Total synthesis and stereochemical reassignment of tasiamide B

Article abstract of DOI:10.1002/psc.1254

The first total synthesis of tasiamide B, an octapeptide bearing 4-amino-3-hydroxy-5-phenylpentanoic acid unit isolated from the marine cyanobacteria Symploca sp. is described. A simple and efficient way was found to avoid the pyroglutamylation of N^α-Me-Gln and led to a reassignment of the N^α-Me-L-Phe of tasiamide B to be N ^α-Me-D-Phe, which was also supported by 1D and 2D NMR. Copyright

Full text of DOI:10.1002/psc.1254

Research Article
Received: 3 January 2010
Revised: 14 April 2010
Accepted: 26 April 2010
Published online in Wiley Interscience: 27 May 2010
(www.interscience.com) DOI 10.1002/psc.1254
Total synthesis and stereochemical
reassignment of tasiamide B
Tiantian Sun,a Wei Zhang,b∗ Chengli Zong,a Peng Wanga and Yingxia Lib∗
The first total synthesis of tasiamide B, an octapeptide bearing 4-amino-3-hydroxy-5-phenylpentanoic acid unit isolated from
the marine cyanobacteria Symploca sp. is described. A simple and efficient way was found to avoid the pyroglutamylation of
Nα-Me-Gln and led to a reassignment of the Nα-Me-L-Phe of tasiamide B to be Nα-Me-D-Phe, which was also supported by 1D
c
and 2D NMR. Copyright ꢀ 2010 European Peptide Society and John Wiley & Sons, Ltd.
Supporting information may be found in the online version of this article
Keywords: tasiamide B; total synthesis; pyroglutamylation; reassignment
Introduction
lished protocols [5,6], the two units were coupled using 1-(3-
dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride (EDCI)
and 1-hydroxybenzotriazole (HOBt) as coupling reagents to afford
dipeptide 11 (62.1%). The product was treated with 4 M HCl in
ethyl acetate to remove the Boc group and then, coupled with
Cbz-Ala-OH (13) to produce tripeptide 14 smoothly in 76.6% yield
over two steps. Debenzyloxycarbonylation of 14 under catalytic
hydrogenolysis gave primary amine 15, which was coupled with
Boc-Leu-OH (16) under the same conditions mentioned above to
give the linear tetrapeptide 3 in 60% yield over two steps.
Ocean is a unique resource that provides a diverse array of
natural products. Since the mid-1980s, mounting evidence has
suggested that marine microorganisms and cyanobacteria might
be the real producers of many of the compounds isolated from
the invertebrates such as sponges, tunicates, bryozoans, and
mollusks [1]. From then on, scientists turned their attention to the
cyanobacteria, and a series of new pharmacologically interesting
natural products were found one after another.
Tasiamide B (1) (Figure 1), an 8-residue acyclic peptide, first
isolated from the marine cyanobacteria Symploca sp. in 2003 by
Moore et al., was found to be cytotoxic against KB cells with an
IC50 value of 0.8 µM [2].
The next effort was to synthesize fragment 6 as shown in
Scheme 2.L-Valinebenzylester(17)whichwaspreparedaccording
to the literature [7] and commercially available L-lactic acid
(18) were coupled using EDCI and 1-hydroxy-7-azabenzotriazole
(HOAt) as coupling reagents to form 6. However, NMR data
indicated that significant racemization occurred during the
condensation. The phenomenon could be explained by keto–enol
tautomerism of the activated L-lactic acid, which was promoted
by the unprotected hydroxy group (Scheme 2). In order to avoid
the racemization, benzylate was employed to protect the hydroxy
group first [8]. To our delight, no racemization was detected and
dipeptide 6b was obtained efficiently (95%).
In order to construct fragment 5, Nα-Fmoc-Nα-Me-glutamine (8)
must be first prepared. Though the majority of naturally occurring
NMAs (N-methyl amino acids) are aliphatic type (MePhe, MeAla,
MeGly, MeVal, MeIle, MeLeu), a number of others contain basic,
acidic, and alcoholic side chains. The syntheses of these NMAs are
often not straightforward. Many methods have been reported to
synthesizeNMAs,suchasN-methylationbyalkylation[9],reductive
amination [10], 5-oxazolidinones [11], asymmetric syntheses [12],
This natural product is structurally similar to tasiamide (2)
isolated from cyanobacterium Symploca sp. in 2002, which is
cytotoxic against KB and LoVo cells with IC50 values of 0.48 and
3.47 µg/ml, respectively [3]. Different from the latter, tasiamide B
contains an Ahppa residue, which is a novel δ-amino-β-hydroxy
acid and found in a metabolite of a marine cyanobacterium
for the first time. Now, we have finished the total synthesis
and structural reassignment of tasiamide [4], and its preliminary
structure–activity relationship study is also ongoing. For a better
understanding of the biological activity and the SAR of those
compounds, we decided to explore an efficient synthesis of
tasiamide B.
Results and Discussion
Our retrosynthetic analysis of 1 is outlined in Figure 2. Considering
the minor steric hindrance, tetrapeptide 3 and 4 were selected
as important synthetic intermediates. For fragment 4, the more
timesaving and economical [2 + 2] strategy rather than linear
synthesis was employed.
∗
Correspondence to: Wei Zhang and Yingxia Li, School of Pharmacy, Fudan
University, Zhangheng Rd. 826, Shanghai, China.
E-mail: zhangw416@hotmail.com; liyx417@fudan.edu.cn
The first effort was to synthesize the easily accessible frag-
ment 3 (Scheme 1). After the preparation of L-proline methyl
ester (10) and Nα-Boc-Nα-Me-phenylalanine (9) following estab-
a
School of Pharmacy, Ocean University of China, Yushan Rd. 5, Qingdao, China
School of Pharmacy, Fudan University, Zhangheng Rd. 826, Shanghai, China
b
c
J. Pept. Sci. 2010; 16: 364–374
Copyright ꢀ 2010 European Peptide Society and John Wiley & Sons, Ltd.

SYNTHESIS AND STEREOCHEMICAL REASSIGNMENT OF TASIAMIDE B
N -Me-L-Gln
N-Me-L-Phe
ever, this method has a low stereoselectivity (anti:syn = 74: 26).
Starting from phenyl acetaldehyde, Kumar et al. carried out the
Wittig reaction and Sharpless aminohydroxylation to afford the
amino alcohol. After several protection group manipulations, the
target γ -amino β-hydroxy acids were furnished by the Wolff rear-
rangement [15]. Apparently, this method has a long route and a
poor yield.
In this article, Ahppa was prepared according to the method re-
ported by Hoffman in 1997 [16] (Scheme 4). First, 24 was obtained
by methyl esterification and N,N-dibenzylation of commercially
available phenylalanine 22. Then, Claisen condensation of 24 with
lithio tert-butyl acetate, followed by the reduction of β-ketoester
25 using sodium borohydride in absolute methanol, gave the
major product syn-26 (dr = 17: 1). It is worth mentioning that the
alkaline hydrolysis of compound 24 was omitted because of the
low yield and significant racemization during saponification. After
dedibenzylationof syn-26underPalladium-catalyzedhydrogenol-
ysis, tert-butyl (3S, 4S)-Ahppa (7) was obtained with satisfactory
yields and high enantioselectivity.
L-Leu
CONH2
L-Lactic Acid
O
O
O
OH
H
H
N
H
N
N
N
N
N
N
OH
H
O
O
O
O
O
OMe
O-Me-L-Pro
L-Ala
L-Val
(28S,29S)-Ahppa
Tasiamide B 1
N-Me-D-Gln
N-Me-D-Phe
L-ILe
L-Leu
O
O
O
H
N
N
OH
N
N
N
N
H
H
O
OMe
O
O
O
L-Gly
H2N
O
L-2-hydroxy-3-
methylvaleric acid
O-Me-L-Pro
Tasiamide (revised structure) 2
Figure 1. Structures of tasiamide B (1) and tasiamide (2).
With fragment 7 and 8 in hand, the stage was now set to
carry out their coupling. First, amino acid 8 is to be activated
by HOAt/EDCI before it was incorporated into the peptide chain.
However, 8-H rather than 5 was obtained as the major product
(Scheme 5). What is worse, the minor but desired dipeptide 5 was
found to be easily lactamizated when the protected group (Fmoc)
was removed. Thus, 4b could not be obtained employing the
present strategy.
As it can be seen from Scheme 6, the undesired result could
be attributed to the unprotected amide of glutamine. Although
glutamine is usually used without side chain protection in peptide
synthesis in solution, in our synthesis, as a flexible molecular, the
presence of N-Me in glutamine not only enhanced the bend of
the peptide chain but also the nucleophilicity of the secondary N
atom. Further more, the amide of glutamine was also vulnerable
and so on. Different strategies are applied for different amino
acids. In this article, the widely used method 5-oxazolidinones was
applied for the synthesis of (8) which has a reactive side chain
amide.
