
39.8/39.7
69.6
134.2
12.5
4/4'-Me
5/5'
15.7/12.4
16.2/12.7
15.8/12.6
12.5/16.1
135.0/140.6 140.8 134.8/137.9
142.7/140.3 134.1/138.5 140/134
140.3
36.7
6/6'
33.0/33.1
77.4b/77.7b
39.5/39.6
69.5
33.2
76.6b
39.8
69.6
33.3/33.4
76.7b
33.6/33.2
78.6/76.6
39.8/39.7
68.9/69.2
33
33.6/33.2
77.2b/76.7b
39.5/39.1
69.4/69.7
7/7'
77.0/75.7
39.6/39.3
69.0/69.6
69.8
40.7a
8/8'
39.1
9/9'
69.2/68.6
72.6
ACS Paragon Plus Environment

Page 17 of 29
The Journal of Organic Chemistry
1
2
3
4
5
6
7
8
9
10/10'
11/11'
12/12'
129.7a/129.6a 129.8 129.6a/129.5a 129.7/129.8 129.6
129.5/129.6 129.8
129.4
124.2
31.3
123.8a/123.7a 123.5 124.0/123.2
123.4/123.7 124.0/123.8 123.9/123.6 123.6
31.5a/31.4a
64.1/63.8
36.2/35.9
77.2b
31.6
63.6
36.9
76
31.4/31.3
64.4/64.3
36.1/35.7
78.0b
31.5
31.3/31.2
64.4/64.3
35.7/35.6
77.0b
31.4
64.5
31.4/31.6
63.7/63.5
36.9/36.8
75.8/76.0
57.5
13/13'
14/14'
63.5/64.1
36.2/36.7
75.7/75.4
57.6/57.5
41.6
64.5
35.9
77.6b
57.2
40.5a
9.8
36.1/35.9
77.0b
15/15'
15/15'-OMe
16/16'
57.1a/57.2a
57.5
41.5
9.3
57.1
57.2/57.1
40.9/40.5
10.2/9.9
74.3/74.2
57.3/56.9
40.8/40.6
9.9
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
41.5
41.0/41.2
10.0/10.2
73.8/74.2
41.5/41.4
9.4/9.3
16/16'-Me
17/17'
9.9a/9.6a
73.9/73.6
9.2/9.0
74
73.6/73.8
74.3/74.1
74.0/74.1
75
18/18'
40.9/41.2
40.8
41.7
40.9/40.6
41.7/41.4
41.7/41.5
40.8
40.8a
19/19'
20/20'
69.3/68.8
41.1/41.5
71.1/71.4
41.1/40.9
10.0/10.2
69.9
42.2
70.6
40.8
10.1
69.5
70.0/69.4
42.4
69
41.8
69.8/68.3
42.1
69.8
69.2
41.7
70.4
40.9a
10.2
41.9/42.1
71.1/70.7
40.5
42.1/42.0
70.7/70.6
40.8/40.7
10.3/10.2
21/21'
70.3/70.5
41.2/40.9
9.9/10.1
71.3/70.8
41.1/40.9
10.3
70.9/71.3
41.3/40.9
10.3/10.2
22/22'
22/22'-Me
10.3/10.5
23/23'
76.6/76.1
76.3
76.5
76.1/76.5
76.7
76.3
76.8b
76.6b
24/24'
33.5
17.6a /17.5a
33.2
17.4
33.0/32.8
17.7/17.6
33.4/33.0
17.5
33.4
17.6
33
33.2/33.3
17.5/17.4
33.3
17.7
24.2
24/24'-Me
17.6
25/25'
26/26'
24.0a /23.8a
29.4/29.3
71.6/71.5
35.1
23.9
29.4
71
24.2/23.9
29.8/29.3
71.7
23.6/24.0
29.8
24
24.2/23.6
29.3/29.2
71.5/71.7
35
24
29.4/29.5
71.1/71.0
35
29.3
71.5
35
29.4
71.6
35
27/27'
71.4/71.3
35.1/35.0
73.4
28/28'
35
35
29/29'
73.4
73.4
55.4
38.7
64.9
22
73.4
73.4
55.4
38.9
64.7
22
73.4
73.4
73.4
55.5
38.8
64.8
22
29/29'-OMe
30/30'
55.4
55.4
55.4
55.4
55.5
38.9
38.9
38.8
38.9
38.7
31/31'
64.8
22.0a/21.9a
64.7
64.8/64.9
21.9/22.0
64.7
64.9
31/31'-Me
32/32'
22
21.9
21.9
―
100.7/101.2 103.3 101.9/102.6
101.4/102.9 100.4/102.1 100.8/102.5 103.2/102.9
―
33/33'
73.4
73.4
83.5
60.2
79.4
58.6
62.5
―
73.4
83.4/83.5
60.3/60.2
79.0/79.3
58.5
73.0/73.3
82.1/83.3
60.1
73.4/72.9
82.0/83.5
60.2/60.3
79.1
73.4/73.0
82.4/83.6
60.2/60.1
79.3
73.1/72.1
83.2/83.3
60.1/59.8
79.3/68.9
58.5/―
62.5/64.2
―
―
34/34'
82.1/82.4
60.2a/60.1a
79.0/79.3
58.6a/58.7a
62.7
―
34/34'-OMe
35/35'
―
79.2
58.7a/58.5a
58.6a/58.5a
―
35/35'-OMe
36/36'
58.5
62.6
62.2/62.5
170.5/―
72.6/―
64.6/―
62.6
62.7
―
―
―
―
―
37/37'
171.4/170.9
72.5/72.4
64.4/64.5
171.5/―
72.4/―
64.3/―
170.8/―
72.4/―
64.5/―
―
―
―
38/38'
―
―
―
39/39'
a Could be exchanged.
b Overlapped with CDCl3, assigned using HMBC, HSQC and HSQC-TOCSY.
c The data at the same carbon atom position could be exchanged.
