Total synthesis of micromide: A marine natural product

Article abstract of DOI:10.1002/ejoc.201402977

This paper describes an efficient procedure for the synthesis of micromide, a natural product that shows anti-solid-tumor activity. Our strategy involved the synthesis of N-nosyl-protected amino acids and their N-methylation with iodo-methane. The hindered oligopeptides containing N-methyl amino acids were synthesized in excellent yields and high purities.[ampi]]

Full text of DOI:10.1002/ejoc.201402977

FULL PAPER
DOI: 10.1002/ejoc.201402977
Total Synthesis of Micromide: a Marine Natural Product
[a]
[a]
[a]
[a]
[a]
Jianrong Han,* Jingtang Lian, Xia Tian, Shengwei Zhou, Xiaoli Zhen, and
Shouxin Liu*[
b]
Keywords: Total synthesis / Natural products / Peptides / Amino acids / Antitumor agents / Sulfonamides
This paper describes an efficient procedure for the synthesis
of micromide, a natural product that shows anti-solid-tumor
methane. The hindered oligopeptides containing N-methyl
amino acids were synthesized in excellent yields and high
activity. Our strategy involved the synthesis of N-nosyl-pro- purities.
tected amino acids and their N-methylation with iodo-
Introduction
connection of a pentapeptide relies on independent con-
struction of dipeptide and tripeptide fragments to be joined
together by a convergent approach. Therefore, a late-stage
construction of micromide by a convergent approach would
lead back to dipeptide and tripeptide fragments. We
planned to produce dipeptide and tripeptide fragments by
standard methods for peptide synthesis. However, this strat-
egy was not successful. The difficulties in this attempted
synthesis may be caused by the sterically hindered N-methyl
amino acids that resist coupling. We then attempted to syn-
thesize a linear peptide in a solution-phase method using
standard Boc (tert-butoxycarbonyl) chemistry and a one-
Micromide (1) is a naturally occurring linear peptide iso-
lated from a species of marine cyanobacterium of the genus
[
1]
Symploca. Structural studies have shown that micromide
contains features common to many cyanobacterial metabo-
lites, including N-methylated amino acids, a d-amino acid,
and a modified cysteine unit in the form of a thiazole ring.
It consists of five amino acid residues and a unique β-meth-
oxy acid unit, 3-methoxyhexanoic acid. Micromide shows
significant selectivity for solid-tumor cell lines. It has an
IC50 value of 0.26 μm against the KB cell line. The chemical
modification of natural products has been a major means
of exploration for more potent analogs.[ Thus, the synthe-
sis of micromide, a structurally simple linear peptide cyto-
toxic alkaloid, and its derivatives could be a significant step
towards the development of a new anti-solid-tumor drug.
In this paper, we report the first total synthesis of micro-
mide.
[3]
by-one stepwise approach. However, when we attempted
2]
the reaction between the tripeptide and N-Boc-N-Me-d-Val
using DIPEA (diisopropylethylamine) as base in the pres-
ence of HATU {1-[bis(dimethylamino)methylene]-1H-1,2,3-
triazolo[4,5-b]pyridinium 3-oxide hexafluorophosphate}
and PyBOP (benzotriazol-1-yl-oxytripyrrolidinophosphon-
ium hexafluorophosphate) as coupling reagents at room
temperature, the reaction did not proceed smoothly (see
Supporting Information). This attempt failed, and the tetra-
peptide was produced as the major product instead. There-
fore, the preparation of N-methylated peptides was a chal-
Results and Discussion
Micromide is a pentapeptide with 3-methoxyhexanoic
acid at its N-terminus and N-Me-Gly-thiazole at its C-ter-
minus. Micromide contains three N-methyl-amino acids
and two natural amino acid residues. A logical initial dis-
[
4]
lenge, and the careful selection of protecting groups and
coupling methods was crucial. The most efficient method
for site-selective N-methylation of peptides was developed
[
5]
[6]
by Fukuyama and Miller. This method is a three-step
procedure involving amine activation by protection with an
o-nitrophenylsulfonyl (o-Ns) group, followed by alkylation
and then deprotection of the o-Ns group on a solid support.
[
a] College of Sciences, Hebei University of Science and
Technology,
Shijiazhuang 050018, People’s Republic of China
E-mail: Han_jianrong@126.com
http://lxy.web.hebust.edu.cn/
[
7]
Ye et al. reported the total synthesis of dragonamide
[
b] State Key Laboratory Breeding Base, Hebei Laboratory of using this method in solution.
Molecular Chemistry for Drug, Hebei University of Science
For our current work, target compound 1 was considered
and Technology,
Shijiazhuang 050018, People’s Republic of China
E-mail: chlsx@hebust.edu.cn
in seven fragments, namely, N-Me-Gly-thiazole, N-Me-Phe,
N-Me-Ile, Val, N-Me-Val, Phe, and 3-methoxyhexanoic
acid. The retrosynthetic analysis of micromide is shown in
Scheme 1. Based on the principles of retrosynthesis, the tar-
http://hgxy.web.hebust.edu.cn/
Supporting information for this article is available on the
WWW under http://dx.doi.org/10.1002/ejoc.201402977.
