Synthesis and Evaluation of Oligomeric Thioether-Linked Carbacyclic β-(1?3)-Glucan Mimetics

Article abstract of DOI:10.1021/acs.joc.9b00504

Extrapolating from lessons learnt with previous low-molecular-weight β-(1?3)-glucan mimetics, we designed a series of minimal 2,4-dideoxy-thioether-linked carbacyclic β-(1?3)-glucan mimetics and synthesized di-, tri-, and tetramers in an enantiomerically

Full text of DOI:10.1021/acs.joc.9b00504

Subscriber access provided by READING UNIV
Article
Synthesis and Evaluation of Oligomeric Thioether-
Linked Carbacyclic #-(1#3)-Glucan Mimetics
Peng Wen, Vaclav Vetvicka, and David Crich
J. Org. Chem., Just Accepted Manuscript • DOI: 10.1021/acs.joc.9b00504 • Publication Date (Web): 01 Apr 2019
Downloaded from http://pubs.acs.org on April 2, 2019
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.
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 37
The Journal of Organic Chemistry
1
1
2
3
4
Synthesis and Evaluation of Oligomeric Thioether-Linked Carbacyclic β-(1→3)-Glucan
5
6
Mimetics
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
Peng Wen,a Václav Větvička,b and David Cricha,*
a) Department of Chemistry, Wayne State University, 5101 Cass Avenue, Detroit, MI
48202, USA
b) Department of Pathology, University of Louisville, 323 East Chestnut Street, Louisville,
KY 40202, USA
Dcrich@chem.wayne.edu
Abstract
Extrapolating from lessons learnt with previous low molecular weight β-(1→3)-mimetics we
designed a series of minimal 2,4-dideoxy-thioether-linked carbacyclic β-(1→3)-mimetics and
synthesized the di-, tri- and tetramers in enantiomerically pure form by an iterative sequence based
on a simple building block readily available from commercial (S)-(-)-3-cyclohexenecarboxylic
acid. These substances were screened for their ability to inhibit anti-CR3-FITC staining of human
neutrophils and anti-Dectin-1-FITC staining of mouse macrophages, as well as for their ability to stimulate
phagocytosis and pinocytosis. In each assay the synthetic compounds displayed comparable activity to the
corresponding native β-(1→3)-glucans, laminaritriose and tetraose suggesting that the exploitation
of hydrophobic patches in the lectin-binding domains of CR3 and Dectin-1 is a promising strategy
for the development of small molecule analogues of the β-(1→3)-glucans.
ACS Paragon Plus Environment

The Journal of Organic Chemistry
Page 2 of 37
2
1
2
3
4
Introduction
5
6
7
8
9
The β-(1→3)-glucans 1 (Fig 1) are a group of widely occurring immunostimulatory
oligosaccharides with the potential for use as therapeutic agents and/or vaccines in a number of
disease areas including cancer and inflammatory bowel disease.1-8 Systematic investigation of
these many beneficial properties is however hindered by the wide structural heterogeneity of β-
(1→3)-glucans obtained from natural sources, which stems from variations in purity, of the degree
of polymerization, and of β-(1→6)-branching. Nevertheless, it has been established that,
following ingestion, the β-(1→3)-glucans are taken up in the small intestine by macrophages
through interaction with the lectin domain of Dectin-1.9 Degradation of the large β-(1→3)-glucans
by the macrophages then provides smaller fragments which are taken up by circulating
granulocytes, monocytes and macrophages, via binding to the lectin domain of complement
receptor 3 (CR3), where they can induce the relevant immune responses.10,11 The relative
inaccessibility of structurally homogeneous β-(1→3)-glucans from nature has been palliated in
recent years by the many advances in carbohydrate chemistry12-19 culminating in numerous
impressive syntheses of both linear and branched oligomeric glucans permitting further biological
evaluation,20-27 and conformational analysis of the glucan chains.24,28 Work with pure oligomers
obtained by degradation and extensive HPLC purification of natural isolates led to the conclusion
that the shortest β-(1→3)-glucans capable of binding to Dectin-1 in a microarray format were the
deca- and undecamers.29 Subsequent work with synthetic material and an SPR-based assay
demonstrated that even the heptasaccharide is capable of binding to recombinant murine Dectin-
1.22 The length of the natural β-(1→3)-glucans is such that multiple Dectin-1 units can bind to a
single polymeric glucan chain through a multivalent interaction with affinity increasing in an
additive manner.22 STD NMR studies showed little or no binding between either recombinant
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

Page 3 of 37
The Journal of Organic Chemistry
3
1
2
3
4
5
6
7
8
9
Dectin-1 or CR3 and a synthetic hexamer, whereas other STD-NMR studies revealed that a
hexadecamer,30 but not a hexamer, binds to the lectin domain of Dectin-1.28 Work with glucans
isolated from yeast cell walls showed the tetrasaccharide to be the smallest unit able to block the
ingestion of zymosan (a natural β-(1→3)-glucan) by monocytes via CR3,6 while study of a series
of homogeneous synthetic oligomers revealed the tetra- and especially the pentasaccharides
possess immunostimulatory effects including the potentiation of phagocytosis similar to those of
phycarine (another natural β-(1→3)-glucan).20 Chemical synthesis also revealed that penta- and
hexameric glucans could be modified by replacement of the terminal reducing end glucopyranose
residue by a gluco and/or manno-configured glucitol unit, by a mannopyranose ring, or by a 4-
deoxyglucopyranose ring 2 (Fig 1) without loss of the ability to promote phagocytosis.31,32
Likewise, it was demonstrated that the glycosidic oxygen could be replaced by a thioglycoside
moiety 3-5 without loss of activity.33
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 lectin domain of Dectin-1 was revealed by X-ray crystallographic studies to consist of a
shallow carbohydrate-binding groove in which the side chains of Trp 221 and His 223 line the
walls of a hydrophobic pocket.34 Further, laminarin, a natural β-(1→3)-glucan, was shown by
STD-NMR studies to bind to this hydrophobic patch in recombinant Dectin-1 by the interaction
with the α-faces of the terminal residues at either its reducing or non-reducing ends (Fig 2).28,30
Overall, the picture is one of a relatively weak interaction of a short carbohydrate epitope with the
lectin domain, as is commonly found in carbohydrate-protein interactions, that is bolstered by the
multivalent effect due to the repetitive presentation of the epitope along the length of the polymeric
oligosaccharide.35-39
ACS Paragon Plus Environment

The Journal of Organic Chemistry
Page 4 of 37
4
1
2
3
4
5
OH
O
OH
O
OH
O
HO
HO
HO
H
O
O
OH
O
OH
OH
OH
n
6
1
-(13)-Glucan (n is highy variable and source dependent)
7
8
9
OH
O
OH
O
OH
O
HO
HO
H
O
O
OH
O
OH
OH
OH
n
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
2
Pentasaccharide with terminal 4-deoxy modification , n = 3
OH
O
OH
O
OH
O
HO
HO
H
O
X
S
HO
OH
OH
OH
OH
n
3
4
5
Thioglucans : n = 1, X =O, : n = 2, X = O, : n = 2, X =S
Figure 1. Structures of β-(1→3)-glucan and synthetic analogs
HO
HO
HO
HO
O
O
O
O
H
H
H
H
HO
HO
H
H
n
hydrophobic
-face
H
H
N
N
N
His 223
Trp 221
Figure 2. Schematic representation of the hydrophobic α-face of a disaccharide unit of a β-(1→3)-
glucan in complex with the hydrophobic binding pocket of the Dectin-1 lectin domain
In our laboratory, pursuing a glycomimetic approach as opposed to a multivalent glycoconjugate
approach to the synthesis of improved β-(1→3)-glucan analogues,40,41 we prepared and evaluated
the properties of the di- and trimeric hydroxylamine linked constructs 6 and 7 (Fig 3).42 We found
both 6 and 7 to display significant affinity for Dectin-1 and CR3, and hypothesized that this arises
from the enhanced hydrophobicity of the α-face arising from removal of the C2-hydroxyl group,
and replacement of the ring oxygen by a methylene group. Building on this hypothesis, and
ACS Paragon Plus Environment

Page 5 of 37
The Journal of Organic Chemistry
5
1
2
3
4
5
6
7
8
9
informed by the previous development of the monothioglucoside analogs 3-5 by the Ferrières
laboratory,33 and by the frequently observed enhancement of protein-small molecule and protein-
carbohydrate interactions on replacement of ether units by thioethers,43-52 we designed and
prepared the di-, tri- and tetrameric 1,5-dithia analogs 8-10 (Fig 3), and were again rewarded by
the observation of significant affinity for Dectin-1 and CR3, particularly for the trimer 9.53
Pursuing this line of investigation further, and noting the extensive history of carbacyclic motifs
as carbohydrate analogs,54-59 we now report the synthesis and evaluation of the thioether-linked
carbasugar glucan mimetics 11-13 that incorporate features of the hydroxylamines 6 and 7, the
dithiaglucans 8-10, and the 4-deoxyglucopyranose moiety in 2.
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
OH
OH
OH
HO
HO
HO
HO
O
O
O
N
N
N
n
6
7
Synthetic oligohydroxylamines : n = 0, : n = 1
OH
S
OH
S
OH
S
HO
HO
HO
HO
S
S
OH
OH
OH
OH
n
8
9
10
Synthetic dithiaglucans : n = 0, : n = 1, : n = 2
OH
OH
OH
SEt
S
S
HO
n
Thioether-linked oligomeric carbasugars
11 12 13
: n = 0, : n = 1, : n = 2
Figure 3. Previous (6-10) and Targeted (11-13) β-(1→3)-Glucan Mimetics.
Results and Discussion
Synthesis. Following the protocol reported by Mori,60 commercially available (S)-(-)-3-
cyclohexenecarboxylic acid 14 was converted to the enone 15 in four known steps and 53% overall
yield (Scheme 1). Installation of a methoxymethyl ether under standard conditions then gave the
central building block 16 in 94% yield (Scheme 1).
ACS Paragon Plus Environment