Starting from commercially available Nα-Fmoc-Nδ-trityl-
glutamine (20), (8) was obtained via oxazolidinone intermediate
(21) and reductive ring-opening following Freidinger’s procedure
[13] as shown in Scheme 3.
The next effort was to construct (3S, 4S)-4-amino-3-hydroxy-
5-phenylpentanoic acid (Ahppa), the most unique residue in
tasiamide B. Several methods have been reported. Pedrosa
et al. used α-aminoaldehyde as the starting material to obtain
γ -amino-β-hydroxy acids via a Reformatsky reaction [14]. How-
CONH
2
O
N
O
OH
O
H
H
H
N
N
N
N
t
1
OH
BuO
N
NHBoc
+
O
O
O
OMe
O
O
3
4
CONH
2
O
O
OH
H
N
H
N
t
NFmoc
BuO
BnO
OH
+
O
O
5
6
CONH
2
OH
O
OtBu
+
NH
N
COOH
2
Fmoc
8
7
Figure 2. Retrosynthetic analysis of 1.
c
J. Pept. Sci. 2010; 16: 364–374 Copyright ꢀ 2010 European Peptide Society and John Wiley & Sons, Ltd. www.interscience.com/journal/psc

SUN ET AL.
O
NHCbz
HO
NH.HCl
OMe
13
O
HOBt/EDC
NaHCO3
N
+
NR
NHR
N
N
HOBt/EDC/NaHCO3
DCM 76.6% for two steps
O
O
O
O
HOOC
N
DCM 12h
62.1%
OMe
O
OMe
Boc
10
9
R=Cbz 14
R=H 15
R=Boc
11
4M
HCl/EtOAc
Pd/C H2
R=H 12
HO
NHBoc
16
O
O
H
N
N
HOBt/EDC DIPEA/DCM
60% for two steps
N
NHBoc
O
O
O
OMe
3
Scheme 1. Synthesis of fragment 3.
O
H
O
H
O
HOAt EDC
DIPEA DCM
73.8%
N
N
NH2.TosOH HO
+
BnO
OH
+
BnO
OH
BnO
OH
HO
O
O
O
18
17
6
6a
6:6a = 7:1 (by 1H NMR)
O
O
O
O
HO
O
Β
+HB
HO
OAt
OAt
OAt
OAt
H
O
H
N
HO
HOAt EDC
BnO
OBn
OBn
+
17
DIPEA DCM
95%
O
O
19
6b
Scheme 2. Synthesis of fragment 6b.
CONHTrt
O
CONH2
CONHTrt
COOH
TES, TFA
(CH2O)n, TsOH
toluene, reflux, 6 h
95.4%
Fmoc
CHCl3, rt, 2 d
91.1%
N
N
COOH
FmocHN
Fmoc
O
20
8
21
Scheme 3. Synthesis of Nα-Fmoc-Nα-Me-glutamine (8).
to nucleophilic attack. So Nα-Me-glutamine could be elongated
neither from the C-terminus nor from the N-terminus, which was
observed for the first time. Therefore, as for the liquid phase
synthesis, the free amide group must be sealed for peptide
elongation.
First, Trt was employed to protect the side chain amide of Fmoc-
Gln-OH. However, desired product was not obtained because of
thesterichindrancebetweenFmocandTrt.Then,thecommercially
available reagent bearing smaller size protected group (29) was
adopted. Trt group was employed to protect its amide and it
did well [17] (Scheme 7). The following N-methylation was carried
out to provide 28 using sodium hydride and methyl iodide [6].
Unavoidably, a small amount of 28b was produced as a byproduct.
The next step was to synthesize fragment 5b. As expected, no
pyroglutamylated byproduct was detected during this coupling
step. Debenzyloxycarbonylation of 5b produced 31 and alkaline
hydrolysisof6busingLiOH·H2Oproduced27,whichwerecoupled
to give linear tetrapeptide 4c with a yield of 65% for three steps.
With tetrapeptides 4c and 3 in hand, the stage was finally set to
carry out their coupling (Scheme 8). First, the Trt group and tert-
butyl ester of 4c were removed using TFA–dichloromethane =
1 : 1, simultaneously under the same condition 33 was prepared by
3. Then, the coupling of 32 and 33 provided octapeptide 34 (38%
for three steps). Finally, the synthetic tasiamide B (1) was obtained
smoothly after debenzylation of 34 under Palladium-catalyzed
hydrogenolysis (91%).
To our surprise, the analytical data of 1 was inconsistent with
those published for natural product. The optical rotation value of
c
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SYNTHESIS AND STEREOCHEMICAL REASSIGNMENT OF TASIAMIDE B
O
O
O
SOCl2 MeOH
BnBr NaHCO3
OMe
NBn2
OMe
OH
HCl
NH2
NH2
THF: DMSO=4:1
reflux 12 h
99%
rt, 2 d
100%
23
22
24
O
O
OH O
OH O
1.HMDS nBuLi -20°C 1 h
NaBH4 MeOH
OtBu
OtBu
OtBu
2.AcOtBu THF -78°C-rt 6 h
+
-20 °C 3.5 h
NBn2
NBn2
tran-26
NBn2
90%
89%
25
syn-26
Pd/C H2
dr = 17:1
8 h 100%
OH O
OtBu
NH2
7
Scheme 4. Synthesis of protected (3S, 4S)-Ahppa (7).
CONH2
CONH2
O
OH O
O
OH
H
N
HOAt EDC
OtBu
tBuO
NH
NFmoc
+
+
FmocN
DIPEA
DCM
29.3%
O
NH2
N
COOH
Fmoc
O
7
8
8-H
5
O
H
O
H
N
N
LiOH.H2O
H3O
OBn
BnO
OBn
HO
1.CH3CN:DEA=1:1
2.HOAt EDC
O
O
27
6b
CONH2
O
OH
H
N
O
O
O
OH
H
H
N
tBuO
N
N
tBuO
N
OBn
O
O
O
5-H
4b
Scheme 5. Unsuccessful synthesis of 4b.
O
CONH2
O
NH2
HOAt EDC
NH
OAt
FmocN
FmocN
FmocN
COOH
O
O
8-H
8
O
CONH2
O
OH
H
O
OH
H
NH2
O
OH
CH3CN:DEA=1:1
O
H
N
N
N
NFmoc
BuO
N
BuO
NH
BuO
O
O
O
5-H
5
Scheme 6. Proposed mechanism for the formation of 8-H and 5-H.
the synthetic sample was much higher than that of the natural
the structure of the natural product might consist of Nα-
Me-D-phenylalanine or D-alanine or both of them. To confirm
our hypothesis, three diastereomers of 1 were designed and
synthesized.
product {[α]20 = −89.6 (c 0.4, MeOH) while lit. [α]21 = −28 (c
D
D
0.4, MeOH)}. According to the HMQC/HMBC analysis, we found
that the 13C NMR signals assigned to Nα-Me-L-phenylalanine and
L-alanine of 1 were quite different from those reported for the
natural product (Table 1).
Meanwhile, we noticed that the structural analog tasiamide
contained a Nα-Me-D-phenylalanine. Therefore, we inferred that
Nα-Me-L-phenylalanine of
1
was replaced by Nα-Me-D-
phenylalanine to get diastereomer 1a; L-alanine was replaced
by D-alanine to give diastereomer 1b; and both were replaced
giving diastereomer 1c (Figure 3).
c
J. Pept. Sci. 2010; 16: 364–374 Copyright ꢀ 2010 European Peptide Society and John Wiley & Sons, Ltd. www.interscience.com/journal/psc

SUN ET AL.
CONTrt
Cbz
N
COOH
CONHTrt
MeI/NaH
15%
CONH2
COOH
28b
Trt-OH Ac2O
Cbz
N
H
COOH
Cbz
THF 12 h
H AcOH 50 °C
OH O
N
H
CONHTrt
72%
OtBu
29
30
NH2
7
Cbz
N
COOH
HOAt/EDC, DIPEA/THF 81.9%
28
75%
CONHTrt
CONHTrt
O
65% for three steps
O
OH
O
OH
H
H
H
HOAt/EDC, DIPEA/THF
R
N
N
N
N
tBuO
tBuO
N
OBn
O
H
O
O
O
N
RO
OBn
4c
O
R=Cbz 5b
31
Pd/C H2
R=Bn 6b
R=H 27
R=H
LiOH.H2O
H3O
Scheme 7. Synthesis of fragment 4c.