7232
© 2014 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
Eur. J. Org. Chem. 2014, 7232–7238

Total Synthesis of Micromide: a Marine Natural Product
get molecule was produced by individually coupling the methylated amino acids in solution[10] or on solid support.
seven fragments from the C-terminus to the N-terminus in We began with the conversion of l-phenylalanine, l-iso-
a one-by-one stepwise approach.
leucine, and d-valine into their tert-butyl esters S2 using
tert-butyl acetate and perchloric acid. Compounds S2 were
then successively protected as their 2-nitrobenzenesulfon-
amides S3 by treatment with o-nosyl chloride in the pres-
ence of a base. The alkylation was achieved by treating S3
with methyl iodide in DMF with potassium carbonate as
base to obtain S4. After alkylation, compounds S4 were
treated with trifluoroacetic acid (TFA) to produce the free
acids S5. The acids were readily converted into their acyl
chlorides 3, 4, and 5 by treatment with oxalyl chloride and
DMF in dichloromethane (Scheme 3).
Fragments 2 and 3 were coupled under mild conditions
to give compound 6. Compound 6 was then treated with
mercaptoacetic acid in DMF under basic conditions
(LiOH) to produce 7, which was then coupled with 4 to
give dipeptide 8 under the same conditions as those used
for the synthesis of 6. However, when 8 was deprotected,
and the free amine (i.e., 9) was treated with l-N-o-Ns-Val
chloride under the same coupling reaction conditions, we
Scheme 1.
Starting material N-Me-Gly-thiazole 2 was prepared in did not obtain the desired tripeptide product (i.e., a pro-
two steps and 83% yield from 1,3-thiazole-2-carbaldehyde. tected N-nosyl form of 11). The actual product of this reac-
First, 1,3-thiazole-2-carbaldehyde was allowed to react with tion may be the product of coupling of the non-N-methyl-
methylamine in the presence of molecular sieves (4 Å) to ated amino acid (l-N-o-Ns-Val chloride) with itself. Given
give the imine. This was then reduced with sodium borohy- the unexpected difficulty of the coupling between 9 and l-
dride to give N-Me-Gly-thiazole 2 (Scheme 2).
N-o-Ns-Val chloride, we modified our original approach.
To obtain tripeptide product 11 in the most efficient way, 9
was coupled with l-N-Boc-Val using DIPEA as base in the
presence of HATU as coupling reagent at room tempera-
ture. This procedure yielded 94.2% of N-Boc-protected tri-
peptide 10. Subsequent deprotection of the amine in the
tripeptide using TFA/CH Cl yielded 11. In the next reac-
Scheme 2.
2
2
tion, d-N-o-Ns-N-Me-Val chloride 5 was treated with 11 in
The synthesis of three N-methyl amino acids was then the presence of Et N to give the desired coupling product.
3
investigated. N-methyl amino acids undergo racemization This was then deprotected with mercaptoacetic acid in
under acidic and basic conditions,[ so racemization-free DMF under basic conditions (LiOH) to obtain 12. A fur-
methods have been developed for their synthesis. Miller et ther iteration of this coupling procedure led to the forma-
al. found base-catalyzed N-alkylation of o-Ns using dimeth- tion of compound 13. Deprotection of the amine in the
8]
ylsulfate as the methylating agent to be a better alterna- pentapeptide using TFA/CH Cl2 produced an insoluble
2
[
9]
tive. A modification of this method based on the Mitsu- TFA salt 14. These steps completed the synthesis of the
nobu reaction has been optimized to obtain all possible N- main peptide portion of the molecule (Scheme 4).
Scheme 3.
Eur. J. Org. Chem. 2014, 7232–7238
© 2014 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
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J. Han, J. Lian, X. Tian, S. Zhou, X. Zhen, S. Liu
FULL PAPER
Scheme 4.
After the completion of the pentapeptide synthesis, we um(II)-catalyzed hydrogenation. Thus, to complete our
focused on the fragment (R)-3-methoxyhexanoic acid. The work towards the total synthesis of the natural product
development of methods for the synthesis of chiral β-hy- micromide, we investigated the asymmetric hydrogenation
droxy acids is highly desirable because of the utility of such of a β-keto ester under the published conditions using a
molecules in organic synthesis. Much effort has gone into RuCl /(R)-MeO-BIPHEP catalyst system (1 mol-%) to give
3
looking for highly selective methods to obtain chiral β- ethyl (R)-3-hydroxyhexanoate. The hydrogenation of the β-
hydroxy esters, such as ethyl 3-hydroxyhexanoate. The keto ester was carried out at 10 bar and 40–60 °C for
[
12]
asymmetric reduction of prochiral keto esters is one alter- 10 h.
native. A report
Under these conditions, ethyl (R)-3-hydroxyhex-
has described a strategy to synthesize anoate (15) was obtained in good yield (99%) with an excel-
chiral β-hydroxy acids based on a stereoselective dirhodi- lent enantiomeric excess (99% ee; the enantiomeric excess
[
11]
7234
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Total Synthesis of Micromide: a Marine Natural Product
Scheme 5.
was determined by HPLC analysis using a Chirapak IA col- Experimental Section
umn, hexane/iPrOH, λ = 210 nm; Scheme 5).
General Remarks: H and 13C NMR spectra were recorded with a
1
Compound 15 was methylated with iodomethane using
Bruker Avance II 500 instrument at 500 and 126 MHz, respectively.
