Total Synthesis of A54145 Factor D

Article abstract of DOI:10.1021/acs.joc.9b01938

An efficient total synthesis of A54145 factor D (A5D), a member of the A54145 family of cyclic lipodepsipeptide antibiotics, is reported. The peptide was constructed by attaching the peptide to the 2′-chlorotrityl polystyrene resin via Sar5 and developing

Full text of DOI:10.1021/acs.joc.9b01938

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Article
Total Synthesis of A54145 Factor D
Braden Kralt, Ryan Moreira, Michael Palmer, and Scott D. Taylor
J. Org. Chem., Just Accepted Manuscript • DOI: 10.1021/acs.joc.9b01938 • Publication Date (Web): 26 Aug 2019
Downloaded from pubs.acs.org on August 27, 2019
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Total Synthesis of A54145 Factor D
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Braden Kralt, Ryan Moreira, Michael Palmer and Scott D. Taylor*
Department of Chemistry, University of Waterloo, 200 University Avenue West,
Waterloo, Ontario, Canada, N2L 3G1
ABSTRACT: An efficient total synthesis of A54145 factor D (A5D), a member of the A54145
family of cyclic lipodepsipeptide antibiotics is reported. The peptide was constructed by attaching
the peptide to the 2’-chlorotrityl polystyrene resin via Sar5, and developing conditions that avoided
diketopiperazine formation upon subsequent elaboration using Fmoc SPPS. This route allowed for
facile formation of the crucial depsi bond. A branched acyclic precursor was cyclized off-resin
then globally deprotected to obtain A5D. Consistent with recent studies by others, we found that
the MeOAsp residue has the 2S,3R configuration. We also established that the configuration of the
stereocenter in the anteiso-undecanoyl lipid tail does not affect biological activity.
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Graphical Abstract
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H2N
8
9
O
O
H
O
O
O
OH
N
O
HO
N
H
N
H
NH
NH
O
10
11
12
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O
N
O
HN
SPPS
OH
OCH3
O
O
O
H
N
CH3
O
FmocHN
O
N
N
H
HN
H
N
H
O
O
H2N
O
O
R
OH
NH
O
NH2
N
H
O
O
R = anteiso-undecanoyl
A54145D
O
OH
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INTRODUCTION
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A54145 (A5, Figure 1) is a family of cyclic lipodepsipeptide antibiotics (cLPAs) consisting
of eight different factors isolated from Streptomyces fradiae.1 This family is a member of the
calcium-dependent cyclic lipopeptide class of antibiotics, of which the best known is daptomycin,
which is used in clinic for treating serious infections caused by Gram-positive bacteria. Like
daptomycin, each member of the A5 family consists of a 10 amino acid macrocycle closed with
an ester (depsi) bond, to which is attached an exocyclic tripeptide bearing an N-terminal lipid.
However, the A5 family differs from daptomycin in amino acids at eight positions. They also
contain L-hydroxyasparagine (HOAsn3) and L-methoxyaspartate (MeOAsp9) which are not
present in daptomycin. The macrocyclic nuclei of the A5 family differ from each other only at
position 13 (Val or Ile) and/or position 12 (Glu or (2S,3R)-3-methylglutamate (MeGlu12)). The
N-terminus is acylated with either an iso-decanoyl (iC10), n-decanoyl (nC10) or anteiso-
undecanoyl (aC11) lipid.
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No members of the A5 family have been developed into clinically useful drugs. The most
potent natural factors (B, B1 and E) contain MeGlu12, and are only about 2-fold less active than
daptomycin but are toxic to mice.1b,2a,b For example, the LD50 for A5B is low at only 28 mg/kg.2b
Factors containing Glu12, such as A5D, are about 2-fold less potent than those containing MeGlu
but are substantially less toxic.1b,2a,b
Daptomycin did not meet noninferiority standards in clinical trials for treating community-
acquired pneumonia (CAP). A possible cause for this failure is its inhibition by lung surfactant.3
Unlike daptomycin, A5D is not severely inhibited by lung surfactant.2a,b,4 This characteristic of
A5D prompted the development of combinatorial biosynthesis methodologies for preparing cLPAs
based on the A5D peptide core.2a,b,4 Although several A5D analogs were obtained that exhibited
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good activity, low toxicity and low inhibition by surfactant, none were sufficiently active in mouse
models for S. pneumoniae to pursue clinically.2a Nevertheless, these results are promising when
one considers that key in vitro criteria (good activity, low toxicity and low inhibition by surfactant)
were met even though the number of analogs prepared represented only a very small fraction of
the potential chemical space. It is possible that the development of an efficient chemical approach
to the synthesis of A5D and its analogs will enable researchers to access a larger portion of the
available chemical space which could ultimately lead to the development of an cLPA for treating
community acquired pneumonia (CAP) that is resistant to conventional therapy. The synthesis of
A54145A, A1, B, B1 and F have very recently been reported.5 Here we report the total synthesis
of A5D.
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H2N
D-Lys8
Ala6
O
O
H
N
O
O
OH
O
N
N
H
NH
NH
Sar5
O
HO
D-Glu2
H
N
O
HN
HN
OH
OCH3
O
O
H
N
MeOAsp9
O
Asp7
Trp1
CH3
O
O
N
N
H
N
Thr4
H
H
O
O
O
R3
Gly10
O
H2N
OH
NH
O
NH2
R1
N
H
O
O
HOAsn3
R2
D-Asn11
Val or Ile13
O
OH
Glu or MeGlu12
Factor R1
R2
R3
A
CH3
CH3
H
H
iso-decanoyl (iC10
n-decanoyl (nC10
)
A1
B
)
CH3 CH3 n-decanoyl (nC10
)
B1
C
D
E
CH3 CH3 iso-decanoyl (iC10
)
H
CH3
CH3
H
CH3 anteiso-undecanoyl (aC11
anteiso-undecanoyl (aC11
CH3 anteiso-undecanoyl (aC11
iso-decanoyl (iC10
)
H
)
)
F
H
)
Figure 1. Structures of the A54145 family of antibiotics
RESULTS AND DISCUSSION
Although the absolute configurations of the side chains of the MeOAsp and HOAsn
residues have been depicted in the literature as having the S configuration (L-threo-MeOAsp and
L-threo-HOAsn),6 in fact, at the time we began these studies, the absolute configuration of the side
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chains of these residues had never been firmly established. While bioinformatic studies strongly
suggested that the HOAsn residue is indeed the L-threo isomer,2b,7 the same could not be said
regarding the configuration of the MeOAsp residue. Nevertheless, we proceeded on the
assumption that the amino acids were synthesized in vivo through a common intermediate and that
both were L-threo amino acids.
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Our initial approach to A5D was to use an on-resin cyclization strategy similar to what we
developed for the synthesis of daptomycin analogs.8 This required that the peptide be attached to
the resin via the side chain of MeOAsp9, and hence the synthesis of Fmoc-L-threo-MeOAspOallyl
(Fmoc-tMeOAspOAllyl, 5 in Scheme 1) was required. Towards this end, an asymmetric
aminohydroxylation reaction, using the conditions of von Eckardstein et al,9 was performed on
ester 110 to give compound 2 in excellent yield. Methylation of the free hydroxyl group using
MeI/Ag2O gave compound 3 in good yield. Oxidation of the phenyl ring followed by esterification
of the resulting acid using allyl bromide gave compound 4 in 52 % yield (2 steps). Finally, removal
of the t-butyl and Boc protecting groups using acid followed by Fmoc protection of the free amino
group gave compound 5 in 87% yield.
O
BocNH2
tBuOCl
(DHDQ)2PHAL
K2[OsO2(OH)4]
BocHN
O
BocHN
O
Ag2O, CH3I
DCM
OtBu
OtBu
OtBu
OH
OMe
MeO
MeO
1
MeO
3
(81%)
2
(91%)
1. 6M HCl in dioxane
2. FmocCl, K2CO3
THF/H2O
1. RuCl3, NaIO4
BocHN
AllylO
O
FmocHN
O
ACN, EtOAc,H2O
AllylO
OtBu
OH
2. allyl bromide
Cs2CO3, DMF
O
OMe
O
OMe
5
4
(87%, 2 steps)
(52%, 2 steps)
Scheme 1. Synthesis of Fmoc-tMeOAspOAllyl (5)
Amino acid 5 was attached to chlorotrityl-Tentagel (TG) resin via its side chain COOH
group (Scheme 2). Removal of the allyl group in peptide 6 followed by installation of GlyOallyl
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gave dipeptide 7 (Scheme 2). However, as the peptide was elongated using standard Fmoc SPPS,
it was found that the amount of peptide remaining attached to the resin decreased with each
successive amino acid coupling. We discovered that aspartimide formation was taking place during
each successive Fmoc deprotection which resulted in cleavage of the peptide from the resin
(Scheme 2). It is worth noting that for our daptomycin synthesis, we started with an Asp9-Gly10
dipeptide attached to the resin via the side chain of the Asp residue; however, no aspartimide
formation was observed upon further elaboration of the peptide.8 The finding that aspartimide
formation occurs with L-tMeOAsp suggests that the presence of the electron withdrawing 3-MeO
group was promoting aspartimide formation.