The Journal of Organic Chemistry
Page 6 of 37
6
1
2
3
i) I2, KI, 98%
O
4
ii) DBU, , 77%
5
6
7
COOH
iii) LiAlH4, 86%
iv) MnO2, 81%
14
OH
15
8
9
O
MOMCl,
iPr2NEt
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
OMOM
, 94%
16
Scheme 1. Synthesis of the Monomeric Building Block 16
Treatment of 16 with sodium ethanethiolate in methanol at room temperature afforded the desired
equatorial Michael adduct 17 and its axial isomer 18 in 56% and 31% yields, respectively.
Resubjecting the axial isomer 18 to the reaction conditions resulted in its conversion to the
equatorial adduct 17 in 64% yield. Reduction with L-Selectride followed by mesylation then
afforded the mesylate 19 in 68% yield. Finally, mesylate displacement by cesium thioacetate in
DMF under microwave irradiation gave the thioester 20 in 41% yield (Scheme 2). Cleavage of
the thioacetyl moiety from 20 with sodium methoxide in methanol followed by exposure of the
resultant thiol to enone 16 in the presence of diazabicycloundecene (DBU) in trifluoroethanol gave
the desired equatorial adduct 21 in 55% yield, together with 33% of its axial isomer 22. Reduction
of 21 with sodium borohydride in methanol afforded 78% of the equatorial alcohol 23 from which
the methoxymethyl ethers were removed with catalytic hydrogen chloride in methanol to give the
mimetic 24 of laminaribiose (Scheme 2).
ACS Paragon Plus Environment

Page 7 of 37
The Journal of Organic Chemistry
7
1
2
3
NaSEt, MeOH
4
5
6
7
8
9
64%
O
OMOM
SEt
OMOM
NaSEt,
MeOH
+
O
O
SEt
OMOM
16
17
18
, 31%
, 56%
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
i) L-Selectride
ii) MsCl. py
OMOM
SEt
OMOM
SEt
CsSAc, W, DMF
AcS
OMs
19
20
, 41%
, 68%
i) NaOMe, MeOH,
16
ii) , DBU, TFE
OMOM
S
OMOM
OMOM
SEt
OMOM
SEt
+
S
O
O
21
, 55%
22
, 33%
NaBH4, MeOH
OMOM
OH
OH
OMOM
HCl (cat),
HO
S
SEt
HO
S
SEt
MeOH,
60 °C
24
, 87%
23
, 78%
Scheme 2. Synthesis of the Laminaribiose Mimetic 24
Reduction of the ketone 21 with L-Selectride in THF afforded the axial alcohol 25 in 82% yield,
and was followed by treatment with mesyl chloride in pyridine to give the mesylate 26 in 92%
yield. Mesylate displacement with cesium thioacetate in DMF under microwave irradiation next
provided the thioacetate 27 in 47% yield which, on deactylation followed by stirring with enone
16 in the trifluoroethanol in the presence of DBU gave the all equatorial trimer 28 in 49% yield
along with the axial isomer 29 in 30% yield. Sodium borohydride reduction of 28 to give the
equatorial alcohol 30 in 61% yield was followed by removal of the MOM groups with HCl in
methanol to afford the laminaritriose mimetic 31 in 76% yield. On the other hand reduction of
ketone 28 with L-Selectride in THF gave 79% of the axial alcohol 32, which was converted to the
mesylate 33 and then to the thioacetate 34 in 96 and 41% yields, respectively. Iteration of the
ACS Paragon Plus Environment

The Journal of Organic Chemistry
Page 8 of 37
8
1
2
3
4
5
6
7
8
9
protocol for the removal of the acetyl moiety followed by conjugate to enone 16 then gave the
isomeric tetramers 35 and 36 in 53% and 28% yields, respectively. Sodium borohydride reduction
of 35 afforded the equatorial alcohol 37 in 54% yield, and was followed by removal of the MOM
groups to give the laminaritetraose mimetic 38 in 76% yield (Scheme 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
OMOM
OMOM
SEt
OMOM
OMOM
SEt
OMOM
OMOM
SEt
MsCl,
Py
CsSAc, W,
L-Selectride,
THF
21
S
AcS
S
S
DMF
OH
OMs
27
, 47%
25
, 82%
26
, 92%
NaBH4, MeOH
i) NaOMe, MeOH,
16
ii) , DBU, TFE
OMOM
S
OMOM
S
OMOM
OMOM
SEt
OMOM
OMOM
OMOM
SEt
OMOM
OMOM
SEt
+
HO
S
S
S
O
S
O
30
, 61%
29
, 30%
28
, 49%
L-Selectride, THF
OMOM OMOM
SEt
HCl (cat), MeOH, 60 °C
OMOM
OMOM
OMOM
SEt
OMOM
OH
OH
OH
MsCl,
Py
S
S
S
S
HO
S
S
SEt
OMs
OH
31
, 76%
33
, 96%
32
, 79%
CsSAc, W, DMF
OMOM
S
i) NaOMe,
MeOH,
OMOM
OMOM
OMOM
SEt
OMOM
OMOM
OMOM
SEt
OMOM
OMOM
SEt
+
O
S
S
AcS
S
S
S
16
ii) , DBU,
2
O
2
TFE
34
, 41%
36
, 28%
35
, 53%
NaBH4, MeOH
OMOM
OH
OH
OH
OH
OMOM
OMOM
OMOM
SEt
HCl (cat), MeOH, 60 °C
HO
S
S
S
SEt
HO
S
S
S
38
, 76%
37
, 54%
Scheme 3. Synthesis of the Laminaritriose and Tetraose Mimetics 31 and 38
Binding to CR3 and Dectin-1 Receptors.
We screened the di-, tri-, and tetrasaccharide mimetics 24, 31, and 38, respectively, for their ability
to inhibit staining of human neutrophils and mouse macrophages by anti-CR3 or anti-Dectin-1
ACS Paragon Plus Environment

Page 9 of 37
The Journal of Organic Chemistry
9
1
2
3
4
5
6
7
8
9
fluorescent antibody conjugates (FITC), as a measure of their affinity for CR3 and Dectin-1,61 as
reported in Table 1. For comparison purposes commercial laminaritriose 39 and tetraose 40 were
screened in parallel as reported in Table 1 together with the values previously obtained for the
hydroxylamine-based mimetics 6 and 7.
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
Stimulation of phagocytosis and pinocytosis. The ability of glycan mimetics 24, 31, and 38 to
stimulate phagocytosis of synthetic polymeric 2-hydroxyethyl methacrylate particles62 by human
macrophage-like RAW 264 cells was examined. In addition the ability of the mimetics to stimulate
pinocytosis, another important mechanism of cellular internalization, was examined by
spectrophotometric measurement of neutral red dye accumulation by mouse macrophages (Table
2).63 Laminaritriose 39 and tetraose 40 were again screened for comparison purposes as was the
commercial highly purified yeast-derived insoluble Glucan #300.64 Also for comparison purposes
the previously recorded phagocytic activity of the di- and trimeric hydroxylamines 6 and 7 is
reproduced in Table.
ACS Paragon Plus Environment

The Journal of Organic Chemistry
Page 10 of 37
10
1
2
3
4
Table 1. Percentage Inhibition of anti-CR3 and anti-Dectin-1-FITC Antibody Staining of Neutrophils and Macrophages by 0.1 μg.mL-
5
6
1 Compound.
7
8
9
Cmpd
Oligomer No % Inhibition of anti-CR3-FITC % Inhibition of anti-Dectin-1-FITC
Staining of Human Neutrophilsa Staining of Mouse Macrophagesa
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
OH
OH
OH
dimer
trimer
40.5 ± 3.6
33.2 ± 2.8
48.9 ± 4.1
41.6 ± 3.6
24
HO
S
S
SEt
OH
31
SEt
HO
2
3
OH
OH
OH
OH
tetramer
dimer
45.3 ± 3.9
26.4 ± 2.7
54.7 ± 3.8
28.2 ± 2.9
S
SEt
O
38
6
HO
HO
HO
O
N
HO
N
OH
OH
trimer
trimer
34.2 ± 3.3
16.1 ± 1.7
43.1 ± 3.5
19.9 ± 0.9
HO
HO
HO
HO
O
7
O
N
HO
HO
HO
N
2
OH
O
OH
O
HO
OH
OH
39
40
O
HO
2
HO
OH
O
OH
O
tetramer
-
31.2 ± 2.1
1.3 ± 0.2
44.8 ± 3.8
0.8 ± 0.3
HO
O
HO
3
HO
PBSb
b
a Mean ± SD. phosphate buffered saline
ACS Paragon Plus Environment

Page 11 of 37
The Journal of Organic Chemistry
11
1
2
3
4
Table 2. Percentage Stimulation of Phagocytosis and Pinocytosis.
5
6
7
Cmpd
Oligomer No % Stimulation of Phagocytosis Stimulation of Pinocytosis
8
9
(RAW 264 macrophages, 10 (Uptake of neutral red dye by mouse
μg/mL, 24 h) a
macrophages after 2 h (ng/L x 105
cells) 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
OH
OH
OH
dimer
trimer
17.7 ± 1.6
20.8 ± 2.9
9.1 ± 2.0
24
HO
S
S
SEt
OH
12.3 ± 1.5
18.3 ± 0.9
7.8 ± 1.1
31
SEt
HO
2
3
OH
OH
OH
OH
tetramer
dimer
16.2 ± 3.0
nd
S
SEt
O
38
6
HO
HO
HO
O
N
HO
N
OH
OH
trimer
trimer
16.6 ± 2.0
7.9 ± 1.0
20.5 ± 1.9
nd
HO
HO
HO
HO
O
7
O
N
HO
HO
HO
N
2
OH
O
OH
O
14.1 ± 1.3
22.4 ± 2.1
HO
OH
OH
39
40
O
HO
2
HO
OH
O
OH
O
tetramer
HO
O
HO
3
HO
34.2 ± 2.6
2.2 ± 0.3
36.8 ± 3.0
1.6 ± 0.2
Glucan #300
PBSb
-
a Mean ± SD at P < 0.05 level. b phosphate buffered saline
ACS Paragon Plus Environment

