Synthetic Studies Toward the Skyllamycins: Total Synthesis and Generation of Simplified Analogues

Article abstract of DOI:10.1021/acs.joc.8b00898

Herein, we report our synthetic studies toward the skyllamycins, a highly modified class of nonribosomal peptide natural products which contain a number of interesting structural features, including the extremely rare α-OH-glycine residue. Before embarking on the synthesis of the natural products, we prepared four structurally simpler analogues. Access to both the analogues and the natural products first required the synthesis of a number of nonproteinogenic amino acids, including three β-OH amino acids that were accessed from the convenient chiral precursor Garner's aldehyde. Following the preparation of the suitably protected nonproteinogenic amino acids, the skyllamycin analogues were assembled using a solid-phase synthetic route followed by a final stage solution-phase cyclization reaction. To access the natural products (skyllamycins A-C) the synthetic route used for the analogues was modified. Specifically, linear peptide precursors containing a C-terminal amide were synthesized via solid-phase peptide synthesis. After cleavage from the resin the N-terminal serine residue was oxidatively cleaved to a glyoxyamide moiety. The target natural products, skyllamycins A-C, were successfully prepared via a final step cyclization with concomitant formation of the unusual α-OH-glycine residue. Purification and spectroscopic comparison to the authentic isolated material confirmed the identity of the synthetic natural products.

Full text of DOI:10.1021/acs.joc.8b00898

Subscriber access provided by Kaohsiung Medical University
Article
Synthetic Studies Towards the Skyllamycins: Total
Synthesis and Generation of Simplified Analogues
Andrew M. Giltrap, F.P. Jake Haeckl, Kenji L. Kurita, Roger G. Linington, and Richard J Payne
J. Org. Chem., Just Accepted Manuscript • DOI: 10.1021/acs.joc.8b00898 • Publication Date (Web): 25 May 2018
Downloaded from http://pubs.acs.org on May 25, 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.
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 39
The Journal of Organic Chemistry
1
2
3
4
5
Synthetic Studies Towards the Skyllamycins: Total Synthesis and Generation of
Simplified Analogues
6
7
Author Information
8
9
Andrew M. Giltrap†, F. P. Jake Haeckl‡, Kenji L. Kurita‡, Roger G. Linington‡ and Richard J.
Payne†*
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
†School of Chemistry, The University of Sydney, Sydney, NSW 2006, Australia
‡Department of Chemistry, Simon Fraser University, Burnaby, British Columbia BC V5A 1S6,
Canada
*richard.payne@sydney.edu.au
O
R2
O
R2
H
N
H
N
H
N
H
N
N
H
COOH
N
COOH
H
O
O
O
O
O
O
O
O
O
N
H
O
O
N
H
OH
OH
HN
HN
O
O
O
O
Cyclization and formation
of -OH-Gly
O
OH
O
OH
HN
HN
α
O
O
N
N
H
N
H2N
O
O
O
O
N
H
O
N
H
O
NH
OH
NH
N
H
N
H
HO
HO
N
H
N
H
O
O
Abstract
Herein we report our synthetic studies toward the skyllamycins, a highly-modified class of
non-ribosomal peptide natural products, which contain a number of interesting structural
features, including the extremely rare a-OH-glycine residue. Before embarking on the
synthesis of the natural products, we prepared four structurally simpler analogues. Access to
both the analogues and the natural products first required the synthesis of a number of non-
proteinogenic amino acids, including three b-OH amino acids that were accessed from the
convenient chiral precursor Garner’s aldehyde. Following the preparation of the suitably
protected non-proteinogenic amino acids, the skyllamycin analogues were assembled using a
solid-phase synthetic route followed by a final stage solution-phase cyclization reaction. In
order to access the natural products – skyllamycins A-C – the synthetic route used for the
analogues was modified. Specifically, linear peptide precursors containing a C-terminal amide
were synthesized via solid-phase peptide synthesis. After cleavage from the resin the N-
terminal serine residue was oxidatively cleaved to a glyoxyamide moiety. The target natural
products, skyllamycins A-C, were successfully prepared via a final step cyclization with
concomitant formation of the unusual a-OH-glycine residue. Purification and spectroscopic
comparison to the authentic isolated material confirmed the identity of the synthetic natural
products.
Introduction
The skyllamycins are a family of non-ribosomal cyclic depsipeptide natural products produced
by Streptomyces sp. Skyllamycin A 1 was first isolated in 2001 by Matsuda and co-workers
who showed that it possessed inhibitory activity against the platelet-derived growth factor
(PDGF) signaling pathway1. This signaling plays a role in cell migration and proliferation and,
importantly, aberrant signaling has been implicated in a number of human disease states,
1
ACS Paragon Plus Environment

The Journal of Organic Chemistry
Page 2 of 39
1
2
3
4
5
6
7
8
9
including cancer2. Skyllamycin A was found to specifically inhibit the interaction of the PDGF
B-type dimer to the PDGF b-receptor. At the time of isolation the stereochemistry of the amino
acids was not assigned. Subsequently, Süssmuth and co-workers re-isolated skyllamycin A (1),
along with the un-methylated skyllamcyin B 2, and carried out a thorough investigation of the
biosynthetic pathways that assemble these interesting natural products3. Skyllamycins A (1), B
(2) and C (3), the latter a reduced congener, were independently isolated in 2014 during our
search for Pseudomonas aeruginosa biofilm inhibitors4. Skyllamycins B and C were both
shown to inhibit the formation of biofilms, while skyllamycin B was also capable of clearing
pre-attached biofilms. Biofilms play a significant role in the ability of bacteria to evade
antibiotics5. Given the established threat of antimicrobial resistance,6 mechanisms such as
biofilm formation that bacteria use to evade antibiotics have become important targets and it is
now recognized that novel methods by which to clear biofilm forming bacterial infections are
urgently needed.
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
R2
R2
R1
H
N
H
N
R1
N
COOH
H
Me Skyllamycin A
(1)
O
O
O
O
O
N
H
OH
HN
O
O
O
O
O
O
OH
HN
Skyllamycin B
(2)
H
H
O
N
H
N
O
N
H
O
OH
NH
N
H
Skyllamycin C
(3)
HO
N
H
O
Figure 1. Structure of skyllamycins A-C 1-3
The skyllamycins are highly modified, and possess a number of unusual amino acids (Figure
1). Specifically, they contain two D-amino acids, D-Leu and D-Trp, as well as b-Me-Asp residue
(blue) and a pseudo N-terminal cinnamic acid residue (green) – unique to this family of natural
products. Related structural motifs are present in other natural products, such as the
pepticinnamins7 and the recently isolated coprisamides8. They are highly hydroxylated,
containing three b-OH amino acids namely Phe, D-Leu and an O-Me Tyr residue (red). Most
unusually they contain and an a-OH-Gly residue (magenta) which to date has only been
reported in the linear natural product spergualin,9-10 but has not been reported in any other
cyclic peptide natural products. Whilst rare in natural products, a-OH-Gly residues are
common in biological systems as precursors to C-terminal amides, particularly amongst
peptide hormones which often require a C-terminal amide for full activity11.
In the second isolation report, Süssmuth and co-workers determined the biosynthetic origins of
the unusual modifications present in the skyllamycins3. Further studies carried out by Cryle
and co-workers revealed that a single P450 monooxygenase was responsible for the installation
of the hydroxyl functionality in the three b-OH amino acids, in each case generating the 3S
stereochemistry12. A crystal structure of this P450 enzyme bound to the peptidyl-carrier protein
domain of the non-ribosomal peptide synthetase was subsequently determined by Cryle and
co-workers13. This important work shed light on the structural and biosynthetic origins of the
stereoselective installation of the hydroxyl moiety. While the gene responsible for the
installation of the a-OH-Gly is known, the exact timing of the formation of this unusual
modification remains elusive. Following their initial biosynthetic studies, Süssmuth and co-
workers confirmed the configuration of the amino acids in the natural products by a
2
ACS Paragon Plus Environment

Page 3 of 39
The Journal of Organic Chemistry
1
2
3
4
5
6
7
8
9
combination of Marfey’s analysis and chiral HPLC/GC14. The unusual a-OH-Gly residue was
not stable to the hydrolytic cleavage conditions employed to convert the natural products back
to their amino acids building blocks. As such, the authors were unable to determine the absolute
stereochemistry of this residue. However, using a combination of intramolecular distances
estimated from NMR-NOESY measurements, and molecular dynamics simulations, the a-OH-
Gly residue was proposed to have the (S)-configuration. Importantly, it was determined that
when in this conformation, the natural products possess a strong internal hydrogen bonding
network involving five strong intramolecular H-bonds, with the a-OH-Gly as well as the
hydroxyl groups of three b-OH residues all participating. When in the (R)-configuration, this
hydrogen bonding network is less extensive, and the a-OH-Gly residue is not involved. The
authors therefore concluded that the hydrogen bonding network stabilizes the a-OH-Gly
residue in the (S)-configuration.
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
Due to their highly unusual and synthetically challenging structures and interesting biological
activities we began a program directed towards the total synthesis of the skyllamycin natural
products as well as the generation of simplified analogues to assess the importance of particular
modifications for activity. We recently reported the first total synthesis of skyllamycins A-C
1-315 and herein we report full details of the total synthesis of these natural products (including
the synthesis of all modified amino acids), as well as the generation of four simplified
analogues.
Results and Discussion
Before we embarked on the synthesis of the natural products we first wanted to investigate the
synthesis of simplified skyllamycin analogues 4-7 (Scheme 1). We envisioned that these
analogues would be easier to access synthetically compared to the natural products, and would
serve to provide insight into the importance of the unusual a-OH-Gly residue for anti-biofilm
activity. We first targeted a simplified skyllamycin analogue 4, in order to validate our
synthetic strategy toward these analogues. Notably, analogue 4 contains only the cinnamic acid
residue and does not possess any of the structurally complex b-OH amino acids or the
challenging a-OH-Gly residue. Following the successful preparation of this simplified
analogue we envisaged the synthesis of the so called deshydroxy skyllamycins 5-7. These
analogues contain all the modified amino acids except for the a-OH-Gly residue. We reasoned
that these molecules would allow us to develop robust synthetic routes to the non-commercially
available amino acids as well as optimize the proposed solid-phase chemistry to access the
natural product skeleton. Furthermore, these analogues would provide initial structure activity
data on biofilm inhibition, particularly the importance of the a-OH-Gly residue.
Retrosynthetically, we envisioned that the simplified analogue 4 and deshydroxy skyllamycins
5-7 could be generated after resin cleavage, cyclization and deprotection from the
corresponding resin-bound linear peptides 8-11. We decided to utilize the Phe-Gly junction as
the cyclization point as the C-terminal Gly residue cannot epimerize. Resin-bound linear
peptides 8-11 could be synthesized beginning from 2-Cl-trityl chloride (2-CTC) resin using a
Fmoc solid-phase peptide synthesis protocol (SPPS). In order to perform the proposed
synthesis a number of suitably protected non-commercially available amino acids, specifically
the three b-OH amino acids, 12-14, two cinnamic acids (15 and 16) and finally a b-Me-Asp 17
required synthesis.
3
ACS Paragon Plus Environment

The Journal of Organic Chemistry
Page 4 of 39
1
2
3
4
5
6
O
R2
H
N
H
N
N
H
COOH
O
O
O
R2 R3 R4
O
O
N
H
R3
H
H
H
4
HN
7
8
9
O
O
Me
Me
deshydroxy skyllamycin A (5)
OH
O
R3
HN
deshydroxy skyllamycin B (6)
H
H
Me
Me
OH
OH
O
N
H
N
O
O
deshydroxy skyllamycin C (7)
N
H
O
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
NH
N
H
R3
R4
O
N
H
1) Resin Cleavage
2) Cyclization
3) Deprotection
O
R2
H
N
H
N
N
H
COOPG
O
O
O
O
O
N
H
R3
R2 R3 R4
O
H
H
H
O
O
8
9
H2N
O
HN
Me
Me
OPG
O
H
N
10
11
H
H
Me
Me
OPG
OPG
O
R3
N
O
O
N
H
O
NH
N
H
R3
NPG
R4
SPPS
O
OPG
O
OPG
O
OPG
HO
HO
FmocHN
13
HO
FmocHN
14
Cl
FmocHN
Cl
O
12
PGOOC
FmocHN
OH
OH
2-CTC
COOH
O
O
17
15
16
Scheme 1. Retrosynthetic analysis of skyllamycin analogues 4-7. PG = protecting group
We started with the preparation of cinnamoyl building block 15, which was inspired by the
synthesis of the related pentenyl phenyl acrylic acid carried out by Jiang and co-workers16
(Scheme 2). Synthesis began with the pyridinium chlorochromate mediated oxidation of o-
iodobenzyl alcohol 18 to afford aldehyde 19. This was reacted with triethyl phosphonacetate
in a Horner-Wadsworth-Emmons reaction to afford the (E)-alkene 20 exclusively in excellent
yield. The key step was a the Sonogashira17 cross-coupling reaction between iodide 20 and
propyne, generated in situ by reaction of 1-bromopropene and n-BuLi18. Gratifyingly this
proceeded in excellent yield providing alkyne 21. The alkyne was subsequently reduced under
Lindlar reduction conditions to afford diene 2219, followed by base-mediated hydrolysis of the
ethyl ester to afford cinnamic acid 15 in excellent overall yield.
4
ACS Paragon Plus Environment