The synthetic methods of the three diastereomers were mostly
similar to the synthesis of 1. Exception was the last condensation
step, in which HATU instead of EDCI was employed as the coupling
reagent to enhance the reaction efficiency (from 40% to 60% for
three steps) and shorten the reaction time (from 48 to 15 h).
After complete NMR analysis (H–H COSY, HMQC, and HMBC;
Supporting information), we found that all of the four synthetic di-
astereomers (1, 1a, 1b, and 1c) showed no significant difference in
their 1H NMRs. However, their 13C NMR data and optical rotations
are quite different (Figure 4). Among them, 1a provided a near-
perfect match with the NMR data reported for tasiamide B, and
the value of optical rotation of this synthetic sample was also the
configurational reassignment of the previously assigned Nα-Me-L-
phenylalanine residue as Nα-Me-D-phenylalanine.
Experimental
General Information
Solventswerepurifiedbystandardmethods. TLCswerecarriedout
on Merck 60 F254 silica gel plates and visualized by UV irradiation
or by staining with iodine absorbed on silica gel, ninhydrin
solution or with aqueous acidic ammonium molybdate solution
as appropriate. Flash column chromatography was performed
on silica gel (200–300 mesh, Qingdao, China). Optical rotations
were obtained using a JASCO P-1020 digital polarimeter. NMR
spectra were recorded on JEOL JNM-ECP 600 MHz spectrometers.
Chemical shifts are reported in parts per million (ppm), relative to
the signals due to the solvent. Data are described as followings:
chemical shift, multiplicity (s = singlet, d = doublet, t = triplet, q
= quartet, br = broad, m = multiplet), coupling constants (Hz),
integration, and assignment. Mass spectra were obtained on a
Q-Tof Ultima Global mass spectrometer.
same as that of the natural product {[α]20 = −30.1 (c 0.4, MeOH)
D
while lit. [α]21 = −28 (c 0.4, MeOH)}. All the details suggested
D
that the Nα-Me-L-phenylalanine residue in tasiamide B has the
D-configuration.
Lastly, all of the four synthesized octapeptides (1, 1a, 1b, and
1c) were subjected to biological test on several cancer cell lines
(A549, LoVo, KB, BEL-7402, P388, and HL-60). The results indicated
that none of these synthetic peptides showed remarkable
cytotoxicity. This was not fully consistent with Williams and
co-workers’ report, as compound 1a (isolated from the marine
cyanobacterium Symploca sp.) displayed an IC50 value of 0.8 µM
against KB cells in their hands [2]. Further studies on tasiamide B
structural modification are currently under way in our laboratory
and will be reported in due course.
Chemistry
Synthesis of 3
Nα-Boc-Nα-Me-Phe-L-Pro-OMe(11). AsolutionofL-prolinemethyl
ester (10) (1.19 g, 7.2 mmol) and Nα-Boc-Nα-Me-D-phenylalanine
(9) (1.30 g, 4.6 mmol) in anhydrous CH2Cl2 (50 ml) was treated
sequentially with HOBt (0.97 g, 7.2 mmol), EDCI (1.38 g, 7.2 mmol),
and NaHCO3 (0.76 g, 9.0 mmol) at 0 ◦C. The mixture was warmed
to room temperature and stirred overnight. After diluted with
Conclusions
In summary, we have finished the first total synthesis of tasiamide
B (1), a promising linear peptide with cytotoxic activity and
c
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SYNTHESIS AND STEREOCHEMICAL REASSIGNMENT OF TASIAMIDE B
CONHR2
O
O
OH
H
H
O
N
N
H
N
OBn
R1O
N
N
N
NHR
O
O
O
O
O
OMe
R1=tBu R2=Trt 4c
R1=H R2=H 32
R=Boc 3
R=H 33
TFA:DCM=1:1
3 h
TFA:DCM=1:1
3 h
EDC/HOAt
DIPEA/THF
2 d 38%
CONH2
O
H
O
OH
H
O
H
N
N
N
N
N
OBn
N
N
H
O
O
O
O
O
OMe
34
Pd/C H2
24 h 91%
CONH2
O
H
O
OH
H
O
H
N
N
N
N
N
OH
N
N
H
O
O
O
O
O
OMe
1
Scheme 8. Synthesis of 1.
Table 1. Main differences of the 13C NMR value between 1 and the reported tasiamide B
C no.
δC (lit.)
δC (compd. 1)
ꢀδC
7
168.1, s
31.8, q
45.2, d
17.3, q
169.2, s
31.3, q
45.8, d
17.8, q
+1.1
−0.5
+0.6
+0.5
16
18
19
N-Me-L-Phe
CONH2
O
O
OH
29 N
O
H
H
H
N
44
N
N
N
6
18
19
7
OH
49
38
8 N
16
21 N
28
2
H
O
O
O
O
42
O
OMe
L-Ala
EtOAc (200 ml), the whole mixture was washed with 10% citric
acid (2 × 30 ml), 5% NaHCO3 (2 × 30 ml), H2O (2 × 30 ml), and
brine (2 × 30 ml), dried over Na2SO4, and concentrated in vacuo.
The residue was purified by flash column chromatography with
petroleum ether–EtOAc (20 : 1–5 : 1) to afford 11 as colorless
3.70, 3.67 (s, 3 H, 5 : 4 : 1, Pro COOCH3), 3.55 (m, 1 H, Pro δ-Ha), 3.36
(m, 1 H, Pro δ-Hb), 3.15, 3.05 (dd, 1 H, 5 : 4, J = 9.4, 18.2 Hz, Phe
β-Ha), 2.97, 2.91 (dd, 1H, 4 : 1, J = 9.9, 14.3 Hz, Phe β-Hb), 2.82,
2.79, 2.69, 2.66 (s, 3H, 5 : 5 : 1 : 1, Phe N-CH3), 2.16–1.92 (m, 4 H,
Pro γ -H + β-H), 1.32, 1.16 (s, 9 H, 1 : 2, C(CH3)3); 13C NMR (CDCl3,
150 MHz) δ172.8, 172.7, 169.2, 138.1 (Ar C-1), 129.5 (Ar C-3 + C-5),
128.5 (Ar C-2 + C-6), 126.5 (Ar C-4), 59.3 (Pro α-C), 56.5 (Phe α-C),
52.3 (OCH3), 46.4 (Pro δ-C), 35.0, 29.9, 29.2, 29.0, 28.1 (C(CH3)3),
24.9; ESI-MS (m/z): 391.2 [M + H]+, 413.2 [M + Na]+, 429.2 [M +
K]+ (calcd. 390.22).
oil (1.128 g, 62.1%). Rf 0.60 (1 : 1, petroleum ether–EtOAc);
1
[α]21 = −130.18 (c 1.0, CHCl3); H NMR (CDCl3, 600 MHz) four
D
rotamers δ: 7.27–7.15 (m, 5 H, ArH), 5.27, 5.13 (dd, 0.5 H, 4 : 1,
J = 6.1, 8.8 Hz, Phe α-H), 4.92, 4.80 (dd, 0.5 H, 4 : 1, J = 4.4, 10.4 Hz,
Phe α-H), 4.48, 4.44 (dd, 1 H, 1 : 1, J = 2.8, 8.2 Hz, Pro α-H), 3.75,
c
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SUN ET AL.
N-Me-D-Phe
N-Me-L-Phe
CONH2
CONH2
O
N
O
N
O
OH
O
O
OH
O
H
N
H
N
H
N
H
N
H
H
N
N
N
N
O
OMe
N
OH
N
H
OH
N
N
H
O
O
O
O
O
O
O
O
O
OMe
L-Ala
L-Ala
1
1a
N-Me-D-Phe
N-Me-L-Phe
CONH2
CONH2
O
N
O
O
OH
O
N
O
O
OH
H
H
H
H
N
H
N
H
N
N
N
N
N
N
N
O
OMe
N
N
OH
N
H
OH
H
O
O
O
O
O
O
O
O
O
OMe
D-Ala
D-Ala
1c
1b
Figure 3. Structures of compound 1 and its three diastereomers (1a, 1b, and 1c).
Figure 4. Differences in 13C NMR shifts between the natural product, synthetic 1,1a, 1b, and 1c.
Cbz-Ala-Nα-Me-Phe-L-Pro-OMe (14). To a solution of 11 (0.40 g,
1.02 mmol) in EtOAc (5 ml) at 0 ◦C, 4 M HCl/EtOAc (15 ml) was
added. The mixture was stirred for 1 h and then evaporated, the
residual oil was dissolved twice in CH2Cl2 (10 ml) with evaporation
each time to give HCl salt 12, which was used directly in the next
step.