The Journal of Organic Chemistry
Page 12 of 37
12
1
2
3
4
5
6
7
8
9
Inspection of Table 1 suggests that the simple thioether-linked carbocyclic di-, tri- and
tetrasaccharide mimetics 24, 31, and 38, respectively, display significant affinity for CR3 and
Dectin-1. This activity is of a comparable magnitude to that of the earlier hydroxylamine based
mimetics 6 and 7,42 of the more recent thiapyranoside mimetics 8-10 (data not shown)53 and
importantly of laminaribiose 39 and tetraose 40 themselves. These affinities are borne out by the
ability of 24, 31, and 38 to stimulate both phagocytosis and pinocytosis (Table 2), again at levels
comparable and even superior to the simple β-(1→3)-glucans of the same length. Considered as
a whole with the earlier analogues prepared in the Ferrières laboratory (Figure 1),31-33 these results
strongly suggest that there is considerable scope for the development of potent small molecule
analogs of the β-(1→3)-glucans.
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
Extrapolating from earlier small molecule β-(1→3)-glucans mimetics reported in the literature or
prepared in our laboratories, we designed and synthesized a series of thioether linked 4-
deoxycarbocyclic analogues of laminaribiose, triose, and tetraose to probe the minimal
requirements for binding to the lectin domains of CR3 and Dectin-1. In the event the di-, tri- and
tetrasaccharide mimetics, 24, 31, and 38 all displayed the ability to inhibit fluorescent antibody
binding to CR3 and Dectin-1 and stimulated phagocytosis and pinocytosis at levels at least
comparable to that of the minimal β-(1→3)-glucans laminaritriose and tetraose.
Experimental Section
ACS Paragon Plus Environment

Page 13 of 37
The Journal of Organic Chemistry
13
1
2
3
4
5
6
7
8
9
General. All reactions were performed using oven-dried glassware under an atmosphere
of argon. All reagents and solvents were purchased from commercial suppliers and were used
without further purification unless otherwise specified. Chromatographic purifications were
performed on silica gel (230-400 mesh) columns (20-50 g of silica gel per gram of crude
compound). Reactions were monitored by analytical thin-layer chromatography on pre-coated
glass backed plates (w/UV 254) and visualized by UV irradiation (254 nm) or by staining with
25% H2SO4 in EtOH or ceric ammonium molybdate (CAM) solution. Specific rotations were
measured on an automatic polarimeter with a path length of 100 mm in the solvent specified.
Concentrations are given in g/100 mL. High resolution mass spectra (HRMS) were recorded with
an electrospray ionization (ESI) source coupled to a time-of-flight (TOF) mass analyzer or with
an electron impact (EI) source coupled to a TOF mass analyzer. 1H, 13C, 19F, spectra were recorded
on a 400, 500 or 600 MHz spectrometer. NMR solvents were used without purification. Chemical
shifts are given in ppm (δ) and coupling constants (J) are given in Hz. Multiplicities are given as
singlet (s), broad singlet (br s), doublet (d), triplet (t), doublet of doublets (dd), triplet of doublets
(td), multiplet (m), apparent quartet (app q), apparent pentet (app p), etc. Reactions requiring
microwave irradiation were performed on a commercial microwave synthesizer with internal
temperature control and magnetic stirring.
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
(5S)-5-((Methoxymethoxy)methyl)cyclohex-2-en-1-one (16). A stirred solution of enone 1560
o
(1.0 g, 7.93 mmol) in N,N-diisopropylethylamine (2 mL) and DCM (2 mL) at 0 C was treated
with chloromethyl methyl ether (1.2 mL, 15.85 mmol), and stirred at room temperature for 6 h
before it was diluted with ethyl acetate, washed with 1 N HCl and brine, dried over Na2SO4, and
concentrated under reduced pressure. Chromatographic purification (33% ethyl acetate/hexanes)
afforded the title compound as a colorless oil (16, 1.27 g, 94%) with spectral data consistent with
ACS Paragon Plus Environment

The Journal of Organic Chemistry
Page 14 of 37
14
1
2
3
4
5
6
7
8
9
the literature values:65 [α]D21 73.0 (c 1.15, CHCl3). 1H NMR (400 MHz, CDCl3) δ 6.96 (ddd, J =
10.1, 5.4, 2.7 Hz, 1H), 6.06 – 5.95 (m, 1H), 4.60 (s, 2H), 3.52 – 3.41 (m, 2H), 3.33 (s, 3H), 2.55 –
2.18 (m, 5H). 13C{1H} NMR (101 MHz, CDCl3) δ 199.1, 149.3, 129.7, 96.5, 70.6, 55.2, 41.0, 35.5,
28.9. HRMS (ESI-TOF) m/z: Calcd. for C9H14O3Na ([M + Na]+): 193.0841; found: 193.0837.
(3R,5R)-3-(Ethylthio)-5-((methoxymethoxy)methyl)cyclohexan-1-one (17) and (3S,5R)-3-
(Ethylthio)-5-((methoxymethoxy)methyl)cyclohexan-1-one (18). To a stirred solution of 16
(300 mg, 1.76 mmol) in MeOH (3.5 mL) at room temperature was added sodium ethanethiolate
(444.8 mg, 5.29 mmol). The reaction mixture was stirred at the same temperature for 25 min before
it was diluted with ethyl acetate, washed with brine, dried over Na2SO4, and concentrated under
reduced pressure. Chromatographic purification (18% ethyl acetate/hexanes) afforded the title
compound as a colorless oil (17, 265.2 mg, 56%): [α]D21 58.6 (c 0.63, CHCl3). 1H NMR (400 MHz,
CDCl3) δ 4.59 (s, 2H), 3.49 – 3.39 (m, 2H), 3.33 (s, 3H), 2.93 (tt, J = 12.7, 4.0 Hz, 1H), 2.68 (ddt,
J = 14.1, 4.2, 2.0 Hz, 1H), 2.58 (q, J = 7.4 Hz, 2H), 2.42 (ddt, J = 13.9, 3.9, 2.0 Hz, 1H), 2.33 –
2.21 (m, 2H), 2.16 (t, J = 13.4 Hz, 1H), 2.10 – 1.98 (m, 1H), 1.51 (app q, J = 12.3 Hz, 1H), 1.24
(t, J = 7.4 Hz, 3H). 13C{1H} NMR (101 MHz, CDCl3) δ 208.2, 96.5, 71.3, 55.3, 48.2, 44.0, 40.9,
37.8, 35.8, 24.4, 14.9. HRMS (ESI-TOF) m/z: Calcd. for C11H20O3SNa ([M + Na]+): 255.1031;
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
found: 255.1036.
21
Further elution (25% ethyl acetate/hexanes) gave 18 as a colorless oil (147.8 mg, 31%): [α]D
-
1
22.9 (c 0.89, CHCl3). H NMR (400 MHz, CDCl3) δ 4.59 (s, 2H), 3.52 – 3.41 (m, 3H), 3.34 (s,
3H), 2.68 (ddd, J = 14.7, 4.7, 0.9 Hz, 1H), 2.59 – 2.42 (m, 5H), 2.23 (ddd, J = 14.3, 9.7, 1.2 Hz,
13
1H), 2.03 – 1.98 (m, 2H), 1.24 (t, J = 7.4 Hz, 3H). C{1H} NMR (101 MHz, CDCl3) δ 208.3,
96.5, 70.9, 55.3, 46.5, 43.9, 40.1, 34.3, 33.1, 24.7, 14.5. HRMS (ESI-TOF) m/z: Calcd. for
C11H20O3SNa ([M + Na]+): 255.1031; found: 255.1038.
ACS Paragon Plus Environment

Page 15 of 37
The Journal of Organic Chemistry
15
1
2
3
4
5
6
7
8
9
(1R,3R,5R)-3-(Ethylthio)-5-((methoxymethoxy)methyl)cyclohexan-1-ol. To a stirred solution
of 17 (252.6 mg, 1.09 mmol) in THF (3 mL) at -78 oC was added a solution of L-Selectride in THF
(1.0 M, 3.26 mL, 3.26 mmol). The reaction mixture was stirred at the same temperature for 5 h
before it was warmed to room temperature. The reaction was quenched by adding MeOH and the
mixture was concentrated under reduced pressure. Chromatographic purification (33% ethyl
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
21
acetate/hexanes) afforded the title compound as a colorless oil (188.6 mg, 74%): [α]D -12.5 (c
1
1.06, CHCl3). H NMR (400 MHz, CDCl3) δ 4.59 (s, 2H), 4.23 (app p, J = 3.0 Hz, 1H), 3.40 –
3.34 (m, 2H), 3.34 (s, 3H), 3.07 (tt, J = 12.5, 3.6 Hz, 1H), 2.58 (q, J = 7.4 Hz, 2H), 2.18 – 2.05 (m,
3H), 1.82 (ddt, J = 13.8, 3.4, 1.3 Hz, 1H), 1.44 (td, J = 13.2, 2.7 Hz, 1H), 1.29 – 1.19 (m, 4H), 1.05
(q, J = 12.8 Hz, 1H). 13C{1H} NMR (101 MHz, CDCl3) δ 96.5, 72.6, 66.7, 55.1, 39.9, 37.1, 36.8,
35.6, 32.2, 24.0, 15.1. HRMS (ESI-TOF) m/z: Calcd. for C11H22O3SNa ([M + Na]+): 257.1187;
found: 257.1189.
(1R,3R,5R)-3-(Ethylthio)-5-((methoxymethoxy)methyl)cyclohexyl Methanesulfonate (19).
To a stirred solution of (1R,3R,5R)-3-(ethylthio)-5-((methoxymethoxy)methyl)cyclohexan-1-ol
(167.4 mg, 0.71 mmol) in pyridine (2 mL) at room temperature was added methanesulfonyl
chloride (0.28 mL, 3.57 mmol). The reaction mixture was stirred at the same temperature for 11 h
before it was diluted with ethyl acetate, washed with 1 N HCl and brine, dried over Na2SO4, and
concentrated under reduced pressure. Chromatographic purification (25% ethyl acetate/hexanes)
afforded the title compound as a colorless oil (19, 204.4 mg, 92%): [α]D21 -1.3 (c 1.19, CHCl3). 1H
NMR (400 MHz, CDCl3) δ 5.11 (app p, J = 2.9 Hz, 1H), 4.58 (s, 2H), 3.37 (d, J = 5.4 Hz, 2H),
3.33 (s, 3H), 3.05 – 2.95 (m, 4H), 2.58 (q, J = 7.4 Hz, 2H), 2.38 (dtt, J = 14.4, 3.7, 2.0 Hz, 1H),
2.17 – 2.03 (m, 3H), 1.52 (ddd, J = 14.8, 12.6, 2.5 Hz, 1H), 1.35 (t, J = 13.7 Hz, 1H), 1.24 (t, J =
7.4 Hz, 3H), 1.12 (app q, J = 13.3, 12.9 Hz, 1H). 13C{1H} NMR (101 MHz, CDCl3) δ 96.5, 78.7,
ACS Paragon Plus Environment