Page 5 of 39
The Journal of Organic Chemistry
1
2
3
OEt
(EtO)2P
4
O
O
5
6
PCC, CH2Cl2,
rt, 4.5 h
NaH, CH2Cl2, 0 ºC,
2 h
I
I
I
7
OH
H
OEt
76%
89%
8
9
O
O
18
19
20
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
Br
1) nBuLi, THF, -78 ºC, 45 min
then H2O -78 ºC to rt, 45 min
2) Pd(PPh3)2Cl2, CuI,
91%
iPr2NH, THF, rt, 16 h
H2, Lindlar Cat,
Quinoline, MeOH,
rt, 50 min
LiOH, EtOH,
0 ºC to rt, 16 h
OH
OEt
OEt
92%
88%
O
O
O
15
22
21
Scheme 2. Synthesis of cinnamic acid 15
With building block 15 in hand, we began the synthesis of simplified analogue 4 (Scheme 3).
Towards this end, Fmoc-Gly-OH was loaded to 2-CTC resin, the resin was capped and Fmoc
deprotection was performed using a solution of 10vol.% piperidine in DMF. Following this,
the peptide was extended utilizing standard Fmoc-SPPS conditions, namely iterative coupling
with (benzotriazol-1-yloxy)tripyrrolidinophosphonium hexafluorophosphate (PyBOP) as the
coupling reagent, N-methylmorpholine (NMM) as the base in DMF, followed by Fmoc
deprotection steps to yield resin-bound tetrapeptide 23. Cinnamic acid 15 was then coupled
using the more active coupling reagent 1-bis(dimethylamino)methylene]-1H-1,2,3-
triazolo[4,5-b]pyridinium
3-oxid
hexafluorophosphate
(HATU),
1-hydroxy-7-
azabenzotriazole (HOAt) and Hünig’s base in DMF to yield resin-bound 24. The key on-resin
esterification reaction with Fmoc-D-Leu-OH was next carried out, mediated by N,N’-
diisopropylcarbodiimide (DIC) and N,N-dimethylamino pyridine (DMAP) to yield branched
resin-bound peptide 25. Further extension of the peptide followed by cleavage from resin under
mildly acidic conditions using 1,1,1,3,3,3-hexafluoro-2-isopropanol (HFIP) in CH2Cl2 yielded
linear side chain protected peptide 26. The crude linear peptide was then treated under
cyclization conditions, namely 4-(4,6-dimethoxy-1,3,5-triazin-2-yl)-4- methylmorpholinium
tetrafluoroborate (DMTMM×BF4) in DMF at high-dilution (0.01 M) to yield protected cyclic
peptide 27. After removal of the solvent the crude cyclic peptide was subjected to acidic
deprotection using a cocktail of trifluoroacetic acid (TFA), triisopropylsilane and water to
remove the side chain protecting groups. This yielded simplified skyllamycin analogue 4 in
9.5% yield (25 steps, 91% per step) after purification by reverse-phase HPLC.
5
ACS Paragon Plus Environment

The Journal of Organic Chemistry
Page 6 of 39
1
2
3
Cl
4
2-CTC resin
5
6
7
8
Loading: 4 eq Fmoc-Gly-OH,
8 eq iPr2EtN,16 h
Capping: CH2Cl2:MeOH:iPr2EtN
(17:2:1 v/v/v), 30 min
Deprotection: 10vol% piperidine
in DMF, 2 x 3 min
Coupling: 4 eq Fmoc-AA-OH,
4 eq PyBOP, 8 eq NMM, 1-2 h
SPPS
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
Coupling 2: 1.2 eq XX,
1.2 eq HATU,2.4 eq HOAt
2.4 eq iPr2EtN, DMF, 16 h
O
O
H
H
N
H
N
N
H2N
HO
N
H
COOtBu
N
H
COOtBu
O
O
O
O
O
HO
O
N
O
N
H
H
24
23
O
O
OH
Fmoc-D-Leu-OH,
DIC, CH2Cl2,30 min
then DMF, DMAP, 16 h
15
O
O
O
H
N
H
N
H
N
H
N
SPPS
N
H
COOtBu
N
H
COOtBu
O
O
O
O
O
O
O
O
O
N
H
O
N
H
O
O
O
H2N
O
O
NHFmoc
O
25
HN
O
H
N
O
N
O
N
H
O
NH
N
H
N
Boc
26
OtBu
30 vol.% HFIP,
CH2Cl2, 2 x 30 min
O
O
H
N
H
N
H
N
H
N
N
H
COOH
N
H
COOtBu
O
O
O
O
O
O
O
O
N
H
O
O
N
H
HN
OH
H2N
O
O
O
O
1) Cyclization:
DMTMM, DMF,
16 h (0.01 M)
2) Deprotection:
TFA:iPr3SiH:H2O
(90:5:5 v/v/v), 2 h
O
HN
O
HN
O
O
N
H
N
H
N
O
N
O
O
O
N
H
O
N
H
O
NH
NH
N
H
N
H
9.5%
N
H
N
Boc
4
27
OH
OtBu
Scheme 3. Synthesis of simplified skyllamycin analogue 4
With this synthetic route to the proposed deshydroxy skyllamycins verified through the
synthesis of 4, we next sought to target deshydroxy skyllamycins A-C 5-7 which differ from
the natural products only by the absence of the unusual a-OH-Gly residue. As such, all the
non-proteinogenic amino acids first required synthesis in suitably protected form Fmoc-SPPS.
We began the synthesis of reduced cinnamic acid 16 present in skyllamycin C 7 from alkene
20 prepared earlier (Scheme 4). Due to the presence of the alkyl iodide, the double bond could
not be reduced using standard Pd-catalyzed hydrogenation. As such, we carried out a three step
procedure to afford reduced aryl iodide 28. Initially, we hydrolyzed the ethyl ester with LiOH,
followed by the key reduction step, mediated by hydrazine hydrate and catalytic guanidine
6
ACS Paragon Plus Environment

Page 7 of 39
The Journal of Organic Chemistry
1
2
3
4
5
6
7
8
9
nitrate20 to yield the reduced carboxylic acid. It was necessary to hydrolyze the ester to prevent
the formation of the corresponding acyl hydrazide during the ensuing reduction reaction.
Finally, the acid was re-esterified affording iodide 28 in 94% yield over the three step sequence.
This was subsequently carried through the same synthetic sequence as described above for
cinnamic acid 15, namely Sonogashira coupling with propyne to afford 29, Lindlar reduction
to diene 30 and ester hydrolysis to yield reduced cinnamic acid 16 in excellent yield over the
seven step synthetic route.
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) LiOH, EtOH,
0 ºC to rt, 16 h
2) N2H4.H2O,
Guanidine.NO3,
O2, EtOH, rt, 40 h
3) SOCl2, EtOH,
0 oC to rt, 16 h
Br
1) nBuLi, THF, -78 ºC,
45 min, then H2O,
-78 ºC to rt, 45 min
2) Pd(PPh3)2Cl2, CuI,
iPr2NH, THF, rt, 16 h
I
I
OEt
OEt
OEt
83%
94%
O
O
20
O
28
29
H2, Lindlar Cat,
Quinoline,
MeOH, rt, 3 h
89%
LiOH, EtOH,
0 ºC to rt, 16 h
OEt
OH
89%
O
O
16
30
Scheme 4. Synthesis of reduced cinnamic acid 16
Having synthesized both cinnamic acids, we next turned our attention to the construction of
the three b-OH amino acids present in the natural product family. As discussed above, all three
contain the 3S stereochemistry at the b-position. Furthermore, both b-OH-Phe and b-OH-O-
Me-Tyr present in the skyllamycins are L-configured, i.e. they possess the 2S stereochemistry.
Based on previous work carried out in our laboratory on the synthesis of b-seleno/thio-Phe for
peptide ligation21-22 we decided to use Garner’s aldehyde23 for the synthesis of (2S,3S)-b-OH-
Phe. Garner’s aldehyde is a convenient chiral starting material for the synthesis of b-OH amino
acids and has been widely used in the synthetic community24. Our synthesis began with
Grignard addition of MgPhBr to (R)-Garner’s aldehyde 31 which led to an inseparable mixture
of diastereomers 32 (Scheme 5). Subsequent oxidation of the alcohol to the ketone 33 under
Swern conditions followed by diastereoselective reduction with DIBAL-H yielded 34 with the
desired anti-stereochemistry25. Removal of the oxazolidine moiety with p-TSA afforded diol
35 in good yield. This was then carried through a reaction sequence requiring only a single
purification. Specifically, the primary alcohol of 35 was first oxidized selectively to the acid
using a TEMPO-mediated oxidation with NaOCl as the stoichiometric oxidant affording acid
36. Following this, Boc-deprotection mediated by HCl in dioxane and a final Fmoc-protection
using Fmoc-OSu in mixed THF and saturated aqueous NaHCO3 yielded suitably protected b-
OH-Phe 37 in excellent yield over the 3 steps.
7
ACS Paragon Plus Environment

The Journal of Organic Chemistry
Page 8 of 39
1
2
3
4
5
6
Oxalyl Chloride, DMSO,
iPr2NEt, CH2Cl2,
-78 ºC, 1.5 h
O
85%
O
OH
O
PhMgBr, Et2O,
0 ºC to rt, 1.5 h
H
O
O
NBoc
NBoc
NBoc
79%
31
32
3:2 anti:syn
33
7
DIBAL-H, THF,
0 ºC, 1 h
8
9
89%
O
10-15% NaOCl,
TEMPO, acetone/
5% aq NaHCO3
(1:1 v/v), rt, 16 h
OH
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
OH
OH
p-TSA, MeOH,
rt, 2.5 h
HO
HO
BocHN
35
84%
NBoc
BocHN
34
36
14:1 anti:syn
1) HCl in dioxane,
CH2Cl2, rt, 2 h
2) Fmoc-OSu,
NaHCO3, THF,
rt, 16 h
64% over 3 steps
O
OH
HO
FmocHN
37
Scheme 5. Synthesis of Fmoc-b-OH-Phe 37
We next turned our attention to the (2R,3S)-b-OH-Leu residue which forms the key ester bond
with the side chain alcohol of the threonine residue in the natural products. As a result, the b-
OH functionality required protection to prevent oligomerization during the esterification
reaction. We envisioned this could be accomplished through the use of a novel pseudo-proline
protecting group strategy. While pseudo-prolines are widely used as turn-inducers in order to
introduce kinks into the growing peptide chain during Fmoc-SPPS26, they are not often
employed as a protecting group for the side-chain hydroxyl groups. To begin the synthesis,
inspired by Joullié and co-workers27, (S)-Garner’s aldehyde 38 was reacted with iPrMgBr to
yield alcohol 39 with exclusive (3S)-stereochemistry in moderate yield (Scheme 6). The
desired protected amino acid could then be accessed in a five-step sequence involving only one
purification step. Specifically, removal of the oxazolidine moiety, in this case using dilute HCl
in THF, afforded diol 40. Selective oxidation of the primary alcohol then yielded acid 41.
Removal of the Boc group, installation of an Fmoc group gave 42, which was then subject to
final protection to yield the desired oxazolidine 43 in 53% over the five-step sequence.
Importantly, the ease and scalability of the final steps allowed for the generation of multi-gram
quantities of this amino acid cassette suitable for direct incorporation into Fmoc-SPPS.
O
OH
OH
HCl (0.5 M), THF,
rt, 16 h
iPrMgBr, THF, -78 ºC
to rt, 3 h
H
HO
BocHN
40
O
O
NBoc
NBoc
39
44%
38
10-15% NaOCl,
TEMPO, acetone/
5% aq NaHCO3,
(1:1 v/v), rt, 16 h
1) HCl in dioxane,
CH2Cl2, rt, 3 h
2) Fmoc-OSu,
NaHCO3,THF,
rt, 16 h
O
OH
O
2,2-DMP,
BF3.OEt2
O
OH
HO
BocHN
HO
Acetone, rt, 4 h HO
O
FmocN
FmocHN
53% over
5 steps
42
41
43
Scheme 6. Synthesis of suitably protected (2R,3S)-b-OH-Leu 43
8
ACS Paragon Plus Environment