HRESIMS analysis of samholide C (3) indicated its molecular formula as C90H152O28, 176
mass units less than that of compound 1, and corresponded to the absence of both glyceric acid
groups. This was consistent with the presence of a monomeric quasi-molecular ion peak at m/z
ACS Paragon Plus Environment

The Journal of Organic Chemistry
Page 18 of 29
1
2
3
4
5
6
7
863.4 in the MS/MS spectrum of 3. The 1H and 13C NMR data of 3 were almost the same as those
of 1, except that the glyceric acid signals were missing and the H-33/H33’ protons were shielded
by 1.5 ppm.
8
9
Samholide D (4) had the same molecular formula as compound 3 from HRMS data. Similar to
the relationship of compounds 1 and 2, the differences in its 1H and 13C NMR spectra were due to
double bond isomerization of the C-2-C-3 olefin in one half of the dimer, as revealed by key
chemical shift differences (e.g. δH 5.69 for H-2, δH 5.82 for H-2’) and twinning of many signals
near this position of variance between the two monomeric halves. Thus, samholide D (4) was
assigned as the C-2 double bond isomer of samholide C (3).
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
A molecular formula of C93H156O31 was established for samholide E (5) from HRESIMS data,
indicating that it was 88 amu less than that of compound 1. This was consistent with the loss of
one of the two glyceric acid residues from samholide A. The presence of ions at m/z 951.5 and
863.5, which result from cleavage of the two ester bonds, further indicated the loss of a glyceric
acid residue from one of the two monomeric units. This change resulted again in a subtle twinning
1
13
of many signals in the H and C NMR of 5; for example, the H-3 and H-3’ protons of the
conjugated diene differed by 0.05 ppm in chemical shift (δH 7.57, d, J = 15.55 Hz, H-3; 7.52, d, J
= 15.56 Hz, H-3’). Thus, samholide E (5) was assigned a structure identical to that of samholide
A (1) but missing one of the two glyceric acid units.
Samholides F (6) and G (7) had the same molecular formula as that of samholide E (5), but
differed in the Z and E geometry of the C-2-C-3 double bond in either the Western or Eastern
halves of the dimer. For compound 6, there was evidence that the C-2-C-3 olefin was Z (δH 6.52,
d, J = 12.62 Hz, H-3, and δH 7.44, d, J = 15.54 Hz, H-3’) whereas in compound 7 the C-2’-C-3’
olefin was Z (δH 6.46, d, J = 12.69 Hz, H-3’, and δH 7.47, d, J = 15.63 Hz, H-3), respectively. The
sugar units, either with or without glyceric acid in the two monomeric halves of compound 6, were
determined by key ROESY correlations between H-3 and H-5 (δH 5.86, t, J = 7.00 Hz), H-5 and
H-7 (δH 4.00, m), and H-7 and H-32 (δH 4.65, J = 6.77 Hz) (Figures S69 and S70 in Supporting
Information). COSY correlations between H-5/H2-6 (δH 2.57, ddd, J = 4.06, 6.48, 13.87; 2.40, dt,
J = 7.02, 15.36 Hz)/H-7 (Figures S63 and S64 in Supporting Information), and HMBC correlations
between the C-4 Me group (1.86, s) with C-3 (δC 148.7), C-4 (δC 134.7) and C-5 (δC 134.1), along
with correlations between H-5 and C-7 (δC 77.0), H-7 and C-32 (δC 100.4), and H-32 and C-7
ACS Paragon Plus Environment

Page 19 of 29
The Journal of Organic Chemistry
1
2
3
4
5
6
7
8
9
(Figures S65 and S66 in Supporting Information), indicated that the glyceric acid residue was
attached to the sugar unit located at C-7 of the 2Z,4E-monomeric structure. Further, lack of a
glyceric acid residue in the 2E,4E-monomeric structure was shown by ROESY correlations
between H-3’ and H-5’ (δH 6.14, t, J = 6.87 Hz), H-5’ and H-7’ (δH 3.97, m), and H-7’ and H-32’
(δH 4.39, J = 6.72 Hz). This was also supported by COSY correlations of H-5’/H2-6’ (δH 2.69,
ddd, J = 4.38, 6.03, 11.73; 2.28, dt, J = 7.45, 15.12 Hz)/H-7’, together with HMBC correlations of
the C-4’ Me group protons (1.81, s) with C-3’ (δC 151.1), C-4’ (δC 134.7) and C-5’ (δC 138.5), the
H-5’ proton with C-7’ (δC 75.7), the H-7’ proton with C-32’ (δC 102.1), and the H-32’ proton with
C-7’. The combination of these NMR data thus confirmed the overall structural assignment for
compound 6 as the 2Z double bond isomer of samholide E (5). Accordingly, a similar analysis for
compound 7 revealed it to be the 2’Z double bond isomer of samholide E (5).