The Journal of Organic Chemistry
Page 16 of 37
16
1
2
3
4
5
6
71.8, 55.2, 38.6, 38.2, 36.6, 36.1, 33.7, 32.5, 24.1, 15.0. HRMS (ESI-TOF) m/z: Calcd. for
C12H24O5S2Na ([M + Na]+): 335.0963; found: 335.0969.
7
8
9
S-((1S,3R,5S)-3-(Ethylthio)-5-((methoxymethoxy)methyl)cyclohexyl) Thioacetate (20).
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
mixture of 19 (60.0 mg, 0.19 mmol) and cesium thioacetate (798.9 mg, 3.84 mmol) in DMF (0.6
mL) was heated to 100 oC by irridation with microwave for 1 h. After cooling to room temperature,
the reaction mixture was diluted with ethyl acetate, washed with brine, dried over Na2SO4, and
concentrated under reduced pressure. Chromatographic purification (6% ethyl acetate/hexanes)
afforded the title compound as a light yellow oil (20, 23.0 mg, 41%): [α]D21 -24.3 (c 0.68, CHCl3).
1H NMR (400 MHz, CDCl3) δ 4.58 (s, 2H), 3.44 (tt, J = 12.7, 3.9 Hz, 1H), 3.36 (d, J = 6.1 Hz,
2H), 3.34 (s, 3H), 2.75 (tt, J = 12.2, 3.7 Hz, 1H), 2.58 (q, J = 7.4 Hz, 2H), 2.32 – 2.24 (m, 4H),
2.09 (ddq, J = 11.1, 3.5, 1.7 Hz, 1H), 2.02 (ddq, J = 12.7, 3.7, 1.9 Hz, 1H), 1.90 – 1.77 (m, 1H),
1.32 (q, J = 12.4 Hz, 1H), 1.24 (t, J = 7.4 Hz, 3H), 1.10 (q, J = 12.5 Hz, 1H), 1.03 (q, J = 12.4 Hz,
1H). 13C{1H} NMR (101 MHz, CDCl3) δ 195.4, 96.5, 72.0, 55.2, 41.5, 40.9, 39.9, 38.5, 36.0, 35.4,
30.7, 24.2, 15.0. HRMS (ESI-TOF) m/z: Calcd. for C13H24O3S2Na ([M + Na]+): 315.1065; found:
315.1070.
(3R,5R)-3-(((1S,3R,5S)-3-(Ethylthio)-5-((methoxymethoxy)methyl)cyclohexyl)thio)-5-
((methoxymethoxy)methyl)cyclohexan-1-one (21) and (3S,5R)-3-(((1S,3R,5S)-3-(Ethylthio)-
5-((methoxymethoxy)methyl)cyclohexyl)thio)-5-((methoxymethoxy)methyl)cyclohexan-1-
one (22). To a stirred solution of 20 (378.6 mg, 1.29 mmol) in MeOH (2 mL) at room temperature
was added sodium methoxide (139.9 mg, 2.59 mmol). After stirring at room temperature for 30
min, the reaction mixture was diluted with ethyl acetate, washed with 1 N HCl and brine, dried
over Na2SO4, and concentrated under reduced pressure to give the free thiol (328.2 mg). To a
solution of the free thiol and enone 16 (330.5 mg, 1.94 mmol) in 2,2,2-trifluoroethanol (3 mL) at
ACS Paragon Plus Environment

Page 17 of 37
The Journal of Organic Chemistry
17
1
2
3
4
5
6
room temperature was added a solution of DBU (197.1 mg, 1.29 mmol) in 2,2,2-trifluoroethanol
(0.5 mL). After stirring at room temperature for 30 min, the reaction mixture was concentrated
7
8
9
o
under reduced pressure at 40 C. Chromatographic purification (33% ethyl acetate/hexanes)
afforded the title compound as a colorless oil (21, 300.0 mg, 55%): [α]D21 19.6 (c 0.25, CHCl3).
1H NMR (600 MHz, CDCl3) δ 4.59 (s, 2H), 4.58 (s, 2H), 3.47 – 3.41 (m, 2H), 3.37 – 3.33 (m, 5H),
3.33 (s, 3H), 3.01 (tt, J = 12.6, 4.0 Hz, 1H), 2.74 (tt, J = 12.2, 3.7 Hz, 1H), 2.70 – 2.61 (m, 2H),
2.57 (q, J = 7.4 Hz, 2H), 2.42 (ddt, J = 14.0, 4.0, 1.9 Hz, 1H), 2.29 (t, J = 13.6 Hz, 1H), 2.26 –
2.18 (m, 2H), 2.16 (t, J = 13.6 Hz, 1H), 2.10 – 2.01 (m, 3H), 1.76 – 1.68 (m, 1H), 1.51 (app q, J =
12.5 Hz, 1H), 1.29 (app q, J = 12.4 Hz, 1H), 1.23 (t, J = 7.4 Hz, 3H), 1.07 – 0.98 (m, 2H). 13C{1H}
NMR (151 MHz, CDCl3) δ 208.0, 96.5 (2 Cs), 72.1, 71.2, 55.3, 55.2, 48.9, 44.0, 41.6, 41.1, 40.9,
39.8, 38.5, 37.8, 36.8, 36.3 (2 Cs), 24.0, 15.0. HRMS (ESI-TOF) m/z: Calcd. for C20H36O5S2Na
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
([M + Na]+): 443.1902; found: 443.1908.
21
Further elution (40% ethyl acetate/hexanes) gave 22 as a colorless oil (177.2 mg, 33%): [α]D
-
28.6 (c 0.21, CHCl3). 1H NMR (600 MHz, CDCl3) δ 4.59 (s, 2H), 4.58 (s, 2H), 3.57 (app p, J =
5.1 Hz, 1H), 3.48 – 3.42 (m, 2H), 3.36 – 3.33 (m, 5H), 3.33 (s, 3H), 2.71 – 2.60 (m, 3H), 2.57 (q,
J = 7.4 Hz, 2H), 2.55 – 2.49 (m, 1H), 2.45 (m, 2H), 2.28 – 2.19 (m, 2H), 2.09 (ddt, J = 12.8, 3.6,
1.9 Hz, 1H), 2.06 – 1.94 (m, 3H), 1.78 – 1.68 (m, 1H), 1.29 (app q, J = 12.3 Hz, 1H), 1.23 (t, J =
7.4 Hz, 3H), 1.08 – 0.98 (m, 2H). 13C{1H} NMR (151 MHz, CDCl3) δ 208.2, 96.51, 96.49, 72.1,
70.9, 55.3, 55.2, 47.1, 43.8, 41.6, 41.1, 40.8, 38.9, 38.5, 36.4, 36.3, 34.4, 33.7, 24.0, 15.0. HRMS
(ESI-TOF) m/z: Calcd. for C20H36O5S2Na ([M + Na]+): 443.1902; found: 443.1913.
(1S,3R,5R)-3-(((1S,3R,5S)-3-(Ethylthio)-5-((methoxymethoxy)methyl)cyclohexyl)thio)-5-
((methoxymethoxy)methyl)cyclohexan-1-ol (23). To a stirred solution of 21 (20.0 mg, 0.05
mmol) in MeOH (0.3 mL) at room temperature was added sodium borohydride (5.4 mg, 0.14
ACS Paragon Plus Environment