Page 9 of 39
The Journal of Organic Chemistry
1
2
3
4
5
6
7
8
9
The final b-OH amino acid that required synthesis was (2S,3S)-b-OH-O-Me-Tyr (Scheme 7).
This was again prepared from (R)-Garner’s aldehyde 31 and was inspired by the work of
Hamada and co-workers in their synthesis of the b-OH-Tyr diastereomers for the synthesis of
papuamides28. The first step involved the addition of the organo-lithium species generated
from p-Br-anisole to (R)-Garner’s aldehyde to yield alcohol 44 as a mixture of diastereomers
(5:1 anti:syn). Removal of the oxazolidine with p-TSA yielded diol 45 which could be
recrystallized to slightly improve the diastereomeric ratio (9:1 anti:syn, 62% recovery).
Subsequent oxidation to acid 46, Boc removal and Fmoc protection to 47 and final oxazolidine
protection yielded the desired final building block 48. Importantly, at this final stage the
diastereomers could be separated by silica column chromatography to yield purified amino
acid 48 as a single diastereomer in good yield over the 4 steps.
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
LiBr, nBuLi,
p-Bromoanisole,
THF, -78 ºC, 4 h
O
OH
p-TSA, MeOH,
rt, 3 h
OH
H
O
O
HO
BocHN
NBoc
75%
NBoc
73%
OMe
OMe
31
44
45
5:1 anti:syn
10-15% NaOCl,
TEMPO, acetone/
5% aq NaHCO3
(1:1 v/v), rt, 3 h
OMe
1) HCl in dioxane,
CH2Cl2, rt, 2 h
2) Fmoc-OSu,
NaHCO3, THF,
rt, 16 h
O
2,2-DMP, Acetone,
BF3.OEt2, rt, 4 h
HO
O
OH
O
OH
HO
FmocHN
HO
O
FmocN
30% over
4 steps
BocHN
OMe
OMe
47
46
48
Scheme 7. Synthesis of suitably protected (2S,3S)-b-OH-O-Me-Tyr 48
Having prepared b-OH amino acids 37, 43 and 48, we investigated the synthesis of deshydroxy
skyllamycin B 6 (Scheme 8). Beginning from resin-bound peptide 24 synthesized above, we
carried out on-resin esterification with b-OH-Leu residue 43 to afford resin-bound branched
peptide 49. Pleasingly, this proceeded efficiently and we could optimize this coupling to reduce
the number of equivalents of this precious building block used. Initially the next amino acid
required, Fmoc-D-Leu-OH was coupled using the PyBOP coupling conditions outlined earlier.
Unfortunately upon cleavage of a small portion of resin and HPLC-MS analysis no coupling
was observed. We turned to other more active coupling reagents such as HATU and COMU,
however unfortunately complete conversion was never observed. We presumed this was due
to the steric bulk of the oxazolidine protecting group and so investigated more forcing
conditions. Pleasingly, treating resin-bound 49 with Fmoc-D-Leu-OH, HATU, HOAt and
Hünig’s base at 50 °C under microwave irradiation for 1 h afforded the desired the desired
resin-bound 50. Extension of the peptide utilizing standard PyBOP conditions for Fmoc-Gly-
OH and Fmoc-D-Trp(Boc)-OH yielded 51, and was followed by coupling with the more active
HATU for precious b-OH-Tyr to afford resin-bound 52. We then coupled Fmoc-Pro-OH using
the microwave conditions described above and analyzed a small portion of cleaved resin by
HPLC-MS. Unfortunately, at this point the chromatogram showed a number of peaks with the
desired peptide. We reasoned that under the strongly acidic cleavage conditions
(TFA:iPr3SiH:H2O, 90:5:5 v/v/v) used to remove the tBu- and Boc-protecting groups the b-
OH-Tyr group was undergoing acid-catalyzed dehydration. Indeed, this issue was reported by
Lipton and co-workers in their synthesis of callipeltin A which also contains a b-OH-Tyr
residue29.
9
ACS Paragon Plus Environment

The Journal of Organic Chemistry
Page 10 of 39
1
2
3
4
5
6
7
8
9
O
O
H
H
N
H
N
H
N
N
N
H
COOtBu
N
H
COOtBu
DIC, CH2Cl2,
then DMF, DMAP,
16 h
O
O
O
O
O
O
O
O
N
H
HO
O
N
H
O
49
24
O
O
O
O
NFmoc
1) 10 vol% piperidine
in DMF 2 x 3 min
2) Fmoc-D-Leu-OH, DIC,
HO
O
FmocN
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
HOAt, DMF, 50 oC, 1 h
43
O
O
H
N
H
H
N
H
N
N
N
H
COOtBu
N
COOtBu
H
O
O
O
O
O
N
O
O
O
N
H
O
O
N
H
50
O
SPPS
O
O
O
O
O
O
N
O
O
H
N
O
NHFmoc
N
H
NHFmoc
51
N
Boc
1) 10 vol% piperidine
in DMF 2 x 3 min
2) 1.2 eq 48,
1.2 eq HATU,
2.4 eq HOAt
2.4 eq iPr2EtN, 16 h
O
O
H
N
H
N
H
N
H
N
N
H
COOH
N
H
COOtBu
O
O
O
O
N
O
O
O
O
N
H
O
O
N
H
OH
OH
HN
O
O
O
O
1) 10 vol% piperidine
in DMF 2 x 3 min
O
HN
O
O
O
H
N
2) 4 eq Fmoc-Pro-OH,
4 eq HATU, 8 eq HOAt,
8 eq iPr2EtN, 50 oC, 1 h
3) TFA:iPr3SiH:H2O
(90:5:5 v/v/v), 2 h
H
N
O
O
O
N
H
O
N
H
O
Fmoc
N
NH
N
H
N
H
O
HO
N
H
N
Boc
O
O
52
Scheme 8. Initial attempted synthesis of deshydroxy skyllamycin B 6
This required us to revise our synthetic strategy, specifically the choice of alternate protecting
groups which would be cleaved under more mildly acidic conditions. With this in mind we
decided to leave the indole ring of the Trp residue unprotected (Scheme 9). We also decided to
employ the hyper acid-labile phenylisopropyl (PhiPr) protecting group for the side chain of the
30
Asp residue, which can be cleaved with 1vol.% TFA in CH2Cl2 . Interestingly, during these
initial studies, the oxazolidine protecting groups were never observed when analyzing peptides
cleaved from the resin by HPLC-MS. This was true even when the peptide was cleaved from
resin using the very mildly acidic HFIP conditions. While the hyper-acid lability of these
protecting groups is not fully understood, it is possible that the bulky amino acid side chains
enhances the acid lability. Importantly, this unexpected acid lability meant that these protecting
groups would still be suitable to be used in the revised synthetic strategy. As such, we targeted
resin-bound protected linear peptide 53.
10
ACS Paragon Plus Environment

Page 11 of 39
The Journal of Organic Chemistry
1
2
3
4
5
6
7
8
9
O
H
O
H
N
H
N
H
N
N
N
H
COOPhiPr
N
H
COOH
O
O
O
O
O
O
O
N
H
O
N
O
O
OH
H
HN
O
H2N
O
O
O
O
O
OH
HN
N
O
O
O
O
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
OH
H
N
H
N
O
N
N
O
O
O
N
H
O
N
H
O
1) Cleavage
2) Cyclization
3) Deprotection
NH
N
H
N
H
O
HO
N
H
N
H
O
O
6
53
Scheme 9. Revised synthetic approach to deshydroxy skyllamycin B 6
The final amino acid required for the synthesis of the deshydroxy skyllamycin analogues was
the (2S,3S)-b-Me-Asp. Having altered our synthetic strategy to the analogues, the more acid-
labile PhiPr protecting group was required on the side-chain carboxylic acid of the building
block (Scheme 10). Towards this end, H2N-b-Me-Asp(tBu)-OH 54 was first synthesized
following a report by Goodman and co-workers31. An Fmoc and an allyl protecting group was
installed on the amine and carboxylic acid, respectively to afford 55, followed by the removal
of the side-chain t-butyl and installation of the desired PhiPr protecting group yielding 56. Final
removal of the allyl ester yielded the suitably protected amino acid 57 ready for incorporation
into Fmoc-SPPS.
1) Fmoc-OSu,
NaHCO3,THF,
rt, 16 h
2) Allyl-Br, iPr2NEt,
DMF, 0 ºC to rt, 16 h
1) TFA/CH2Cl2, rt, 3 h
2) PhiPr-trichloroacetimidate,
CH2Cl2, hexanes, rt, 40 h
tBuOOC
tBuOOC
PhiPrOOC
H2N
COOH
49% over 4 steps
FmocHN
COOAll
FmocHN
COOAll
quant.
54
55
56
Pd(PPh3)4,
PhSiH3,
THF, rt, 2 h
PhiPrOOC
FmocHN
COOH
57
Scheme 10. Synthesis of suitably protected b-Me-Asp 57
With all the required suitably protected non-proteinogenic amino acids in hand we next
embarked on the synthesis of deshydroxy skyllamycins A-C 5-7 via our revised synthetic
strategy. We began by loading Fmoc-Gly-OH to 2-CTC resin (Scheme 11). To this was coupled
either Asp or b-Me-Asp, both suitably protected with the PhiPr protecting group. The peptide
chains were then extended under PyBOP coupling conditions followed by coupling of the
appropriate cinnamic acid to yield resin-bound 58-60. The key on-resin esterification was then
carried out using b-OH-Leu building block 43, followed by the optimized microwave coupling
of Fmoc-D-Leu-OH to the resin. Extension of the peptide using standard PyBOP conditions for
Fmoc-Gly-OH and Fmoc-D-Trp-OH, and the more active HATU for precious b-OH-Tyr.
Microwave coupling of Fmoc-Pro-OH under the above described conditions was then carried
11
ACS Paragon Plus Environment

The Journal of Organic Chemistry
Page 12 of 39
1
2
3
4
5
6
out followed by final coupling of the Fmoc-b-OH-Phe building block 37 to yield resin-bound
linear peptides 61, 53 and 62. Pleasingly, when a small amount of peptide was cleaved from
resin with side chain deprotection using 1vol.% TFA in CH2Cl2 and analyzed by HPLC-MS,
we observed the desired deprotected product without any decomposition.
7
8
9
Initial experiments carried out to cleave the peptides from resin under standard conditions (30%
HFIP in CH2Cl2, 30 mins) led to partial removal of the hyper acid labile PhiPr group. The
cleavage conditions were thus optimized to 20% HFIP in CH2Cl2 for 4 x 5 min treatments,
which resulted in no unwanted PhiPr cleavage and generation of protected linear peptides 63-
65. With the linear peptides in hand we then carried out the cyclization under the optimized
reaction conditions described for simplified skyllamycin analogue 4. Pleasingly, these
conditions (DMTMM×BF4, DMF, 0.01 M) resulted in complete consumption of starting
material and conversion to the desired product. Removal of the DMF, followed by mild
cleavage of the PhiPr with 1% TFA in CH2Cl2, with iPr3SiH as a cation scavenger and final
purification by reverse-phase HPLC yielded the desired deshydroxy skyllamycins A-C 5-7 in
4-8% over 25 steps (88-90% per step).
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
Cl
SPPS
O
R2
O
R2
H
N
H
N
H
H
N
N
SPPS
N
H
COOPhiPr
N
H
COOPhiPr
O
N
O
O
O
O
O
O
O
N
H
HO
O
N
H
O
H2N
O
R2
O
O
O
Me
H
58
59
60
O
O
OH
H
N
O
N
N
H
O
N
H
O
N
H
R2
O
Me
H
61
N
H
53
62
H
O
20 vol.% HFIP
in CH2Cl2,
4 x 5 min
O
R2
O
R2
H
N
H
N
H
N
H
N
N
H
COOPhiPr
N
H
COOH
O
O
O
O
O
O
O
O
N
H
O
O
N
H
OH
OH
OH
HN
H2N
O
O
O
O
O
O
OH
HN
HN
1) DMTMM, iPr2NEt,
DMF, 16 h (0.01 M)
2) TFA:iPr3SiH:CH2Cl2
(1:5:94 v/v/v)
O
O
N
H
N
H
N
OH
N
O
O
O
O
N
H
O
N
H
O
NH
NH
N
H
N
H
5 5.2% over 25 steps
6 7.5% over 25 steps
7 3.9% over 25 steps
HO
HO
N
H
N
H
O
O
R2
R2
Me
H
Me
H
63
64
65
deshydroxy skyllamycin A 5
deshydroxy skyllamycin B 6
H
deshydroxy skyllamycin C 7
H
Scheme 11. Synthesis of deshydroxy skyllamycins A-C 5-7
12
ACS Paragon Plus Environment

Page 13 of 39
The Journal of Organic Chemistry
1
2
3
4
5
6
7
8
9
Having successfully completed the synthesis of these skyllamycin analogues we next sought
to apply the optimized synthetic route to the native natural products. The most interesting
feature of these natural products is the unusual a-OH-Gly residue which is known to be stable
in the mature cyclic peptide3. Indeed, the intramolecular hydrogen bonding network present in
the natural products is thought to stabilize this unusual residue. However, we reasoned that this
residue would not be sufficiently stable to repeated SPPS conditions and a final acidic
cleavage32 which was verified during attempts to synthesize the amino acid (not shown). As
such, in order to assemble the natural products we envisaged we could carry out a final
cyclization and concomitant installation of the a-OH-Gly residue in one step (Scheme 12).
This could be carried out by reaction between the C-terminal amide and an N-terminal aldehyde
66-68, which in turn could be accessed via an oxidative cleavage reaction from the N-terminal
serine residue. Resin-bound linear peptide precursors 69-71 in turn could be synthesized on
Sieber amide resin, beginning from the Trp residue, which would reveal a C-terminal amide
upon cleavage33. In contrast to the synthesis of deshydroxy skyllamycins A-C 5-7, the key on-
resin esterification step in this synthetic sequence would now be carried out after installation
of both the b-OH-Tyr and b-OH-Phe residues. As such, in order to prevent unwanted
esterification during the synthesis, we protected the b-OH-Phe as the oxazolidine to yield 72
(Scheme 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
13
ACS Paragon Plus Environment