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1. cat,. Pd(PPh3)4
O
DMBA
O
O
FmocHN
O
O
Cl
2. GlyOallyl, HOBt,
DIC, DMF
FmocHN
O
O
FmocHN
O
OH
O
O
NH
O
O
DIPEA, DCM
Oallyl
Oallyl
5
7
6
O
O
O
O
N
Fmoc
SPPS
H
H
peptide
N
peptide
N
O
O
O
N
O
O
O
O
O
O
aspartimide
O
Scheme 2. Aspartimide formation during Fmoc SPPS of A5D via an on-resin cyclization
approach.
To overcome the problem of aspartimide formation we prepared dipeptide 8 in which the
amide nitrogen of Gly10 was protected with a dimethoxybenzyl (DMB) group (Scheme 3).11
Unfortunately, we found that upon removal of the Fmoc group, the resulting dipeptide 9 underwent
extremely rapid diketopiperazine (DKP) formation. Attempts to minimize this problem by using
shorter deprotection times were unsuccessful.
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O
O
O
O
O
H
N
H2N
N
O
O
O
FmocHN
allylO
O
allylO
O
O
O
piperidine
O
N
N
O
O
H3CO
OCH3
H3CO
OCH3
H3CO
OCH3
9
8
9
10
11
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Scheme 3. DKP formation upon deprotection of peptide 8.
Due to the difficulties encountered using the on-resin cyclization route, we decided to
pursue an off-resin cyclization strategy. For this approach we preferred to attach the peptide to the
resin via Gly10 or Sar5 in order to avoid potential epimerization issues during peptide cyclization.
Hence, Fmoc-L-threo-MeOAsp(tBu)OH12 was coupled to resin-supported Gly10 or DMB-
protected Gly10 to give dipeptides of type 10 (Scheme 4). Not surprisingly, aspartimide or DKP
formation were again major problems upon further elaboration via Fmoc SPPS.
O
FmocHN
OtBu
O
FmocHN
OtBu
O
O
HO
O
NH
R
O
O
N
O
O
O
R
R = H or DMB
10
DKP or
Fmoc SPPS
aspartimide formation
Scheme 4. Attempted synthesis of A5D starting from Gly10.
There are both advantages and disadvantages to attaching the peptide to the resin via Sar5.
If the peptide is attached to the resin via Sar5 then the ester bond between Thr4 and Ile13 would
be formed at an early stage in the synthesis and, therefore, on a relatively short peptide. This is an
advantage, as we have shown that formation of the ester bond in daptomycin became more difficult
as the length of the peptide containing the Thr residue increased, and was very difficult when the
lipid tail was present.13 Another advantage is aspartimide formation could be avoided through the
use of DMB-Gly while also avoiding DKP formation between MeOAsp9 and Gly10. However, a
disadvantage is that Fmoc-AA-Sar esters (AA is any amino acid) can undergo DKP formation
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during deprotection of the terminal amino group.14
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8
It has been reported that DKP formation can be minimized or eliminated by employing a
one-pot, tandem deprotection/coupling protocol on resin bound N-Alloc dipeptides and activated
Fmoc amino acids.15a,b This procedure was attempted using Alloc-protected peptide 11 and
FmocAsn(Trt)OH as reactants and the conditions reported by Thieriet et al;15a however, none of
the desired tripeptide product 13 was formed (Table 1, entry 1). Performing the reaction
sequentially using DCM as solvent for both the deprotection and coupling reactions resulted in
only trace amounts of peptide 13 (entry 2); however, when the reaction was performed sequentially
using DMF as solvent for the coupling reaction, peptide 13 was obtained in 44 or 49 % yield
depending upon whether TG or polystyrene (PS) resin was used (entry 3).
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Siow et al reported that using 2-methylpiperidine (2-MP) for Fmoc removal can help
reduce DKP formation during Fmoc SPPS.16 Subjecting peptide 12 to 20% 2-MP in DMF for 3 x
10 min followed by coupling of FmocAsn(Trt)OH using DIC/HOBt gave peptide 13 in 26% yield
on PS (entry 4, Table 1). Interestingly, no peptide 13 was obtained using TG resin. Decreasing the
deprotection time to 2 x 5 min gave peptide 13 in 73 % yield on PS resin but in only 34% yield on
TG (entry 5).17 Surprisingly, decreasing the deprotection time to just 2 x 1 min (PS resin) resulted
in almost complete deprotection of peptide 12 without DKP formation as evidenced by the fact
that peptide 13 was obtained in almost quantitative yield (entry 6).
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Table 1. Conditions examined for deprotecting and coupling FmocAsn(Trt)OH to peptides
5
6
11 or 12.
7
8
9
O
O
O
H
N
1. deprotection
TrtHN
Fmoc
O
N
O
H
PG
N
NH
HO
N
O
or
+
O
2. FmocAsn(Trt)OH
conditions
N
N
O
10
11
12
13
14
15
16
17
18
19
20
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22
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HO
H
tentagel
O
O
11
PG = Alloc ( ) or
HO
HO
or polystyrene
12
13
Fmoc (
)
sequential
yield of 13
entry
1
PG
deprotection conditions
coupling reagents
or tandema (%)
Alloc
12 eq DABCO or PhSiH3, 0.2 eq
Pd(PPh3)4, DCM
3 eq AA/HOBt/EDC or
DIC
Tandem
0b
2
3
4
5
6
Alloc
Alloc
Fmoc
Fmoc
Fmoc
10 eq DMBAe, 0.2 eq Pd(PPh3)4,
DCM, 1 h
4 eq AA/HOBt/DIC,
DCM, 4 h
Sequential tracec
Sequential 44c, 49d
Sequential 0c, 26d
Sequential 34c, 73d
Sequential 99%d
10 eq DMBAe, 0.2 eq Pd(PPh3)4,
DCM, 1 h
4 eq AA/HOBt/DIC,
DMF, 4 h
4 eq AA/HOBt/DIC,
DMF, 4 h
20% 2-MP, DMF, 3 x 10 min
20% 2-MP, DMF, 2 x 5 min
20% 2-MP, DMF, 2 x 1 min
4 eq AA/ HOBt/DIC,
DMF, 4 h
4 eq AA/ HOBt/DIC,
DMF, 4 h
aAll tandem reactions were conducted for 2 h. bTentagel or polystyrene resin. cTentagel resin.
dPolystyrene resin. eDMBA = dimethylbarbituric acid.
With conditions in hand that enabled us to remove the Fmoc group from peptide 12 without
DKP formation, we were now ready to begin the synthesis of A5D starting from peptide 12. First,
(±)-8-methyldecanoic (16) was prepared (Scheme 5). This was achieved by performing a Wittig
reaction between phosphonium salt 1418 and (±)-2-methylbutanal which gave alkene 15 in 66%
yield. Lipid 16 was obtained in good yield by subjecting 15 to hydrogenolysis and then NaOH.
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O
O
1. NaHMDS, THF, -78oC, 30 min
Ph3P
Br
O
O
2. 2-methylbutanal
THF, -78oC, warm to rt, o/n
14
(+)-15
(66%)
-
6
7
8
O
1. Pd/C, H2, ethanol
2. NaOH, MeOH/H2O 3h
OH
9
(+)-16
(74%, 2 steps)
-
10
11
12
13
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Scheme 5. Synthesis of (±)-8-methyldecanoic acid (16)
We initially used Fmoc-tHOAsn(OTBS,NTrt)OH19 for the synthesis of A5D. Thus, peptide
12 was deprotected using the conditions outlined in entry 6 in table 1 and Fmoc-
tHOAsn(OTBS,NTrt)OH was coupled to the resulting deprotected dipeptide using DIC/HOBt.