Chemical shifts are expressed as δ values [ppm] using tetramethyl-
silane as internal standard, and coupling constants (J) are given in
Hertz (Hz). Melting points were determined with an X4 apparatus.
Optical rotations were measured using a polarimeter at the sodium
sodium ethoxide as base in CH Cl . When the methylation
2
2
of the hydroxy group (to give 16) was complete, hydrolysis
and acidification processes were carried out to give acid 18
in 21% yield. In addition to the production of 16, elimi-
nation product 17 was also obtained in the methylation re- D line. HPLC–MS was carried out with a Thermo Finnigan LCQ-
Advantage mass spectrometer. Reactions were monitored by thin-
action.
Having synthesized fragments 14 and 18, we focused on layer chromatography on silica gel plates (GF254). Flash
chromatography was carried out with silica gel (200–400 mesh). El-
the completion of the synthesis. Fragment 14 was coupled
with acid 18 under HATU activation to give micromide 1
emental analyses were carried out with a Perkin–Elmer 2400C in-
1
13
strument.
in moderate yield (Scheme 6). The H and C NMR spec-
tra were consistent with those reported by Moore.[
1]
All of the organic solvents used in this study were dried with appro-
priate drying agents and distilled before use. All other chemicals
used in this study were commercially available.
N-Me-Gly-Thiazole (2): 1,3-Thiazole-2-carbaldehyde (0.45 g,
4
.0 mmol) and methylamine (solution in ethanol; 1 mL, 8.0 mmol)
were dissolved in CH Cl (60 mL). Molecular sieves (4 Å) and an-
hydrous Na SO (1.42 g, 10 mmol) were added, and the mixture
2
2
2
4
was left overnight. The solid particles were removed by filtration,
and the solvent was removed under reduced pressure.
The crude product was dissolved in EtOH (40 mL), and then
NaBH
mixture was stirred for 8 h, and then H
solvent was evaporated, and the residue was suspended in saturated
NaCl (40 mL), and this mixture was extracted with CHCl (3ϫ
0 mL). The combined organic extracts were dried with MgSO
filtered, and concentrated. The residue was purified by column
chromatography (CH Cl /MeOH, 100:1) to give 2 (0.43 g, 83%).
H NMR (500 MHz, CDCl ): δ = 2.52 (s, 3 H), 4.09 (s, 2 H), 4.96
(s, 1 H), 7.27 (s, 1 H), 7.71 (s, 1 H) ppm. C H N S (128.04): calcd.
4
(0.23 g, 6.0 mmol) was added portionwise. The reaction
2
O (8 mL) was added. The
3
5
4
,
2
2
1
3
Scheme 6.
5
8
2
C 46.85, H 6.29, N 21.85; found C 46.91, H 6.27, N 21.92.
Amino Acid tert-Butyl Esters S2: HClO (75 mmol) was slowly
4
added to a solution of amino acid (50.0 mmol) in tert-butyl acetate
120 mL) at 8 °C. The reaction mixture was stirred at room tem-
perature for 12 h, and then it was washed with H O (250 mL) and
hydrochloric acid (1.0 m; 150 mL). The pH of the resulting aqueous
solution was adjusted to 9 by the addition of K CO (10% aq.).
The solution was then extracted with dichloromethane (3ϫ
Conclusions
(
2
We have accomplished the stereoselective synthesis of
micromide (found in marine cyanobacterium) from easily
available starting materials. Our synthetic route enables the
2
3
preparation of various peptides containing N-methyl-sub- 50 mL). The combined organic extracts were dried with anhydrous
stituted amino acids.
sodium sulfate, filtered, and concentrated to give S2 as a yellow
Eur. J. Org. Chem. 2014, 7232–7238
© 2014 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
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7235

J. Han, J. Lian, X. Tian, S. Zhou, X. Zhen, S. Liu
FULL PAPER
oil. This compound was used in the next step without further puri-
fication.
CDCl
3
): δ = 2.86 (s, 3 H), 2.97 (m, 1 H), 3.22 (s, 3 H), 3.25 (m, 1
H), 4.62 (d, J = 15.0 Hz, 1 H), 4.79 (d, J = 15.0 Hz, 1 H), 5.21 (m,
H), 7.16 (m, 5 H), 7.28 (d, J = 8.0 Hz, 1 H), 7.65 (m, 4 H), 7.86
1
N-o-Nitrophenylsulfonyl-Substituted Amino Acid tert-Butyl Esters
S3: Triethylamine (6.0 mL, 39.6 mmol) and o-Ns chloride (10.5 g,
1
3
(
d, J = 8.0 Hz, 1 H) ppm. C NMR (126 MHz, CDCl
3
): δ =
69.80, 165.50, 147.54, 142.83, 135.22, 133.58, 131.87, 131.65,
30.18, 128.93 (2 C), 128.27, 128.20, 126.77, 124.12, 119.98, 55.16,
8.65, 35.61, 35.05, 30.73 ppm. C21 (474.10): calcd. C
3.15, H 4.67, N 11.81; found C 53.21, H 4.59, N 11.87.