The Journal of Organic Chemistry
Page 14 of 39
1
2
3
4
5
6
7
8
9
O
R2
O
H
R2
H
N
H
N
H
N
N
N
H
N
COOH
COOH
H
O
O
O
O
O
O
O
O
N
H
O
O
N
H
OH
OH
HN
HN
O
O
O
O
O
OH
O
OH
HN
HN
O
O
N
N
H
N
H2N
O
O
O
O
N
H
O
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
N
H
O
OH
NH
O
NH
cyclization
N
H
N
H
HO
HO
N
N
H
O
O
H
R2
R2
Me
H
66
Me
H
Skyllamycin A 1
Skyllamycin B 2
Skyllamycin C 3
67
68
H
H
1) resin cleavage
2) oxidative cleavage
O
H
N
R2
H
N
N
COOPhiPr
H
O
O
N
O
O
O
N
H
N
O
O
O
O
O
O
N
NHFmoc
O
NH2
O
N
H
HO
H
N
O
O
N
SPPS
N
H
O
Sieber amide resin
NH
O
R2
Me
H
69
70
71
H
Scheme 12. Retrosynthetic analysis of skyllamycins A-C 1-3
O
OH
O
2,2-DMP, acetone,
BF3.OEt2, rt, 4 h
HO
HO
FmocHN
37
94%
O
FmocN
72
Scheme 13. Synthesis of oxazolidine protected b-OH-Phe 72
Construction of the linear peptide began with loading of acid-labile Sieber amide resin with
Fmoc-D-Trp-OH followed by coupling of b-OH-Tyr 48 to yield resin-bound dipeptide 73
(Scheme 14). Subsequent Fmoc-deprotection and microwave-assisted coupling of Fmoc-Pro-
OH yielded resin-bound tripeptide 74. This was followed by coupling of protected b-OH-Phe
72 and microwave-assisted coupling of Fmoc-Gly-OH. This yielded the key resin-bound
pentapeptide 75 which was used as the key intermediate for the synthesis of the three natural
products.
14
ACS Paragon Plus Environment

Page 15 of 39
The Journal of Organic Chemistry
1
2
3
4
5
6
NHFmoc
1) 10 vol% piperidine in DMF, 2 x 3 min
2) Fmoc-D-Trp-OH, PyAOP, NMM, DMF, 16 h
3) Ac2O:Pyridine (1:9, v/v), 10 min
4) 10 vol% piperidine in DMF, 2 x 3 mn
5) 48, HATU, HOAt, iPr2EtN, DMF, 16 h
H
N
O
O
Fmoc
N
N
H
O
O
7
8
9
NH
NHFmoc
O
73
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) 10 vol% piperidine
in DMF, 2 x 3 min
2) Fmoc-Pro-OH, HATU,
HOAt, iPr2EtN, DMF
50 oC, 1 h
O
FmocHN
N
O
O
1) 10 vol% piperidine in DMF,
2 x 3 min
2) 72, HATU, HOAt, iPr2EtN,
DMF, 16 h
3) 10 vol% piperidine in DMF
2 x 3 min
4) Fmoc-Gly-OH, HATU, HOAt,
iPr2EtN, DMF, 50 oC, 1 h
N
FmocN
O
O
H
N
H
N
O
O
O
O
N
N
N
H
N
H
O
O
NH
NH
O
O
74
75
SPPS
O
R2
O
R2
H
H
N
H
N
H
N
N
N
H
COOPhiPr
O
N
H
COOH
1) 43, DIC, CH2Cl2,
30 min, then DMF,
DMAP, 16 h
2) 10 vol% piperidine
in DMF 2 x 3 min
3) Fmoc-D-Leu-OH, DIC,
HOAt, DMF, 50 oC, 1 h
4) 10 vol% piperidine
in DMF 2 x 3 min
5) Fmoc-Ser-OH, PyBOP,
NMM, DMF, 1 h
O
O
O
O
O
O
N
H
O
O
N
H
HO
OH
HN
N
O
O
O
O
OH
O
HN
O
N
N
NH2
H2N
O
O
N
H
H
N
O
O
O
O
N
NH
HO
N
H
N
H
O
6) 10 vol% piperidine
in DMF 2 x 3 min
7) TFA:iPr3SiH:CH2Cl2
(1:5:94), 30 min
HO
O
NH
NH
O
R2
R2
Me 11%
Me
H
79
80
81
76
12%
12%
H
H
77
78
H
Scheme 14. Synthesis of linear peptides 79-81
Resin-bound 75 was then coupled with either Fmoc-Asp(PhiPr)-OH or Fmoc-b-Me-
Asp(PhiPr)-OH 57 and the peptide further elongated before coupling of the appropriate
cinnamic acid to yield resin-bound 76-78. The key on-resin esterification was then performed
to yield the desired branched peptides. At this stage when analyzing a sample of the cleaved
peptide we noticed the presence of a small number of peptides containing two or three b-OH-
Leu moieties. Considering the lability of the oxazolidine protecting groups to acid, we
hypothesize that these were removed at some point during the SPPS. Whilst surprising, this is
potentially due to conformational effects exerted by the peptide backbone. However, by
reducing the number of equivalents (to 3 equiv) and then retreating the peptide with a further
0.5 equiv we were able to achieve near quantitative esterification with minimal over-
esterification.
15
ACS Paragon Plus Environment

The Journal of Organic Chemistry
Page 16 of 39
1
2
3
4
5
6
7
8
9
Next, we carried out microwave-assisted coupling of Fmoc-D-Leu-OH under the previously
optimized conditions – namely HATU, HOAt and Hünig’s base. Pleasingly, this reaction
proceeded efficiently for the synthesis of skyllamycins B and C. Unfortunately, when the same
conditions were used for skyllamycin A and a small portion of crude peptide was analyzed, we
noticed the presence of a large amount of an unwanted by-product, with a mass corresponding
to that of the target peptide with the loss of water. This was presumably due to the formation
of an aspartimide, a common by-product during Fmoc-SPPS34. We hypothesize that this was
only the case during the synthesis of skyllamycin A due a conformational effect induced by the
b-Me group on the Asp residue. Interestingly, this was never observed during the synthesis of
deshydroxy skyllamycin A 5 and this may be attributed to the proximity of the b-Me-Asp
residue to the resin, with the steric bulk of the 2-Cl-Trt resin linker preventing formation of the
aspartimide. Aspartimides are generally formed due to excess base present in the Fmoc
deprotection reaction, however in this case it was proposed that the large excess of Hünig’s
base and extended microwave heating had led to the formation of this by-product. As such, we
trialled base-free conditions to affect the coupling of Fmoc-D-Leu-OH to the oxazolidine
protected b-OH-Leu residue. Specifically, the transformation was carried out using DIC and
HOAt with microwave heating at 50 °C with no external base added. Pleasingly, this
successfully forged the desired bond with no aspartimide formation. In subsequent syntheses
of skyllamycins B 2 and C 3 these optimized base-free microwave conditions were used when
carrying out this coupling. A final coupling of Fmoc-Ser-OH followed by cleavage from the
resin and PhiPr cleavage under mildly acidic conditions furnished the desired unprotected
linear peptides 79-81.
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
Having prepared the linear peptides 79-81 we were able to embark on the end-game of the
synthesis of the skyllamycins. Specifically, we treated peptides 79-81 with NaIO4 in a mixture
of mildly basic Na2HPO4 buffer (pH = 9) and MeCN35 (Scheme 15). Pleasingly, this led to
rapid oxidative cleavage to the aldehyde in 10 min, at which point a solution of excess ethylene
glycol was added to quench the reaction. It was important to keep the pH of the solution
controlled, as initial attempts using aqueous NaHCO3 led to hydrolysis of the ester bond. The
linear peptide aldehydes were then immediately purified by RP-HPLC and lyophilized to
furnish aldehydes 66-68 in yields of 62-68%.
O
R2
O
R2
H
N
H
N
H
N
H
N
N
COOH
N
H
COOH
H
O
O
O
O
O
O
O
O
N
H
O
O
O
N
H
OH
OH
HN
NH
HN
O
O
O
O
OH
O
OH
O
HN
HN
O
N
O
N
NaIO4, Na2HPO4 buffer,
10 min, then (CH2OH)2
NH2
H2N
H2N
O
O
O
N
H
N
H
O
O
O
NH
HO
N
H
N
H
HO
HO
O
N
H
O
NH
79
R2
Me
H
R2
Me 62%
66
80
81
63%
68%
67
68
H
H
H
Scheme 15. Synthesis of aldehydes 66-68
Having successfully obtained linear peptide aldehydes 66-68, the proposed cyclization reaction
was first investigated on skyllamycin B. We reasoned that the C-terminal amide and N-terminal
16
ACS Paragon Plus Environment

Page 17 of 39
The Journal of Organic Chemistry
1
2
3
4
5
6
7
8
9
aldehyde would react under mildly acidic conditions and, as such, incubated peptide 67 in a
solution of 1% TFA in MeCN for 20 h. Pleasingly, monitoring by HPLC-MS revealed the
consumption of starting material and the presence of two new peaks, both with the desired
mass of the natural product. The two compounds were then isolated and analyzed by 1H-NMR,
however unfortunately both compounds had very different spectra to the isolated natural
product. We hypothesized that these products might be cyclic hemi-acetals, formed by attack
of one of the b-OH groups onto the aldehyde which would afford an identical mass to the target
natural products.
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
Gratifyingly, the next cyclization conditions we attempted proved successful. Specifically, we
incubated linear aldehyde 67 in MeCN at 60 °C and analyzed the reaction by HPLC-MS
(Scheme 16). After 25 h all starting material 67 was consumed and a new peak with the desired
mass and different retention time to those observed previously appeared. This peak was isolated
1
in 42% yield after HPLC, analyzed by H-NMR and the compared to the authentic natural
material. Unfortunately, the NMR analysis revealed that the isolated peak was in fact a 1:1
mixture of two compounds, however one set of signals overlapped with the isolated material.
This mixture was then subject to careful UPLC-MS co-injection studies with the isolated
natural product which revealed that the synthetic mixture did indeed contain the natural
product. Using a highly optimized HPLC protocol we were able to separate the mixture and
isolated skyllamycin B 2 in 19% yield. This purified material was further subject to
comprehensive NMR studies and possessed almost identical spectra to the isolated material.
While the identity of the compound which co-eluted with skyllamycin B was not definitively
confirmed, we suspect that it was the epimer at the newly formed a-OH-Gly position.
O
R2
O
R2
H
N
H
N
H
N
H
N
N
H
COOH
N
H
COOH
O
O
O
O
O
O
O
O
N
H
O
O
N
H
OH
OH
HN
HN
O
O
O
O
O
OH
O
OH
HN
HN
MeCN, 60 oC, 25 h
O
O
N
N
H
H2N
O
O
O
N
O
O
N
H
O
N
H
O
NH
OH
NH
N
H
N
H
HO
HO
N
H
N
H
O
O
R2
R2
Me
H
Skyllamycin A 1
Skyllamycin B 2
Skyllamycin C 3
Me
H
32%
19%
33%
66
67
68
H
H
Scheme 16. Synthesis of skyllamycins A-C 1-3
Having successfully synthesized skyllamycin B we next applied the optimized conditions to
the synthesis of skyllamycins A 1 and C 3. Pleasingly, this resulted in the formation of new
peaks with the desired mass by HPLC-MS. Interestingly, in both of these cases the HPLC-MS
profiles were very different to those observed for skyllamycin B, implying that slight changes
in structure made a large change in the elution profile of the natural products. Importantly, the
peaks resolved much more efficiently, and skyllamycins A and C could be isolated in 32% and
33% yields respectively. The synthesized natural products were then compared to the isolated
material by UPLC-MS co-injection and shown to have identical elution times. Synthetic
skyllamycins A 1 and C 3 were also comprehensively analyzed by NMR and produced spectra
17
ACS Paragon Plus Environment

The Journal of Organic Chemistry
Page 18 of 39
1
2
3
4
5
6
which were extremely consistent with the isolated natural products suggesting the synthetic
material was identical to the isolated natural products. All three natural products exhibited
highly analogous circular dichroism spectra which strongly suggests that the synthetic and
isolated material are the same enantiomer.
7
8
9
Given our ongoing interest in the development of anti-biofilm lead compounds against
Pseudomonas aeruginosa, the simplified analogue 4, deshydroxy skyllamycins A-C 5-7 and
both isolated and synthetic skyllamycins A-C 1-3 were tested in our image-based biofilm
inhibition assay4. Evaluation of biofilm coverage from these images revealed moderate
activities for all compounds at the top tested concentrations. Surprisingly, neither removal of
the hydroxyl group from the hydroxyglycine motif (compounds 5-7) nor removal of the b-OH
moieties from the three b-OH amino acids and elimination of the methyl group from the -OMe
tyrosine (compound 4) dramatically impacted activity. This result suggests that variation at
these positions is well tolerated, paving the way for the development of simplified skyllamycin
analogue libraries using standard SPPS reagents and methods.
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
Conclusion
In summary, the total synthesis of skyllamycins A-C, as well as the generation of four
simplified analogues is described. We were able to efficiently synthesize the non-proteinogenic
amino acids present in the natural products that were suitable for direct incorporation into
Fmoc-SPPS. Simplified skyllamycin analogue 4 and deshydroxy skyllamycins A-C 5-7 were
first rapidly assembled using an efficient SPPS protocol and solution phase macrolactamization
strategy. An alternate SPPS strategy needed to be employed for the synthesis of the linear
precursors 79-81 of the skyllamycin natural products. The linear peptide precursors were
successfully converted to skyllamycins A-C 1-3 using a novel cyclization strategy that
proceeded with concomitant formation of the unusual a-OH-Gly residue in the final step. The
synthetic natural products were consistent with the structures of the isolated natural products
as confirmed by spectroscopic studies. The reported study provides insight into these highly
modified natural products, and the synthetic route is amenable to the generation of further
analogues for detailed structure-activity relationships against bacterial biofilms as well as other
phenotypic screens. Studies towards this end will serve as future research efforts in our
laboratories.
Experimental Section
All reactions were carried out in dried glassware under an argon atmosphere and at room
o
temperature (22 C) unless aqueous conditions were used or unless otherwise specified.
Reactions undertaken at -78 °C utilized a bath of dry ice and acetone. Reactions carried out at
0 °C employed a bath of water and ice. Anhydrous THF, CH2Cl2, MeCN, DMF, toluene and
MeOH were obtained using a PureSolv® solvent purification system with water detectable only
in low ppm levels. Reactions were monitored by thin layer chromatography (TLC) on
aluminium backed silica plates (Merck Silica Gel 60 F254). Visualization of TLC plates was
undertaken with an ultraviolet (UV) light at l = 254 nm and staining with solutions of vanillin,
ninhydrin, phosphomolybdic acid (PMA), potassium permanganate or sulfuric acid, followed
by exposure of the stained plates to heat. Silica flash column chromatography (Merck Silica
Gel 60 40 – 63 µm) was undertaken to purify crude reaction mixtures using solvents as
specified.
18
ACS Paragon Plus Environment