The Journal of Organic Chemistry
Page 18 of 37
18
1
2
3
4
5
6
7
8
9
mmol). The reaction mixture was stirred at room temperature for 20 min before it was diluted with
ethyl acetate, washed with 1 N HCl and brine, dried over Na2SO4, and concentrated under reduced
pressure. Chromatographic purification (50% ethyl acetate/hexanes) afforded the title compound
as a colorless oil (23, 15.7 mg, 78%): [α]D21 -17.8 (c 0.32, CHCl3). 1H NMR (600 MHz, CDCl3) δ
4.60 (s, 2H), 4.59 (s, 2H), 3.65 (tt, J = 10.9, 4.3 Hz, 1H), 3.41 – 3.37 (m, 2H), 3.37 – 3.35 (m, 2H),
3.34 (s, 3H), 3.33 (s, 3H), 2.79 – 2.71 (m, 2H), 2.66 (tt, J = 12.2, 3.7 Hz, 1H), 2.58 (q, J = 7.4 Hz,
2H), 2.30 – 2.23 (m, 2H), 2.12 – 1.99 (m, 4H), 1.77 – 1.68 (m, 2H), 1.52 (d, J = 4.8 Hz, 1H), 1.30
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
13
(app q, J = 12.3 Hz, 1H), 1.26 – 1.21 (m, 4H), 1.07 – 0.95 (m, 4H). C{1H} NMR (151 MHz,
CDCl3) δ 96.53, 96.51, 72.2, 72.1, 69.6, 55.2 (2 Cs), 43.1, 41.7, 41.1, 40.7, 38.6, 38.5, 38.4, 36.9,
36.7, 36.4 (2 Cs), 24.0, 15.0. HRMS (ESI-TOF) m/z: Calcd. for C20H38O5S2Na ([M + Na]+):
445.2058; found: 445.2070.
(1S,3R,5R)-3-(((1S,3R,5S)-3-(Ethylthio)-5-(hydroxymethyl)cyclohexyl)thio)-5-
(hydroxymethyl)cyclohexan-1-ol (24). A mixture of alcohol 23 (22.6 mg, 0.05 mmol), MeOH
o
(1 mL) and concentrated HCl (10 µL) was heated to 60 C for 6 h. After cooling to room
temperature, the reaction mixture was concentrated under reduced pressure. Chromatographic
purification (9% methanol/dichloromethane) afforded the title compound as a colorless oil (24,
15.5 mg, 87%): [α]D21 -16.4 (c 0.14, CH3OH). 1H NMR (600 MHz, CD3OD) δ 3.58 (tt, J = 11.1,
4.2 Hz, 1H), 3.44 – 3.35 (m, 4H), 2.84 – 2.74 (m, 2H), 2.70 (tt, J = 12.1, 3.6 Hz, 1H), 2.57 (q, J =
7.4 Hz, 2H), 2.26 (dtd, J = 14.4, 3.6, 1.7 Hz, 1H), 2.23 – 2.18 (m, 1H), 2.08 – 1.99 (m, 3H), 1.99
– 1.93 (m, 1H), 1.66 – 1.54 (m, 2H), 1.22 (t, J = 7.4 Hz, 3H), 1.20 – 1.10 (m, 2H), 0.96 – 0.83 (m,
4H). 13C{1H} NMR (151 MHz, CD3OD) δ 68.9, 66.33, 66.31, 42.9, 41.4 (2 Cs), 40.6, 40.5, 38.6,
38.5, 37.5, 36.6, 36.6, 36.0, 23.4, 14.0. HRMS (ESI-TOF) m/z: Calcd. for C16H30O3S2Na ([M +
Na]+): 357.1534; found: 357.1543.
ACS Paragon Plus Environment

Page 19 of 37
The Journal of Organic Chemistry
19
1
2
3
4
(1R,3R,5R)-3-(((1S,3R,5S)-3-(Ethylthio)-5-((methoxymethoxy)methyl)cyclohexyl)thio)-5-
5
6
((methoxymethoxy)methyl)cyclohexan-1-ol (25). To a stirred solution of 21 (277.7 mg, 0.66
7
8
9
o
mmol) in THF (3 mL) at -78 C was added a solution of L-Selectride in THF (1.0 M, 1.98 mL,
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
1.98 mmol). The reaction mixture was stirred at the same temperature for 4 h before it was warmed
to room temperature. The reaction was quenched by adding MeOH and the mixture was
concentrated under reduced pressure. Chromatographic purification (15% acetone/hexanes)
afforded the title compound as a colorless oil (25, 228.1 mg, 82%): [α]D21 -22.2 (c 0.36, CHCl3).
1H NMR (600 MHz, CDCl3) δ 4.58 (s, 2H), 4.57 (s, 2H), 4.22 (app p, J = 2.9 Hz, 1H), 3.38 – 3.30
(m, 10H), 3.15 (tt, J = 12.4, 3.7 Hz, 1H), 2.74 (tt, J = 12.1, 3.7 Hz, 1H), 2.65 (tt, J = 12.1, 3.6 Hz,
1H), 2.57 (q, J = 7.5 Hz, 2H), 2.30 (dtd, J = 14.6, 3.6, 1.7 Hz, 1H), 2.17 – 2.01 (m, 5H), 1.80 (dtt,
J = 13.9, 3.7, 2.1 Hz, 1H), 1.77 – 1.68 (m, 1H), 1.42 (td, J = 13.3, 2.7 Hz, 1H), 1.28 (app q, J =
12.5 Hz, 1H), 1.25 – 1.20 (m, 4H), 1.09 – 0.98 (m, 3H). 13C{1H} NMR (151 MHz, CDCl3) δ 96.50,
96.47, 72.5, 72.2, 66.7, 55.2, 55.1, 41.7, 41.3, 40.7, 40.5, 38.6, 37.7, 36.9, 36.4, 36.0, 35.6, 32.2,
24.0, 15.0. HRMS (ESI-TOF) m/z: Calcd. for C20H38O5S2Na ([M + Na]+): 445.2058; found:
445.2066.
(1R,3R,5R)-3-(((1S,3R,5S)-3-(Ethylthio)-5-((methoxymethoxy)methyl)cyclohexyl)thio)-5-
((methoxymethoxy)methyl)cyclohexyl Methanesulfonate (26). To a stirred solution of 25 (88.4
mg, 0.21 mmol) in pyridine (2 mL) at room temperature was added methanesulfonyl chloride (80.9
µL, 1.05 mmol). The reaction mixture was stirred at the same temperature for 20 h before it was
diluted with ethyl acetate, washed with 1 N HCl and brine, dried over Na2SO4, and concentrated
under reduced pressure. Chromatographic purification (40% ethyl acetate/hexanes) afforded the
title compound as a colorless oil (26, 95.9 mg, 92%): [α]D21 -11.6 (c 0.56, CHCl3). 1H NMR (600
MHz, CDCl3) δ 5.08 (app p, J = 3.0 Hz, 1H), 4.57 (s, 2H), 4.56 (s, 2H), 3.37 – 3.33 (m, 4H), 3.32
ACS Paragon Plus Environment

The Journal of Organic Chemistry
Page 20 of 37
20
1
2
3
4
5
6
7
8
9
(s, 3H), 3.31 (s, 3H), 3.07 (tt, J = 12.4, 3.7 Hz, 1H), 3.01 (s, 3H), 2.74 (tt, J = 12.2, 3.7 Hz, 1H),
2.65 (tt, J = 12.2, 3.6 Hz, 1H), 2.57 (q, J = 7.4 Hz, 2H), 2.36 (dtt, J = 14.4, 3.9, 2.0 Hz, 1H), 2.26
(ddt, J = 12.6, 3.8, 1.9 Hz, 1H), 2.14 – 2.02 (m, 5H), 1.77 – 1.68 (m, 1H), 1.51 (ddd, J = 14.8, 12.6,
2.5 Hz, 1H), 1.37 – 1.25 (m, 2H), 1.23 (t, J = 7.4 Hz, 3H), 1.12 (app q, J = 12.9 Hz, 1H), 1.06 –
0.98 (m, 2H). 13C{1H} NMR (151 MHz, CDCl3) δ 96.5 (2 Cs), 78.6, 72.1, 71.8, 55.20, 55.17, 41.6,
41.1, 41.0, 38.8, 38.7, 38.5, 36.9, 36.8, 36.3, 35.7, 33.6, 32.5, 24.0, 15.0. HRMS (ESI-TOF) m/z:
Calcd. for C21H40O7S3Na ([M + Na]+): 523.1834; found: 523.1841.
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
S-((1S,3R,5S)-3-(((1S,3R,5S)-3-(Ethylthio)-5-((methoxymethoxy)methyl)cyclohexyl)thio)-5-
((methoxymethoxy)methyl)cyclohexyl) Thioacetate (27). A mixture of 26 (10.0 mg, 0.02
mmol) and cesium thioacetate (12.5 mg, 0.06 mmol) in DMF (0.05 mL) was heated to 100 oC by
irridation with microwave for 50 min. After cooling to room temperature, the reaction mixture was
diluted with ethyl acetate, washed with brine, dried over Na2SO4, and concentrated under reduced
pressure. Chromatographic purification (14% ethyl acetate/hexanes) afforded the title compound
as a light yellow oil (27, 4.5 mg, 47%): [α]D21 -16.7 (c 0.18, CHCl3). 1H NMR (600 MHz, CDCl3)
δ 4.59 (s, 2H), 4.57 (s, 2H), 3.42 (tt, J = 12.6, 3.9 Hz, 1H), 3.37 – 3.33 (m, 7H), 3.32 (s, 3H), 2.81
(tt, J = 12.1, 3.8 Hz, 1H), 2.72 (tt, J = 12.2, 3.7 Hz, 1H), 2.66 (tt, J = 12.2, 3.6 Hz, 1H), 2.57 (q, J
= 7.4 Hz, 2H), 2.29 (s, 3H), 2.27 – 2.20 (m, 2H), 2.11 – 1.97 (m, 4H), 1.88 – 1.78 (m, 1H), 1.77 –
1.69 (m, 1H), 1.36 – 1.26 (m, 2H), 1.24 (t, J = 7.4 Hz, 3H), 1.12 – 0.97 (m, 4H). 13C{1H} NMR
(151 MHz, CDCl3) δ 195.3, 96.52 (2 Cs), 96.50, 72.2, 72.0, 55.2, 41.7, 41.1, 40.9, 40.8, 40.6, 40.5,
38.5 (2 Cs), 37.0, 36.6, 36.3, 35.4, 30.7, 24.0, 15.0. HRMS (ESI-TOF) m/z: Calcd. for
C22H40O5S3Na ([M + Na]+): 503.1936; found: 503.1946.
(3R,5R)-3-(((1S,3R,5R)-3-(((1S,3R,5S)-3-(Ethylthio)-5-
((methoxymethoxy)methyl)cyclohexyl)thio)-5-((methoxymethoxy)methyl)cyclohexyl)thio)-
ACS Paragon Plus Environment