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
Samholide H (8) had a molecular formula of C89H150O28 based on HRESIMS analysis. MS/MS
data of 8 showed two different ions for the two monomeric halves of the molecule at m/z 863.4
and 849.4, indicating the lack of a glyceric acid residue in one half and the lack of a methyl or
methylene group in the other, relative to the monomeric structures present in compound 5. Further,
an m/z 703.3 fragment ion derived from the m/z 849.4 fragment, indicating the lack of a methyl
group on the sugar residue. HSQC-TOCSY revealed the signals of both sugar units (Tables 2 and
3); however, the unit lacking the glyceric acid residue also showed an up-field shift for C-35’ (δC
79.3 for C-35 vs δC 68.9 for C-35’). Combined with the absence of this mid-field methyl group
signal in its 1H and 13C NMR spectra, it was deduced that samholide H (8) possessed one half of
the samholide C structure combined with a second half in which the sugar lacked the C-35 methoxy
methyl group. This was further supported by COSY, HMBC, and ROESY correlation networks
for these two different sugar residues (Figures S86-S93 in Supporting Information).
The molecular formula of samholide I (9) was determined as C76H128O20 from HRESIMS data.
MS/MS fragmentation gave a prominent ion at m/z 703.4, suggesting that 9 was an analogue of
1
compound 1 but lacked both the xylose sugar and glyceryl groups. Accordingly, the H NMR
spectrum of compound 9 showed no sugar or glyceric acid signals, and the 2D NMR data set was
fully consistent with the aglycone structure of samholide A 1 (see Supporting Information).
Because the samholides (1-9) are structurally similar to the highly cytotoxic swinholides, they
were evaluated for activity against the H-460 human lung carcinoma cell line. All of the
ACS Paragon Plus Environment

The Journal of Organic Chemistry
Page 20 of 29
1
2
3
4
5
6
7
8
9
samholides showed significant activity with IC50 values less than 1 μM (Table 4), although it
should be noted that these IC50 values are all considerably higher than those for the original
swinholides.5 However, compound 9 (samholide I), which lacks both the glyceric acid and sugar
units, showed the highest IC50 of 0.9 μM, indicating that sugar and glyceryl moieties enhance the
cytotoxicity of the samholides. Compound 2 with the 2Z double bond, showed reduced activity
compared to 1; however, compounds 3 and 4 as well as 5 and 6, two pairs which also differ only
in the 2E versus 2Z geometry, showed equivalently high potency (0.17-0.21 μM). Compound 7
with the 2’Z configuration, was also a highly potent compound (0.21 μM). Compound 8, which
had a less methylated xylose sugar, was of lower potency in this assay. In summary, it appears
that highest potency among these analogs was obtained when the dimethoxylated xylose sugar was
present, with or without glyceric acid residues attached.
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
Table 4. Cytotoxicity of compounds 1-9 to H-460 human lung carcinoma cells.a
Compounds
IC50, mean ± standard errors (μM)
0.17 ± 0.01
Samholide A (1)
Samholide B (2)
Samholide C (3)
Samholide D (4)
Samholide E (5)
Samholide F (6)
Samholide G (7)
Samholide H (8)
Samholide I (9)
Doxorubicin
0.52 ± 0.02
0.21 ± 0.08
0.17 ± 0.06
0.17 ± 0.01
0.17 ± 0.00
0.21 ± 0.01
0.47 ± 0.04
0.91 ± 0.05
0.30 ± 0.02
a Cytotoxicity was assayed in triplicate and doxorubicin was used as the positive control.
The samholides (1-9) possess several notable structural features, including the uncommon 20-
demethyl 44-membered lactone ring, methoxylated xylose sugar, and unusual glyceric acid moiety.
The previously described swinholide compounds isolated from sponges and cyanobacteria have
been remarkably consistent in the structures of their fundamental carbon skeleton, and structural
diversity has been observed in the side chains, position of lactonization and overall ring size.
Variations in methyl appendages have only been observed at positions C-16 and C-20. The glyceric
acid group, generally a rare structural feature in natural products, is relatively common in
ACS Paragon Plus Environment

Page 21 of 29
The Journal of Organic Chemistry
1
2
3
4
5
cyanobacterium-derived peptides.23 However, the occurrence of such a residue in a polyketide
such as the samholides, and as a substituent on a sugar unit, is quite unique.
6
7
8
9
In 1993, Carme1i et al.24 found that tolytoxin was a polyketide assembled from a glycine starter
unit and 15 acetate repeater units, and that the one-carbon branches originated from the
tetrahydrofolate Cl pool. Recently, Ueoka et al.11 showed that the architecture of the cyanobacterial
PKS megasynthase which produces scytophycin and related compounds was highly similar to the
trans-AT PKS cluster responsible for misakinolide production by “Entotheonella sp.”, a symbiont
in the sponge Theonella swinhoei. Thus, the biosynthesis of the samholides most likely follows a
similar PKS pathway for construction of the carbon chains but with extra sugar and glyceric acid
units attached as novel substituents.