1
1
4
5
4
5.0 mmol) were added to a solution of tert-butyl ester S2
36.0 mmol) in dichloromethane (120 mL) at 8 °C. The reaction
mixture was stirred at room temperature overnight, and then it was
washed with H O (3ϫ 15 mL). The organic phase was dried with
Na SO and concentrated under reduced pressure to give S3 as a
(
22 4 5 2
H N O S
2
N-Me-Gly-thiazole-N-Me-Phe (7): Solid LiOH·H
2
O
(8.82 g,
2
4
yellow oil.
210 mmol) and mercaptoacetic acid (3.86 g, 42.0 mol) were added
to a solution of 6 (9.76 g, 21 mmol) in DMF (250 mL) at 0 °C. The
reaction mixture was stirred at room temperature for 3 h, then it
N-o-Nitrophenylsulfonyl-N-methyl-Substituted Amino Acid tert-
Butyl Esters S4: K
of S3 (27.6 mmol) in DMF (60.0 mL) at 0 °C under a protective
flow of N . After 10 min, iodomethane (7.0 mL, 110.4 mmol) was
2 3
CO (7.7 g, 55.2 mmol) was added to a solution
was quenched by adding saturated NH
was extracted with ethyl acetate (3ϫ 120 mL). The combined or-
ganic extracts were washed with saturated NaHCO (2ϫ 100 mL)
and brine (2ϫ 100 mL), dried with Na SO , and concentrated to
4
Cl (20 mL). The mixture
2
3
added by syringe. The reaction mixture was stirred at room tem-
perature overnight, then it was quenched by the addition of satu-
rated aqueous NH Cl (20 mL). The mixture was extracted with
4
ethyl acetate (3ϫ 80 mL). The combined organic extracts were
washed with HCl (0.5 m; 2ϫ 50 mL) and brine (2ϫ 50 mL), dried
2
4
give 7 (5.60 g, 92.1%) as a yellow oil, which was used in the next
step without further purification.
3
N-Me-Gly-thiazole-L-N-Me-Phe-L-N-Me-Ile-N-(o-Ns) (8): Et N
with Na
2
SO
4
, and then concentrated to give S4.
-phenylalanine tert-Butyl Ester:
Yellow oil, 90% yield. H NMR (500 MHz, CDCl ): δ = 1.32 (s, 9
H), 2.92–2.97 (m, 1 H), 3.02 (s, 3 H), 3.29–3.34 (m, 1 H), 4.81–4.84
m, 1 H), 7.19–7.36 (m, 5 H), 7.51–7.56 (m, 2 H), 7.61–7.64 (m, 1
H), 7.75 (d, 1 H) ppm. C20 S (420.14): calcd. C 57.13, H
.75, N 6.66; found C 57.10, H 5.73, N 6.70.
N-o-Nitrophenylsulfonyl-N-methyl- -isoleucine tert-Butyl Ester: Yel-
low oil, 92% yield. H NMR (500 MHz, CDCl ): δ = 0.90 (t, J =
.5 Hz, 3 H), 0.96 (d, J = 6.5 Hz, 3 H), 1.09–1.15 (m, 1 H), 1.30
(14 mL) was added to a solution of 4 (6.97 g, 20 mmol) in dichloro-
methane (40 mL) at 0 °C, and then a solution of 7 (5.22 g,
N-o-Nitrophenylsulfonyl-N-methyl-
L
18 mmol) in dichloromethane (20 mL) was added. The reaction
1
3
mixture was stirred at room temperature overnight, and then it was
concentrated to dryness. The residue was dissolved in EtOAc
(
(
150 mL), and this solution was successively washed with HCl (5%
aq.; 2ϫ 30 mL), saturated NaHCO (2ϫ 30 mL), and brine (2ϫ
0 mL). The solution was dried with Na SO , and concentrated to
give 8, which was used in the next step without further purification.
24 2 6
H N O
3
5
3
2
4
L
1
3
N-Me-Gly-thiazole- -N-Me-Phe- -N-Me-Ile (9): Solid LiOH·H
2.1 g, 87.2 mmol) and mercaptoacetic acid (1.60 g, 17.5 mol) were
added to a solution of 8 (5.00 g, 8.72 mmol) in DMF (100 mL) at
°C. The reaction mixture was stirred at room temperature for 3 h,
then it was quenched by adding saturated NH Cl (20 mL). The
mixture was extracted with ethyl acetate (3ϫ 120 mL). The com-
bined organic extracts were washed with saturated NaHCO (2ϫ
): δ = 0.97 (d, J = 100 mL) and brine (2ϫ 100 mL), dried with Na SO , and concen-
L
L
2
O
2
(
(
(
s, 9 H), 1.54–1.61 (m, 1 H), 1.92–1.97 (m, 1 H), 3.06 (s, 3 H), 4.11
d, J = 10.5 Hz, 1 H), 7.62 (m, 1 H), 7.68 (m, 2 H), 8.04 (m, 1 H)
0
26 2 6
ppm. C17H N O S (386.15): calcd. C 52.83, H 6.78, N 7.25; found
C 52.79, H 6.81, N 7.30.