Page 21 of 37
The Journal of Organic Chemistry
21
1
2
3
4
5-((methoxymethoxy)methyl)cyclohexan-1-one
(28)
and
(3S,5R)-3-(((1S,3R,5R)-3-
5
6
(((1S,3R,5S)-3-(Ethylthio)-5-((methoxymethoxy)methyl)cyclohexyl)thio)-5-
7
8
9
((methoxymethoxy)methyl)cyclohexyl)thio)-5-((methoxymethoxy)methyl)cyclohexan-1-one
(29). To a stirred solution of 27 (24.6 mg, 0.05 mmol) in MeOH (0.6 mL) at room temperature
was added sodium methoxide (5.5 mg, 0.10 mmol). After stirring at room temperature for 25 min,
the reaction mixture was diluted with ethyl acetate, washed with 1 N HCl and brine, dried over
Na2SO4, and concentrated under reduced pressure to give the free thiol (21.8 mg). To a solution of
the free thiol and enone 16 (10.5 mg, 0.06 mmol) in 2,2,2-trifluoroethanol (0.6 mL) was added a
solution of DBU (7.8 mg, 0.05 mmol) in 2,2,2-trifluoroethanol (0.1 mL). After stirring at room
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
o
temperature for 30 min, the reaction mixture was concentrated under reduced pressure at 40 C.
Chromatographic purification (40% ethyl acetate/hexanes) afforded the title compound as a
colorless oil (28, 15.4 mg, 49%): [α]D21 13.6 (c 0.39, CHCl3). 1H NMR (600 MHz, CDCl3) δ 4.59
(s, 2H), 4.58 (s, 2H), 4.57 (s, 2H), 3.48 – 3.41 (m, 2H), 3.38 – 3.29 (m, 13H), 3.01 (tt, J = 12.6,
4.1 Hz, 1H), 2.78 – 2.70 (m, 3H), 2.70 – 2.62 (m, 2H), 2.57 (q, J = 7.4 Hz, 2H), 2.42 (ddt, J = 14.0,
3.9, 1.9 Hz, 1H), 2.33 – 2.12 (m, 5H), 2.10 – 2.01 (m, 5H), 1.77 – 1.67 (m, 2H), 1.52 (app q, J =
12.5 Hz, 1H), 1.33 – 1.20 (m, 5H), 1.07 – 0.97 (m, 4H). 13C{1H} NMR (151 MHz, CDCl3) δ 208.0,
96.5 (3 Cs), 72.2, 72.0, 71.2, 55.3, 55.22, 55.20, 48.9, 44.0, 41.7, 41.3, 41.11, 41.07, 40.6, 40.5,
39.8, 38.51, 38.48, 37.8, 36.94, 36.86, 36.7, 36.32, 36.28, 24.0, 15.0. HRMS (ESI-TOF) m/z:
Calcd. for C29H52O7S3Na ([M + Na]+): 631.2773; found: 631.2780.
Further elution (50% ethyl acetate/hexanes) gave 29 as a colorless oil (9.4 mg, 30%): [α]D21 -18.3
(c 0.30, CHCl3). 1H NMR (600 MHz, CDCl3) δ 4.60 – 4.56 (m, 6H), 3.57 (app p, J = 5.1 Hz, 1H),
3.50 – 3.42 (m, 2H), 3.40 – 3.28 (m, 13H), 2.78 – 2.62 (m, 5H), 2.57 (q, J = 7.4 Hz, 2H), 2.55 –
2.48 (m, 1H), 2.48 – 2.41 (m, 2H), 2.29 – 2.17 (m, 3H), 2.11 – 1.92 (m, 6H), 1.78 – 1.67 (m, 2H),
ACS Paragon Plus Environment

The Journal of Organic Chemistry
Page 22 of 37
22
1
2
3
4
5
6
7
8
9
1.32 – 1.20 (m, 5H), 1.07 – 0.97 (m, 4H). 13C{1H} NMR (151 MHz, CDCl3) δ 208.2, 96.53, 96.52,
96.49, 72.2, 72.0, 70.9, 55.3, 55.2 (2 Cs), 47.1, 43.8, 41.6, 41.2, 41.13, 41.09, 40.6, 40.5, 39.0,
38.53, 38.49, 37.0, 36.9, 36.33, 36.26, 34.4, 33.7, 24.0, 15.0. HRMS (ESI-TOF) m/z: Calcd. for
C29H52O7S3Na ([M + Na]+): 631.2773; found: 631.2766.
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
(1S,3R,5R)-3-(((1S,3R,5R)-3-(((1S,3R,5S)-3-(Ethylthio)-5-
((methoxymethoxy)methyl)cyclohexyl)thio)-5-((methoxymethoxy)methyl)cyclohexyl)thio)-
5-((methoxymethoxy)methyl)cyclohexan-1-ol (30). To a stirred solution of 28 (5.7 mg, 0.009
mmol) in MeOH (0.1 mL) at room temperature was added sodium borohydride (1.1 mg, 0.03
mmol). The reaction mixture was stirred at room temperature for 20 min before it was diluted with
ethyl acetate, washed with 1 N HCl and brine, dried over Na2SO4, and concentrated under reduced
pressure. Chromatographic purification (20% acetone/hexanes) afforded the title compound as a
colorless oil (30, 3.5 mg, 61%): [α]D21 -11.4 (c 0.14, CHCl3). 1H NMR (600 MHz, CDCl3) δ 4.62
– 4.55 (m, 6H), 3.65 (tt, J = 11.1, 4.2 Hz, 1H), 3.43 – 3.29 (m, 15H), 2.80 – 2.70 (m, 4H), 2.66 (tt,
J = 12.2, 3.6 Hz, 1H), 2.58 (q, J = 7.4 Hz, 2H), 2.31 – 2.20 (m, 3H), 2.11 – 1.97 (m, 6H), 1.78 –
1.68 (m, 3H), 1.34 – 1.20 (m, 6H), 1.08 – 0.94 (m, 6H). 13C{1H} NMR (151 MHz, CDCl3) δ 96.54
(2 Cs), 96.51, 72.22, 72.16, 72.1, 69.6, 55.22 (2 Cs), 55.19, 43.3, 41.70, 41.65, 41.1, 40.8, 40.6,
40.5, 38.7, 38.6, 38.5, 38.3, 36.92, 36.89, 36.8, 36.7, 36.4, 36.3, 24.0, 15.0. HRMS (ESI-TOF)
m/z: Calcd. for C29H54O7S3Na ([M + Na]+): 633.2929; found: 633.2931.
(1S,3R,5R)-3-(((1S,3R,5R)-3-(((1S,3R,5S)-3-(Ethylthio)-5-(hydroxymethyl)cyclohexyl)thio)-
5-(hydroxymethyl)cyclohexyl)thio)-5-(hydroxymethyl)cyclohexan-1-ol (31). A mixture of
alcohol 30 (5.2 mg, 0.009 mmol), MeOH (0.5 mL) and concentrated HCl (5 µL) was heated to 60
oC for 5 h. After cooling to room temperature, the reaction mixture was concentrated under reduced
pressure. Chromatographic purification (9% methanol/dichloromethane) afforded the title
ACS Paragon Plus Environment

Page 23 of 37
The Journal of Organic Chemistry
23
1
2
3
4
5
6
7
8
9
compound as a colorless oil (31, 3.1 mg, 76%): [α]D21 -11.4 (c 0.07, CH3OH). 1H NMR (600 MHz,
CD3OD) δ 3.58 (tt, J = 10.9, 4.1 Hz, 1H), 3.45 – 3.35 (m, 6H), 2.88 – 2.75 (m, 4H), 2.72 (tt, J =
12.1, 3.6 Hz, 1H), 2.58 (q, J = 7.4 Hz, 2H), 2.31 – 2.23 (m, 2H), 2.20 (dtt, J = 12.1, 4.0, 2.1 Hz,
1H), 2.11 – 1.99 (m, 5H), 1.96 (d, J = 12.1 Hz, 1H), 1.73 – 1.52 (m, 3H), 1.22 (t, J = 7.4 Hz, 3H),
1.20 – 1.11 (m, 3H), 1.01 – 0.78 (m, 6H). 13C{1H} NMR (151 MHz, CD3OD) δ 68.9, 66.3 (3 Cs),
42.8, 42.0, 41.44, 41.42, 40.6, 40.52, 40.51, 40.47 (2 Cs), 38.6, 38.5, 37.4, 36.64, 36.56 (2 Cs),
36.5, 35.9, 23.4, 14.1. HRMS (ESI-TOF) m/z: Calcd. for C23H42O4S3Na ([M + Na]+): 501.2143;
found: 501.2155.
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
(1R,3R,5R)-3-(((1S,3R,5R)-3-(((1S,3R,5S)-3-(Ethylthio)-5-
((methoxymethoxy)methyl)cyclohexyl)thio)-5-((methoxymethoxy)methyl)cyclohexyl)thio)-
5-((methoxymethoxy)methyl)cyclohexan-1-ol (32). To a stirred solution of 28 (14.9 mg, 0.02
mmol) in THF (0.3 mL) at -78 oC was added a solution of L-Selectride in THF (1.0 M, 73.4 µL,
0.07 mmol). The reaction mixture was stirred at the same temperature for 12 h before it was
warmed to room temperature. The reaction was quenched by adding MeOH and the mixture was
concentrated under reduced pressure. Chromatographic purification (14% acetone/hexanes)
afforded the title compound as a colorless oil (32, 11.8 mg, 79%): [α]D21 -18.7 (c 0.47, CHCl3). 1H
NMR (600 MHz, CDCl3) δ 4.59 (s, 2H), 4.58 (s, 2H), 4.57 (s, 2H), 4.22 (br s, 1H), 3.42 – 3.28 (m,
15H), 3.14 (tt, J = 12.4, 3.7 Hz, 1H), 2.78 – 2.69 (m, 3H), 2.66 (tt, J = 12.2, 3.6 Hz, 1H), 2.57 (q,
J = 7.4 Hz, 2H), 2.31 – 2.22 (m, 2H), 2.17 – 1.99 (m, 7H), 1.80 (dtd, J = 14.1, 3.6, 1.8 Hz, 1H),
1.77 – 1.69 (m, 2H), 1.42 (td, J = 13.5, 2.6 Hz, 1H), 1.33 – 1.18 (m, 6H), 1.10 – 0.94 (m, 5H).
13C{1H} NMR (151 MHz, CDCl3) δ 96.51 (2 Cs), 96.47, 72.5, 72.21, 72.19, 66.6, 55.2 (2 Cs),
55.1, 41.9, 41.7, 41.2, 40.9, 40.7, 40.6 (2 Cs), 38.6, 38.5, 37.8, 37.0, 36.92, 36.85, 36.3, 36.2, 35.5,
ACS Paragon Plus Environment