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
Conclusions
The swinholide family of compounds has attracted much attention due to their intriguing structural,
biosynthetic and pharmacological features. In the present study, nine new swinholide-related
compounds, samholides A-I (1-9), were obtained from an American Samoa field collection of the
cyanobacterium cf. Phormidium sp. (Figure 6). A 20-demethyl 44-membered lactone ring
distinguished these new compounds, with structural diversification occurring from geometrical
isomerization of double bonds, presence of xylose sugar units with unique glyceric acid moieties
and varied O-methylations. The sugar and glyceric acid units appeared to enhance the relative
potency of these agents in a cytotoxicity assay. Discovery of these additional representatives of
the swinholide family from another cyanobacterium expands on the number of divergent species
that contain this biosynthetic capacity, and deepens questions concerning its evolutionary origins
and history among the producing species of heterotrophic bacteria, cyanobacteria and possibly
additional sources.
ACS Paragon Plus Environment

The Journal of Organic Chemistry
Page 22 of 29
1
2
3
4
5
6
7
8
9
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
Figure 6. Photomicrograph of voucher sample of cf. Phormidium sp.
Experimental methods
Optical rotation was measured on a Jasco P-2000 polarimeter. IR spectra were measured on a
Thermo Electron Corporation Nicolet IR 100 FT-IR. UV/visual-light spectra were recorded on a
Beckman Coulter DU 880 spectrophotometer.1H NMR and 2D NMR spectra were collected on a
Bruker Avance III DRX-600 NMR with a 1.7 mm dual tune TCI cyroprobe (600 and 150 MHz for
1H and 13C NMR, respectively). 13C NMR spectra were run on a Varian X-Sens 500 MHz NMR
13
(125 MHz) equipped with a 5 mm Xsens C{1H} cryoprobe. NMR spectra were referenced to
residual solvent CDCl3 signals (δH 7.26 ppm and δC 77.16 ppm as internal standards). High-
resolution mass spectra were carried out on an Agilent 6230 TOF-MS under positive ion ESI-TOF-
MS conditions in the University of California, San Diego (UCSD) Small Molecule MS Facility.
MS fragmentation experiments were run with a Biversa Nanomate electrospray source for a
Finnigan LTQ-FTICR-MS instrument running Tune Plus software version 1.0. HPLC was
performed using Chromeleon 7 software with Thermo Dionex UltiMate 3000 pump and a RS diode
array detector. All solvents were HPLC grade except for water, which was produced by a Millipore
Milli-Q system.
Cyanobacterial Collection and Taxonomy.
ACS Paragon Plus Environment

Page 23 of 29
The Journal of Organic Chemistry
1
2
3
4
5
6
The marine cyanobacterium cf. Phormidium sp. (voucher specimen available from W. H. G. as
collection number ASX22Jul14-1) was found growing in 1.0-2.0 m of water at Fagaalu Park in
American Samoa, USA. The sample was hand collected in July 2014, preserved with a 1:1 2-
7
8
9
o
propanol–seawater solution and stored in the laboratory at -20 C until extraction. Microscopic
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
examination indicated that this collection was morphologically consistent with the genus
Phormidium sp.
Extraction and Isolation
The preserved cyanobacterium was filtered through cheesecloth and the biomass (101.7 g dry wt)
was extracted repeatedly by soaking in 500 mL 2:1 CH2Cl2/MeOH with warming (<30 oC) for 30
min to afford 1.8 g of dried extract. A portion of the extract was fractionated by silica gel vacuum
liquid chromatography (VLC) using a stepwise gradient solvent system of increasing polarity
starting from 100% hexanes to 100% MeOH [nine fractions, 100% hexanes (Fr. A, 51.7 mg), 90%
hexanes/10% EtOAc (Fr. B, 132.8 mg), 80% hexanes/20% EtOAc (Fr. C 597.7 mg), 60%
hexanes/40% EtOAc (Fr. D, 358.5 mg), 40% hexanes/60% EtOAc (Fr. E, 68.2 mg), 20%
hexanes/80% EtOAc (Fr. F, 78.2 mg), 100% EtOAc (Fr. G, 38.6 mg), 75% EtOAc/25% MeOH
(Fr. H, 180.6 mg) and 100% MeOH (Fr. I, 248.1 mg)]. Fr. I was dissolved in 50% CH3CN/50%
H2O, subjected to chromatography on C18 solid-phase extraction (SPE) with a Strata 6 mL column
and 1 g of C18-E (55 μm, 70 Å), and eluted sequentially with 30 mL 50% CH3CN/50% H2O (Fr.
I1, 15.2 mg), 65% CH3CN/35% H2O (Fr. I2, 10.4 mg) , 80% CH3CN/20% H2O (Fr. I3, 73.2 mg) ,
and 100% CH3CN (Fr. I4, 15.4 mg). Fr. I2 was further purified by HPLC using a Synergi 4 μm
Hydro-RP 80 Å column (10.00 × 250 mm) and isocratic elution using 97% ACN/3% H2O at the
flow rate of 3 mL/min over 45 min. This yielded six sub-fractions: Fr. I2A, Fr. I2B, Fr. I2C, Fr.