Page 19 of 39
The Journal of Organic Chemistry
1
2
3
4
5
6
7
8
9
All commercially available reagents were used as obtained from Sigma-Aldrich, Merck or
Acros Organics. Amino acids, coupling reagents and resins were obtained from NovaBiochem
or GL Biochem and peptide synthesis grade DMF was obtained from Merck or Labscan. All
non-commercially available reagents were synthesized according to literature procedures as
referenced. Microwave-assisted peptide couplings were carried out using a Biotage Initiator+
Alstra microwave peptide synthesizer equipped with an inert gas manifold. Fmoc-strategy
solid-phase peptide synthesis (Fmoc-SPPS) procedures were employed using HMPB
functionalized polyethylene glycol resin (HMPB-NovaPEG), 2-CTC functionalized
polystyrene resin or Sieber amide functionalized resin within fritted syringes (purchased from
Torviq). All reagent equivalents are in regard to the amount of amino acid loaded to resin.
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
1H NMR spectra were obtained using a Bruker DRX 400 or DRX 500 at frequencies of 400
MHz or 500 MHz respectively in CDCl3, acetone-d6, CD3OD or DMSO-d6. Chemical shifts
are reported in parts per million (ppm) and coupling constants in Hertz (Hz). The residual
solvent peaks were used as internal standards without the use of tetramethylsilane (TMS). 1H
NMR data is reported as follows: chemical shift values (ppm), multiplicity (s = singlet, d =
doublet, t = triplet, q = quartet, m = multiplet, br. = broad, ap. = apparent), coupling constant(s)
and relative integral. 13C NMR spectra were obtained using a Bruker DRX 400 or DRX 500 at
100 MHz or 125 MHz in CDCl3, MeOD, acetone-d6 or DMSO-d6 unless otherwise specified.
13C NMR data is reported as chemical shift values (ppm). Any rotamers were confirmed by
saturation transfer experiments, or the presence of in-phase cross-peaks in a NOESY spectrum.
Low-resolution mass spectra for novel compounds were recorded on a Bruker amaZon SL mass
spectrometer (ESI) operating in positive mode or on a Shimadzu 2020 (ESI) mass spectrometer
operating in positive mode. High resolution mass spectra were recorded on a Bruker-Daltronics
Apex Ultra 7.0T Fourier transform (FTICR) mass spectrometer. Circular dichroism spectra
were recorded on a Chirascan qCD from 600 nm to 200 nm at a 1 nm resolution at a scan rate
of 0.5 scans/sec.
LC-MS was performed either on a Shimadzu 2020 LC-MS instrument with an LC-M20A
pump, SPD-20A UV/Vis detector and a Shimadzu 2020 (ESI) mass spectrometer operating in
positive mode or on a Shimadzu UHPLC-MS equipped with the same modules as above but
with an SPD-M30A diode array detector. Separations on the LC-MS system were performed
on a Waters Sunfire 5 µm, 2.1 x 150 mm (C18) column. On the UHPLC-MS system,
separations were performed on a Waters Acquity 1.7 µm, 2.1 x 50 mm (C18) column. These
separations were performed using a mobile phase of 0.1 vol% formic acid in water (Solvent A)
and 0.1 vol% formic acid in MeCN (Solvent B) using linear gradients. Preparative reverse-
phase HPLC was performed using a Waters 500 pump with a 2996 photodiode array detector
and a Waters 600 Multisolvent Delivery System.
Fmoc-SPPS General Protocols
Fmoc Deprotection
A given resin-bound peptide was washed with CH2Cl2 (x 5) and DMF (x 5) before being treated
with a solution of 10 vol% piperidine in DMF (2 x 3 min). The resin was again washed with
DMF (x 5), CH2Cl2 (x 5) and DMF (x 5).
PyBOP Coupling Conditions
A given resin-bound peptide was washed with CH2Cl2 (x 5) and DMF (x 5) before being treated
with a solution of 10 vol% piperidine in DMF (2 x 3 min). The resin was again washed with
DMF (x 5), CH2Cl2 (x 5) and DMF (x 5). The resin was shaken for 1 h at room temperature
with a solution of the desired Fmoc-protected amino acid (4 equiv.), PyBOP (4 equiv.) and 4-
19
ACS Paragon Plus Environment

The Journal of Organic Chemistry
Page 20 of 39
1
2
3
4
5
6
methylmorpholine (NMM) (8 equiv.) in DMF (0.1 M in regard to loaded peptide). The
coupling solution was discharged and the resin washed with DMF (x 5), CH2Cl2 (x 5) and DMF
(x 5).
7
8
9
HATU Coupling Conditions
A given resin-bound peptide was washed with CH2Cl2 (x 5) and DMF (x 5) before being treated
with a solution of 10 vol% piperidine in DMF (2 x 3 min). The resin was again washed with
DMF (x 5), CH2Cl2 (x 5) and DMF (x 5). The resin was shaken for 16 h at room temperature
with a solution of the desired Fmoc-protected amino acid (1.1-1.5 equiv.), HATU (1.1-1.5
equiv.), HOAt (2.2-3 equiv.) and iPr2NEt (2.2-3 equiv.) in DMF (0.1 M in regard to loaded
peptide). The coupling solution was discharged and the resin washed with DMF (x 5), CH2Cl2
(x 5) and DMF (x 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
On-resin esterification conditions
To a solution of oxazolidine protected Fmoc-D-Leu-OH or Fmoc-β-OH-D-Leu-OH 43 (4 equiv.
compared to the resin-bound peptide) in CH2Cl2 (0.06 M) at 0 ºC was added DIC (1 equiv.
compared to amino acid). The solution was warmed to room temperature and stirred for 30 min
before being concentrated under a stream of N2. The resultant slurry was dissolved in DMF
(0.1 M in regard to loaded peptide) and sucked into the fritted syringe containing resin-bound
peptide. Subsequently, a solution of DMAP (catalytic, ~6 small crystals) in DMF (0.15 mL)
was sucked up and the resin was shaken at room temperature for 16 h. The coupling solution
was discharged and the resin washed with DMF (x 5), CH2Cl2 (x 5) and DMF (x 5).
Microwave coupling conditions to oxazolidine protected amino acid
The resin was transferred to a Biotage microwave peptide synthesis reaction vessel and treated
with a solution of the desired Fmoc-protected amino acid (4 equiv.), HATU (4 equiv.), HOAt
(8 equiv.) and iPr2NEt (8 equiv.) in DMF (0.1 M in regard to loaded peptide) under microwave
irradiation at 50 ºC for 1 h (sealed reaction vessel, temperature monitored via internal probe).
The resin was then transferred to a fritted syringe, the coupling solution was discharged and
the resin washed with DMF (x 5), CH2Cl2 (x 5) and DMF (x 5).
2-CTC resin loading
2-CTC resin (maximum loading 0.9 mmol/g) in a fritted syringe was swollen in CH2Cl2 (5 mL)
for 30 min before being washed with a solution of 20 vol% iPr2NEt in CH2Cl2 (2 x 5 mL). The
resin was then shaken with a solution of Fmoc-Gly-OH (0.6 equiv. relative to resin
functionalization) and iPr2NEt (1.2 equiv. relative to resin functionalization) in CH2Cl2 at room
temperature for 16 h. The resin was then washed CH2Cl2 (x 5), DMF (x 5) and CH2Cl2 (x 5),
and then treated with a solution of CH2Cl2:MeOH: iPr2NEt (v/v/v, 17:2:1, 5 mL) for 30 min.
The resin was then washed CH2Cl2 (x 5), DMF (x 5) and CH2Cl2 (x 10) before being dried
under high vacuum and accurately split by weighing before the resin loading was determined.
Loaded resin was treated with a solution of 10 vol% piperidine in DMF (2 x 3 min) which was
then diluted (100 µL in 10 mL) and the absorbance analyzed at λ = 301 nm to determine number
of µmol of amino acid loaded to resin.
2-CTC resin cleavage conditions
Resin-bound peptide was washed with CH2Cl2 (x 5) and DMF (x 5) before being treated with
a solution of 10 vol% piperidine in DMF (2 x 3 min). The resin was again washed with DMF
(x 5), CH2Cl2 (x 5), DMF (x 5) and CH2Cl2 (x 20). The resin was treated with a 20 vol%
solution of HFIP in CH2Cl2 (~5 mL, 4 x 4 min) and the solution transferred to a round bottom
20
ACS Paragon Plus Environment

Page 21 of 39
The Journal of Organic Chemistry
1
2
3
4
5
flask and diluted with CH2Cl2 (30 mL) before being concentrated under a stream of N2 and
dried in vacuo.
6
7
Sieber amide loading
8
9
Sieber amide resin (550 mg, maximum loading 0.73 mmol/g) was placed in a fritted syringe
and swollen in CH2Cl2 (10 mL) for 30 min before being washed with DMF (x 5). The resin
was then treated with a solution of 10 vol% piperidine in DMF (2 x 3 min) and washed with
DMF (x 5), CH2Cl2 (x 5) and DMF (x 5) before being shaken with a solution of Fmoc-D-Trp-
OH (255 mg, 600 µmol), PyAOP (312 mg, 600 µmol) and N-methylmorpholine (131 µL, 1.2
mmol) in DMF (4 mL) at room temperature for 16 h. The resin was then washed DMF (x 5)
and CH2Cl2 (x 5), and then treated with a solution of pyridine:Ac2O (v/v, 9:1, 5 mL) for 10
min. The resin was then washed with DMF (x 5), CH2Cl2 (x 5), and DMF (x 5) before the resin
loading was determined. Loaded resin was treated with a solution of 10 vol% piperidine in
DMF (2 x 3 min) which was then diluted and the absorbance analyzed at λ = 301 nm to
determine number of µmol of amino acid loaded to resin.
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
Sieber amide resin cleavage conditions
Resin-bound peptide was washed with CH2Cl2 (x 5) and DMF (x 5) before being treated with
a solution of 10 vol% piperidine in DMF (2 x 3 min). The resin was again washed with DMF
(x 5), CH2Cl2 (x 5), DMF (x 5) and CH2Cl2 (x 20). The resin was treated with a solution of
TFA:iPr3SiH:CH2Cl2 (1:5:94, v/v/v) in CH2Cl2 (0.001 M wrt. resin loading) and the solution
transferred to a round bottom flask and diluted with CH2Cl2 (30 mL) before being concentrated
under a stream of N2 and dried in vacuo. Linear peptides were purified via HPLC using a
Waters Sunfire C18 OBD 19x150 mm column, using a 0-50 vol% MeCN in H2O (0.1% TFA)
focused gradient (0-40 vol% MeCN over 5 min, 40-50 vol% over 15 min) at a flow rate flow
rate of 16 mL min-1 and lyophilized.
General procedures for modified amino acid synthesis
General Procedure A: Selective oxidation of the primary alcohol
To a solution of amino-diol (1 equiv.) in acetone (0.2 M) was added TEMPO (0.2 equiv.)
followed by aqueous NaHCO3 (5% g/100 mL) to bring the solution to 0.1 M. The mixture was
cooled to 0 ºC and sodium hypochlorite (3.5 equiv., 10-15% by weight) was added portionwise
over 25 min. The mixture was warmed to room temperature and stirred for 3 h – 16 h (until
judged complete by TLC analysis). The reaction mixture was diluted with water and the
aqueous phase was washed with Et2O (2 x) then acidified to pH = 2 with aqueous HCl (1 M).
The aqueous phase was extracted with EtOAc (3 x) and the combined organic layers were dried
(MgSO4), filtered and concentrated in vacuo.
General Procedure B: Boc deprotection followed by Fmoc protection
To a solution of amino acid (1 equiv.) in CH2Cl2 (0.16 M) was added HCl in dioxane (10 equiv.,
4 M). The solution was stirred at room temperature for 1.5 -3 h (until judged complete by TLC
analysis) and the solvent was removed in vacuo. To a solution of the crude residue in a mixture
of THF: saturated aqueous NaHCO3 (2:1 v/v, 0.1 M) was added Fmoc-OSu (1.05 equiv.). The
reaction was stirred for 16 h at room temperature before being poured onto water and washed
with Et2O (2 x). The aqueous phase was then acidified to pH = 1 with aqueous HCl (1 M) and
extracted with EtOAc (3 x) and the combined organic layers were dried (MgSO4), filtered and
concentrated in vacuo.
General Procedure C: Oxazolidine protection of b-OH amino acid.
21
ACS Paragon Plus Environment