However, the coupling was sluggish and, based upon the lower than expected signal intensities by
analytical RP-HPLC of subsequent amino acid couplings, we suspected that increased DKP
formation was again an issue. Therefore, we decided to try the less sterically encumbered Fmoc-
tHOAsn(OTBS)OH building block.12 In this case, the coupling proceeded smoothly and tripeptide
17 was formed in almost quantitative yield (Scheme 6).
Ester bond formation between peptide 17 and N3IleOH proved to be straightforward using
DIC and cat. DMAP, going almost to completion after one 12 h coupling. No epimerization was
evident by RP-HPLC analysis of the resulting peptide 18 (Scheme 6). Fmoc SPPS was used to
introduce Glu2, Trp1 and the racemic lipid 16 into peptide 18 to give peptide 19.
Attempts to reduce the N3 group in peptide 19 using PBu3 in 95:5 THF/H2O in the presence
of the symmetric anhydride of FmocGlu(tBu)OH8 gave a complex mixture of products. However,
it was found that if the TBS group was removed prior to N3 reduction then the desired peptide 20
could be obtained almost quantitatively. It is worth noting that, despite the presence of the
symmetric anhydride of FmocGlu(tBu)OH, the major peptide product after the azide reduction
was indeed peptide 20, and not the Fmoc-Glu(tBu)-coupled product, consistent with our previous
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studies.8 The presence of the symmetric anhydride FmocGlu(tBu)OH during azido group reduction
prevents cleavage of the ester bond during this step.8 Accordingly, subsequent Fmoc-SPPS still
required a standard coupling of Fmoc-Glu(tBu)OH using DIC/HOBt.
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Standard Fmoc SPPS was used to introduce residues 12 to 6 to give peptide 21. DMB-
protected Gly was used to prevent aspartimide formation. Peptide 21 was cleaved from the resin
using 1% TFA in DCM (v/v) to give peptide 22 which was cyclized using PyAOP, HOAt, and
2,4,6-collidine to give peptide 23. Global deprotection of peptide 23 with a solution of
TFA:TIPS:H2O (95:2.5:2.5, v/v) and purification by semi-preparative RP-HPLC gave A5D-
tMeOAsp in a 8% yield based on resin loading.
O
O
O
PS resin
1. 20% 2-MP in DMF
O
O
OtBu
10 equiv N3IleOH
10 equiv DIC, 10 mol %DMAP
DCM, 12 h
O
O
NBoc
O
O
O
O
N
2 x 1 min
O
N
O
N
Fmoc SPPS
using DIC/HOBt
O
N
O
O
O
H
N
2. Fmoc-tHOAsn(OTBS)OH
DIC/HOBt
CH3
OH
12
FmocHN
CH3
OH
CH3
O
FmocHN
CH3
FmocHN
N
H
N
N
H
N
H
H
NH
O
O
O
O
H2N
O
H2N
H2N
O
OTBS
17
OTBS
19
O
OTBS
18
N3
N3
O
1. TBAF, AcOH, THF
2. 5 equiv (FmocGlu(tBu)CO)2
3 equiv PBu3, 5 min, THF
3. H2O (5 %), 18 h
O
BocHN
BocHN
O
O
O
O H2N
O
O
O H2N
O
O
OtBu
O
OtBu
O
OtBu
HN
O
O
OtBu
O
OtBu
HN
NBoc
OH
tBuO2C
CH3
N
NBoc
O
O
NBoc
O
O
N
N
H
H
HN
O
N
O
N
O
HN
OCH3
1% TFA in DCM
O
O
O
OCH3
O
H
N
H
N
Fmoc SPPS
using DIC/HOBt
H
N
tBuO2C
DMB
CH3
O
CH3
DMB
N
N
H
N
O
N
N
H
N
N
H
N
O
H
H
H
H
O
H
NH
O
O
O
N
O
NH
O
O
NH
O
O
O
N
O
O
H2N
H2N
H2N
OH
NH
O
OH
20
O
OH
O
NH
O
N
H
O
NH2
NTrt
N
H
O
NTrt
O
O
O
O
22
21
O
OtBu
O
OtBu
PyAOP, HOAt
2,4,6-collidine, DMF
BocHN
H2N
O
O
O
O
H
H
N
O
O
OtBu
O
OtBu
N
O
OH
O
OH
O
NBoc
O
N
N
NH
O
N
H
N
H
H
H
HN
O
N
O
TFA/TIPS/H2
95:2.5:2.5 (v/v)
O
O
N
O
HN
HN
OCH3
O
OCH3
O
H
N
tBuO2C
H
N
HO2C
CH3
DMB
CH3
N
N
N
O
N
N
H
O
H
H
H
H
O
H
NH
O
O
O
N
O
NH
O
O
O
N
O
O
H2N
H2N
OH
NH
O
NTrt
OH
O
NH
O
NH2
N
H
O
N
H
O
O
O
O
23
O
OtBu
A5D-tMeOAsp
O
OH
Scheme 6. Synthesis of A5D-tMeOAsp by attaching the peptide to the resin via Sar5.
The antimicrobial activities of authentic A5D and our synthesized A5D-tMeOAsp were
determined against Bacillus subtilis 1046 (Table 2). The synthesized A5D-tMeOAsp exhibited an
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MIC that was at least 64 times greater than that of authentic A5D which suggested to us that the
configuration of the side chain of the HOAsn residue and/or the MeOAsp residue in the synthetic
peptide was incorrect. As bioinformatic studies strongly suggested that the HOAsn residue is the
L-threo isomer,2b,7 we assumed that the problem was most likely with the MeOAsp residue.
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
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41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
To determine if the problem was with the MeOAsp residue, we prepared Fmoc-L-erythro-
MeOAsp(tBu)OH(compound 28 in Scheme 7). The configuration of the alcohol in compound 2412
was inverted by first treating 24 with DIAD, PPh3 and p-nitrobenzoic acid (PNBCOOH), and the
resulting PNB ester 25 was then subjected to NaN3.20 This gave the corresponding erythro isomer
26 in excellent yield. Methylation of the alcohol in 26 with MeI/Ag2O in refluxing ether gave ether
27 in almost quantitative yield. Removal of the benzyl group in 27 using Bobbit’s salt12 and
oxidation of the in situ generated alcohol using NaO2Cl/NaOCl gave acid Fmoc-L-erythro-
MeOAsp(tBu)OH (28) in 80% yield.
DIAD
PNBCOOH
PPh3
THF 0 oC to rt
OH
OPNB
OtBu
OH
OtBu
OtBu
NaN3
MeOH, 45 o
BnO
FmocHN
24
BnO
FmocHN
25
BnO
FmocHN
26
C
O
O
O
(70%)
(95%)
(88%)
o/n
O
O
O
Bobbit's salt
MeI
Ag2O
OtBu
OtBu
9:1 ACN:H2O rt, o/n
then NaO2Cl,
BnO
FmocHN
27
HO
ether, reflux
o/n
O
FmocHN
28
O
NaOCl, phosphate
buffer:ACN (1:1), 0 oC 6h
(96%)
(80%)
Scheme 7. Synthesis of Fmoc-L-erythro-MeOAsp(tBu)OH
A5D-erythro-MeOAsp was prepared using the same route (Scheme 6) that was used for
the threo isomer. The biological activity of A5D-erythro-MeOAsp was the same as authentic A5D
(Table 2), and the two compounds co-eluted when analyzed by RP-HPLC (see Figure S4 in the
Supporting Information). Comparing the NMR spectra of our synthesized A5D-eMeOAsp in D2O
to the literature spectrum (reported in D2O) was not particularly helpful in terms of characterization
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due to significant line broadening which was probably due to aggregation.21 Therefore, the A5D
nucleus was synthesized using the route described in Scheme 6 except that Boc-Trp(Boc)-OH was
used instead of Fmoc-Trp(Boc)-OH, and no lipid tail was coupled. The synthetic A5D-nucleus
was characterized by 1-D and 2-D NMR spectroscopy (see Figures S19-S23 and Table S2 in the
Supporting Information) and its 1H-NMR spectrum was consistent with the literature spectrum of
the A5D nucleus (see Figure S18 in the Supporting Information).22 These results indicated that the
MeOAsp residue in A5D has the 2S,3R (L-erythro) configuration and also strongly suggested that
the HOAsn residue is indeed the L-threo isomer. On the basis that the literature 1H NMR spectrum
of A5D-nucleus matched our 1H NMR spectrum, we also completely characterized A5-eMeOAsp
by 1D and 2D NMR, with DMSO-d6 as the solvent, which suppressed the line-broadening
previously noticed when D2O was used (See Figures S12-S17 and Table S1 in the Supporting
Information).