I2D, Fr. I2E and Fr. I2F at 13.0-16.0 min, 19.5-26.8 min, 27.2-31.0 min, 32.6-35.0 min, 36.2-38.8
min and 41.0-44.0 min, respectively. Three fractions (Fr. I2C, Fr. I2E and Fr. I2F) after reversed-
phase HPLC were purified on the same type of column (10.00 × 250 mm Synergi 4 μm Hydro-RP
80 Å column) with different eluent solvents applied. Fr. I2C was purified using a gradient from
70% ACN/30% H2O to 90% ACN/10% H2O over 30 min, then 90% ACN/10% H2O for 10 min,
finally ramping back to 70% ACN/30% H2O over 5 min, detection at 269 nm], giving compound
2 (1.4 mg) at 27.8-29.2 min. Fr. I2E was purified using 97% ACN/3% H2O, (detection at 269 nm),
giving compound 5 (5.2 mg) at 32.0-34.5 min. Fr. I2F was purified using 97% ACN/3% H2O
(detection at 269 nm) to yield 1 (5.5 mg) at 35.0-37.4 min and 3 (2.6 mg) at 39.5-42.2 min. In the
ACS Paragon Plus Environment

The Journal of Organic Chemistry
Page 24 of 29
1
2
3
4
5
6
7
8
9
same way, Fr. H was dissolved in 50% CH3CN/50% H2O, purified over C18 solid-phase extraction
(SPE) with a Strata 6 mL column and 1 g of C18-E (55 μm, 70 Å), and eluted sequentially with 30
mL 50% CH3CN/50% H2O (Fr. H1, 15.2 mg), 65% CH3CN/35% H2O (Fr. H2, 10.4 mg), 80%
CH3CN/20% H2O (Fr. H3, 73.2 mg), and 100% CH3CN (Fr. H4, 15.4 mg). Fr. H3 was separated
further using RP HPLC (4μ Phenomenex Kinetex column, isocratic 70% ACN/30% H2O for 30
min) to yield six sub-fractions (Fr. H3A, Fr. H3B, Fr. H3C, Fr. H3D, Fr. H3E and Fr. H3F at 5.6-
6.7 min, 8.0-10.0 min, 10.2-14.5 min, 14.5-16.8 min, 16.8-22.0 min and 22.5-24.5 min,
respectively. Three fractions (Fr. H3B, Fr. H3C and Fr. H3D) were further purified by RP-HPLC
using a Synergi Hydro-RP 80 Å column (10.00 × 250 mm, 4 μm) or Phenomenex Luna phenyl-
hexyl column with different elution solvents at a flow rate of 3 mL/min. Fr. H3B was subjected to
HPLC purification with Synergi Hydro-RP 80 Å using a gradient from 88% MeOH/12% H2O to
94% MeOH/6% H2O over 30 min, then 94% MeOH/6% H2O for 5 min, finally ramping back to
88% MeOH/12% H2O over 2 min, detection at 269 nm, giving compound 8 (0.5 mg) at 29.0-31.0
min. Fr. H3C was subjected to HPLC purification using a Synergi Hydro-RP 80 Å column
[isocratic 80% ACN/20% H2O for 40 min, then a gradient from 80% ACN/20% H2O to 90%
ACN/10% H2O over 5 min, isocratic 90% ACN/10% H2O for 10 min, finally ramping back to 80%
ACN/20% H2O over 5 min, detection at 269 nm], giving compounds 9 (0.3mg), 6 (0.8 mg) and 7
(0.8 mg). Fr. H3D was purified to yield compound 4 (0.3 mg) after reversed-phase HPLC using
the same mobile condition.
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
LC–MS Analysis and Molecular Networking Generation
The crude extract and fractions A-I were dissolved in MeOH and passed through a Bond Elut-C18
OH cartridge (Agilent Technologies, USA) that was prewashed with 3 mL of CH3CN.
Subsequently, the Bond Elut-C18 OH cartridge was washed with 3 mL of CH3CN, the solvent
dried under N2 and the residue dissolved in MeOH. A 0.020 mL aliquot of each sample was
injected and analyzed via LC–MS/MS on a ThermoFinnigan Surveyor Autosampler-Plus/LC-
MS/MS/PDA-Plus system coupled to a Thermo Finnigan LCQ Advantage Max mass spectrometer
with a gradient of 30–100% CH3CN in water with 0.1% formic acid. The MS/MS spectra of the
crude extract and nine fractions were used to generate a molecular network following previously
described methodology and visualized using Cytoscape (www.cytoscape.org).15 Algorithms
ACS Paragon Plus Environment

Page 25 of 29
The Journal of Organic Chemistry
1
2
3
4
5
6
assumed a cosine threshold set at 0.6 and nodes were color-coded according to the fractions from
VLC isolation.
7
8
Cytotoxicity assay
9
Cytotoxicity to H-460 human lung carcinoma cells was measured as cell viability using the 3-(4,5-
dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) reduction method.17 Cells were
cultured in Roswell Park Memorial Institute (RPMI) 1640 medium containing L-glutamine
(Mediatech, Manassas, VA) and supplemented with 1 nM sodium pyruvate, 100 μg/mL
streptomycin, 100 units penicillin, 0.15% sodium bicarbonate and 10% fetal bovine serum (FBS)
at 37 ℃, 5% CO2. Cells were seeded in 96-well plates at 6660 cells/well in 180 μL. After 24 h,
samholides A-I (1-9) were dissolved in DMSO at 1 mg/mL. The dose-response assays used 20 uL
of this DMSO stock and were diluted with 180 μL of RPMI-1640 medium (without fetal bovine
serum) to 100 ug/mL followed by nine serial 25 μL:54 μL (logarithmic scale) dilutions with RPMI-
1640. Subsequently, 20 μL/well of all 10 mixtures were added to cells in duplicate, resulting in a
final maximal DMSO concentration of 1%. Equal volumes of RPMI-1640 medium were added to
10 wells designated as negative controls for each plate. After 48 h, the medium was removed by
aspiration and cell viability determined by MTT staining. All assays were validated using
doxorubicin at 1.0 and 0.1 μg/mL as the positive control. OD values were measured on
ThermoElectron Multiskan Ascent plate reader at 570 and 630 nm. Dose response graphs were
generated using GraphPad Prism (GraphPad Software Inc., San Diego, CA) for IC50 values
determination.