4
N-o-Nitrophenylsulfonyl-N-methyl-
D-valine tert-Butyl Ester: Yellow
3
oil, 90% yield. 1H NMR (500 MHz, CDCl
3
2
4
7
.0 Hz, 3 H), 1.03 (d, J = 7.0 Hz, 3 H), 1.30 (s, 9 H), 2.15 (m, 1
H), 3.07 (s, 3 H), 4.07 (q, J = 10.5 Hz, 1 H), 7.64 (m, 1 H), 7.71
m, 2 H), 8.05 (m, 1 H) ppm.
trated. The residue was purified by flash chromatography on silica
gel (ethyl acetate/hexane, 1:1) to give 9 (3.27 g, 90.2%) as a yellow
oil. [α] 2D 0 = –172.6 (c = 0.5, MeOH). H NMR (500 MHz, CDCl
1
):
(
3
δ = 0.63 (t, J = 8.0 Hz, 3 H), 0.71 (d, J = 6.5 Hz, 3 H), 0.90 (m, 1
H), 1.35 (m, 2 H), 2.00 (br., 1 H), 2.88 (s, 3 H), 2.91 (s, 6 H), 2.90
N-o-Nitrophenylsulfonyl-N-methyl-Substituted Amino Acid Chlor-
ides 3, 4, and 5: TFA (5.0 mL, 67.3 mmol) was added dropwise to
a stirred solution of S4 (2.6 mmol) in dichloromethane (10 mL) at
(
m, 1 H), 3.05 (m, 1 H), 4.02 (d, J = 15 Hz, 1 H), 4.91 (d, J =
1
5 Hz, 1 H), 5.85 (t, J = 7.5 Hz, 1 H), 7.00 (m, 1 H), 7.10 (m, 5
0
°C. The reaction was monitored by TLC, and when the starting
1
3
H), 7.14 (d, J = 3.5 Hz, 1 H), 7.51 (d, J = 3.5 Hz, 1 H) ppm.
C
material had disappeared, the mixture was concentrated to give S5
as a dark red oil.
NMR (126 MHz, CDCl
3
): δ = 174.89, 170.22, 165.88, 142.22,
136.82, 129.38, 129.32, 128.44 (2 C), 126.70, 120.06, 64.90, 53.40,
COCl
2
(0.56 mL, 6.5 mmol) and DMF (20 mL, 0.3 mmol) were 49.06, 37.92, 35.32, 35.27, 34.89, 30.48, 23.93, 16.24, 11.60 ppm.
C H N O S (416.22): C 63.43, H 7.74, N 13.45; found C 63.39,
added to a solution of S5 in dichloromethane (10 mL) at 0 °C. The
2
2
32
4
2
reaction was monitored by TLC, and when the starting material H 7.80, N 13.50.
had disappeared, the reaction mixture was concentrated to give 3,
N-Me-Gly-thiazole-
Compound 9 (8.52 g, 20 mmol) was dissolved in CH
and DMF (50 mL), and then l-N-Boc-Val (5.21 g, 24 mmol) and
L
-N-Me-Phe-
L
-N-Me-Ile-
L
-Val-N-Boc
(10):
(500 mL)
4, or 5 as a brown-yellow oil. These three compounds were used in
2
Cl
2
the next step without further purification.
N-Me-Gly-thiazole- -N-Me-Phe-(o-Ns) (6): A solution of Et
L
3
N
HATU (15.2 g, 6 mmol) were added. DIPEA (ca. 30 mL) was then
(0.84 mL, 6 mmol) and 2 (192 mg, 1.5 mmol) in dichloromethane gradually added to the solution. The mixture was stirred at room
(
3 mL) was added to a solution of 3 (458 mg, 1.2 mmol) in dichlo-
temperature overnight. The solution was concentrated in vacuo,
then water (100 mL) was added, and the aqueous solution was ex-
tracted with ethyl acetate (5ϫ 30 mL). The combined organic ex-
romethane (3 mL) at 0 °C. The reaction mixture was stirred at
room temperature overnight, and then it was washed with HCl (5%
aq.; 2ϫ 30 mL), saturated NaHCO
3
(2ϫ 30 mL), and brine (2ϫ
SO and then concen-
tracts were washed with water, saturated Na
(2% aq.; 2ϫ 30 mL), and brine (30 mL). The solution was dried
with Na SO , and the solvent was removed under reduced pressure.