δ-Ha), 3.35 (m, 1 H, Pro δ-Hb), 3.26 (dd, 1 H, J = 7.1, 14.3 Hz,
Phe β-Ha), 3.11, 3.09 (s, 3 H, 1 : 5, Phe N-CH3), 2.98 (dd, 1 H,
J = 8.3, 14.3 Hz, Phe β-Hb) 2.15 (m, 1H, Pro β-Ha), 2.04 (s, 3H,
Ala β-CH3), 1.95–1.85 (m, 3H, Pro γ -H + β-Hb); 13C NMR (CDCl3,
150 MHz) δ 173.1, 172.1, 169.4, 155.3 (NHCOOCH2Ph), 136.6, 136.5,
129.2 (two), 128.6 (two), 128.5 (two), 128.2, 128.1 (two), 126.9,
66.8 (NHCOOCH2Ph), 59.1 (α-C), 55.5 (α-C), 52.3 (OCH3), 47.2, 46.9,
34.9 (Phe α-C), 31.1 (N-CH3), 29.1 (Pro β-C), 24.9 (Pro γ -C), 18.7
(Ala β-C); ESI-MS (m/z): 518.2 [M + Na]+, 534.2 [M + K]+ (calcd.
495.24).
A solution of 12 and Cbz-Ala-OH (13) (0.27 g, 1.23 mmol) in
anhydrous CH2Cl2 (25 ml) was treated sequentially with HOBt
(0.21g, 1.54 mmol), EDCI (0.29 g, 1.54 mmol), and NaHCO3 (0.17 g,
2.05 mmol) at 0 ◦C. The solution was stirred for 30 min, warmed
to room temperature and stirred for another 12 h. And then, the
mixture was treated as described for 11. The residue was purified
by flash column chromatography with petroleum ether–EtOAc
(10 : 1–1 : 1) to afford 14 as a white solid (388.3 mg, 76.6%). Rf 0.45
Nα-Boc-Leu-Ala-Nα-Me-Phe-L-Pro-OMe (3). Hydrogenation of 14
(373 mg, 0.75 mmol) was carried out in EtOAc–EtOH (1 : 4, 40 ml)
in the presence of a catalytic amount of Pd-C (10%) under
hydrogen at room temperature. Pd-C was removed by filtration
and concentrated under reduced pressure to yield the amine 15,
which was used directly in the next step.
(3 : 2, petroleum ether–EtOAc); [α]20 = −72.25 (c 0.5, CHCl3);
D
1H NMR (CDCl3, 600 MHz) two rotamers δ: 7.37–7.14 (m, 10 H,
ArH), 5.59 (t, 1 H, J = 7.7 Hz, Phe α-H), 5.47 (d, 1H, J = 8.3 Hz,
Ala α-H), 5.08 (s, 2H, PhCH2O), 4.43 (dd, 1 H, J = 4.4, 8.8 Hz,
Pro α-H), 3.70, 3.66 (s, 3 H, 9 : 2, Pro COOCH3), 3.54 (m, 1 H, Pro
A solution of 15 and Boc-Leu-OH (16) (225 mg, 0.90 mmol)
in anhydrous CH2Cl2 (40 ml) was treated sequentially with HOBt
c
www.interscience.com/journal/psc Copyright ꢀ 2010 European Peptide Society and John Wiley & Sons, Ltd. J. Pept. Sci. 2010; 16: 364–374

SYNTHESIS AND STEREOCHEMICAL REASSIGNMENT OF TASIAMIDE B
(152 mg, 1.13 mmol), EDCI (216 mg, 1.13 mmol), and NaHCO3
(126 mg, 1.50 mmol) at 0 ◦C. The solution was stirred for 30 min,
warmed to room temperature and stirred for another 12 h. And
then, the reaction mixture was treated as described for 11.
The residue was purified by flash column chromatography with
petroleum ether–EtOAc (5 : 1–1 : 2) to afford 3 as a white solid
24 (4.32 g, 12 mmol) in 24 ml THF was added dropwise to the
colorless solution of the lithium enloate at −78 ◦C under argon.
The resulting mixture was stirred for 30 min, warmed to room
temperature and stirred for another 6 h. The solution was then
quenched with 1 M HCl (200 ml), and extracted with ethyl acetate
(3 × 100 ml). The organic extracts were combined, washed with
brine, dried (Na2SO4), and concentrated under reduced pressure.
The residue was purified by flash column chromatography with
petroleum ether-EtOAc (80 : 1–60 : 1) to afford 25 as colorless
transparent oil (4.73 g, 89%). Rf 0.70 (20 : 1, petroleum ether-
EtOAc); 1H NMR (CDCl3, 600 MHz) δ:7.34–7.15 (m, 15H, ArH), 3.81
(d, 2H, J = 13.2 Hz, 2PhCHaN), 3.62 (dd, 1H, J = 3.3, 8.8 Hz, Phe
α-H), 3.56 (d, 1H, J = 18.2 Hz, COCHaCO), 3.54 (d, 2H, J = 13.7 Hz,
2PhCHbN), 3.37 (d, 1H, J = 15.4 Hz, COCHbCO), 3.20 (dd, 1H,
J = 8.8, 13.2 Hz, Phe β-Ha), 2.92 (m, 1H, Phe β-Hb), 1.25 (s, 9H,
C(CH3)3).
(257 mg, 60.0%). Rf 0.50 (EtOAc); [α]20 = −87.63 (c 1.0, CHCl3);
D
1H NMR (CDCl3, 600 MHz) δ: 7.27–7.19 (m, 5 H, ArH), 6.74 (d, 1H,
J = 7.1 Hz, Ala NH), 5.60 (t, 1 H, J = 8.2 Hz, Phe α-H), 4.73 (t, 1H,
J = 7.1 Hz, Ala α-H), 4.43 (dd, 1 H, J = 4.4, 8.8 Hz, Pro α-H), 4.05
(d, 1H, J = 3.8 Hz, Leu α-H), 3.70, 3.69 (s, 3 H, 6 : 1, Pro COOCH3),
3.55 (m, 1 H, Pro δ-Ha), 3.37 (m, 1 H, Pro δ-Hb), 3.26 (dd, 1 H,
J = 7.1, 14.3 Hz, Phe β-Ha), 3.13, 3.07 (s, 3 H, 1 : 5, Phe N-CH3),
2.97 (dd, 1 H, J = 8.3, 14.3 Hz, Phe β-Hb), 2.15 (m, 1H, Pro β-Ha),
1.97–1.88 (m, 3H, Pro γ -H × 2 + β-Hb), 1.61 (m, 1H, Leu γ -H),
1.51 (m, 1H, Leu β-Ha), 1.40–1.44 (m, 10H, Leu β-Hb + C(CH3)3),
1.26 (d, 3H, J = 6.6 Hz, Ala β-CH3), 0.92 (d, 6H, J = 6.6 Hz, Leu
δ-CH3); 13C NMR (CDCl3, 150 MHz) δ 172.7, 172.4, 171.7, 169.3,
155.6 (NHCOOC(CH3)3), 136.6 (Ar C-1), 129.1 (Ar C-3 + C-5), 128.6
(Ar C-2 + C-6), 126.9 (Ar C-4), 80.0 (NHCOOC(CH3)3), 59.1 (Pro α-C),
55.3 (Phe α-C), 53.2 (Leu α-C), 52.4 (OCH3), 46.9 (Pro δ-C), 45.7 (Ala
α-C), 41.8 (Leu β-C), 34.8 (Phe β-C), 31.0 (N-CH3), 29.1 (Pro β-C),
28.4 (C(CH3)3), 25.0 (Leu γ -C), 24.8 (Pro γ -C), 23.1 (Leu δ-C × 2),
18.5 (Ala β-C); ESI-MS (m/z): 575.4 [M + H]+, 597.3 [M + Na]+, 613.3
[M + K]+ (calcd. 574.34).
(3S,4S)-tert-Butyl 4-(N, N-dibenzylamino)-3-hydroxy-5-phenyl-
pentanoate (syn-26). 3-Oxo ester 25 (463 mg, 1.25 mmol) was
dissolved in anhydrous methanol (25 ml). The resulting solution
was then cooled to −22 ◦C and treated with NaBH4 (166 mg,
4.38 mmol). The reaction was monitored by TLC. After 3.5 h,
the solution was quenched with H2O (100 ml) at pH = 5–6
(adjusted by 1 M HCl), extracted with ether (3 × 100 ml), washed
by brine (100 ml), dried (Na2SO4), and concentrated under
reduced pressure. The residue was purified by flash column
chromatography with petroleum ether–EtOAc (60 : 1–20 : 1) to
Synthesis of 4c
afford syn-26 as a colorless oil (397 mg, 85%); Rf 0.55 (10 : 1,
1
petroleum ether–EtOAc); [α]21 = 14.3 (c 1.35, CHCl3); H NMR
H-Phe-OMe(23). ToasolutionofH-Phe-OH(22)(6.61 g, 40 mmol)
in dry MeOH (40 ml) at −10 ◦C under argon, 12 ml of SOCl2 was
added dropwise. After stirring for 30 min at −10 ◦C, the reaction
was allowed to warm to room temperature and stirred for another
2 days. Then, the reaction mixture was dissolved twice in MeOH
(30 ml) with evaporation each time to yield the 23 as a white solid
(8.6 g, 100%). Rf 0.70 (10 : 1, CHCl3-MeOH); 1H NMR (DMSO-d6,
600 MHz) δ: 8.74 (s, 2H, NH), 7.34–7.23 (m, 5H, ArH), 4.23 (t, 1H,
J = 7.8 Hz, α-H), 3.65 (s, 3H, OCH3), 3.21 (dd, 1H, J = 5.5, 14.3 Hz,
β-Ha), 3.10 (dd, 1H, J = 7.7, 14.3 Hz, β-Hb).