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
Samholide A (1):
White, amorphous solid; [α]27D −48 (c 0.1, MeOH); UV (MeOH) λmax (log ε) 269 (4.72); 1H NMR
(600 MHz, CDCl3) and 13C NMR (125 MHz, CDCl3), see Table 1; HRESIMS m/z 1880.0657 [M
+ Na]+ (calcd for C96H160O34Na+, 1880.0683).
Samholide B (2):
White, amorphous solid; [α]27D −50 (c 0.1, MeOH); UV (MeOH) λmax (log ε) 268 (4.68); 1H NMR
(600 MHz, CDCl3) and 13C NMR (125 MHz, CDCl3), see Tables 2 and 3; HRESIMS m/z
1880.0654 [M + Na]+ (calcd for C96H160O34Na+, 1880.0683).
Samholide C (3):
ACS Paragon Plus Environment

The Journal of Organic Chemistry
Page 26 of 29
1
2
3
4
5
White, amorphous solid; [α]27D −52 (c 0.1, MeOH); UV (MeOH) λmax (log ε) 269 (4.70); 1H NMR
(600 MHz, CDCl3) and 13C NMR (125 MHz, CDCl3), see Tables 2 and 3; HRESIMS m/z
1704.0327 [M + Na]+ (calcd for C90H152O28Na+, 1704.0362).
6
7
8
Samholide D (4):
9
White, amorphous solid; [α]27D −59 (c 0.1, MeOH); UV (MeOH) λmax (log ε) 268 (4.66); 1H NMR
(600 MHz, CDCl3) and 13C NMR (125 MHz, CDCl3), see Tables 2 and 3; HRESIMS m/z
1704.0359 [M + Na]+ (calcd for C90H152O28Na+, 1704.0362).
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
Samholide E (5):
White, amorphous solid; [α]27D −66 (c 0.1, MeOH); UV (MeOH) λmax (log ε) 269 (4.71); IR (neat,
1
KBr) 3445, 3298, 2931, 1730, 1685, 1620, 1459, 1379, 1297, 1080, 1028 cm-1; H NMR (600
13
MHz, CDCl3) and C NMR (125 MHz, CDCl3), see Tables 2 and 3; HRESIMS m/z 1792.0508
[M + Na]+ (calcd for C93H156O31Na+, 1792.0523).
Samholide F (6):
White, amorphous solid; [α]27D −25 (c 0.1, MeOH); UV (MeOH) λmax (log ε) 269 (4.69); 1H NMR
(600 MHz, CDCl3) and 13C NMR (125 MHz, CDCl3), see Tables 2 and 3; HRESIMS m/z
1792.0494 [M + Na]+ (calcd for C93H156O31Na+, 1792.0523).
Samholide G (7):
White, amorphous solid; [α]27D −32 (c 0.1, MeOH); UV (MeOH) λmax (log ε) 269 (4.70); 1H NMR
(600 MHz, CDCl3) and 13C NMR (125 MHz, CDCl3), see Tables 2 and 3; HRESIMS m/z
1792.0503 [M + Na]+ (calcd for C93H156O31Na+, 1792.0523).
Samholide H (8):
White, amorphous solid; [α]27D −10 (c 0.1, MeOH); UV (MeOH) λmax (log ε) 270 (4.70); 1H NMR
(600 MHz, CDCl3) and 13C NMR (125 MHz, CDCl3), see Tables 2 and 3; HRESIMS m/z
1690.0211 [M + Na]+ (calcd for C89H150O28Na+, 1690.0206).
Samholide I (9):
White, amorphous solid; [α]27D −17 (c 0.1, MeOH); UV (MeOH) λmax (log ε) 269 (4.66); 1H NMR
(600 MHz, CDCl3) and 13C NMR (125 MHz, CDCl3), see Tables 2 and 3; HRESIMS m/z
1383.8881 [M + Na]+ (calcd for C76H128O20Na+, 1383.8891).
Absolute Configuration of Glyceric Acid Residue.
An aliquot (0.2 mg) of compound 5 was hydrolyzed with 6 N HCl (0.3 mL) for 16 h at 90 °C. The
hydrolysate was concentrated to dryness and subjected to chiral HPLC analysis (Phenomenex
Chirex 3126 (D)-penicillamine (150 x 4.6 mm) HPLC column; flow rate 1 mL/min; UV detection
at 254 nm; solvent 2 mM CuSO4:MeOH 85:15). The retention time of glyceric acid from the
hydrolyzate was 17.4 min. This was compared to authentic standards whose retention times were
14.9 min for L-glyceric acid and 17.4 min for D-glyceric acid.