The Journal of Organic Chemistry
Page 22 of 39
1
2
3
4
5
6
7
8
9
To a solution of alcohol (1 equiv.) in acetone (0.14 M) was added 2,2-dimethoxypropane (10
equiv.) followed by BF3.OEt2 (0.1 equiv.). The reaction mixture was stirred at room
temperature for 5 - 16 h (until judged complete by TLC analysis). The reaction mixture was
poured onto saturated aqueous NH4Cl and extracted with EtOAc (3 x) and the combined
organic layers were dried (MgSO4), filtered and concentrated in vacuo.
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
General procedures for peptide cyclization
General Procedure D: Peptide cyclization conditions
Crude cleaved peptide was suspended in DMF (0.01 M) before a solution of DMTMM.BF4
(1.5 equiv.) in DMF (0.01 M) with respect to peptide) was added, to give an overall
concentration of 0.005 M of crude peptide, followed by iPr2NEt (2 equiv.) and stirred at room
temperature for 16 h. The reaction mixture was analyzed by HPLC-MS and concentrated under
a stream of N2. The crude solid was diluted with CH2Cl2 (~30 mL) and further concentration
by a stream of N2 and dried in vacuo.
General Procedure E: Cyclic peptide deprotection conditions
To a solution of crude cyclized peptide in CH2Cl2 (0.001 M wrt. crude linear peptide) was
added iPr3SiH (5% v/v of CH2Cl2) followed by TFA (1% v/v of CH2Cl2). The reaction mixture
was stirred for 30 min, then diluted with CH2Cl2 (equal to initial volume) and concentrated
under a stream of N2 and dried in vacuo. Cyclic peptides were purified via HPLC using a
Waters Sunfire C18 OBD 19x150 mm column, using a 0-60 vol% MeCN in H2O (0.1% TFA)
focussed gradient (0-50 vol% MeCN over 5 min, 50-60 vol% over 15 min) at a flow rate flow
rate of 16 mL min-1.
General procedures for the synthesis of skyllamycins A-C (1-3)
General Procedure F: Oxidative cleavage reaction
To a solution of linear peptide bearing an N-terminal serine residue (79-81) (6.5-7 µmol) in a
mixture of aqueous Na2HPO4 (1 M, pH = 8.4) (400 µL) and MeCN (400 µL) was added an
aqueous solution of NaIO4 (53-58 µL, 2 equiv., 10 mg in 200 µL of H2O). The mixture was
mixed using a vortex mixer before being allowed to react for 10 min. To the mixture was added
an aqueous solution of ethylene glycol (35-38 µL, 10 equiv., 20 µL in 200 µL of H2O). The
mixture was then diluted up to 3.5 mL with a mixture of MeCN and H2O (with 0.1% TFA) and
purified via HPLC using a Waters Sunfire C18 OBD 19x150 mm column, using a 0-60 vol%
MeCN in H2O (0.1% TFA) focussed gradient (0-50 vol% MeCN over 5 min, 50-60 vol% over
15 min) at a flow rate flow rate of 16 mL min-1 and lyophilized.
General Procedure G: Cyclization reaction
Linear peptide aldehyde (66-68) (5.8-6.8 mg) was dissolved in MeCN (4 mL) in 2 separate
microcentrifuge tubes. The solutions were incubated at 60 °C and the reaction was monitored
by HPLC-MS. After 25 h, each microcentrifuge tube was diluted up to 3.5 mL total volume
with H2O and then purified over two runs via HPLC using a Waters Sunfire C18 OBD 19x150
mm column, using a 0-60 vol% MeCN in H2O (0.1% TFA) focussed gradient (0-50 vol%
MeCN over 5 min, 50-60 vol% over 15 min) at a flow rate flow rate of 16 mL min-1. All peaks
were collected and lyophilized.
Synthesis of cinnamic acid 15
2-iodobenzaldehyde (19)
22
ACS Paragon Plus Environment

Page 23 of 39
The Journal of Organic Chemistry
1
2
3
4
5
6
7
8
9
To a solution of m-iodo benzyl alcohol 18 (3.00 g, 12.8 mmol) in CH2Cl2 (30 mL) was added
pyridinium chlorochromate (3.04 g, 15.14 mmol). The mixture was left to stir at room
temperature for 3.5 h at which point TLC showed the presence of starting material. A further
portion of pyridinium chlorochromate (0.5 g, 2.5 mmol) was added and the mixture was stirred
for a further 1 h. The mixture was concentrated in vacuo and crude residue was purified by
flash chromatography (eluent: 3:7 v/v EtOAc/petroleum benzines) to yield aldehyde 19 as a
pale yellow solid (2.42 g, 76%).
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
1H-NMR (CDCl3, 500 MHz) δ (ppm) 10.07 (d, J = 0.7 Hz, 1H), 7.95 (dd, J = 7.90, 0.75 Hz
1H), 7.88 (dd, J = 7.7, 1.8 Hz, 1H), 7.46 (t, J = 7.5 Hz, 1H), 7.28 (td, J = 7.6, 1.8 Hz, 1H);
LRMS (+ESI) m/z 233 [M+Na]+; mp. 38.2-39.9 ºC. These data are in agreement with those
previously reported by Tummatorn and Dudley36.
(E)-ethyl 3-(2-iodophenyl)acrylate (20)
To a solution of NaH (265 mg, 6.65 mmol, 60% dispersion in oil) in CH2Cl2 (24 mL) at 0 ºC
was added triethyl phosphonoacetate (0.96 mL, 4.87 mmol) dropwise. The mixture was stirred
at this temperature for 5 min. A solution of aldehyde 19 (1.1g, 4.43 mmol) in CH2Cl2 (19 mL)
was added dropwise to the above mixture and the resultant mixture was stirred at 0 ºC for 2 h.
The reaction mixture was quenched with cold H2O (10 mL) and poured onto H2O (20 mL).
The aqueous layer was extracted with EtOAc (4 x 20 mL). The combined organic layers were
dried (MgSO4), filtered and concentrated in vacuo. The crude product was purified by flash
chromatography (eluent: 3:97 → 5:95 v/v EtOAc/petroleum benzines) to yield iodide 20 as a
yellow oil which solidified on freezing (1.2 g, 89%).
1H-NMR (CDCl3, 400 MHz) δ (ppm) 7.90 (d, J = 15.8 Hz, 1H), 7.90 (dd, J = 8.0, 1.2 Hz, 1H),
7.56 (dd, J = 7.8, 1.6 Hz, 1H), 7.38-7.34 (m, 1H), 7.05 (td, J = 7.6, 1.6 Hz, 1H), 6.31 (d, J =
15.8 Hz, 1H), 4.29 (q, J = 7.1 Hz, 2H), 1.35 (t, J = 7.1 Hz, 3H); LRMS (+ESI) m/z 325
[M+Na]+. These data are in agreement with those previously reported by Sun et al.16
(E)-ethyl 3-(2-(prop-1-yn-1-yl)phenyl)acrylate (21)
To a solution of 1-bromo-1-propene (0.46 mL, 5.35 mmol) in THF (7.1 mL) at -78 ºC was
added n-BuLi (2.8 mL, 7.08 mmol, 2.5 M in hexanes) dropwise. The solution was stirred at
this temperature for 45 min. H2O (128 µL, 7.08 mmol) was added and the resultant solution
was warmed to room temperature. After 45 min, a solution of iodide 20 (535 mg, 1.77 mmol)
in THF (2.2 mL) was added dropwise, followed by CuI (34 mg, 0.18 mmol), iPr2NH (5.2 mL)
and Pd(PPh3)2Cl2 (62 mg, 0.089 mmol). The resultant mixture was stirred at room temperature
for 16 h. The reaction mixture was then poured onto saturated aqueous NH4Cl (30 mL), and
extracted with Et2O (3 x 30 mL). The combined organic layers were dried (MgSO4), filtered
and concentrated in vacuo. The crude product was purified by flash chromatography (eluent:
2:98 → 5:95 v/v EtOAc/petroleum benzines) to yield alkyne 21 as a yellow solid (344 mg,
91%).
1H-NMR (CDCl3, 500 MHz) δ (ppm) 8.21 (d, J = 16.1 Hz, 1H), 7.62-7.61 (m, 1H), 7.47-7.45
(m, 1H), 7.31-7.29 (m, 2H), 6.51 (d, J = 16.1 Hz, 1H), 4.29 (q, J = 7.1 Hz, 2H), 2.15 (s, 3H),
13
1.37 (t, J = 7.1 Hz, 3H); C-NMR (CDCl3, 125 MHz) δ (ppm) 167.1, 143.0, 135.8, 133.1,
129.7, 127.9, 126.1, 125.1, 119.5, 92.5, 77.2, 60.6, 14.5, 4.7; LRMS (+ESI) m/z 215 [M+H]+;
HRMS (+ESI) m/z: [M+Na]+ Calcd for C14H14O2Na 237.0886; Found: 237.0886; IR νmax
(ATR) 2980, 2916, 2247, 2212, 1710, 1633, 1478, 1315, 1268, 1176 cm-1; mp. 37.6-39.4 ºC.
(E)-ethyl 3-(2-((Z)-prop-1-en-1-yl)phenyl)acrylate (22)
23
ACS Paragon Plus Environment

The Journal of Organic Chemistry
Page 24 of 39
1
2
3
4
5
6
7
8
9
To a solution of alkyne 21 (257 mg, 1.2 mmol) in MeOH (11.8 mL) was added quinoline (154
µL, 1.24 mmol) and Lindlar’s catalyst (258 mg, 0.12 mmol, 5% Pd by weight). The solution
was stirred under an atmosphere of H2 for 20 min. A further portion of Lindlar’s catalyst (200
mg) was added and the mixture was stirred under an atmosphere of H2 for 50 min. The mixture
was then filtered over Celite® and concentrated in vacuo. The crude product was purified by
flash chromatography (eluent: 1:99 → 5:95 v/v EtOAc/petroleum benzines) to yield diene 22
as a colourless oil (230 mg, 88%).
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
1H-NMR (CDCl3, 400 MHz) δ (ppm) 7.89 (d, J = 16.0 Hz, 1H), 7.60 (d, J = 7.8 Hz, 1H), 7.36-
7.21 (m, 3H), 6.58 (dd, J = 11.4, 1.5 Hz, 1H), 6.39 (d, J = 16.0 Hz, 1H), 5.97-5.92 (m, 1H),
13
4.26 (q, J = 7.1 Hz, 2H), 1.66 (dd, J = 7.0, 1.8 Hz, 3H), 1.33 (t, J = 7.1 Hz, 3H); C-NMR
(CDCl3, 100 MHz) δ (ppm) 167.2, 143.1, 138.1, 133.0, 130.2, 129.6, 129.4, 127.9, 127.2,
126.6, 119.2, 60.5, 14.5 14.4; LRMS (+ESI) m/z 217 [M+H]+; HRMS (+ESI) m/z: [M+Na]+
Calcd for C14H16O2Na 239.1043; Found 239.1043; IR νmax (ATR) 2979, 1708, 1632, 1477,
1445, 1311, 1268, 1165 cm-1.
(E)-3-(2-((Z)-prop-1-en-1-yl)phenyl)acrylic acid (15)
To a solution of diene 22 (189 mg, 0.87 mmol) in ethanol (2.8 mL, 95%) at 0 ºC was added
LiOH (84 mg, 3.5 mmol). The solution was warmed to room temperature and stirred for 16 h.
The mixture was cooled to 0 ºC and acidified to pH = 1 with aqueous HCl (1 M). The mixture
was extracted with EtOAc (4 x 20 mL). The combined organic layers were dried (Na2SO4),
filtered and concentrated in vacuo to yield acid 15 as an off-white solid (152 mg, 92%).
1H-NMR (CDCl3, 400 MHz) δ (ppm) 8.01 (d, J = 16.0 Hz, 1H), 7.66 (dd, J = 7.6, 0.7 Hz, 1H),
7.38-7.24 (m, 4H), 6.59 (dd, J = 11.4, 1.5 Hz, 1H), 6.42 (d, J = 16.0, 1H), 5.98 (dq, J = 11.4,
7.0 Hz, 1H), 1.66 (dd, J = 7.0, 1.8 Hz, 3H); 13C-NMR (CDCl3, 100 MHz) δ (ppm) 172.4, 145.7,
138.5, 132.6, 130.3, 130.2, 129.8, 127.8, 127.3, 126.8, 118.1, 14.6; LRMS (+ESI) m/z 189
[M+H]+; HRMS (+ESI) m/z: [M+Na]+ Calcd for C12H12O2Na 211.0730; Found 211.0730; IR
ν
max (ATR) 3014 (broad), 1678, 1623, 1479, 1422, 1332, 1300, 1288, 1224 cm-1; mp. 128.7-
132.2 ºC.
Synthesis of reduced cinnamic acid 16
ethyl 3-(2-iodophenyl)propanoate (28)
To a solution of ester 20 (500 mg, 1.65 mmol) in ethanol (5.3 mL, 95%) at 0 ºC was added
LiOH (159 mg, 6.62 mmol). The solution was warmed to room temperature and stirred for
16 h. The mixture was cooled to 0 ºC and acidified to pH = 1 with aqueous HCl (0.1 M). The
mixture was extracted with EtOAc (3 x 20 mL). The combined organic layers were dried
(MgSO4), filtered and concentrated in vacuo to yield acid as a white solid (447 mg) which was
used without further purification.
To a solution of the above crude acid (447 mg, 1.63 mmol) in a solution of ethanol (5 mL) and
EtOAc (5 mL) was added guanidine nitrate (36.5 mg, 0.163 mmol) followed by hydrazine
hydrate (217 µL, 4.89 mmol). The reaction mixture was stirred under an atmosphere of O2 for
16 h, at which point an aliquot was analyzed by 1H-NMR. Starting material was detected, and
so the reaction mixture was stirred under O2 for a further 16 h. The mixture was then
concentrated under a stream of N2. Water (30 mL) was added and then acidified to pH = 1 with
aqueous HCl (1 M). The mixture was extracted with EtOAc (3 x 20 mL). The combined organic
layers were dried (MgSO4), filtered and concentrated in vacuo to yield reduced acid as a white
solid (446 mg) which was used without further purification.
24
ACS Paragon Plus Environment