D
(CDCl3,600 MHz)δ:7.19–7.36(m,15H,ArH),4.01(m,2H,2PhCHaN),
3.95 (m, 1H, Ahppa H-3), 3.43 (d, 2H, J = 13.1 Hz, 2PhCHbN), 3.11
(d, 1H, J = 10.4 Hz, Ahppa H-5a), 2.86 (m, 1H, Ahppa H-5b), 2.78
(m, 1H, Ahppa H-4), 2.40 (dd, 1H, J = 9.9, 16.0 Hz, Ahppa H-2a),
2.04 (m, 1H, Ahppa H-2b), 1.39 (s, 9H, C(CH3)3); 13C NMR (CDCl3,
150 MHz) δ 192.5 (C-1), 134.6 (Ar C-1×3), 129.8, 129.4, 129.3, 129.2,
129.1, 128.8, 128.7, 81.9 (C(CH3)3), 63.0 (C-4), 54.9 (N(CH2Ph)2), 40.9
(C-2), 34.8 (C-5), 28.1 (C(CH3)3). ESI-MS (m/z): 446.2 [M + H]+ (Calcd
445.3).
N, N-Bn2-Phe-OMe (24). H-Phe-OMe (23) (4.31g, 20 mmol), BnBr
(7.14 ml, 60 mmol), and NaHCO3 (8.4 g, 100 mmol), were sus-
pended in 50 ml THF and 12 ml DMSO and refluxed for 12 h. Then,
the reaction was allowed to cool off to room temperature, diluted
with water (50 ml), 5 ml of MeOH was added and stirred for 10 min.
The solution was extracted with EtOAc (3 × 70 ml). The organic
layer was then washed with brine, dried over Na2SO4, filtered, and
concentrated under reduced pressure. The residue was purified
by flash column chromatography with petroleum ether–EtOAc
(80 : 1–60 : 1) to afford 24 as colorless transparent oil (7.1 g, 99%).
Rf 0.70 (20 : 1, petroleum ether–EtOAc); 1H NMR (CDCl3, 600 MHz)
δ: 7.33–7.05 (m, 15H, ArH), 3.99 (d, 2H, J = 13.7 Hz, PhCHaN), 3.77
(s, 3H, OCH3), 3.71 (t, 1H, J = 7.7 Hz, α-H), 3.58 (d, 2H, J = 13.7 Hz,
PhCHbN), 3.16 (dd, 1H, J = 7.1, 13.7 Hz, β-Ha), 3.04(dd, 1H, J = 8.2,
13.2 Hz, β-Hb).
(3S,4S)-tert-Butyl 4-amino-3-hydroxy-5-phenylpentanoate (Ahppa)
(7). Hydrogenation of syn-26 (490 mg, 1.1 mmol) was carried out
inEtOAc(40 ml)inthepresenceofacatalyticamountofPd-C(10%)
under hydrogen at room temperature. The reaction mixture was
stirred for 8 h and the catalyst Pd-C was removed by filtration, then
the resulting filtrate was reconcentrated under reduced pressure
to yield the amine 7 as a white solid (292 mg, 100%); 1H NMR
(CDCl3, 600 MHz) δ: 7.20–7.31 (m, 5H, ArH), 3.90 (m, 1H, H-3), 2.99
(m, 1H, H-5a), 2.92 (m, 1H, H-5b), 2.64 (m, 1H, H-4), 2.56 (dd, 1H,
J = 8.8, 13.8 Hz, H-2a), 2.45 (dd, 1H, J = 8.8, 16.0 Hz, H-2b).
N-Cbz-Gln(Trt)-OH(30). Cbz-Gln-OH (29) (4.2 g, 15 mmol), Trt-OH
(7.8 g, 30 mmol), Ac2O (3 ml, 30 mmol), and 75.3 µl conc. H2SO4
were suspended in 50 ml of glacial acetic acid and stirred for
3 h at 50 ◦C, The solution was then slowly added to 150 ml cold
H2O, the precipitate filtered off, dissolved in 150 ml ethyl acetate,
washed with H2O, dried, evaporated and crystallized from ethyl
acetate-hexane to afford 30 as a white solid (5.6 g, 72%). Rf 0.25
(10 : 1, CHCl3 –MeOH); 1H NMR (DMSO-d6, 600 MHz) δ: 8.62 (s, 1H,
COOH), 7.54 (d, 1H, J = 7.7 Hz, NH), 7.15–7.36 (m, 20H, ArH), 5.04
(m, 2H, PhCH2O), 3.94 (m, 1H, α-H), 2.31–2.41 (m, 2H, γ -H × 2),
1.90 (m, 1H, β-Ha), 1.68 (m, 1H, β-Hb).
tert-Butyl 4-(N, N-dibenzylamino)-3-oxo-5-phenylpentanoate (25).
To a stirred solution of hexamethyldisiloxane (HMDS) (8 ml,
38.4 mmol) in 24 ml THF, nBuLi (14.4 ml, 36 mmol, 2.5 M in hexane)
was added slowly at −20 ◦C under argon. The solution was stirred
for 0.5 h, cooled down to −78 ◦C, then tert-butyl acetate (4.65 ml,
36 mmol) was added and stirred for 1 h. Then, the solution of
c
J. Pept. Sci. 2010; 16: 364–374 Copyright ꢀ 2010 European Peptide Society and John Wiley & Sons, Ltd. www.interscience.com/journal/psc

SUN ET AL.
N-Cbz-N-Me-Gln(Trt)-OH (28). To a solution of N-Cbz-Gln (Trt)-
OH (30) (1.04 g, 2 mmol) in dry THF (25 ml) at 0 ◦C under argon,
NaH (60 wt % in mineral oil, 160 mg, 4 mmol) was added. After
stirring for 30 min at 0 ◦C, MeI (186.8 µl, 3 mmol) was added. The
reaction was allowed to warm to room temperature and stirred
for another 16 h. The reaction mixture was quenched with water
(2 ml) and then concentrated under reduced pressure. The residue
was diluted with water (100 ml), washed twice with n-hexane
(15 ml). The aqueous layer was acidified with HCl (0.1 M) until
pH = 1–2 and extracted with EtOAc (3 × 50 ml). The organic
layer was then washed with saturated Na2S2O3 and then brine,
dried over Na2SO4, filtered, and concentrated under reduced
Val γ -CH3), 0.87 (d, 3H, J = 7.1 Hz, Val γ -CH3); 13C NMR (CDCl3,
150 MHz) δ: 173.4, 171.6, 137.2, 135.3, 128.6, 128.5, 128.4, 128.3,
128.0, 127.9, 76.3 (La α-C), 72.2 (PhCH2OCH), 67.0 (PhCH2OCO),
56.4 (Val α-C), 31.3 (Val β-C), 19.2 (Val γ -C), 19.0 (Val γ -C), 17.4 (La
β-C); ESI-MS (m/z): 370.2 [M + H]+, 392.2 [M + Na]+, 408.1 [M +
K]+ (Calcd 369.19).
BnO-Lac-Val-N-Me-Gln(Trt)-Ahppa-OtBu (4c). Hydrogenation of
5b (350 mg, 0.447 mmol) was carried out in EtOAc (30 ml) in
the presence of a catalytic amount of Pd-C (10%) under hydrogen
at room temperature for 5 h. Pd-C was removed by filtration and
the resulting filtrate was reconcentrated under reduced pressure
to yield the amine 31. Meanwhile, to a solution of 6b (190 mg,
0.514 mmol) in THF–MeOH–H2O (4 : 1 : 2, 21 ml) at 0 ◦C, LiOH·H2O
(86.3 mg, 2.0 m mol) was added and stirred for 20 min, warmed
to room temperature and stirred for another 1 h. The reaction
mixture was then concentrated under reduced pressure. The
residue was diluted with brine (100 ml), washed thrice with DCM
(15 ml) to remove BnOH. The aqueous layer was acidified with HCl
(0.1 M) until pH = 2–3 and extracted with EtOAc (3 × 50 ml), then
concentrated under reduced pressure to yield 27. A solution of
residue 31 and 27 in THF (50 ml) was treated sequentially with
HOAt (121.8 mg, 0.894 mmol), EDCI (171.4 mg, 0.894 mmol), and
DIPEA (156.1 µl, 0.894 mmol). The solution was stirred at 0 ◦C for
30 min, warmed to room temperature and stirred for another 15 h.