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11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
Table 2. MIC’s of A5D peptides
MIC (g/mL)a
entry
Peptide
1
2
3
4
5
Authentic A5D
A5D-tMeOAsp
A5D-eMeOAsp
A5D-(R)-anteiso
A5D-(S)-anteiso
0.5
>32
0.5
0.5
0.5
aAgainst B. subtilis 1046. 1.25 mM Ca2+ present.
The fact that our synthetic A5D, which contained a racemic lipid, exhibited the same
activity as authentic A5D suggests that the stereochemistry of the lipid does not affect activity or
that the lipid in A5D is racemic.23 To determine if this is indeed the case, we prepared A5D
(containing the correct L-erythro-MeOAsp isomer) bearing either (R)-8-methyldecanoic or (S)-8-
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methyldecanoic acid (A5D-(8R)-anteiso and A5D-(8S)-anteiso). (8R)- and (8S)-methyldecanoic
acid were prepared using the same route used to prepare (±)-8-methyldecanoic (Scheme 5) except
(R) or (S)-2-methylbutanal was used instead of (±)-2-methylbutanal. (S)-2-methylbutanal was
readily prepared by TEMPO oxidation of commercially available (S)-2-methylbutanol.24
However, (R)-2-methylbutanol and (R)-2-methylbutanal are either not commercially available or
prohibitively expensive. We prepared (R)-2-methylbutanal using a route similar to that developed
by Yang et al for the synthesis of (R)-2-methylpentanal (Scheme 8).25 (1S)-2,10-camphorsultam,
29, was treated with butyryl chloride in the presence of TEA and DMAP which gave sulfonamide
30 in 69% yield. Methylation at the 2-position of the butyryl portion of 30 with NaHMDS/MeI
gave compound 31 in 79 % yield.26 Treatment of 31 with DIBAL gave (R)-2-methylbutanal (32)
in 33 % yield.27
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19
20
21
22
23
24
25
26
27
28
29
30
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60
butyryl chloride
TEA, DMAP
THF, 0 oC, 30 min
1. NaHMDS, THF
HMPA, - 78 o
H
C
H
H
2. MeI, - 78 oC - rt
NH
N
O
N
O
S
S
S
18 h
O
O
O
O
O
O
29
30
31
(69%)
(79%)
DIBAL, toluene/DCM
- 78 oC, 90 min
O
Scheme 5
(R)-16
H
32
(33%)
Scheme 8. Synthesis of (R)-2-methylbutanal
A5D-(8R)-anteiso and A5D-(8S)-anteiso were prepared using the same route outlined in
Scheme 6 except Fmoc-L-eMeOAsp(tBu)OH, and (8R)- or (8S)-methyldecanoic acid was used.
The biological activities of A5D-(8R)-anteiso and A5D-(8S)-anteiso were determined against B.
subtilis (Table 2), and it was found that the biological activities of A5D-(R)-anteiso and A5D-(S)-
anteiso were identical to those of authentic A5D and A5D-eMeOAsp revealing that the
configuration of the lipid does not affect biological activity.
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CONCLUSIONS
5
6
7
8
Very recently, and while the work described here was nearing completion, Chen et al
reported the total synthesis of A54145A, A1, B, B1 and F, the stereochemical revision of the
MeOAsp, and confirmed that the HOAsn residue is the L-threo isomer.5 Their approach was to
attach the peptide to the resin via Gly10 (as in Scheme 4); however, the yields were low (3%). Our
studies suggest that the low yields may have been due to DKP and aspartimide formation. Their
synthesis involved making the ester bond on a 10-mer peptide containing the lipid tail, which may
also have contributed to the low yields. In contrast, we have devised a more efficient synthesis of
A5D. Consistent with the studies of Chen et al,5 we found that the MeOAsp residue has the 2S,3R
configuration. We show that the configuration of the lipid does not affect biological activity. This
opens the door for the synthesis of A5D analogs to establish SARs, which could lead to the
development of novel antibiotics for treating CAP.
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
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59
60
EXPERIMENTAL SECTION
General experimental information
All reagents used for peptide synthesis were obtained from commercial sources including coupling
reagents, resins, and Fmoc amino acids: AllocThrOH was prepared according to a literature
procedure.28 N3IleOH was prepared according to the procedure of Kim et al29 except imidazole-1-
sulfonyl azide hydrochloride30 was used in place of triflic azide. ACS grade, N,N’-
dimethylformamide (DMF), 4-methylpiperidine (4-MP), 2-methylpiperidine (2-MP), TFA,
triisopropylsilane (TIPS) were purchased from commercial suppliers and used without further
purification. CH2Cl2 (DCM) was distilled from calcium hydride under nitrogen. THF was distilled
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from sodium metal and benzophenone under nitrogen. Peptide synthesis was performed manually
using a rotary mixer for agitation.
7
8
9
Peptide syntheses were monitored by treating small aliquots of resin with 95:2.5:2.5
TFA/TIPS/H2O for 1.5 h, removing the solvent by N2 stream, re-dissolving the peptide in 1:1
CH3CN/H2O and analyzing by RP-HPLC and LRMS using a linear ion trap mass spectrometer.
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
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41
42
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45
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53
54
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59
60
Analytical HPLC was accomplished with a reversed-phase C18 column (10 µm, 250 mm
× 4.6 mm, 1 ml/min flow rate). Peptides were purified by reversed-phase semi-preparative HPLC
using a C18 column (10µm, 150 mm × 20 mm, 10 mL/min flow rate). High resolution positive ion
electrospray (ESI+) mass spectra were obtained using a hybrid quadrupole-orbitrap mass
spectrometer, , dissolving samples in 1:1 MeOH/H2O + 0.1% formic acid.
Resin loading was estimated using a procedure modified from Gude et al.31 Resin
containing the first amino acid, di- or tripeptide were subjected to 2 mL of 2% DBU in DMF (v/v)
for 30 minutes. The suspension was diluted to 10 mL with acetonitrile, of which 1 mL was further
diluted to 12.5 mL with acetonitrile. Absorbance of this solution was observed in triplicate at 304
nm with a reference solution as a blank. Resin loading was estimated by substitution of appropriate
values into the equation below:
퐴푏푠304 ∗ 0.0164
푙표푎푑푖푛푔 (푚푚표푙푒/푔)304 푛푚
=
푀푊 ― 35.45
(
푚푟푒푠푖푛 (푔) ― 퐴푏푠304
∗
∗ 0.0164
)
1000
where Abs304 is the average absorbance of the three trials, mresin is the weight of the aliquot of resin,
and MW is the molecular weight of the amino acid or peptide on the resin. The modified equation
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above accounts for the mass of the amino acid or peptide on the resin as well as the displacement
of chlorine.
7
8
9
Antibacterial Assays. The antibacterial activity of the synthetic peptides and authentic A54145-
D was determined using a broth microdilution assay.32 Overnight bacterial cultures were grown in
LB broth then diluted to approximately 1 × 106 CFU/mL according to the measured optical density
at 600 nm (OD600). Two-fold serial dilutions were used to prepare a series of peptide solutions in
LB broth which were then inoculated with an equal volume of diluted bacterial culture in 96-well
microplates. Plates were incubated for 24 h at 37 °C, then the MIC was determined by the lowest
concentration at which there was no visible growth of bacteria.