Page 25 of 39
The Journal of Organic Chemistry
1
2
3
4
5
6
7
8
9
To a solution of the above crude reduced acid (398 mg, 1.44 mmol) in ethanol (6 mL) at 0 ºC
was added SOCl2 (526 µL, 7.20 mmol) dropwise. The solution was warmed to room
temperature and stirred for 16 h. The solution was concentrated in vacuo. The crude product
was purified by flash chromatography (eluent: 5:95 v/v EtOAc/petroleum benzines) to yield
reduced ethyl ester 28 as a colourless oil (413 mg, 94%).
1H-NMR (CDCl3, 400 MHz) δ (ppm); 7.82 (d, J = 8.4 Hz, 1H), 7.30-7.22 (m, 2H), 6.90 (ddd,
J = 7.9, 6.6, 2.5, 1H), 4.14 (q, J = 7.2 Hz, 2H), 3.05 (t, J = 7.6 Hz, 2H), 2.62 (t, J = 8.2 Hz,
2H), 1.25 (d, J = 7.1 Hz, 3H); LRMS (+ESI) m/z 327 [M+Na]+. These data are in agreement
with those previously reported by Tummatorn and Dudley36.
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
ethyl 3-(2-(prop-1-yn-1-yl)phenyl)propanoate (29)
To a solution of 1-bromo-1-propene (0.57 mL, 6.71 mmol) in THF (5 mL) at -78 ºC was added
n-BuLi (3.58 mL, 8.96 mmol, 2.5M in hexanes) dropwise. The solution was stirred at this
temperature for 45 min. H2O (161 µL, 8.96 mmol) was added and the resultant solution was
warmed to room temperature. After 45 min, a solution of iodide 28 (340 mg, 1.12 mmol) in
THF (1.5 mL) was added dropwise, followed by CuI (43 mg, 0.22 mmol), iPr2NH (3.12 mL)
and Pd(PPh3)2Cl2 (79 mg, 0.112 mmol). The resultant mixture was stirred at room temperature
for 16 h. The reaction mixture was then poured onto saturated aqueous NH4Cl (30 mL), and
extracted with Et2O (3 x 30 mL). The combined organic layers were dried (MgSO4), filtered
and concentrated in vacuo. The crude product was purified by flash chromatography (eluent:
5:95 v/v EtOAc/petroleum benzines) to yield alkyne 29 as a colourless oil (202 mg, 83%).
1H-NMR (CDCl3, 500 MHz) δ (ppm) 7.37 (d, J = 7.6 Hz, 1H), 7.21-7.11 (m, 3H), 4.13 (q, J =
7.2 Hz, 2H), 3.08 (t, J = 7.6, 2H), 2.65 (t, J = 8.1 Hz, 2H), 2.09 (s, 3H), 1.24 (t, J = 7.3 Hz,
13
3H); C-NMR (CDCl3, 125 MHz) δ (ppm) 173.3, 142.4, 132.4, 128.8, 127.9, 126.3, 123.6,
90.1, 78.1, 60.5, 35.0, 30.1, 14.4, 4.6; LRMS (+ESI) m/z 239 [M+Na]+; HRMS (+ESI) m/z:
[M+Na]+ Calcd for C14H16O2Na 239.1043; Found 239.1043; IR νmax (ATR) 2980, 2917, 2252,
1730, 1485, 1447, 1371, 1293, 1181, 1155 cm-1.
(Z)-ethyl 3-(2-(prop-1-en-1-yl)phenyl)propanoate (30)
To a solution of alkyne 29 (102 mg, 0.47 mmol) in MeOH (4.7 mL) was added quinoline (58
µL, 0.47 mmol) and Lindlar’s catalyst (100 mg, 0.047 mmol, 5% Pd by weight). The solution
was stirred under an atmosphere of H2 for 1 h. A further portion of Lindlar’s catalyst (100 mg)
was added and the mixture was stirred under an atmosphere of H2 for 45 min. The reaction was
monitored by TLC every 20 min, and if the reaction was not complete a further portion of
Lindlar’s catalyst (200 - 300 mg) was added. The reaction was dosed a total of 6 times in order
to push it to completion. The mixture was then filtered over Celite® and concentrated in vacuo.
The crude product was purified by flash chromatography (eluent: 5:95 v/v EtOAc/petroleum
benzines) to yield alkene 30 as a colourless oil (85 mg, 83%).
1H-NMR (CDCl3, 400 MHz) δ (ppm) 7.22-7.17 (m, 4H), 6.53 (ap. dd, J = 11.5, 1.7 Hz, 1H),
5.87 (dq, J = 11.5, 7.0 Hz, 1H), 4.13 (q, J = 7.1 Hz, 2H), 2.93 (t, J = 7.8 Hz, 2H), 2.54 (t, J =
8.3 Hz, 2H), 1.73 (dd, J = 7.0, 1.8 Hz, 3H), 1.24 (t, J = 7.2 Hz, 3H); 13C-NMR (CDCl3, 100
MHz) δ (ppm) 173.2, 138.8, 136.4, 129.8, 129.0, 128.4, 127.8, 127.1, 126.1, 60.5, 35.1, 28.9,
14.4, 14.3; LRMS (+ESI) m/z 241 [M+Na]+; HRMS (+ESI) m/z: [M+Na]+ Calcd for
C14H18O2Na 241.1199; Found 241.1199; IR νmax (ATR) 2980, 1730, 1447, 1371, 1288, 1252,
1178, 1157, 1113 cm-1.
(Z)-3-(2-(prop-1-en-1-yl)phenyl)propanoic acid (16)
25
ACS Paragon Plus Environment

The Journal of Organic Chemistry
Page 26 of 39
1
2
3
4
5
6
7
8
9
To a solution of alkene 30 (79 mg, 0.36 mmol) in ethanol (4 mL, 95%) at 0 ºC was added LiOH
(35 mg, 1.45 mmol). The solution was warmed to room temperature and stirred for 16 h. The
mixture was cooled to 0 ºC and acidified to pH = 1 with aqueous HCl (1 M). The mixture was
extracted with EtOAc (3 x 20 mL). The combined organic layers were dried (Na2SO4), filtered
and concentrated in vacuo to yield acid 16 as an off-white solid (62 mg, 89%).
1H-NMR (CDCl3, 500 MHz) δ (ppm) 7.24-7.14 (m, 4H), 6.53 (dq, J = 11.4, 1.8 Hz, 1H), 5.88
(dq, J = 11.4, 7.0 Hz, 1H), 2.94 (t, J = 7.7 Hz, 2H), 2.61 (t, J = 8.3 Hz, 2H), 1.73 (dd, J = 7.0,
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
1.8Hz, 3H); C-NMR (CDCl3, 125 MHz) δ (ppm) 179.6, 138.4, 136.4, 129.9, 129.0, 128.3,
128.0, 127.2, 126.3, 34.8, 28.5, 14.4; LRMS (+ESI) m/z 213 [M+Na]+; HRMS (+ESI) m/z:
[M+Na]+ Calcd for C12H14O2Na 213.0886; Found 213.0886; IR νmax (ATR) 3014 (broad),
1701, 1435, 1407, 1319, 1275, 1215 cm-1; mp. 62.1-65.0 ºC.
b
Synthesis of oxazolidine protected Fmoc- -OH-Leu-OH 43
(S)-tert-butyl 4-((S)-1-hydroxy-2-methylpropyl)-2,2-dimethyloxazolidine-3-carboxylate
(39)
Magnesium granules (3.9 g, 159.5 mmol) were heated under vacuum with a heat gun for 2
minutes. THF (10 mL) and iodine (10 crystals) were then added and the mixture was stirred
vigorously under argon for 15 min. A solution of isopropyl bromide (9 mL, 05.9 mmol) in THF
(35 mL) was added dropwise over 20 min by which point the mixture turned a dull grey colour
and began to self reflux. It was heated at reflux for 1 h and then cooled to room temperature.
The solution was then added dropwise via cannula over 15 min, to a solution of (S)-Garner’s
aldehyde (38) (7.3 g, 31.9 mmol) at -78 ºC. The solution was stirred for 2 h at this temperature
and then warmed to room temperature and stirred for 1 h. The mixture was then diluted with
Et2O (100 mL), and quenched with saturated aqueous NH4Cl (200 mL). The aqueous phase
was extracted with Et2O (2 x 100 mL) and the combined organic layers were washed with
saturated aqueous NH4Cl (2 x 150 mL), dried (MgSO4), filtered and concentrated in vacuo.
The crude product was purified by flash chromatography (eluent: 8:92 → 10:90 v/v
EtOAc/petroleum benzines) to yield a single diastereomer of alcohol 39 as a white crystalline
solid (3.57 g, 41%).
1H-NMR (CDCl3, 500 MHz) δ (ppm) 4.09-3.96 (br. s, 1H), 3.93 (dd, J = 9.3, 5.8 Hz, 1H), 3.76
(d, J = 9.1 Hz, 1H), 3.50 (br. d, J = 7.4 Hz, 1H), 1.66 (septd, J = 6.8, 2.4 Hz, 1H), 1.60 (s, 3H),
1.51 (s, 3H), 1.49 (s, 9H), 1.03 (d, J = 6.8 Hz, 3H), 0.90 (d, J = 6.7 Hz, 3H); LRMS (+ESI)
+
a]
m/z 296 [M+Na] ; [
= -51.1° (c 0.45, CH2Cl2); mp. 84.6-87.3 ºC. These data are in
D
agreement with those previously reported by Williams et al.27
(4R,5S)-3-(((9H-fluoren-9-yl)methoxy)carbonyl)-5-isopropyl-2,2-dimethyloxazolidine-4-
carboxylic acid (43)
To a solution of alcohol 39 (3.46 g, 12.65 mmol) in THF (400 mL) was added aqueous HCl
(17.0 mL, 0.5 M). The solution was stirred at room temperature for 16 h at which point it was
dried (MgSO4), filtered and concentrated in vacuo to yield diol 40 as a colourless oil which
was used without further purification. Crude diol 40 (3.49 g, 12.65 mmol) was oxidized by
general procedure A to produce carboxylic acid 41 which was used without further purification.
Crude acid 41 (2.48 g, 10 mmol) was subject to protecting group manipulation via general
procedure B to produce Fmoc amino acid 42 which was used without further purification.
Crude Fmoc amino acid 42 (3.20 g, 8.65 mmol) was oxazolidine protected using general
procedure C. Crude oxazolidine protected amino acid 43 was purified by flash chromatography
26
ACS Paragon Plus Environment

Page 27 of 39
The Journal of Organic Chemistry
1
2
3
4
5
6
7
8
9
(eluent: 10:90 → 60:40 v/v EtOAc/petroleum benzines) to yield oxazolidine protected amino
acid 43 as an off-white crystalline solid (2.72 g, 53% from alcohol 39).
1H-NMR (CDCl3, 500 MHz, rotamers) δ (ppm) 8.52 (br. s, 1H), 7.78-7.67 (m, 2H), 7.58-7.49
(m, 2H), 7.40-7.25 (m, 4H), 4.66 (d, J = 4.2 Hz, 1H), 4.42 (m, 1H), 4.22-4.05 (m, 2H), 3.87
(br. t, J = 6.1 Hz, 0.5H) and 3.79 (br. t, J = 6.4 Hz, 0.5H), 1.91-1.71 (m, 1H), 1.60 (m, 3H),
13
1.09-0.87 (m, 9H); C-NMR (CDCl3, 125 MHz, rotamers) δ (ppm) 176.6 and 176.1, 153.1
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
and 152.0, 144.2 and 144.0, 143.8 and 143.7, 141.7 and 141.6, 141.5 and 141.4, 127.8, 127.3
and 127.2, 127.2 and 127.2, 125.0, 124.5 and 124.5, 120.0 and 120.0, 96.0 and 95.0, 83.9 and
83.9, 67.2 and 66.9, 62.6 and 61.8, 47.4, 32.1 and 31.7, 26.9, 24.8 and 24.7, 18.8 and 18.5, 17.7
and 17.6 LRMS (+ESI) m/z 432 [M+Na]+; HRMS (+ESI) m/z: [M+Na]+ Calcd for
C24H27NO5Na 432.1781; Found 432.1782; IR νmax (ATR) 2961, 2924, 1714, 1697, 1411, 1348,
-1
a]
1259 cm ; [ D = +29.6° (c 0.48, CH2Cl2); mp. 59.5-60.5 ºC.
b
Synthesis of Fmoc- -OH-Phe-OH (37)
(4R)-tert-butyl 4-(hydroxy(phenyl)methyl)-2,2-dimethyloxazolidine-3-carboxylate (32)
Magnesium granules (545 mg, 22.4 mmol) were heated under vacuum with a heat gun for 2
minutes. Ether (4 mL) and iodine (2 crystals) were then added and the mixture was stirred
vigorously under argon for 20 min. A solution of bromobenzene (1.24 mL, 11.8 mmol) in THF
(12 mL) was added dropwise over 10 min by which point the mixture turned a dull grey colour
and began to self reflux. The reaction was refluxed for 1 h and then cooled to 0 ºC. To the
reaction mixture was then added dropwise a solution of (R)-Garner’s aldehyde (31) (2.00 g,
8.72 mmol). The reaction mixture was stirred for 20 min at this temperature and then warmed
to room temperature and stirred for 1 h. The mixture was then quenched with saturated aqueous
NH4Cl (100 mL). The aqueous phase was extracted with EtOAc (3 x 70 mL) and the combined
organic layers were dried (MgSO4), filtered and concentrated in vacuo. The crude product was
purified by flash chromatography (eluent: 20:80 v/v EtOAc/petroleum benzines) to yield
alcohol 32 as a white solid (2.12 g, 79%, syn/anti 2:2.7).
1H-NMR (CDCl3, 400 MHz) δ (ppm) 7.43-7.18 (m, 10H), 5.05 (m, 1H), 4.74 (d, J = 8.8 Hz,
+
a]
1H), 4.40-3.50 (m, 3H), 1.60-1.33 (m, 15H); LRMS (+ESI) m/z 330 [M+Na] ; [ D = +8.0° (c
0.5, CH2Cl2); mp. 79.9-84.0 ºC. These data are in agreement with those previously reported by
Malins et al.21
(R)-tert-butyl 4-benzoyl-2,2-dimethyloxazolidine-3-carboxylate (33)
To a solution of oxalyl chloride (0.90 mL, 10.6 mmol) in CH2Cl2 (16 mL) at -78 ºC was added
a solution of dimethylsulfoxide (1.20 mL, 18.1 mmol) in CH2Cl2 (4.3 mL) dropwise. The
reaction mixture was stirred at this temperature for 10 min. A solution of alcohol 32 (1.63 g,
5.3 mmol) in CH2Cl2 (4.3 mL) was added dropwise and the mixture was stirred at -78 ºC for
1 h. iPr2NEt (3.25 mL, 18.6 mmol) was added and the mixture was stirred at this temperature
for 10 min and then warmed to room temperature. The reaction mixture was poured onto
saturated aqueous NH4Cl (100 mL). The aqueous phase was extracted with CH2Cl2 (3 x 70 mL)
and the combined organic layers were dried (MgSO4), filtered and concentrated in vacuo. The
crude product was purified by flash chromatography (eluent: 20:80 v/v EtOAc/petroleum
benzines) to yield ketone 33 as a white solid (1.38 g, 85%).
1H-NMR (CDCl3, 400 MHz, rotamers) δ (ppm) 7.93-7.89 (m, 2H), 7.62-7.44 (m, 3H),
5.47 (dd, J = 7.4, 2.9 Hz, 0.4H) and 5.36 (dd, J = 7.7, 3.7 Hz, 0.6H), 4.31 (ddd, J = 9.0, 7.6,
4.8, 1H) and 3.98-3.90 (m, 1H), 1.76 (s, 2H) and 1.73 (s, 1H), 1.60 (s, 2H) and 1.56 (s, 1H),
+
a]
1.50 (s, 4H) and 1.28 (s, 5H); LRMS (+ESI) m/z 328 [M+Na] ; [ D = +59.6° (c 0.26, CH2Cl2);
27
ACS Paragon Plus Environment