And then, the mixture was treated as described for 11. The residue
was purified by flash column chromatography with CHCl3 –MeOH
(100 : 1–50 : 1) to afford the 4c as a white solid (264 mg, 65%). Rf
pressure to give 28 as a white solid (0.80 g, 75%). Rf 0.32 (10 : 1,
1
CHCl3 –MeOH); [α]20 = −13.23 (c 1.0, CHCl3); H NMR (CDCl3,
D
600 MHz) two coformers δ: 7.14–7.36 (m, 20H, ArH), 6.79, 6.59 (s,
1H, 2 : 1, CONHTrt), 5.01–5.20 (m, 2H, PhCH2O), 4.77, 4.68 (d, 1H,
J = 5.5, 9.9 Hz, 1 : 2, α-H), 2.88, 2.86 (s, 3H, N-Me), 2.72, 2.65 (m, 1H,
γ -Ha), 2.20–2.35 (m, 3H, γ -Hb, β-H × 2). ESI-MS (m/z): 537.2976
[M + H]+, 559.2601 [M + Na]+ (Calcd 536.23).
N-Cbz-N-Me-Gln(Trt)-Ahppa-OtBu (5b).
A
solution of 28
(536.2 mg, 1.0 mmol) and 7 (292 mg, 1.1 mmol) in anhydrous THF
(50 ml) was treated sequentially with HOAt (204.3 mg, 1.5 mmol),
EDCI (287.6 mg, 1.5 mmol), and DIPEA (349.2 µl, 2.0 mmol) at 0 ◦C.
The solution was stirred for 30 min, warmed to room temperature
and stirred for another 12 h. And then, the mixture was treated
as described for 11. The residue was purified by flash column
chromatography with petroleum ether–EtOAc (10 : 1–2 : 1) to af-
ford 5b as a white solid (642 mg, 81.9%). Rf 0.52 (1 : 1, petroleum
0.63 (20 : 1, CHCl3 –MeOH); [α]20 = −61.47 (c 0.5, CHCl3); 1H NMR
D
ether–EtOAc); [α]20 = −64.63 (c 0.5, CHCl3); 1H NMR (CDCl3,
D
(CDCl3, 600 MHz) δ: 7.15–7.41 (m, 26 H, ArH + Val α-H), 6.82 (d,
1H, J = 9.4 Hz, Ahppa NH), 6.76 (s, 1H, CONHTrt), 4.95 (m, 1H, Gln
α-H), 4.50 (d, 1H, J = 11.6 Hz, PhCHaO), 4.33 (d, 1H, J = 11.0 Hz,
PhCHbO), 4.25 (t, 1H, J = 7.7 Hz, Val α-H), 4.20 (dd, 1H, J = 7.7,
13.7 Hz, Ahppa H-4), 4.04 (m, 1H, La α-H), 3.96 (m, 1H, Ahppa H-3),
2.92 (m, 1H, Ahppa H-5a), 2.87 (s, 3H, N-CH3), 2.78 (m, 1H, Ahppa
H-5b), 2.26–2.35 (m, 4H, Ahppa H-2 × 2 + Gln γ -H × 2), 1.93–1.99
(m, 2H, Val β-H + Gln β-Ha), 1.81 (m, 1H, Gln β-Hb), 1.43 (d, 3H,
J = 6.6 Hz, La β-CH3), 1.41 (s, 9H, C(CH3)3), 0.99 (d, 3H, J = 7.1 Hz,
Val γ -CH3), 0.92 (d, 3H, J = 6.6 Hz, Val γ -CH3); 13C NMR (CDCl3,
150 MHz) δ: 175.1, 174.1, 172.8, 171.0, 169.9, 144.7, 144.6, 137.8,
137.2, 129.3, 128.7, 128.6, 128.4, 128.2, 128.0, 127.9, 127.7, 127.0,
126.8, 126.4, 81.6, 75.7, 72.1, 71.9, 70.4, 54.7, 54.2, 53.4, 40.9, 39.6,
38.0, 33.2, 31.0, 28.0 (C(CH3)3), 23.1, 18.5, 17.6; ESI-MS (m/z): 911.5
[M + H]+, 933.4 [M + Na]+, 949.5[M + K]+ (Calcd 910.49).
600 MHz) two coformers δ: 716–7.39 (m, 25H, ArH), 6.72, 6.57 (s,
1H, CONHTrt), 6.36, 6.05 (d, 1H, J = 8.2 Hz, Ahppa NH), 5.15 (s, 2H,
PhCH2O), 4.61, 4.52 (m, 1H, Ahppa H-3), 4.07 (m, 1H, Ahppa H-4),
4.00 (m, 1H, Gln α-H), 2.74–2.89 (m, 2H, Ahppa H-5×2), 2.51 (s, 3H,
N-CH3), 2.14–2.34 (m, 5H, Ahppa H-2a + Gln γ -H × 2 + β-H × 2),
1.92 (m, 1H, Ahppa H-2b), 1.44 (s, 9H, C(CH3)3); 13C NMR (CDCl3,
150 MHz) δ: 172.8, 170.8, 157.4 (PhCH2OCO), 144.7, 137.8, 136.4,
129.3, 128.8, 128.6, 128.3, 128.1, 127.1, 126.5, 81.8 (C(CH3)3), 70.7
(Ahppa C-3), 67.8 (PhCH2O), 58.3 (Glu α-C), 53.6 (Ahppa C-4), 39.6
(Ahppa C-2), 38.2 (Ahppa C-5), 33.8 (Glu γ -C), 29.6 (N-CH3), 28.2
(C(CH3)3), 23.7 (Glu β-C); ESI-MS (m/z): 784.1 [M + H]+, 806.1 [M +
Na]+ (Calcd 783.39).
(S)-Benzyl
2-((S)-2-(benzyloxy)propanamido)-3-methylbutanoate
(BnO-Lac-Val-OBn) (6b). A solution of H-Val-OBn·TosOH (17)
(242.0 mg, 0.666 mmol) and (S)-2-(benzyloxy)propanoic acid (19)
(100.0 mg, 0.555 mmol) in anhydrous DCM (10 ml) was treated
sequentially with HOAt (113.4 mg, 0.833 mmol), EDCI (159.7 mg,
0.833 mmol), and DIPEA (193.8 µl, 1.110 mmol) at 0 ◦C. The
solution was stirred for 30 min, warmed to room temperature
and stirred for another 12 h. And then, the mixture was treated
as described for 11. The residue was purified by flash column
chromatography with petroleum ether–EtOAc (40 : 1–5 : 1) to
afford 6b as a pale-yellow viscous solid (194.8 mg, 95.0%). Rf 0.65
Synthesis of 1
BnO-Lac-Val-N-Me-Gln-Ahppa-Leu-Ala-N-Me-Phe-Pro-OMe
(34).
To a solution of 4c (50.0 mg, 0.055 mmol) and 3 (37.8 mg,
0.066 mmol) in DCM (2 ml) at 0 ◦C, TFA (2 ml) was added and
stirred for 20 min, warmed to room temperature and stirred for
another 1.5 h. The solution was then dissolved twice in CH2Cl2
(2 ml) with evaporation each time to yield the acid 32 and the
amine 33. A solution of them in anhydrous THF (5 ml) was treated
sequentially with HOAt (15.0 mg, 0.11 mmol l), EDCI (21.1 mg,
0.11 mmol), and DIPEA (50.0 µl, 0.14 mmol). The solution was
stirred at 0 ◦C for 30 min, warmed to room temperature and stirred
for another 16 h. And then, the mixture was treated as described
for 11. The residue was purified by flash column chromatography
with CHCl3 –MeOH (80 : 1–20 : 1) to afford the 34 as a white solid
(22.3 mg, 38%). Rf 0.60 (10 : 1, CHCl3 –MeOH).