2 3
CO (2ϫ 30 mL), HCl
30 mL). The mixture was dried with Na
2
4
trated. The crude oil was purified by flash chromatography on silica
2
4
gel (ethyl acetate/hexane, 1:2) to give compound 6 (0.52 g, 90.8%)
as a yellow oil. [α]20 = –46.8 (c = 0.5, MeOH). H NMR (500 MHz,
The crude product was purified by silica gel chromatography (pe-
troleum ether/ethyl acetate, 8:1) to give 10 (11.6 g, 94.2%) as a
D
1
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Eur. J. Org. Chem. 2014, 7232–7238

Total Synthesis of Micromide: a Marine Natural Product
1
white solid. H NMR (500 MHz, CDCl
(
3
): δ = 0.79 (m, 3 H), 0.83
(2ϫ 30 mL), HCl (5% aq.; 2ϫ 30 mL), and brine (30 mL). The
solution was dried with Na SO , and the solvent was removed un-
der reduced pressure. The crude product was purified by silica gel
s, 3 H), 2.97 (dd, J = 6.5, J = 8.0 Hz, 1 H), 3.06 (s, 3 H), 3.21 (dd, chromatography (petroleum ether/ethyl acetate, 6:1) to give 13
m, 3 H), 0.86 (m, 3 H), 0.90 (m, 3 H), 1.21 (m, 1 H), 1.41 (s, 9
H), 1.73 (m, 1 H), 2.03 (m, 1 H), 2.35 (m, 1 H), 2.53 (s, 3 H), 2.96
2
4
(
J = 6.5, J = 8.0 Hz, 1 H), 4.28 (m, 1 H), 4.63 (d, J = 15.0 Hz, 1 (0.59 g, 91.4%) as a white solid. [α] 2D 0 = –98.0 (c = 0.5, MeOH). H
H), 5.05 (d, J = 15 Hz, 1 H), 5.11 (m, 2 H), 5.96 (m, 1 H), 7.22 NMR (500 MHz, CDCl ): δ = 0.53 (d, J = 6.5 Hz, 3 H), 0.75 (t, J
m, 5 H), 7.30 (d, J = 3.5 Hz, 1 H), 7.69 (d, J = 3.5 Hz, 1 H) ppm. = 6.5 Hz, 6 H), 0.85 (d, J = 7.0 Hz, 3 H), 0.91 (q, J = 3.0 Hz, 6
C NMR (126 MHz, CDCl
55.40, 141.62, 136.16, 128.86 (2 C), 127.72 (2 C), 126.04, 119.55,
8.65, 56.62, 54.93, 52.45, 48.57, 34.77, 34.53, 32.28, 29.70, 29.16,
1
3
(
1
3
3
): δ = 172.38, 169.19, 169.14, 165.25,
H), 1.13 (m, 1 H), 1.38 (s, 9 H), 1.75 (m, 1 H), 1.99 (m, 2 H), 2.15
(m, 1 H), 2.48 (s, 3 H), 2.74 (m, 1 H), 2.94 (s, 3 H), 3.01–3.05 (m,
2 H), 3.07–3.10 (m, 1 H), 3.08 (s, 6 H), 3.22 (dd, J = 6.5, J =
1
7
2
7.69 (4 C), 22.97, 19.36, 16.84, 15.16, 10.37 ppm. HRMS (ESI): 8.0 Hz, 1 H), 4.41 (m, 1 H), 4.48 (m, 1 H), 4.75 (m, 1 H), 5.05 (m,
+
calcd. for C32
N-Me-Gly-thiazole-
pound 10 (6.15 g, 10 mmol) was dissolved in a mixture of TFA
50 mL) and CH Cl (50 mL), and the resulting solution was stirred
for 2 h. The solvent and TFA were removed in vacuo to give crude
1 (Ͼ95% yield), which was used in the next step reaction without
further purification.
N-Me-Gly-thiazole-
Et N (8 mL) was added to a solution of 5 (4.02 g, 12 mmol) in
dichloromethane (24 mL) at 0 °C. A solution of 11 (5.15 g,
0 mmol) in dichloromethane (20 mL) was then added. The reac-
tion mixture was stirred at room temperature overnight, and then
it was concentrated to dryness. The residue was dissolved in EtOAc
150 mL), and this solution was successively washed with HCl (5%
aq.; 2ϫ 30 mL), saturated NaHCO (2ϫ 30 mL), and brine (2ϫ
0 mL). The solution was dried with Na SO , and then concen-
H
50
N
5
O
5
S [M + H] 616.3533; found 616.3538.
2 H), 5.60 (m, 1 H), 5.94 (m, 1 H), 6.87 (m, 2 H), 7.19–7.25 (m, 10
1
3
H), 7.27–7.30 (m, 1 H), 7.70 (m, 1 H) ppm. C NMR (126 MHz,
CDCl ): δ = 172.861, 171.901, 170.035, 169.848, 169.599, 165.717,
55.149, 142.203, 136.657, 129.529, 129.428 (3 C), 128.371, 128.224
2 C), 126.684, 120.214, 120.090, 115.428, 113.888, 79.492, 62.986,
L
-N-Me-Phe- -N-Me-Ile- -Val (11): Com-
L
L
3
1
(
(
2
2
5
3
2
7.180, 53.937, 53.155, 52.655, 50.587, 49.325, 39.132, 35.391,
5.191, 32.711, 30.656, 30.393, 29.928, 29.694, 28.377 (2 C), 25.304,
1
3.575, 19.941, 19.652, 18.433, 17.105, 15.744, 11.034 ppm. HRMS
+
L-N-Me-Phe-L-N-Me-Ile-
L-Val-D-N-Me-Val (12):
(ESI): calcd. for C H N O S [M + H] 876.5057; found 876.5084.
4
7
70
7
7
3
Ethyl (R)-3-Hydroxyhexanoate (15): (R)-MeO-BIPHEP (116 mg,
.2 mmol) and anhydrous RuCl (52 mg, 0.2 mmol) were placed in
0
3
1
a stainless steel autoclave and degassed with three vacuum/nitrogen
cycles at room temperature. Ethyl 3-oxohexanoate (6.32 g,
4
0 mmol) was added, followed by degassed methanol (500 mL).
The solution was purged with hydrogen and pressurized under
0 bar, then it was heated to 40–60 °C, and stirred for 10 h. The
(
3
1
3
2
4
autoclave was cooled to room temperature, and the hydrogen was
vented. The reaction mixture was concentrated in vacuo, and the
residue was purified by flash chromatography (ethyl acetate/hexane,
trated to give the coupled intermediate, which was used without
further purification.