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11
12
13
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17
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19
20
21
22
23
24
25
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42
43
44
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57
58
59
60
tert-Butyl
(2S,3R)-3-((tert-butoxycarbonyl)amino)-2-hydroxy-3-(4-
methoxyphenyl)propanoate (2). This was prepared using the procedure that was reported by von
Eckardstein et al.9 To a solution of tert-butylcarbamate (3.67 g, 31.0 mmol) in nPrOH (40 mL)
was added a solution of NaOH (1.2 g, 30 mmol) in water (70 mL) followed by the addition of tert-
butylhypochlorite (3.74 mL, 33.0 mmol). The resulting mixture was stirred for 6 minutes then
cooled to 0 oC (ice bath). A solution of (DHQD)2PHAL in iPrOH (36 mL) was added followed
by a solution of 110 (2.34 g, 10.0 mmol) in nPrOH (70 mL) which was followed by a solution of
K2OsO2(OH)4 in aq. NaOH (36 mg NaOH in 30 mL water). The ice bath was removed and the
mixture was stirred for 1.5 h. The mixture was concentrated to 1/3 the volume by rotary
evaporation and then diethyl ether (150 mL) and water (150 mL) was added with stirring. The
layers were separated and the aqueous layer was extracted with diethyl ether (2 x 50 mL). The
combined organics were washed with 0.05 M HCl (100 ml), sat. NaHCO3 (100 mL) and sat. brine
(100 ml). The organic layer was dried (Na2SO4), filtered and the solvent removed by rotary
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evaporation. The resulting crude product was subjected to FC (20% EtOAc/80% hexane, Rf = 0.2)
which gave pure 2 as a white foam (3.56 g, 91% yield). [α]22D = -32.3 (c. 0.840, CH2Cl2). 1H NMR
(300 MHz, CDCl3): δ 7.26 (2H, d, J = 9.0 Hz), 6.86 (2H, d, J = 8.3 Hz), 5.34 (2H, d, J = 10.5 Hz),
5.15 (2H, d, J = 9.0 Hz), 4.30 (1H, s), 3.78 (3H, s), 3.14 (1H, d, J = 2.9 Hz), 1.50 (9H, s), 1.39
(9H, s); 13C{1H} NMR (75 MHz, CDCl3): δ 172.0, 158.8, 154.9, 131.9, 127.8, 113.7, 83.4, 79.3,
73.7, 55.1, 28.2, 27.7; HRMS (ESI+) m/z:: [M + H]+ Calcd for C19H30O6N 368.2068; Found
368.2068.
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
tert-Butyl
(2S,3R)-3-((tert-butoxycarbonyl)amino)-2-methoxy-3-(4-
methoxyphenyl)propanoate (3). To a solution of 2 (0.500 g, 1.36 mmol) in dry dichloromethane
(7 mL) was added a few beads of 4 Å MS followed by the addition of Ag2O (0.42 g, 1.8 mmol)
then methyl iodide (0.235 mL, 4.10 mmol). The mixture was stirred for 3 days with the exclusion
of light. The mixture was filtered through Celite, then the filtrate was concentrated by rotary
evaporation. The crude material was subjected to FC (5% EtOAc, 95% hexane, Rf = 0.2) which
1
gave pure 3 as a white foam (421.5 mg, 81% yield). [α]22D = -21.6 (c. 0.204, CH2Cl2). H NMR
(300 MHz, CDCl3): δ 7.18 (2H, d, J = 7.8 Hz); 6.77 (2H, d, J = 7.8 Hz); 5.45 (1H, bs), 5.03 (1H,
bs); 3.76 (1H, s), 3.67 (3H, s), 3.22 (3H, s), 1.40 (9H, s), 1.30 (9H, s); 13C{1H} NMR (75 MHz,
CDCl3): δ 169.0, 158.6, 154.7, 132.0, 127.6, 113.4, 83.0, 81.8, 79.0, 58.4, 55.1, 54.8, 28.0, 27.6;
HRMS (ESI+)m/z: [M + H]+ Calcd for C20H32O6N 382.2224; Found 382.2224.
1-Allyl 4-(tert-butyl) (2S,3S)-2-((tert-butoxycarbonyl)amino)-3-methoxysuccinate
(4). To a solution of 3 (2.0 g, 5.2 mmol) in EtOAc/H2O/CH3CN (2:1:1, 125 mL) was added NaIO4
(19 g, 89 mmol) then RuCl3 (54 mg, 0.26 mmol). The mixture was stirred vigorously for 4 h.
iPrOH (10 mL) was added and the mixture stirred for 10 min. The mixture was gravity filtered
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through Celite twice and then filtered again without Celite (filter paper). The filtrate was
concentrated to dryness. The residue was dissolved in dry methanol (25 mL) then gravity filtered
(filter paper) and the filtrate concentrated. The residue was dissolved in dry methanol (25 mL) then
CsCO3 (1.15 g, 3.00 mmol) was added and the mixture stirred for 2 h. The mixture was
concentrated to dryness. The residue was dissolved in dry DMF (20 mL) and allyl bromide (0.70
mL, 6.8 mmol) was added. The mixture was stirred for 16 h. Ether (200 mL) was added and then
mixture was washed with water (3 x 100 mL) then concentrated to dryness. The crude material
was subjected to FC (7.5 % EtOAc/92.5 % hexane then 10% EtOAc/90% hexane, Rf = 0.2) which
gave pure 4 as a white foam (0.971 g, 52% yield). 1H NMR (300 MHz, CDCl3): δ 5.84-5.92 (1H,
m), 5.30 (1H, d, J = 16.6 Hz), 5.21-5.25 (2H, m), 4.80 (1H, dd, J = 2.2, 10.3 Hz), 4.69 (1H, dd, J
= 5.6, 13.3 Hz), 4.59 (1H, dd, J = 5.8, 13.3 Hz); 4.21 (1H, 2, J = 2.2 Hz), 3.38 (3H, s), 1.44 (9H,
s), 1.37 (9H, s); 13C{1H} NMR (75 MHz, CDCl3): δ 169.3, 167.9, 155.2, 131.4, 118.3, 82.5, 79.8,
79.7, 66.0, 58.8, 55.9, 28.0, 27.7; HRMS (ESI+) m/z: [M + H]+ Calcd for C17H30O7N 360.2017;
Found 360.2016.
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
Fmoc-tMeOAspOAllyl (5). To 4 (1.00 g , 2.87 mmol) was added a solution of 6 M HCl
in dry dioxane (35 mL). The mixture was stirred for 16 h then concentrated to dryness. The
residue was dissolved in THF (15 mL) then concentrated to dryness (2x). The residue was
dissolved in THF (15 mL) and water (25 mL) then cooled (ice bath). A solution of Fmoc-Cl in
THF (35 mL) was added and the mixture was stirred for 2 h. Most of the THF was removed by
rotary evaporation. Water (30 mL) was added. The aqueous solution was washed with diethyl ether
(3 x 50 mL). The aqueous layer was acidified to pH = 2 then extracted with EtOAc (4 x 50 mL).
The combined organics were dried (Na2SO4), filtered, then concentrated to dryness. The residue
was subjected to FC (30% EtOAc/68% hexane/2% AcOH, Rf = 0.2) which gave pure 5 as a white
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foam (1.03 g, 87% yield). [α]22D = -18.3 (c. 1.00, CH2Cl2). 1H NMR (300 MHz, CDCl3): δ 10.64
(1H, bs), 7.76 (2H, d, J = 7.6 Hz), 7.62 (2H, d, J = 4.0 Hz), 7.41 (2H, app t, J = 7.4 Hz), 7.27-7.35
(2H, m), 5.80-5.97 (1H, m), 5.80 (1H, d, J = 9.9 Hz), 5.37 (1H, d, J = 17.0 Hz), 5.30 (1H, d, J =
10.3 Hz), 5.03 (1H, d, J = 9.9 Hz); 4.76 (1H, dd, J = 5.8, 12.9 Hz), 4.70 (1H, dd, J = 5.8, 13.5 Hz),
4.47 (1H, s), 4.36-4.41 (2H, m), 4.25 (1H, t, J = 7.4 Hz); 3.48 (3H, s); 13C{1H} NMR (75 MHz,
CDCl3): δ 172.2, 168.7, 156.8, 143.8, 143.6, 141.3, 131.2, 127.7, 127.1, 125.3, 119.9, 119.1, 79.3,
67.9, 66.7, 59.4, 56.3, 46.9; HRMS (ESI+)m/z: [M + H]+ Calcd for C23H24O7N 426.1547; Found
426.1541.