(2 : 1, petroleum ether–EtOAc); [α]20 = −51.99 (c 0.5, CHCl3); 1H
D
NMR (CDCl3, 600 MHz) δ: 7.29–7.37 (m, 10 H, ArH), 7.12 (d, 1 H,
J = 9.4 Hz, Val NH), 5.21 (d, 1 H, J = 12.1 Hz, PhCHaOCO), 5.16 (d,
1H, J = 12.1 Hz, PhCHbOCO), 4.63 (dd, 1 H, J = 5.0, 9.4 Hz, Val
α-H), 4.62 (d, 1H, J = 11.6 Hz, PhCHaOCH), 4.49 (d, 1H, J = 11.5 Hz,
PhCHbOCH), 3.97 (q, 1 H, J = 6.6, Hz, La α-H), 2.23 (m, 1 H, Val
β-H), 1.43 (d, 3 H, J = 7.1 Hz, La β-CH3), 0.93 (d, 3H, J = 6.6 Hz,
c
www.interscience.com/journal/psc Copyright ꢀ 2010 European Peptide Society and John Wiley & Sons, Ltd. J. Pept. Sci. 2010; 16: 364–374

SYNTHESIS AND STEREOCHEMICAL REASSIGNMENT OF TASIAMIDE B
HO-Lac-Val-N-Me-Gln-Ahppa-Leu-Ala-N-Me-Phe-Pro-OMe(1). Hy-
drogenation of 34 (22.3 mg, 0.021 mmol) was carried out in
EtOAc-EtOH (4 : 1, 5 ml) in the presence of a catalytic amount
of Pd-C (10%) under hydrogen at room temperature for 17 h.
The catalyst Pd-C was removed by filtration and the resulting
filtrate was reconcentrated under reduced pressure to afford the
(C-10), 129.5 (C-11, 15), 129.1 (C-32, 36), 128.4 (C-12, 14), 128.2
(C-33, 35), 126.6 (C-13), 126.4 (C-34), 69.3 (C-28), 68.5 (C-49), 59.2
(C-2), 55.7 (C-38), 55.6 (C-8), 54.2 (C-44), 53.9 (C-29), 52.3 (C-6), 52.1
(C-21), 46.9 (C-5), 45.3 (C-18), 40.8 (C-22), 40.6 (C-27), 37.6 (C-30),
34.7 (C-9), 31.8 (C-16), 31.1 (C-40), 30.8 (C-42), 30.4 (C-45), 28.8
(C-3), 25.3 (C-4), 24.7 (C-23), 22.8 (C-24, 39), 21.9 (C-25), 20.8 (C-50),
19.5 (C-47), 17.8 (C-46), 17.2 (C-19); HRESIMS calcd for C50H75N8O12
[M + H]+ 979.5504, found 979.5470.
1 as a white solid (18.6 mg, 91%). Rf 0.28 (10 : 1, CHCl3 –MeOH);
1
[α]20 = −89.6 (c = 0.45, MeOH); H NMR (CDCl3, 600 MHz) δ:
D
7.49 (m, 1H, Leu NH), 7.32 (d, 1H, J = 7.3 Hz, Val NH), 7.16–7.25
(m, 10H, ArH), 7.10 (m, 1H, Ala NH), 7.07 (m, 1H, Ahppa NH), 5.51
(t, 1H, J = 7.3 Hz, Phe α-H), 5.04 (m, 1H, Gln α-H), 4.69 (t, 1H,
J = 6.4 Hz, Ala α-H), 4.47 (t, 1H, J = 7.3 Hz, Val α-H), 4.41 (dd, 1H,
J = 4.1, 9.1 Hz, Pro α-H), 4.34 (m, 1H, Leu α-H), 4.24 (m, 1H, Ahppa
H-4), 4.19 (m, 1H, La α-H), 4.05 (m, 1H, Ahppa H-3), 3.70 (s, 3H, Pro
OMe), 3.53 (m, 1H, Pro δ-Ha), 3.34 (m, 1H, Pro δ-Hb), 3.25 (dd, 1H,
J = 6.9, 14.2 Hz, Phe β-Ha), 3.06 (s, 3H, Phe N-CH3), 2.94–3.00 (m,
2H, Phe β-Hb + Ahppa H-5a), 2.85 (m, 1H, Ahppa H-5b), 2.81 (s, 3H,
Gln N-CH3), 2.49 (m, 1H, Ahppa H-2a), 2.36 (m, 1H, Ahppa H-2b),
2.11–2.24 (m, 4H, Pro β-Ha + Gln β-Ha + Gln γ -H × 2), 1.85–1.96
(m, 5H, Gln β-Hb + Pro β-Hb + Pro γ -H × 2 + Val β-H), 1.59 (m,
1H, Leu γ -H), 1.51 (m, 2H, Leu β-H × 2), 1.39 (d, 3H, J = 6.4 Hz, La
β-CH3), 1.25 (d, 3H, J = 6.4 Hz, Ala β-CH3), 0.94 (d, 3H, J = 6.4 Hz,
Val γ -CH3), 0.91 (t, 6H, J = 5.5 Hz, Val γ -CH3 + Leu δ-CH3), 0.89
(d, 3H, J = 6.4 Hz, Leu δ-CH3); 13C NMR (CDCl3, 150 MHz) δ 176.3
(C-48), 175.6 (C-41), 173.5 (C-43), 172.7 (C-1), 172.3 (C-17), 171.8
(C-20), 171.6 (C-26), 170.1 (C-37), 169.2 (C-7), 138.0 (C-31), 136.6
(C-10), 129.1 (C-11, 15, 32, 36), 128.5 (C-12, 14), 128.4 (C-33, 35),
126.8 (C-13), 126.4 (C-34), 69.4 (C-28), 68.6 (C-49), 59.1 (C-2), 55.9
(C-38), 55.7 (C-8), 54.2 (C-44), 53.9 (C-29), 52.2 (C-6), 52.1 (C-21),
46.9 (C-5), 45.8 (C-18), 41.0 (C-27), 40.8 (C-22), 37.6 (C-30), 34.7
(C-9), 31.3 (C-16), 31.0 (C-40), 30.9 (C-42), 30.3 (C-45), 29.0 (C-3),
24.9 (C-4), 24.7 (C-23), 23.0 (C-24), 21.9 (C-25), 20.9 (C-50), 19.5
(C-47), 18.1 (C-46), 17.8 (C-19); HRESIMS calcd for C50H74N8O12Na
[M + Na]+ 1001.5324, found 1001.5328.
HO-Lac-Val-N-Me-Gln-Ahppa-Leu-D-Ala-N-Me-Phe-Pro-OMe (1b).
Compound 1b was obtained as a white solid as described
for the synthesis of compound 1 but using the correspond-
ing configuration of amino acids and HATU instead of DECI
at the last condensation step. Rf 0.28 (10 : 1, CHCl3 –MeOH);
[α]20 = −126.41 (c = 0.4, MeOH); 1H NMR (CDCl3, 600 MHz)
D
δ: 7.40 (d, 1H, J = 5.9 Hz, Ala NH), 7.37 (d, 1H, J = 6.8 Hz, Leu NH),
7.30 (d, 1H, J = 7.8 Hz, Val NH), 7.16–7.24 (m, 10H, ArH), 6.99 (d,
1H, J = 8.7 Hz, Ahppa NH), 6.88 (brs, 1H, CONHa), 6.72 (brs, 1H,
CONHb), 5.67 (dd, 1H, J = 5.5, 10.6 Hz, Phe α-H), 5.08 (m, 1H, Gln
α-H), 4.59 (t, 1H, J = 6.9 Hz, Ala α-H), 4.41–4.45 (m, 2H, Val α-H
+ Pro α-H), 4.36 (m, 1H, Leu α-H), 4.23 (m, 1H, Ahppa H-4), 4.15
(m, 1H, La α-H), 4.05 (m, 1H, Ahppa H-3), 3.72 (s, 3H, Pro OMe),
3.48–3.51 (m, 2H, Pro δ-H × 2), 3.16 (dd, 1H, J = 5.5, 14.6 Hz, Phe
β-Ha), 3.00–3.05 (m, 4H, Phe N-CH3 + Phe β-Hb), 2.94 (dd, 1H,
J = 7.3, 14.6 Hz, Ahppa H-5a), 2.81 (dd,1H, J = 9.7, 15.6 Hz, Ahppa
H-5b), 2.73 (s, 3H, Gln N-CH3), 2.42 (dd, 1H, J = 7.8, 13.7 Ahppa
H-2a), 2.35 (m, 1H, Ahppa H-2b), 2.12–2.21 (m, 4H, Pro β-Ha + Gln
β-Ha + Gln γ -H×2), 1.85–1.95 (m, 5H, Gln β-Hb + Pro β-Hb + Pro
γ -H ×2+ Val β-H), 1.44–1.57 (m, 3H, Leu γ -H + Leu β-H ×2), 1.38
(d, 3H, J = 6.9 Hz, La β-CH3), 0.94 (d, 3H, J = 6.8 Hz, Val γ -CH3),
0.91 (d, 3H, J = 6.4 Hz, Val γ -CH3), 0.87 (d, 3H, J = 5.9 Hz, Leu
δ-CH3), 0.85 (d, 3H, J = 5.9 Hz, Leu δ-CH3), 0.77 (d, 3H, J = 6.8 Hz,
Ala β-CH3); 13C NMR (CDCl3, 150 MHz) δ 176.5 (C-48), 176.0 (C-41),
173.5 (C-43), 172.6 (C-1), 172.1 (C-17), 171.6 (C-20), 171.5 (C-26),
170.0 (C-37), 168.5 (C-7), 137.9 (C-31), 136.5 (C-10), 129.2 (C-11,
15), 129.0 (C-32, 36), 128.4 (C-12, 14), 128.3 (C-33, 35), 126.7 (C-13),
126.5 (C-34), 69.4 (C-28), 68.5 (C-49), 59.2 (C-2), 55.7 (C-38), 55.0
(C-8), 54.2 (C-44), 53.4 (C-29), 52.3 (C-6), 51.6 (C-21), 46.9 (C-5),
45.7 (C-18), 41.0 (C-27), 40.6 (C-22), 37.7 (C-30), 34.6 (C-9), 31.2
(C-16), 30.8 (C-40), 30.6 (C-42), 30.2 (C-45), 28.9 (C-3), 24.8 (C-4),
24.7 (C-23), 23.5 (C-24), 23.0 (C-39), 21.7 (C-25), 20.7 (C-50), 19.5
(C-47), 17.8 (C-46), 16.5 (C-19); HRESIMS calcd for C50H75N8O12 [M
+ H]+ 979.5504, found 979.5478.