The Journal of Organic Chemistry
Page 24 of 37
24
1
2
3
4
5
6
32.2, 24.0, 15.0. HRMS (ESI-TOF) m/z: Calcd. for C29H54O7S3Na ([M + Na]+): 633.2929; found:
633.2932.
7
8
9
(1R,3R,5R)-3-(((1S,3R,5R)-3-(((1S,3R,5S)-3-(Ethylthio)-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
((methoxymethoxy)methyl)cyclohexyl)thio)-5-((methoxymethoxy)methyl)cyclohexyl)thio)-
5-((methoxymethoxy)methyl)cyclohexyl Methanesulfonate (33). To a stirred solution of 32
(10.5 mg, 0.02 mmol) in pyridine (0.25 mL) at room temperature was added methanesulfonyl
chloride (6.7 µL, 0.09 mmol). The reaction mixture was stirred at the same temperature for 11 h
before it was diluted with ethyl acetate, washed with 1 N HCl and brine, dried over Na2SO4, and
concentrated under reduced pressure. Chromatographic purification (45% ethyl acetate/hexanes)
afforded the title compound as a colorless oil (33, 11.4 mg, 96%): [α]D21 -9.0 (c 0.41, CHCl3). 1H
NMR (600 MHz, CDCl3) δ 5.09 (br s, 1H), 4.60 – 4.56 (m, 6H), 3.43 – 3.28 (m, 15H), 3.07 (tt, J
= 12.4, 3.6 Hz, 1H), 3.01 (s, 3H), 2.80 – 2.70 (m, 3H), 2.67 (tt, J = 12.2, 3.6 Hz, 1H), 2.57 (q, J =
7.4 Hz, 2H), 2.38 (dd, J = 14.2, 3.7 Hz, 1H), 2.30 – 2.20 (m, 2H), 2.16 – 2.00 (m, 7H), 1.79 – 1.69
(m, 2H), 1.51 (ddd, J = 14.8, 12.7, 2.5 Hz, 1H), 1.34 (t, J = 13.5 Hz, 1H), 1.31 – 1.25 (m, 2H),
1.23 (t, J = 7.4 Hz, 3H), 1.12 (app q, J = 12.7 Hz, 1H), 1.07 – 0.97 (m, 4H). 13C{1H} NMR (151
MHz, CDCl3) δ 96.52, 96.51 (2 Cs), 78.6, 72.2, 72.1, 71.8, 55.20 (2 Cs), 55.19, 41.63, 41.59, 41.1,
41.0, 40.6, 40.5, 38.9, 38.7, 38.49, 38.47, 37.0, 36.9 (2 Cs), 36.8, 36.4, 35.7, 33.6, 32.6, 24.0, 15.0.
HRMS (ESI-TOF) m/z: Calcd. for C30H56O9S4Na ([M + Na]+): 711.2705; found: 711.2727.
S-((1S,3R,5S)-3-(((1S,3R,5S)-3-(((1S,3R,5S)-3-(Ethylthio)-5-
((methoxymethoxy)methyl)cyclohexyl)thio)-5-((methoxymethoxy)methyl)cyclohexyl)thio)-
5-((methoxymethoxy)methyl)cyclohexyl) Thioacetate (34).
A mixture of 33 (11.4 mg, 0.02 mmol) and cesium thioacetate (10.3 mg, 0.05 mmol) in DMF (0.1
o
mL) was heated to 100 C by irridation with microwave for 35 min. After cooling to room
ACS Paragon Plus Environment

Page 25 of 37
The Journal of Organic Chemistry
25
1
2
3
4
5
6
7
8
9
temperature, the reaction mixture was diluted with ethyl acetate, washed with brine, dried over
Na2SO4, and concentrated under reduced pressure. Chromatographic purification (22% ethyl
acetate/hexanes) afforded the title compound as a light yellow oil (34, 4.5 mg, 41%): [α]D21 -16.1
(c 0.18, CHCl3). 1H NMR (600 MHz, CDCl3) δ 4.59 (m, 4H), 4.57 (s, 2H), 3.43 (tt, J = 12.6, 3.9
Hz, 1H), 3.39 – 3.29 (m, 15H), 2.81 (tt, J = 12.9, 4.1 Hz, 1H), 2.77 – 2.70 (m, 3H), 2.67 (tt, J =
12.2, 4.0 Hz, 1H), 2.58 (q, J = 7.4 Hz, 2H), 2.29 (s, 3H), 2.28 – 2.19 (m, 3H), 2.12 – 1.96 (m, 6H),
1.89 – 1.79 (m, 1H), 1.79 – 1.69 (m, 2H), 1.36 – 1.25 (m, 3H), 1.24 (t, J = 7.4 Hz, 3H), 1.09 (app
q, 1H), 1.06 – 0.97 (m, 5H). 13C{1H} NMR (151 MHz, CDCl3) δ 195.3, 96.54 (2 Cs), 96.51, 72.22,
72.16, 72.0, 55.21, 55.19 (2 Cs), 41.7, 41.5, 41.1, 40.83, 40.80, 40.5 (4 Cs), 38.5 (3 Cs), 36.98,
36.96, 36.9, 36.6, 36.4, 35.3, 30.7, 24.0, 15.0. HRMS (ESI-TOF) m/z: Calcd. for C31H56O7S4Na
([M + Na]+): 691.2807; found: 691.2817.
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
(3R,5R)-3-(((1S,3R,5R)-3-(((1S,3R,5S)-3-(((1S,3R,5S)-3-(Ethylthio)-5-
((methoxymethoxy)methyl)cyclohexyl)thio)-5-((methoxymethoxy)methyl)cyclohexyl)thio)-
5-((methoxymethoxy)methyl)cyclohexyl)thio)-5-((methoxymethoxy)methyl)cyclohexan-1-
one
(35)
and
(3S,5R)-3-(((1S,3R,5R)-3-(((1S,3R,5S)-3-(((1S,3R,5S)-3-(Ethylthio)-5-
((methoxymethoxy)methyl)cyclohexyl)thio)-5-((methoxymethoxy)methyl)cyclohexyl)thio)-
5-((methoxymethoxy)methyl)cyclohexyl)thio)-5-((methoxymethoxy)methyl)cyclohexan-1-
one (36). To a stirred solution of 34 (3.3 mg, 0.005 mmol) in MeOH (0.2 mL) at room temperature
was added sodium methoxide (0.5 mg, 0. 01 mmol). After stirring at room temperature for 10 min,
the reaction mixture was diluted with ethyl acetate, washed with 1 N HCl and brine, dried over
Na2SO4, and concentrated under reduced pressure to give the free thiol (4.5 mg). To a solution of
the free thiol and enone 16 (1.0 mg, 0.006 mmol) in 2,2,2-trifluoroethanol (0.1 mL) was added a
solution of DBU (750 µg, 0.005 mmol) in 2,2,2-trifluoroethanol (10 µL). After stirring at room
ACS Paragon Plus Environment

The Journal of Organic Chemistry
Page 26 of 37
26
1
2
3
4
5
6
7
8
9
o
temperature for 30 min, the reaction mixture was concentrated under reduced pressure at 40 C.
Chromatographic purification (45% ethyl acetate/hexanes) afforded the title compound as a
colorless oil (35, 2.1 mg, 53%): [α]D21 2.0 (c 0.15, CHCl3). 1H NMR (600 MHz, CDCl3) δ 4.61 –
4.58 (m, 8H), 3.49 – 3.41 (m, 2H), 3.41 – 3.30 (m, 18H), 3.02 (tt, J = 12.6, 4.1 Hz, 1H), 2.81 –
2.71 (m, 5H), 2.71 – 2.63 (m, 2H), 2.58 (q, J = 7.4 Hz, 2H), 2.43 (ddt, J = 13.9, 4.0, 1.9 Hz, 1H),
2.30 (t, J = 13.6 Hz, 1H), 2.29 – 2.20 (m, 4H), 2.17 (t, J = 13.7 Hz, 1H), 2.13 – 2.00 (m, 7H), 1.79
– 1.69 (m, 3H), 1.52 (t, J = 12.5 Hz, 1H), 1.32 – 1.22 (m, 6H), 1.09 – 0.97 (m, 6H). 13C{1H} NMR
(151 MHz, CDCl3) δ 208.0, 96.6, 96.5 (3 Cs), 72.20, 72.17, 72.0, 71.2, 55.3, 55.23 (2 Cs), 55.20,
48.9, 44.0, 41.7, 41.5, 41.3, 41.2, 41.1, 40.6 (2 Cs), 40.54, 40.46, 39.8, 38.53, 38.49, 38.47, 37.8,
36.94 (3 Cs), 36.88, 36.8, 36.4, 36.3, 24.0, 15.0. HRMS (ESI-TOF) m/z: Calcd. for C38H68O9S4Na
([M + Na]+): 819.3644; found: 819.3643.
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
Further elution (50% ethyl acetate/hexanes) gave 36 as a colorless oil (1.1 mg, 28%): [α]D21 -10.9
(c 0.11, CHCl3). 1H NMR (600 MHz, CDCl3) δ 4.62 – 4.56 (m, 8H), 3.58 (app p, J = 4.9 Hz, 1H),
3.50 – 3.42 (m, 2H), 3.42 – 3.30 (m, 18H), 2.81 – 2.62 (m, 7H), 2.58 (q, J = 7.4 Hz, 2H), 2.56 –
2.49 (m, 1H), 2.48 – 2.41 (m, 2H), 2.32 – 2.17 (m, 4H), 2.13 – 1.93 (m, 8H), 1.80 – 1.69 (m, 3H),
1.34 – 1.21 (m, 6H), 1.09 – 0.96 (m, 6H). 13C{1H} NMR (151 MHz, CDCl3) δ 208.2, 96.6, 96.54
(2 Cs), 96.50, 72.20, 72.17, 72.1, 70.9, 55.3, 55.23, 55.22, 55.21, 47.1, 43.8, 41.7, 41.5, 41.2,
41.14, 41.08, 40.6 (2 Cs), 40.53, 40.46, 39.0, 38.53 (2 Cs), 38.48, 37.1, 37.0, 36.9 (2 Cs), 36.4,
36.3, 34.4, 33.7, 24.0, 15.0. HRMS (ESI-TOF) m/z: Calcd. for C38H68O9S4Na ([M + Na]+):
819.3644; found: 819.3656.
(1S,3R,5R)-3-(((1S,3R,5R)-3-(((1S,3R,5S)-3-(((1S,3R,5S)-3-(Ethylthio)-5-
((methoxymethoxy)methyl)cyclohexyl)thio)-5-((methoxymethoxy)methyl)cyclohexyl)thio)-
5-((methoxymethoxy)methyl)cyclohexyl)thio)-5-((methoxymethoxy)methyl)cyclohexan-1-ol
ACS Paragon Plus Environment