Solid LiOH·H
2
O (1.05 g, 43.6 mmol) and mercaptoacetic acid
1
9
:1) to give (R)-ethyl 3-hydroxyhexanoate (15; 6.34 g, 99.0%,
(
0.80 g, 8.8 mol) were added to a solution of the crude coupled
intermediate (3.55 g, 4.36 mmol) in DMF (50 mL) at 0 °C. The re-
action mixture was stirred at room temperature for 3 h, then it was
quenched by adding saturated NH
extracted with ethyl acetate (3ϫ 120 mL). The combined organic
extracts were washed with saturated NaHCO (2ϫ 100 mL) and
brine (2ϫ 100 mL), dried with Na SO , and concentrated to give
9.0% ee) as a yellow oil. [α] 2D 0 = –6.9 (c = 0.5, EtOH).
(
R)-3-Methoxyhexanoic Acid (18): EtONa (0.68 g, 10 mmol) was
added to (R)-ethyl 3-hydroxyhexanoate (15; 1.60 g, 10 mmol) in
CH Cl (50 mL). Then CH I (5 mL, 80 mmol) was slowly added
dropwise. The solution was stirred under reflux for 12 h, then the
solvent was removed under a stream of N . H O (exactly 20 mL),
4
Cl (20 mL). The mixture was
2
2
3
3
2
2
2
4
NaOH (0.4 g), and MeOH (20 mL) were added to the mixture,
which was then stirred overnight. The mixture was acidified to pH
2 with HCl (5% aq.), and then it was extracted with ethyl acetate
crude 12 as a yellow oil. The crude product was purified by silica
gel chromatography (petroleum ether/ethyl acetate, 5:1) to give 12
1
(
5.3 g, 84.2%) as a white solid. H NMR (500 MHz, CDCl
3
): δ =
(
4ϫ 30 mL). The combined organic extracts were washed with sat-
0
0
.70–0.77 (m, 6 H), 0.80–0.83 (m, 3 H), 0.86–0.89 (m, 3 H), 0.91–
.96 (m, 6 H), 1.12–1.14 (m, 1 H), 1.23–1.25 (m, 1 H), 1.83 (m, 1
urated NaCl, dried with MgSO , and concentrated in vacuo. The
4
products were purified by flash chromatography (ethyl acetate/hex-
H), 2.00–2.05 (m, 2 H), 2.39 (s, 3 H), 2.54 (s, 3 H), 2.79 (m, 1 H),
.93 (dd, J = 6.5, J = 8.0 Hz, 1 H), 2.97 (s, 3 H), 3.05 (s, 3 H), 3.22
ane, 1:1) to give (R)-3-methoxyhexanoic acid (18; 0.31 g, 21.0%) as
2
1
a colorless oil. H NMR (500 MHz, CDCl
3
): δ = 0.937 (t, J =
(dd, J = 6.5, J = 8.0 Hz, 1 H), 4.63 (m, 2 H), 5.03 (m, 1 H), 5.10
7
.5 Hz, 3 H), 1.33–1.44 (m, 2 H), 1.46–1.53 (m, 1 H), 1.56–1.63 (m,
1 H), 2.48 (dd, J = 5.0, J = 10.5 Hz, 1 H), 2.56 (dd, J = 5.0, J =
0.5 Hz, 1 H), 3.38 (s, 3 H), 3.66 (m, 2 H), 10.28 (br., 1 H) ppm.
(146.09): calcd. C 57.51, H 9.65; found C 57.46, H 9.68.
(
7
m, 1 H), 5.95 (m, 1 H), 7.19–7.25 (m, 5 H), 7.27–7.29 (m, 1 H),
.64 (m, 1 H), 7.69 (m, 1 H) ppm. 1 C NMR (126 MHz, CDCl
3
3
):
1
δ = 173.396, 172.244, 169.786, 169.661, 165.746, 142.272, 136.654,
1
5
3
1
29.417 (2 C), 128.371 (2 C), 126.715, 120.065, 70.673, 57.155,
3.090, 49.279, 36.245, 35.452, 35.198, 34.124, 32.816, 31.517,
0.800, 30.363, 29.833, 23.706, 19.948, 19.486, 17.550, 17.282,
7 14 3
C H O
Micromide (1): (R)-3-Methoxyhexanoate (44 mg, 0.3 mmol) was
dissolved in a mixture of DMF (0.8 mL) and CH Cl (8 mL), and
2
2
53 6 4
5.624, 10.859 ppm. HRMS (ESI): calcd. for C33H N O S [M +
then HATU (190 mg, 6 mmol) and DIPEA (0.5 mL) were added.
+
H] 629.3849; found 629.3851.
N-Me-Gly-thiazole- -N-Me-Phe-
-Phe-N-Boc (13): N-Boc-phenylalanine (0.206 g, 0.8 mmol) was
dissolved in a mixture of DMF (2 mL) and CH Cl (20 mL), then added, and the aqueous solution was extracted with ethyl acetate
2 (0.42 g, 0.67 mmol) and HATU (0.51 g, 0.2 mmol) were added, (5ϫ 30 mL). The combined organic extracts were washed with
The mixture was stirred for 0.5 h, then 14 (193 mg, 0.25 mmol) was
L
L
-N-Me-Ile-
L
-Val-
D
-N-Me-Val- added, and the mixture was stirred at room temperature overnight.