ACS Paragon Plus Environment

Page 27 of 29
The Journal of Organic Chemistry
1
2
3
4
Acknowledgements
5
6
7
8
We thank Y. Su (UCSD Chemistry and Biochemistry Mass Spectrometry Facility) for some of
the HRMS data, N. Moss for help in obtaining an IR spectrum, and B. Duggan and A. Mrse for
assistance with NMR technical support. This work was supported by National Institutes of
Health grant CA100851 (WHG), Guangdong Natural Science Foundation (2016A030313588),
Special Fiscal Fund of Guangdong Provincial Oceanic and Fishery Administration in 2017, Fund
of the Education Bureau of Guangzhou City (1201610155), National Natural Science Foundation
of China (Grant No. 41522605), the project of “AoShan” excellent scholar for Qingdao National
Laboratory for Marine Science and Technology, and the Chinese Scholarship Council (CSC) for
financial support of this work. We thank the National Fisheries and Parks Service for permits for
sample collection in American Samoa.
9
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
Supporting Information.
The Supporting Information is available free of charge on the ACS Publications website at DOI:
XXXX.
Full NMR and MS data for samholides A-I (compounds 1-9), NMR data comparisons with
known compounds, MS2-based molecular network for cf. Phormidium sp.
References
1. Komarek, J.; Kastovsky, J.; Mares, J.; Johansen, J. R. Preslia 2014, 86, 295-335.
2. Nunnery, J. K.; Mevers, E.; Gerwick, W. H. Curr. Opin. Biotechnol. 2010, 21, 787-793.
3. Blunt, J. W.; Copp, B. R.; Keyzers, R. A.; Munro, M. H.; Prinsep, M. R. Nat. Prod. Rep. 2015, 32,
116-211.
4. Mayer, A. Marine Pharmaceuticals: The Clinical Pipeline.
http://marinepharmacology.midwestern.edu/clinPipeline.htm
5. Carmeli, S.; Kashman, Y. Tetrahedron Lett. 1985, 26, 511-514.
6. Moore, R. E. In Marine Natural Products: Chemical and Biological Perspectives, P. J. Scheuer, Ed.,
Vol. 4, Academic Press, 1981, pp. 1-52.
7. (a) Ishibashi, M.; Moore, R. E.; Patterson, G. M. L.; Xu, C.; Clardy, J. J. Org. Chem. 1986, 51, 5300-
5306; (b) Moore, R. E.; Ishibashi, M.; Patterson, G. M. L.; Xu, C.; Clardy, J. Tennen Yuki Kagobutsu
Toronkai Koen Yoshishu 1986, 28, 184-191; (c) Moore, R. E.; Patterson, G. M.; Mynderse, J. S.; Barchi,
J.; Norton, T.; Furusawa, E.; Furusawa, S. Pure Appl. Chem. 1986, 58, 263-271.
8. Andrianasolo, E. H.; Gross, H.; Goeger, D.; Musafija-Girt, M.; McPhail, K.; Leal, R. M.; Mooberry, S.
L.; Gerwick, W. H. Org. Lett. 2005, 7, 1375-1378.
9. Kitagawa, I.; Kobayashi, M.; Katori, T.; Yamashita, M.; Tanaka, J.; Doi, M.; Ishida, T. J. Am. Chem.
Soc. 1990, 112, 3710-3712.
10. Bewley, C. A.; Holland, N. D.; Faulkner, D. J. Experientia 1996, 52, 716-722.
ACS Paragon Plus Environment

The Journal of Organic Chemistry
Page 28 of 29
1
2
3
4
5
11. Ueoka, R.; Uria, A. R.; Reiter, S.; Mori, T.; Karbaum, P.; Peters, E. E.; Helfrich, E. J. N.; Morinaka,
B. I.; Gugger, M.; Takeyama, H.; Matsunaga, S.; Piel, J. Nat. Chem. Biol. 2015, 11, 705-712.
6
7
8
12. Humisto, A.; Jokela, J.; Liu, L.; Wahlsten, M.; Wang, H.; Permi, P.; Machado, J. P.; Antunes, A.;
Fewer, D. P.; Sivonen, K. Appl. Environ. Microbiol. 2018, 84, e02321-17
9
13. Patterson, G. M.; Carmeli, S. Arch. Microbiol. 1992, 157, 406-410.
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
14. Smith, C. D.; Carmeli, S.; Moore, R. E.; Patterson, G. M. Cancer Res. 1993, 53, 1343-1347.
15. (a) Yeung, K.-S.; Paterson, I. Angew. Chem. Int. Ed. 2002, 41, 4632-4653; (b) Allingham, J. S.;
Klenchin, V. A.; Rayment, I. Cell. Mol. Life Sci. 2006, 63, 2119-2134.
16. Wang, M.; Carver, J. J.; Phelan, V. V.; Sanchez, L. M.; Garg, N.; Peng, Y.; Nguyen, D. D.; Watrous,
J.; Kapono, C. A.; Luzzatto-Knaan, T.; Porto, C.; Bouslimani, A.; Melnik, A. V.; Meehan, Mi. J.; Liu,
W.-T.; Crusemann, M.; Boudreau, P. D.; Esquenazi, E.; Sandoval-Calderon, M.; Kersten, R. D.; Pace, L.
A.; Quinn, R. A.; Duncan, K. R.; Hsu, C.-C.; Floros, D. J.; Gavilan, R. G.; Kleigrewe, K.; Northen, T.;
Dutton, R. J.; Parrot, D.; Carlson, E. E.; Aigle, B.; Michelsen, C. F.; Jelsbak, L.; Sohlenkamp, C.;
Pevzner, P.; Edlund, A.; McLean, J.; Piel, J.; Murphy, B. T.; Gerwick, L.; Liaw, C.-C.; Yang, Y.-L.;
Humpf, H.-U.; Maansson, M.; Keyzers, R. A.; Sims, A. C.; Johnson, A. R.; Sidebottom, A. M.; Sedio, B.