The Journal of Organic Chemistry
Page 28 of 39
1
2
3
4
5
mp. 114.6-117.5 ºC These data are in agreement with those previously reported by Malins et
al.21
6
7
8
9
(R)-tert-butyl 4-((S)-hydroxy(phenyl)methyl)-2,2-dimethyloxazolidine-3-carboxylate (34)
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 a solution of ketone 33 (1.38 g, 4.51 mmol) in THF (190 mL) at 0 ºC was added diisobutyl
aluminium hydride (13.5 mL, 1 M in hexanes, 13.5 mmol) dropwise over 30 min. The solution
was stirred at this temperature for 30 min. MeOH (10 mL) was added and the reaction mixture
was poured onto ice cold HCl (150 mL, 1M). The aqueous phase was extracted with EtOAc (3
x 70 mL) and the combined organic layers were dried (MgSO4), filtered and concentrated in
vacuo. The crude product was purified by flash chromatography (eluent: 20:80 v/v
EtOAc/petroleum benzines) to yield a 14:1 mixture of diastereomers of alcohol 34 as a white
solid (1.23 g, 89%).
1H-NMR (CDCl3, 500 MHz, 328 K) δ (ppm) 7.41-7.26 (m, 5H), 5.11 (s, 1H), 4.24-4.21 (m,
1H), 4.07 (dd, J = 9.1, 2.0 Hz, 1H), 3.86-3.84 (m, 1H), 1.55-1.45 (m, 15H); LRMS (+ESI) m/z
+
a]
330 [M+H] ; [ D = +22.9° (c 0.48, CH2Cl2); mp. 96.2-102.0 ºC. These data are in agreement
with those previously reported by Malins et al.21
tert-butyl ((1S,2R)-1,3-dihydroxy-1-phenylpropan-2-yl)carbamate (35)
To a solution of oxazolidine 34 (800 mg, 2.6 mmol) in MeOH (27 mL) was added p-toluene
sulfonic acid (198 mg, 0.46 mmol). The mixture was stirred at room temperature for 2.5 h. The
reaction mixture was poured onto saturated aqueous NaHCO3 (50 mL). The aqueous phase was
extracted with EtOAc (3 x 30 mL) and the combined organic layers were dried (MgSO4),
filtered and concentrated in vacuo. The crude product was purified by flash chromatography
(eluent: 50:50 v/v EtOAc/petroleum benzines) to yield a 14:1 mixture of diastereomers of diol
35 as a white solid (510 mg, 74%).
1H-NMR (CDCl3, 400 MHz, 328K) δ (ppm) 7.41-7.24 (m, 5H), 5.30 (d, J = 7.6 Hz, 1H), 5.02-
4.96 (m, 1H), 3.83-3.73 (m, 1H), 3.65-3.57 (m, 1H), 3.34 (d, J = 3.7 Hz, 1H), 2.67 (brs, 1H),
+
a]
1.43 (s, 9H); LRMS (+ESI) m/z 290 [M+Na] ; [ D = +2.1° (c 0.27, CH2Cl2); mp. 80.8-84.2
ºC. These data are in agreement with those previously reported by Williams et al.27
(2S,3S)-2-((((9H-fluoren-9-yl)methoxy)carbonyl)amino)-3-hydroxy-3-phenylpropanoic
acid (37)
Diol 35 (479 mg, 1.79 mmol) was oxidized by general procedure A to produce carboxylic acid
36 which was used without further purification. Crude acid 36 (371 mg, 1.32 mmol) was
subject to protecting group manipulation via general procedure B to afford Fmoc amino acid
37 which was purified by flash chromatography (eluent: 70:30 → 100:0 + 0.1% AcOH v/v
EtOAc/petroleum benzines) to yield Fmoc protected amino acid 37 as an off-white crystalline
solid (463 mg, 64% from diol 35).
1H-NMR (MeOD, 400 MHz) δ (ppm) 7.76 (d, J = 7.6 Hz, 2H), 7.57-7.50 (m, 2H), 7.46-7.19
13
(m, 9H), 4.99 (d, J = 7.3 Hz, 1H), 4.52 (d, J = 7.3 Hz, 1H), 4.30-4.04 (m, 3H); C-NMR
(MeOD, 100 MHz) δ (ppm) 174.0, 158.1, 145.2, 145.1, 142.5, 142.4, 142.0, 129.2, 128.9,
128.7, 128.1, 128.0, 126.3, 126.2, 120.9, 75.1, 68.2, 61.3 48.2; LRMS (+ESI) m/z 426
[M+Na]+; HRMS (+ESI) m/z: [M+Na]+ Calcd for C24H21NO5Na 426.1312; Found 436.1313;
-1
a]
IR νmax (ATR) 3408, 3314, 3064, 3083, 1711, 1522, 1450, 1414, 1332, 1260, 1233 cm ; [
= +24.1° (c 0.19, CH2Cl2:MeOH, 0.91:0.9); mp. 164.1-173.0 ºC
D
28
ACS Paragon Plus Environment

Page 29 of 39
The Journal of Organic Chemistry
1
2
3
4
b
Synthesis of oxazolidine protected Fmoc- -OH-Phe-OH (72)
5
6
7
(4S,5S)-3-(((9H-fluoren-9-yl)methoxy)carbonyl)-2,2-dimethyl-5-phenyloxazolidine-4-
carboxylic acid (72)
8
9
Fmoc-β-OH-Phe-OH (37) (450 mg, 1.15 mmol) was protected via general procedure C. Crude
oxazolidine protected amino acid 72 was purified by flash chromatography (eluent: 30:70 →
45:55 v/v EtOAc/petroleum benzines) to yield oxazolidine protected amino acid 72 as an off-
white crystalline solid (465 mg, 94%).
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
1H-NMR (CDCl3, 500 MHz, rotamers) δ (ppm) 7.78 (ap. d, J = 7.88 Hz, 0.7H) and 7.68 (ap.
dd, J = 5.7, 7.5 Hz, 1.3H), 7.57 (dd, J = 3.5, 7.5 Hz, 0.7H) and 7.47 (ap. t, J = 7.0 Hz, 1.3H),
7.44-7.16 (m, 9H), 5.34 (d, J = 6.8 Hz, 0.65H) and 5.20 (d, J = 6.7 Hz, 0.35H), 4.76 (dd, J =
4.2, 10.8 Hz, 0.35H) and 4.39 (dd, J = 6.3, 10.8 Hz, 0.65H), 4.69 (dd, J = 4.4, 11.0 Hz, 0.35H)
and 4.30 (dd, J = 6.8, 10.8 Hz), 4.52-4.45 (m, 1H), 4.23 (ap. t, J = 4.2 Hz, 0.35H) and 4.02 (ap.
t, J = 6.6 Hz, 0.65H), 1.87 (s, 2H) and 1.21 (s, 1H), 1.63 (s, 2H) and 1.02 (s, 1H); 13C-NMR
(CDCl3, 125 MHz, rotamers) δ (ppm) 173.6 and 173.4, 152.9 and 151.8, 144.0, 143.9 and
143.6, 141.8 and 141.4, 141.6 and 141.3, 134.2, 129.0 and 128.8, 128.5 and 128.4, 127.9 and
127.9, 127.8 and 127.8, 127.4 and 127.2, 127.3 and 127.2, 126.5 and 126.4, 125.1 and 125.0,
124.6 and 124.5, 120.1 and 120.1, 120.0 and 120.0, 95.5 and 94.7, 78.2 and 77.4, 67.3 and
66.9, 64.6 and 64.0, 47.4 and 47.3, 25.5 and 25.4, 25.0 and 24.3 (extra signals due to further
rotational conformations in the Fmoc-region); LRMS (+ESI) m/z 466 [M+Na]+; HRMS
(+ESI) m/z: [M+Na]+ Calcd for C27H25NO5Na 466.1624; Found 466.1624; IR νmax (ATR)
-1
a]
2923, 1744, 1706, 1451, 1410, 1348, 1248, 1216, 1190, 1159 cm ; [
CH2Cl2); mp. 60.6-71.2 ºC
= +21.4° (c 0.5,
D
b
Synthesis of oxazolidine protected Fmoc- -OH-O-Me-Tyr-OH (48)
(R)-tert-butyl
4-((S)-hydroxy(4-methoxyphenyl)methyl)-2,2-dimethyloxazolidine-3-
carboxylate (44)
To a solution of dry LiBr (6.66 g, 88.8 mmol) in THF (140 mL) was added p-bromoanisole
(3.97 mL, 31.53 mmol). The reaction mixture was cooled to -78 ºC and n-BuLi (13.2 mL, 33.02
mmol, 2.5 M in hexanes) was added dropwise. The reaction mixture was stirred at this
temperature for 45 min. To the reaction mixture was added a solution of (R)-Garner’s aldehyde
(31) (3.44 g, 15.01 mmol) in THF (16 mL) dropwise. The reaction mixture was stirred at -78
ºC for 4 h before being quenched with saturated aqueous NH4Cl (30 mL). The mixture was
warmed to room temperature and poured onto saturated aqueous NH4Cl (100 mL). The aqueous
phase was extracted with EtOAc (3 x 100 mL) and the combined organic layers were dried
(Na2SO4), filtered and concentrated in vacuo. The crude product was purified by flash
chromatography (eluent: 15:85 → 25:75 v/v EtOAc/petroleum benzines) to yield a 5.1:1
mixture of diastereomers of alcohol 44 as a colourless oil (3.80 g, 75%, anti/syn 5.1:1).
1H-NMR (CDCl3, 400 MHz, 328 K) major diastereomer (anti) δ (ppm) 7.27 (d, J = 8.6 Hz,
2H), 6.87 (d, J = 8.7 Hz, 2H), 5.04-4.98 (m, 1H), 4.23-4.07 (m, 1H), 4.02 (dd, J = 9.4, 2.2 Hz,
1H), 3.88-3.80 (m, 1H), 3.78 (s, 3H), 1.56-1.42 (m, 15H); minor diastereomer (syn) δ (ppm)
7.27 (d, J = 8.6 Hz, 2H), 6.87 (d, J = 8.7 Hz, 2H), 4.70 (dd, J = 8.6, 3.5 Hz, 1H), 4.23-4.07 (m,
1H), 3.78 (s, 3H), 3.73-3.61 (m, 2H), 1.56-1.42 (m, 15H); 13C-NMR (CDCl3, 100 MHz, 328
K) major diastereomer (anti) δ (ppm) 159.7, 159.3, 133.6, 127.4, 114.0, 94.8, 80.8, 74.0, 64.0,
63.5, 55.4, 28.6 24.0; minor diastereomer (syn) δ (ppm) 159.7, 159.3, 133.6, 128.5, 114.1, 94.8,
80.8, 77.2, 65.0, 63.5, 55.4, 28.6, 26.7 LRMS (+ESI) m/z 360 [M+Na]+; HRMS (+ESI) m/z:
29
ACS Paragon Plus Environment