Page 27 of 37
The Journal of Organic Chemistry
27
1
2
3
4
5
6
7
8
9
(37). To a stirred solution of 35 (4.9 mg, 0.006 mmol) in MeOH (0.1 mL) at room temperature
was added sodium borohydride (0.7 mg, 0.018 mmol). The reaction mixture was stirred at room
temperature for 20 min before it was diluted with ethyl acetate, washed with 1 N HCl and brine,
dried over Na2SO4, and concentrated under reduced pressure. Chromatographic purification (25%
acetone/hexanes) afforded the title compound as a colorless oil (37, 2.7 mg, 54%): [α]D21 -10.0 (c
0.11, CHCl3). 1H NMR (600 MHz, CDCl3) δ 4.61 – 4.57 (m, 8H), 3.65 (tt, J = 10.9, 3.8 Hz, 1H),
3.44 – 3.31 (m, 20H), 2.80 – 2.70 (m, 6H), 2.66 (tt, J = 12.2, 3.6 Hz, 1H), 2.58 (q, J = 7.4 Hz, 2H),
2.32 – 2.19 (m, 4H), 2.13 – 1.98 (m, 8H), 1.79 – 1.69 (m, 4H), 1.34 – 1.20 (m, 7H), 1.09 – 0.94
(m, 8H). 13C{1H} NMR (151 MHz, CDCl3) δ 96.6, 96.54 (2 Cs), 96.51, 72.20 (2 Cs), 72.16, 72.1,
69.6, 55.3, 55.22, 55.20, 55.19, 43.3, 41.8, 41.7, 41.5, 41.2, 40.8, 40.60, 40.57 (3 Cs), 40.5, 38.8,
38.6, 38.54, 38.50, 38.3, 37.0, 36.92 (2 Cs), 36.88, 36.7, 36.37, 36.36, 24.0, 15.0. HRMS (ESI-
TOF) m/z: Calcd. for C38H70O9S4Na ([M + Na]+): 821.3800; found: 821.3766.
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
(1S,3R,5R)-3-(((1S,3R,5R)-3-(((1S,3R,5S)-3-(((1S,3R,5S)-3-(Ethylthio)-5-
(hydroxymethyl)cyclohexyl)thio)-5-(hydroxymethyl)cyclohexyl)thio)-5-
(hydroxymethyl)cyclohexyl)thio)-5-(hydroxymethyl)cyclohexan-1-ol (38). A mixture of
alcohol 37 (2.2 mg, 0.003 mmol), MeOH (0.3 mL) and concentrated HCl (3 µL) was heated to 60
oC for 6 h. After cooling to room temperature, the reaction mixture was concentrated under reduced
pressure. Chromatographic purification (11% methanol/dichloromethane) afforded the title
compound as a colorless oil (38, 1.3 mg, 76%): [α]D21 -22.5 (c 0.04, CH3OH). 1H NMR (600 MHz,
CD3OD) δ 3.62 – 3.55 (m, 1H), 3.44 – 3.35 (m, 8H), 2.88 – 2.75 (m, 6H), 2.72 (tt, J = 12.2, 3.6
Hz, 1H), 2.58 (q, J = 7.4 Hz, 2H), 2.30 – 2.23 (m, 3H), 2.21 (d, J = 11.9 Hz, 1H), 2.10 – 1.99 (m,
7H), 1.96 (d, J = 12.3 Hz, 1H), 1.68 – 1.54 (m, 4H), 1.22 (t, J = 7.4 Hz, 3H), 1.20 – 1.10 (m, 4H),
0.98 – 0.83 (m, 8H). 13C{1H} NMR (151 MHz, CD3OD) δ 68.9, 66.3 (4 Cs), 42.9, 42.09, 42.05,
ACS Paragon Plus Environment

The Journal of Organic Chemistry
Page 28 of 37
28
1
2
3
4
5
6
7
8
9
42.0, 41.5, 41.4, 40.54 (4 Cs), 40.48 (4 Cs), 38.7, 38.5, 37.4, 36.7, 36.6 (4 Cs), 36.0, 23.4, 14.1.
HRMS (ESI-TOF) m/z: Calcd. for C30H54O5S4Na ([M + Na]+): 645.2752; found: 645.2753.
Laminaritriose 39 and tetraose 40 were purchased from commercial sources. They were found
1
13
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
to be pure by H and C NMR spectroscopy and ESI mass spectrometry, and had spectral data
consistent with the literature:20
39: 1H NMR (600 MHz, D2O) δ 5.09 (d, J = 3.7 Hz, 0.42H, H-1α), 4.65-4.60 (m, 2H), 4.53 (d, J
= 8.1 Hz, 0.58H, H-1β), 3.81 – 3.55 (m, 9H), 3.44 – 3.22 (m, 9H).13C{1H} NMR (151 MHz, D2O)
δ 102.7, 102.55, 102.46, 95.6, 92.0, 84.4, 84.21, 84.17, 82.2, 75.9, 75.6, 75.55, 75.52, 73.8, 73.4,
73.20, 73.17, 71.2, 71.0, 69.5, 68.05, 68.01, 60.6, 60.5. ESIHRMS calculated for C18H32O16
[M+Na]+, 527.1588; found, 527.1578.
40: 1H NMR (600 MHz, D2O) δ 5.09 (d, J = 3.7 Hz, 0.37H, H-1α), 4.66 – 4.61 (m, 3H), 4.53 (d, J
= 8.0 Hz, 0.63H, H-1β), 3.81 – 3.54 (m, 12H), 3.45 – 3.22 (m, 12H). 13C{1H} NMR (151 MHz,
D2O) δ 102.7, 102.6, 102.5, 102.4, 95.6, 91.9, 84.4, 84.2, 84.03, 83.99, 82.2, 75.9, 75.6, 75.56,
75.54, 75.51, 73.8, 73.4, 73.3, 73.25, 73.22, 71.2, 71.0, 69.5, 68.05, 68.01, 60.62, 60.59, 60.5.
ESIHRMS calculated for C24H42O21 [M+Na]+, 689.2116; found, 689.2111.
Inhibition of anti-CR3-FITC antibody staining of human neutrophils
and of anti-Dectin 1-FITC antibody staining of mouse macrophages.
For fluorescent staining, anti-CR3-FITC antibodies (MN-41 donated by
Drs. Allison Eddy and Alfred Michael of the University of Minnesota,
Minneapolis, MN, and rat anti Mouse Dectin-1 antibody labeled with FITC
(purchased from AbD Serotec, Raleigh, NC) were employed. Either human
neutrophils or mouse peritoneal macrophages were incubated with 0.1
g.mL-1 of tested samples for 0.5 h on ice and washed. Subsequently, the
ACS Paragon Plus Environment

Page 29 of 37
The Journal of Organic Chemistry
29
1
2
3
4
5
6
7
8
cells were stained with antibodies on ice using standard techniques.
After centrifugation of cells through a 3 mL cushion of 12% BSA in PBS,
the cells were re-suspended in PBS containing 1% BSA and 10 mM sodium
9
azide. Cell cytometry was performed with
a
Becton Dickinson-LSRII
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
instrument. The inhibition of CR3 receptor and Dectin-1 receptor staining
was calculated as described.61
Stimulation of phagocytosis. The technique employing phagocytosis of synthetic polymeric
microspheres was described earlier.66 Human cells (cell line RAW 264) were incubated in vitro with 10
μg.mL-1 of tested samples for 24 h at 37 oC. After washing, 0.05 mL of 2-hydroxyethyl methacrylate
particles (HEMA; 5x108/mL) was added. The test tubes were incubated at 37 oC for 1 h, with intermittent
shaking. Smears were stained with Wright stain. Cells with three or more HEMA particles were
considered positive. The insoluble glucan Glucan #300 used as comparison standard was obtained from
Yeast-derived insoluble Glucan #300 (>85% dry w/w basis) was purchased from Transfer Point
(Columbia, SC, USA). This glucan contains 96% carbohydrates and 2.1% proteins. Neutral
sugar analysis confirmed 91.3% glucose and 8% mannose.
Stimulation of pinocytosis. Stimulation of pinocytosis was determined spectrophotometrically as
described.63
Supporting Information. The Supporting Information is available free of charge on the ACS
Publications website at DOI: 10.1021/acs.joc.8b01645.
Copies of the 1H and 13C NMR spectra of all new compounds (PDF)
Acknowledgments. We thank the NIH (GM62160) for support of this work and Vikram A
Sarpe for assistance with the checking of commercial glycans.
References
ACS Paragon Plus Environment