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Ethyl (Z)-8-methyldec-6-enoate (15). The following procedure was used to prepare (±)-
15, (S)-15 and (R)-15 using either (±)-, (S)- or (R)-2-methylbutanal (31). To a 100 mL round-
bottom flask was added 1418 (1.02 g, 2.10 mmol) and purged with argon gas. Dry THF (50 mL)
was added and the mixture was cooled to -78 oC in a slurry of dry ice in acetone. A 1.0 M solution
of NaHMDS in THF (2.1 mL, 2.1 mmol) was slowly added over five min and the resulting brown
solution was stirred for 30 min at -78 oC. A solution of (±)-2-methylbutanal (231 µL, 2.00 mmol)
in THF (5 mL) was transferred to the reaction mixture. The solution was allowed to warm to room
temperature and stirred overnight. The reaction was quenched with saturated sodium bicarbonate
and the product was extracted with ethyl acetate (2 x 50 mL). The organic layers were combined,
dried with sodium sulfate, and concentrated in vacuo. The residue was subjected to FC (95%
hexane/5% ethyl acetate, Rf = 0.3) which provided pure (±)-15 as a colorless oil (0.305 g, 66%
1
yield). The yields of (S)-15 and (R)-15 were 71% and 69% respectively. H NMR (300 MHz,
CDCl3) δ: 5.22 (1H, dt, J = 7.2 Hz, 10.8 Hz), 5.04 (1H, app t, J = 9.9 Hz), 4.04 (2H, q, J = 7.2 Hz),
2.22 (2H, t, J = 7.5 Hz), 1.97 (2H, q, J = 7.3 Hz), 1.57 (2H, app quint, J = 7.6 Hz), 1.36-1.04 (7H,
m), 0.85 (3H, d, J = 6.7 Hz), 0.76 (3H, t, J = 7.4 Hz). 13C{1H} NMR (75 MHz, CDCl3) δ: 173.5,
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136.4, 127.8, 60.0, 34.1, 33.3, 30.2, 29.3, 27.0, 24.5, 20.9, 14.1, 11.8. HRMS (ESI+)m/z: [M + H]+
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Calcd for C13H25O2213.1849; Found 213.1849. The H NMR spectra of (S)-15 and (R)-15 were
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identical to that of (±)-15.
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(±)-8-methyldecanoic acid (16). The following procedure was used to prepare (±)-16,
(S)-16 and (R)-16 using either (±)-15, (S)-15 and (R)-15. Compound (±)-15 (201 mg, 0.95 mmol)
was added to a 50 mL round-bottom flask. To this was added ethanol (25 mL) and 10% Pd/C (40
mg) and the flask was purged with argon gas. The flask was then flushed with hydrogen gas and
allowed to stir overnight at room temperature. The mixture was filtered over Celite and the filtrate
was concentrated yielding a colorless oil (1H-NMR (300 MHz, CDCl3) δ: 4.06 (2H, q, J = 7.0 Hz),
2.22 (2H, t, J = 7.4 Hz), 1.56 (2H, m), 1.35-0.95 (15H, m), 0.88-0.72 (6H, m)). The oil (176 mg,
0.82 mmol) was dissolved in a solution of 1:1 0.6 M NaOH (aq) and MeOH (25 mL). After 3 h,
the reaction was acidified to pH 1 with 1M HCl and the product extracted into ethyl acetate (2 x
50 mL). The organic layers were dried with Na2SO4 and the solvent removed by rotary evaporation
yielding pure 16 as a colorless oil (153 mg, 77% yield). The yields of (S)-16 and (R)-16 were 78%
and 79% respectively. 1H-NMR (300 MHz, CDCl3) δ: 11.74 (1H, br s), 2.33 (2H, t, J = 7.5 Hz),
1.59 (2H, app. quint, J = 7.3 Hz), 1.39-1.00 (11H, m), 0.88-0.76 (6H, m); 13C{1H} NMR (75 MHz,
CDCl3) δ: 183.6, 39.4, 37.2, 37.0, 32.5, 32.3, 32.0, 29.7, 27.5, 22.0, 14.2; HRMS (ESI-) m/z: [M-
1
H]- Calcd for C11H21O2 185.1547; Found 185.1552. The H NMR spectra of (S)-16 and (R)-16
were identical to that of (±)-16. (S)-16: [α]22 +4.7 (c 0.20, CHCl3) (lit.,33 [α]25 +6.1 (1.14,
D
D
CHCl3)). (R)-16: [α]22D -4.4 (c 0.20, CHCl3).
Fmoc-tHOAsn(OTBS,NTrt)OH. A solution of H2SO4:acetic anhydride (1:9, 300 µL,
0.557 mmol) was added dropwise to a suspension of trityl alcohol (5.37 g, 20.6 mmol, 10 equiv)
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in acetic anhydride (8.3 mL) cooled in a room temperature water bath. After stirring for 5 min, the
water bath was removed and after stirring for an additional 10 min at room temperature, Fmoc-
tHOAsn(OTBS)OH12 was added in a single portion. After 30 min, the reaction was diluted with
ethyl acetate (20 mL), cooled in an ice water bath, and quenched through a dropwise addition of
saturate aqueous NaHCO3 (10 mL). After separation, the aqueous layer was cooled (ice-water
bath) and acidified (pH ca. 3) with 1N HCl. The cold solution was extracted with ethyl acetate (2
x 30 mL) and the combined organic layer was dried over MgSO4, filtered and concentrated. Silica
gel column chromatography (5% ethyl acetate/94% hexanes/1% AcOH then 40% ethyl
acetate/59% hexanes/1% AcOH) yielded Fmoc-tHOAsn(OTBS,NTrt)OH as a colorless solid (1.05
g, 70% yield). All spectroscopic data matched that previously reported.34 [α]22D = -22.2 (c. 0.724,
CH2Cl2). 1H NMR (300 MHz, DMSO-d6): δ 8.31 and 8.20 (1H, s), 7.90-7.20 (24H, m) 4.68 and
4.64 (1H, s), 4.56-4.49 (1H, m), 4.42 (1H, d, J = 9.0 Hz), 4.30-4.16 (2H, m) 0.882 (9H, s) 0.084-
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0.020 (6H, m); C{1H} NMR (75 MHz, DMSO-d6): 171.4, 168.7, 156.5, 144.3, 143.6, 143.6,
140.7, 140.6, 128.6, 128.5, 127.6, 127.5, 127.1, 127.0, 126.7, 125.6, 125.3, 120.0, 73.9, 69.5, 66.2,
57.5, 46.6, 25.6, 17.7, -5.0, -5.4; HRMS (ESI-) m/z: [M - H]- Calculated for C44H45N2O6Si
725.3052; Found 725.3039.
(2R,3R)-3-((((9H-fluoren-9-yl)methoxy)carbonyl)amino)-4-(benzyloxy)-1-(tert-
butoxy)-1-oxobutan-2-yl 4-nitrobenzoate(25). Compound 2412 (er = 95:5, 3.09 g, 6.14 mmol),
p-nitrobenzoic acid (1.54 g, 9.20 mmol) and triphenylphosphine (2.41 g, 9.20 mmol) were
dissolved in THF (31 mL) and this solution was cooled (ice-water bath). Diisopropyl
azodicarboxylate (1.81 mL, 9.20 mmol) was added and the solution was left for 24 h at room
temperature under an inert atmosphere. After concentrating the reaction mixture, a single re-
crystallization of the crude solid in MeOH afforded 25 as a colorless, crystalline solid (3.52 g, 88%
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yield). [α]22D = -7.8 (c. 1.03, CH2Cl2). H NMR (300 MHz, CDCl3): δ 8.20 (2H, d, J = 8.4 Hz),
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8.10 (2H, d, J = 8.4 Hz), 7.75 (2H, m), 7.58 (2H, m), 7.41-7.25 (9H, m), 5.37-5.29 (2H, m), 4.58-
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4.42 (5H, m), 3.72-3.64 (2H, m) 1.45 (9H, s); C{1H} NMR (75 MHz, CDCl3): 166.3, 163.8,
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155.7, 150.6, 143.7, 141.3, 137.3, 134.5, 131.0, 128.5, 128.0, 127.7, 127.0, 125.0, 124.9, 123.5,
120.0, 83.3, 73.3, 67.8, 67.0, 51.1, 47.1, 27.9; HRMS (ESI+) m/z: [M + H]+ Calcd for C37H37O9N2
653.2494; Found 653.2507.
tert-Butyl (2R,3R)-3-((((9H-fluoren-9-yl)methoxy)carbonyl)amino)-4-(benzyloxy)-2-
hydroxybutanoate (26). Compound 25 (1.95 g, 3.00 mmol) and NaN3 (584 mg, 9.00 mmol) were
dissolved in MeOH (50 mL) at room temperature. The colorless suspension was stirred at 45 oC
for 4 hours. The homogenous mixture was concentrated and the crude residue subjected to FC
(20% ethyl acetate/80% hexanes) which provided pure 26 as a colorless foam (1.45 g, 95% yield).