The solution was concentrated in vacuo, then water (100 mL) was
L
2
2
1
followed by the gradual addition of DIPEA (1 mL). The mixture
was stirred at room temperature overnight. The solution was con-
centrated in vacuo, then water (50 mL) was added, and the aqueous
solution was extracted with ethyl acetate (5ϫ 30 mL). The com-
water, saturated Na
and brine (30 mL). The solution was dried with Na
solvent was removed under reduced pressure. The crude product
was purified by silica gel chromatography (petroleum ether/ethyl
acetate, 8:1) to give micromide (1; 0.15 g, 67.2%) as a yellow solid.
2
CO
3
(2ϫ 30 mL), HCl (2% aq.; 2ϫ 30 mL),
SO , and the
2
4
2 3
bined organic extracts were washed with water, saturated Na CO
Eur. J. Org. Chem. 2014, 7232–7238
© 2014 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
www.eurjoc.org
7237

J. Han, J. Lian, X. Tian, S. Zhou, X. Zhen, S. Liu
FULL PAPER
D
1
[α]20 = –26.9 (c = 0.5, MeOH). H NMR (500 MHz, CDCl
3
): δ =
and the Foundation of the Education Department of Hebei Prov-
ince (grant number ZH2012025).
0
9
.564 (t, J = 6.5 Hz, 3 H), 0.73 (m, 6 H), 0.84 (m, 3 H), 0.91 (m,
H), 1.15 (m, 1 H), 1.33 (m, 2 H), 1.45–1.60 (m, 1 H), 1.75 (m, 2
H), 1.87–2.03 (m, 2 H), 2.15 (m, 1 H), 2.32 (m, 2 H), 2.54 (s, 3 H),
.75 (s, 3 H), 2.94 (s, 3 H), 2.95 (m, 1 H), 2.97 (m, 1 H), 3.04 (s, 3
H), 3.10 (m, 1 H), 3.22 (m, 1 H), 3.34 (s, 3 H), 3.55 (m, 1 H), 4.43–
.50 (m, 2 H), 4.65 (m, 1 H), 4.97–5.05 (m, 2 H), 5.07–5.10 (m, 1
H), 5.93 (t, J = 8.0 Hz, 1 H), 6.89 (m, 1 H), 7.05 (m, 1 H), 7.17–
.25 (m, 10 H), 7.29 (d, J = 3.5 Hz, 1 H), 7.68 (d, J = 3.5 Hz, 1 H)
[
[
1] P. G. Williams, W. Y. Yoshida, R. E. Moore, V. J. Paul, J. Nat.
Prod. 2004, 67, 49.
2
2] a) A. L. Demain, Med. Res. Rev. 2009, 29, 821; b) Y. Liu, Z.
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1
6, 7185; c) P. Stoilov, C. H. Lin, R. Damoiseaux, J. Nikolic,
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7
13
ppm. C NMR (126 MHz, CDCl
3
1
1
1
5
3
2
1
9
70.126, 169.932, 169.853, 166.090, 142.135, 136.754, 136.573,
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[
29.554 (3 C), 129.512 (C), 128.498 (4 C), 126.944, 126.822,
20.367, 119.774, 77.820, 62.877, 57.401, 57.025, 56.785, 54.161,
3.197, 51.226, 49.347, 41.649, 40.815, 38.678, 36.098, 35.547,
5.358, 34.520, 32.946, 30.866, 30.472, 30.258, 29.897, 25.390,
3.764, 19.999, 19.942, 18.581, 18.475, 17.238, 15.769, 14.256,
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5] T. Fukuyama, C. K. Jow, M. Cheung, Tetrahedron Lett. 1995,
36, 6373.
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74 7 7
H N O
S: [M + H]+
1.236 ppm. HRMS (ESI): calcd. for C49
04.5370; found 904.5348.
2
301; b) S. C. Miller, T. S. Scanlan, Tetrahedron Lett. 1998, 39,
6613.
[
[
7] H. Chen, Y. Feng, Z. Xu, T. Ye, Tetrahedron 2005, 61, 11132.
Supporting Information (see footnote on the first page of this arti-
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8] J. R. McDermott, N. L. Benoiton, Can. J. Chem. 1973, 51,
1
13
2562.
[
[
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9] S. C. Miller, T. S. Scanlan, J. Am. Chem. Soc. 1997, 119, 2301.
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Acknowledgments
The authors are grateful for the financial assistance received from
the National Natural Science Foundation of China (NSFC) (grant
number 1272052), the National Basic Research Program of China
[
12] a) O. Labeeuw, P. Phansavath, J. P. Genet, Tetrahedron 2004,
1
5, 1899; b) C. Mordant, S. Reymond, V. R. Vidal, J. P. Genet,
Tetrahedron 2004, 60, 9715.
(grant numbers 2011CB512007 and 2012CB723501), the Hebei
Received: July 23, 2014
Province Natural Science Fundation (grant number B2014208138),
Published Online: September 30, 2014
7238
www.eurjoc.org
© 2014 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
Eur. J. Org. Chem. 2014, 7232–7238