E.; Klitgaard, A.; Larson, C. B.; Boya C. A.; Torres-Mendoza, D.; Gonzalez, D. J.; Silva, D. B.; Marques,
L. M.; Demarque, D. P.; Pociute, E.; O'Neill, E. C.; Briand, E.; Helfrich, E. J. N.; Granatosky, E. A.;
Glukhov, E.; Ryffel, F.; Houson, H.; Mohimani, H.; Kharbush, J. J.; Zeng, Y.; Vorholt, J. A.; Kurita, K.
L.; Charusanti, P.; McPhail, K. L.; Nielsen, K. F.; Vuong, L.; Elfeki, M.; Traxler, M. F.; Engene, N.;
Koyama, N.; Vining, O. B.; Baric, R.; Silva, R. R.; Mascuch, S. J.; Tomasi, S.; Jenkins, S.; Macherla, V.;
Hoffman, T.; Agarwal, V.; Williams, P. G.; Dai, J.; Neupane, R.; Gurr, J.; Rodriguez, A. M. C.; Lamsa,
A.; Zhang, C.; Dorrestein, K.; Duggan, B. M.; Almaliti, J.; Allard, P.-M.; Phapale, P.; Nothias, L.-F.;
Alexandrov, T.; Litaudon, M.; Wolfender, J.-L.; Kyle, J. E.; Metz, T. O.; Peryea, T.; Nguyen, D.-T.; Van
Leer, D.; Shinn, P.; Jadhav, A.; Muller, R.; Waters, K. M.; Shi, W.; Liu, X.; Zhang, L.; Knight, R.;
Jensen, P. R.; Palsson, B. O.; Pogliano, K.; Linington, R. G.; Gutierrez, M.; Lopes, N. P.; Gerwick, W.
H.; Moore, B. S.; Dorrestein, P. C.; Bandeira, N. Nat. Biotechnol. 2016, 34, 828-837.
17. Alley, M. C.; Scudiero, D. A.; Monks, A.; Hursey, M. L.; Czerwinski, M. J.; Fine, D. L.; Abbott, B.
J.; Mayo, J. G.; Shoemaker, R. H.; Boyd, M. R. Cancer Res.1988, 48, 589-601.
18. Dravid, S. M.; Murray, T. F. Brain Res. 2004, 1006, 8-17.
19. Paterson, I.; Yeung, K.-S.; Ward, R. A.; Smith, J. D.; Cumming, J. G.; Lamboley, S. Tetrahedron
1995, 51, 9467-9486.
20. Nicolaou, K. C.; Ajito, K.; Patron, A. P.; Khatuya, H.; Richter, P. K.; Bertinato, P. J. Am. Chem. Soc.
1996, 118, 3059-3060.
21. Paterson, I.; Watson, C.; Yeung, K.-S.; Ward, R. A.; Wallace, P. A. Tetrahedron 1998, 54, 11955-
11970.
22. Tsukamoto, S.; Ishibashi, M.; Sasaki, T.; Kobayashi, J. J. Chem Soc., Perkin Trans. 1, 1991, 3185-
3188.
23. Ishida, K.; Matsuda, H.; Murakami, M.; Yamaguchi, K. Tennen Yuki Kagobutsu Toronkai Koen
Yoshishu 1997, 39, 667-672.
ACS Paragon Plus Environment

Page 29 of 29
The Journal of Organic Chemistry
1
2
3
4
5
24. Carmeli, S.; Moore, R. E.; Patterson, G. M. L.; Yoshida, W. Y. Tetrahedron Lett. 1993, 34, 5571-
5574.
6
7
25. Todd, J. S.; Alvi, K. A.; Crews, P. Tetrahedron Lett. 1992, 33, 441-442.
8
9
26. Kubanek, J.; Jensen, P. R.; Keifer, P. A.; Sullards, M. C.; Collins, D. O.; Fenical, W. Proc. Natl.
Acad. Sci. U. S. A. 2003, 100, 6916-6921.
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
27. Kobayashi, M.; Tanaka, J.; Katori, T.; Kitagawa, I. Chem. Pharm. Bull. 1990, 38, 2960-2966.
28. Dumdei, E. J.; Blunt, J. W.; Munro, M. H. G.; Pannell, L. K. J. Org. Chem. 1997, 62, 2636-2639.
29. Youssef, D. T. A.; Mooberry, S. L. J. Nat. Prod. 2006, 69, 154-157.
30. De Marino, S.; Festa, C.; D'Auria, M. V.; Cresteil, T.; Debitus, C.; Zampella, A. Mar. Drugs 2011, 9,
1133-1141.
31. Sinisi, A.; Calcinai, B.; Cerrano, C.; Dien, H. A.; Zampella, A.; D'Amore, C.; Renga, B.; Fiorucci, S.;
Taglialatela-Scafati, O. Bioorganic Med. Chem. 2013, 21, 5332-5338.
32. Cui, J.; Morita, M.; Ohno, O.; Kimura, T.; Teruya, T.; Watanabe, T.; Suenaga, K.; Shibasaki, M.
Chem. Eur. J. 2017, 23, 8500-8509.
ACS Paragon Plus Environment