The er was determined to be 97:3 by chiral RP-HPLC (see Figure S24 in the Supporting
Information). 1H NMR (300 MHz, CDCl3): δ 7.76 (2H, d, J = 6.9 Hz), 7.60 (2H, d, J = 6.9 Hz),
7.42-7.25 (9H, m), 5.45 (1H, m), 4.48-4.40 (4H, m), 4.23-4.22 (3H, m), 3.56-3.35 (3H, m), 1.44
(9H, s); 13C{1H} NMR (75 MHz, CDCl3): 171.4, 155.8, 143.8, 141.3, 137.4, 128.4, 127.9, 127.9,
127.7, 127.0, 119.9, 83.0, 73.4, 71.7, 68.4, 66.9, 52.4, 47.1, 27.9; HRMS (ESI+) m/z: [M + H]+
Calcd for C30H34O6N 504.2381; Found 504.2374.
tert-Butyl (2R,3R)-3-((((9H-fluoren-9-yl)methoxy)carbonyl)amino)-4-(benzyloxy)-2-
methoxybutanoate (27). To a solution of 26 (1.20 g, 2.38 mmol) in diethyl ether (48 mL) was
added freshly prepared, dry silver oxide (1.65 g, 7.15 mmol) and MeI (2.97 mL, 47.7 mmol). The
suspension was vortexed briefly then refluxed overnight. The suspension was cooled to room
temperature and then filtered through Celite. The filtrate was concentrated which provided pure
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27 as a white foam (1.18 g, 96% yield). 1H NMR (300 MHz, CDCl3): δ 7.76 (2H, d, J = 7.5 Hz),
7.60 (2H, d, J = 7.5 Hz), 7.40 (2H, t, J = 7.2 Hz), 7.33-7.25 (9H, m), 5.28-5.25 (1H, m), 4.51 (2H,
m), 4.38 (2H, m), 4.23 (2H, m), 3.82 (1H, d, J = 5.7 Hz), 3.62 (1H, dd, J = 5.1, 9.6 Hz) 3.50-3.41
(5H, m), 1.43 (9H, s); 13C{1H} NMR (75 MHz, CDCl3): 169.4, 155.8, 143.9, 141.3, 137.9, 128.4,
127.7, 127.7, 127.7, 127.0, 125.1, 120.0, 82.1, 80.4, 73.1, 68.0, 68.8, 58.7, 52.2, 47.2, 27.9; HRMS
(ESI+) m/z: [M + H]+ Calcd for C31H36O6N 518.2537; Found 518.2539.
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Fmoc-eMeOAsp(tBu)OH (28). Bobbit’s salt (2.33 g, 7.79 mmol) was added to a solution
of 30 (1.30 g, 2.51 mmol) in MeCN (9 mL) and H2O (1 mL) and the mixture was stirred overnight.
The mixture was cooled (ice-water bath) and diluted with a solution of NaO2Cl (80% w/w; 880
mg, 7.78 mmol) in phosphate buffer (0.67 N, pH ca. 7; 16.8 mL). The pH of the resulting solution
was adjusted to 7 with 1 N NaOH, at which point MeCN (15.7 mL) was added, followed by a
dropwise addition of NaOCl (0.81 N bleach; 2.17 mL, 1.76 mmol) over 30 min. After 8 hours, sat.
aqueous NaSO3 was added until the dark red color faded to a pale orange. The two layers were
separated, and the aqueous layer was acidified to pH 2 with conc. aqueous HCl and extracted with
DCM (3 x 50 mL). The combined organic layer was dried over MgSO4, filtered and concentrated.
The crude residue was subjected to FC (40% ethyl acetate/59% hexanes/1% AcOH) which
provided pure 28 as a colorless foam (891 mg, 80% yield). [α]22D = 44.80 (c. 0.800, CH2Cl2). 1H
NMR (300 MHz, CDCl3): δ 11.30 (1H, br. s), 7.76 (2H, d, J = 7.5 Hz), 7.61 (2H, d, J = 7.2), 7.40
(2H, t, J = 7.5 Hz), 7.31 (2H, t, J = 7.5 Hz), 6.16 and 5.78 (1H, d, J = 8.4 Hz), 4.94 and 4.67 (1H,
dd, J = 2.4, 8.4 Hz), 4.52-4.37 (2H, m), 4.25 (1H, t, J = 6.9 Hz), 4.07 (1H, d, J = 2.4 Hz), 3.53 and
13
3.46 (3H, s), 1.51 (9H, s); C{1H} NMR (75 MHz, CDCl3): 172.7, 168.0, 155.9, 143.7, 143.5,
141.2, 127.6, 127.0, 125.0, 119.9, 83.1, 80.7, 67.3, 59.6, 55.8, 46.9, 27.8; HRMS (ESI+) m/z: [M
+ H]+ Calcd for C24H28O7N 442.1860; Found 442.1857.
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1-((3aR,6R)-8,8-dimethyl-2,2-dioxidotetrahydro-3H-3a,6-
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methanobenzo[c]isothiazol-1(4H)-yl)butan-1-one (30). (1S)-(−)-2,10-Camphorsultam (29, 10.0
g, 46.4 mmol) and DMAP (624 mg, 5.11 mmol) were dissolved in dry THF (60 mL) at room
temperature. The solution was cooled (ice bath) and triethylamine (7.04 mL, 69.7 mmol) was
added followed by the dropwise addition of butyryl chloride (5.3 mL, 51.1 mmol). The formed
suspension was stirred for 30 min then quenched with 1N HCl (20 mL) and extracted with ethyl
acetate (3 x 60 mL). The combined organic layer was washed once with 1N NaOH (100 mL), dried
over MgSO4 and filtered. The filtrate was concentrated, and the crude solid was purified by a single
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recrystallization in methanol, yielding 30 as a colorless, crystalline solid (9.2 g, 69% yield). H
NMR (300 MHz, CDCl3): δ 3.83 (1H, dd, J = 5.7, 6.9 Hz), 3.47 (1H, d, J = 13.8 Hz), 3.39 (1H, d,
J = 13.8 Hz), 2.74-2.57 (2H, m), 2.06-2.01 (2H, m), 1.86-1.83 (2H, m), 1.68 (2H, sext, J = 7.2 Hz),
1.41-1.28 (2H, m), 1.12 (3H, s), 0.95-0.90 (6H, m); 13C{1H} NMR (75 MHz, CDCl3): 171.9, 65.1,
52.9, 48.3, 47.7, 44.6, 38.5, 37.3, 32.8, 26.4, 20.8, 19.8, 17.9, 13.5; HRMS (ESI+) m/z: [M + H]+
Calcd for C14H24O3NS 286.1471; Found 286.1465.
S(2R)-1-((3aR,6R)-8,8-dimethyl-2,2-dioxidotetrahydro-3H-3a,6-
methanobenzo[c]isothiazol-1(4H)-yl)-2-methylbutan-1-one (31). NaHMDS (40.1 mL, 1M in
THF, 40.1 mmol) was added dropwise to a solution 30 (8.81 g, 30.9 mmol) in dry THF (221 mL)
o
cooled to -78 C, under an inert atmosphere. The resulting solution was stirred for 1 h, then dry
HMPA (16.1 mL) and MeI (5.76 mL, 92.6 mmol) were added and cooling was removed. The
solution was stirred at room temperature for 18 h, then quenched with sat. aqueous NH4Cl (40 mL)
and extracted with ethyl acetate (3 x 100 mL). The combined organic layer was dried with MgSO4,
filtered and concentrated. The crude solid was purified by a single recrystallization in methanol
which yielded 31 (7.30 g, 79% yield) as a colorless, crystalline solid. 1H NMR (300 MHz, CDCl3):
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δ 3.85 (1H, t, J = 6.3 Hz), 3.47 (1H, d, J = 13.5 Hz), 3.39 (1H, d, J = 13.8 Hz), 2.93 (1H, sex., J =
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6.9 Hz), 2.01-1.99 (2H, m), 1.89-1.70 (4H, m), 1.40-1.27 (3H, m), 1.16-1.11 (6H, m), 0.93-0.84
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(6H, m); C{1H} NMR (75 MHz, CDCl3): 176.2, 64.9, 53.1, 48.1, 47.6, 44.5, 41.7, 38.4, 32.7,
26.3, 25.6, 20.7, 19.8, 18.5, 11.7; HRMS (ESI+) m/z: [M + H]+ Calcd for C15H26O3NS 300.1628;
Found 300.1621. The absolute configuration of 31 was confirmed by X-ray crystallography (see
Figure S25 in the Supporting Information).
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(R)-2-methylbutanal (32). DIBAL (1M in toluene; 36.6 mL, 36.6 mmol) was added
dropwise to a solution of 31 (7.31 g, 24.4 mmol) in dry DCM at -78 oC under an inert atmosphere.
The resulting solution was stirred for 90 min, then quenched with a solution of NaHSO3 (8.77 g)
in H2O (90 mL) and allowed to come to room temperature. The mixture was extracted with pentane
(3 x 100 mL) and the combined organic layer concentrated by rotary evaporation (no heating) until
only toluene remained. The residue was subjected to FC (30% DCM/70% pentane then 45%
DCM/55% pentane). The combined fractions that contained 32 were carefully concentrated by
rotary evaporation (no heating) until only a solution of 32 in DCM remained (40% w/w). The 1H-
NMR spectrum was identical to that of (S)-2-methylbutanal.25 This solution was used immediately
for the synthesis of (R)-16.
Synthesis of A5D-tMeOAsp. 2’-Cl-TrtCl polystyrene resin (theoretical substitution = 1.5
mmol/g, 66.7 mg, 0.1 mmol, 1 equiv) was activated in dry DCM with thionyl chloride (26 µL, 3.6
equiv) and pyridine (58 µL, 7.2 equiv) under reflux for 2 h. The resin was transferred to a
disposable peptide cartridge and rinsed 6x with dry DCM, followed by loading with Fmoc-Sar-
OH (4 equiv) and DIPEA (8 equiv) in 1 mL dry DCM for 2 h, twice. The resin was capped with
17:2:1 DCM/MeOH/DIPEA (3 x 10 min) and the loading efficiency determined to be 1.40 mmol/g.
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Removal of the Fmoc group from Fmoc-Sar-OH was performed with four 30-minute iterations of
20% 4-methylpiperidine in DMF (v/v). All Fmoc-amino acids (4 equiv) and the lipid tail (4 equiv)
were activated for 5 min using HOBt (4 equiv) and DIC (4 equiv) in DMF (2 mL) and then coupled
for 4 h, except for Fmoc-tMeOAsp(tBu)OH, which was coupled for 12 h. Removal of the Fmoc-
group from peptide 12 was performed with two 1-minute iterations of 20% 2-methylpiperidine in
DMF (v/v), and all other Fmoc-removals were performed with three 10-minute iterations of 20%
2-methylpiperidine in DMF. Following installation of Fmoc-tHOAsn(OTBS)OH, yielding peptide
17, peptide 18 was prepared by treating peptide 17 with N3-Ile-OH (10 equiv), pre-activated with
DIC (10 equiv) by stirring in DCM (2 mL) for 1 h, followed by the addition of DMAP (0.1 equiv),
for 12 h. Depsi-bond completion was monitored by HPLC. The next amino acids, D-Glu2, Trp1,
and 8-methyldecanoic acid, were introduced using the above general procedure, yielding peptide
19. The TBS group was removed by treating peptide 19 with TBAF (3 equiv), and acetic acid (3
equiv) in THF (2 mL) for 1 h. The azide group in the resulting peptide was removed by treatment
of the peptide with PBu3 (3 equiv), and (FmocGlu(tBu)CO)2O (5 equiv) (formed by the addition
of 10 equiv FmocGlu(tBu)OH and 5 eq DIC to 2 mL dry THF and stirring for 1 h) for 5 minutes,
followed by the addition of 0.1 mL H2O and further stirring for 16 h, yielding peptide 20. The
remaining amino acids were introduced using the coupling and deprotection protocol mentioned
above, affording peptide 21. The peptide was cleaved from the resin by treatment with 1% TFA in
DCM (5 x 10 min) affording peptide 22, which was converted to peptide 23 off-resin by cyclizing
with PyAOP (5 equiv), HOAt (5 equiv), and 2,4,6-collidine (10 equiv) in DMF (100 mL, 1 mM)
overnight. The solvent was removed by high-vacuum rotary evaporation, and the crude solid was
treated with 95:2.5:2.5 TFA/TIPS/H2O (v/v, 15 mL) for global deprotection of side-chain
protecting groups. The TFA was removed under stream of N2, and the crude product dissolved in
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9:1 H2O/CH3CN. The target compound was purified by preparative RP-HPLC employing an
isocratic gradient of 40% CH3CN/60% H2O (+0.1% TFA). Fractions containing A5D-tMeOAsp
were pooled and lyophilized giving A5D-tMeOAsp as a white powder (12.3 mg, 8 % yield based
on resin loading), judged to be > 95% pure by analytical RP-HPLC (See Figure S1 in the
Supporting Information). HRMS (ESI+) m/z: [M+2H]2+ Calcd for C73H113N17O27 829.8990; Found
829.8992.
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A5D-eMeOAsp. A5D-eMeOAsp was synthesized according to the procedure used for
A5D-tMeOAsp, except that Fmoc-eMeOAsp(tBu)OH was used in place of Fmoc-
tMeOAsp(tBu)OH. The desired compound was isolated as a white powder (12.4 mg, 8 % yield),
judged to be >95% pure by analytical RP-HPLC (see Figure S3 in the supporting information).
HRMS (ESI+) m/z: [M+2H]2+ Calcd for C73H113N17O27 829.8990; Found 829.9010. The peptide
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was also characterized by NMR (1-D H, as well as 2-D: HSQC, HMBC, COSY, TOCSY,
ROESY. See Figures S12-S17 and Table S1 in the Supporting Information).
A5D-(S)-anteiso. A5D-(S)-anteiso was synthesized according to the procedure used for
A5D-tMeOAsp, except that Fmoc-eMeOAsp(tBu)OH was used in place of Fmoc-t-
MeOAsp(tBu)OH, and (S)-8-methyldecanoic acid were used in place of (±)-8-methyldecancoic
acid. A5D-(S)-anteiso was isolated as a white powder (16.6 mg, 11 % yield based on resin loading),
judged to be >95% pure by analytical RP-HPLC (see Figure S6 in the supporting information).
HRMS (ESI+) m/z: [M+2H]2+ Calcd for C73H113N17O27 829.8990; Found 829.8986.
A5D-(R)-anteiso. A5D-(R)-anteiso was synthesized according to the procedure used for
A5D-tMeOAsp, except that Fmoc-eMeOAsp(tBu)OH was used in place of Fmoc-
tMeOAsp(tBu)OH, and (R)-8-methyldecanoic acid were used in place of (±)-8-methyldecancoic
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acid. A5D-(R)-anteiso was isolated as a white powder (16.7 mg, 11% yield based on resin loading),
judged to be >95% pure by analytical RP-HPLC (see Figure S7 in the supporting information).
HRMS (ESI+) m/z: [M+2H]2+ Calcd for C73H113N17O27 829.8990; Found 829.8991.
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A5D-nucleus. The nucleus of A5D, used for characterization purposes, was synthesized
according to the procedure used for A5D-tMeOAsp, except that Fmoc-eMeOAsp(tBu)OH was
used in place of Fmoc-tMeOAsp(OBu)OH, and BocTrp(Boc)OH was used instead of
FmocTrp(Boc)OH. No lipid tail was coupled to the peptide. The peptide was purified using
preparative RP-HPLC employing an isocratic solution of 16% CH3CN/84% H2O (+ 0.1% TFA),
yielding the A5D-nucleus as a white powder (12.0 mg, 9% based on resin-loading). The compound
was judged to be >95% pure based on analytical HPLC (see Figure S10 in the supporting
information). HRMS (ESI+) m/z: [M+2H]2+ Calcd for C62H93N17O26 745.8233; Found 745.8244.
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The peptide was also characterized by NMR (1-D H, as well as 2-D: HSQC, HMBC, COSY,
TOCSY, ROESY. See Figures S19-S23 and Table S2 in the Supporting Information).
ASSOCIATED CONTENT
Supporting Information
The Supporting Information is available free of charge on the ACS Publications website at DOI:
HPLC chromatograms and HRMS spectra of all A5D peptides. 1-D and 2-D NMR spectra of the
A5D nucleus and A5D-eMeOAsp and assignment of residue chemical shifts. Comparison of 1-D
1H NMR spectrum of the synthetic A5D-nucleus to the 1-D 1H-NMR spectrum of the A5D nucleus
reported in the literature. Chiral HPLC of compound 26. 1H- and 13C-NMR spectra for compounds
2-5, (±)15, (±)-16, (S)-16, (R)-16, 25-28, 30, 31 and Fmoc-tHOAsn(OTBS,NTrt)OH. X-ray
crystal structure of compound 31.
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