Synthesis of 1a, 1b, 1c
HO-Lac-Val-N-Me-Gln-Ahppa-Leu-Ala-N-Me-D-Phe-Pro-OMe (1a).
Compound 1a was obtained as a white solid as described for
the synthesis of compound 1 but using the corresponding con-
figuration of amino acids and HATU instead of DECI at the last
condensation step. Rf 0.28 (10 : 1, CHCl3 –MeOH); [α]20 = −30.1
D
(c = 0.4,MeOH);1HNMR(CDCl3,600 MHz)δ:7.44(d,1H,J = 7.3 Hz,
Leu NH), 7.32 (d, 1H, J = 7.8 Hz, Val NH), 7.15–7.23 (m, 10H, ArH),
7.11–7.14 (m, 2H, Ala NH + Ahppa NH), 7.01 (brs, 1H, CONHa), 6.85
(brs, 1H, CONHb), 5.63 (dd, 1H, J = 6.8, 9.2 Hz, Phe α-H), 5.03 (m,
1H, Gln α-H), 4.68 (q, 1H, J = 6.9 Hz, Ala α-H), 4.42–4.46 (m, 2H,
Val α-H + Pro α-H), 4.32 (dd, 1H, J = 8.2, 14.2 Hz, Leu α-H), 4.20
(m, 1H, Ahppa H-4), 4.16 (q, 1H, J = 6.4 Hz, La α-H), 4.02 (m, 1H,
Ahppa H-3), 3.72 (s, 3H, Pro OMe), 3.41 (m, 1H, Pro δ-Ha), 3.26 (m,
1H, Pro δ-Hb), 3.20 (dd, 1H, J = 6.4, 8.2 Hz, Phe β-Ha), 3.01 (s, 3H,
Phe N-CH3), 2.92–2.96 (m, 2H, Ahppa H-5a + Phe β-Hb), 2.78–2.84
(m, 4H, Ahppa H-5b + Gln N-CH3), 2.43 (dd, 1H, J = 10.1, 13.7 Hz,
AhppaH-2a), 2.30(dd, 1H, J = 3.7, 14.6 Hz, AhppaH-2b), 2.17–2.21
(m, 4H, Pro β-Ha + Gln β-Ha + Gln γ -H × 2), 1.79–1.95 (m, 5H, Gln
β-Hb + Pro β-Hb + Pro γ -H × 2 + Val β-H), 1.48–1.61 (m, 3H, Leu
γ -H + Leu β-H × 2), 1.37 (d, 3H, J = 6.9 Hz, La β-CH3), 0.94 (d, 3H,
J = 6.4 Hz, Val γ -CH3), 0.91 (d, 3H, J = 6.9 Hz, Val γ -CH3), 0.87 (d,
3H, J = 6.4 Hz, Leu δ-CH3), 0.84 (d, 3H, J = 6.4 Hz, Leu δ-CH3), 0.82
(d, 3H, J = 7.3 Hz, Ala β-CH3); 13C NMR (CDCl3, 150 MHz) δ: 176.2
(C-48), 175.4 (C-41), 173.5 (C-43), 172.6 (C-1), 172.4 (C-17), 171.9
(C-20), 171.8 (C-26), 170.0 (C-37), 168.1 (C-7), 137.9 (C-31), 136.7
HO-Lac-Val-N-Me-Gln-Ahppa-Leu-D-Ala-N-Me-D-Phe-Pro-OMe (1c).
Compound 1c was obtained as a white solid as described for
the synthesis of compound 1 but using the corresponding
configuration of amino acids and HATU instead of DECI at the last
condensation step. Rf 0.28 (10 : 1, CHCl3 –MeOH); [α]20 = −50.01
D
1
(c = 0.4, MeOH); H NMR (CDCl3, 600 MHz) δ: 7.30–7.32 (m, 2H,
Leu NH + Val NH), 7.27 (m, 1H, Ala NH), 7.10–7.23 (m, 10H, ArH),
7.03 (d, 1H, J = 8.2 Hz, Ahppa NH), 6.76 (brs, 1H, CONHa), 6.60 (brs,
1H, CONHb), 5.43 (dd, 1H, J = 5.9, 9.2 Hz, Phe α-H), 5.08 (m, 1H,
Gln α-H), 4.82 (m, 1H, Ala α-H), 4.43–4.46 (m, 2H, Val α-H + Leu
α-H), 4.39 (dd, 1H, J = 5.0, 8.8 Hz, Pro α-H), 4.29 (m, 1H, Ahppa
H-4), 4.15 (d, 1H, J = 5.9 Hz, La α-H), 4.08 (m, 1H, Ahppa H-3), 3.70
(s, 3H, Pro OMe), 3.26–3.31 (m, 3H, Pro δ-H × 2 + Phe β-Ha), 3.12,
3.08 (s, 3H, 1 : 5, Phe N-CH3), 2.95 (m, 1H, Ahppa H-5a), 2.83 (m,
1H, Ahppa H-5b), 2.73–2.76 (m, 4H, Phe β-Hb + Gln N-CH3), 2.46
(dd, 1H, J = 7.7, 13.7 Hz, Ahppa H-2a), 2.40 (m, 1H, Ahppa H-2b),
2.08–2.18 (m, 4H, Pro β-Ha + Gln β-Ha + Gln γ -H × 2), 1.81–1.93
c
J. Pept. Sci. 2010; 16: 364–374 Copyright ꢀ 2010 European Peptide Society and John Wiley & Sons, Ltd. www.interscience.com/journal/psc

SUN ET AL.
(m, 4H, Gln β-Hb + Pro β-Hb + Pro γ -Ha + Val β-H), 1.78 (m, 1H,
Pro γ -Hb), 1.59–1.61 (m, 2H, Leu γ -H + Leu β-Ha), 1.51 (m, 1H, Leu
β-Hb), 1.38 (d, 3H, J = 6.4 Hz, La β-CH3), 1.32 (d, 3H, J = 6.9 Hz,
2 Williams PG, Yoshida WY, Moore RE, Paul VJ. The isolation and
structure elucidation of tasiamide B,
a 4-amino-3-hydroxy-5-
phenylpentanoic acid containing peptide from the marine
cyanobacterium Symploca sp. J. Nat. Prod. 2003; 66: 1006–1009.
3 Williams PG, Yoshida WY, Moore RE, Paul VJ. Tasiamide, a cytotoxic
peptide from the marine cyanobacterium Symploca sp. J. Nat. Prod.
2002; 65: 1336–1339.
4 Ma ZH, Song N, Li CX, Zhang W, Wang P, Li YX. Total synthesis and
stereochemical reassignment of tasiamide. J. Pept. Sci. 2008; 14:
1139–1147.
Ala β-CH3), 0.89–0.96 (m, 12H, Val γ -CH3 ×2+ Leu δ-CH3 ×2); 13
C
NMR (CDCl3, 150 MHz) δ 176.4 (C-48), 175.8 (C-41), 173.5 (C-43),
172.8 (C-1), 172.6 (C-17), 171.8 (C-20), 170.0 (C-37), 167.9 (C-7),
137.9 (C-31), 137.2 (C-10), 129.6 (C-11, 15), 129.0 (C-32, 36), 128.4
(C-12, 14, 33, 35), 126.5 (C-13), 126.5 (C-34), 69.5 (C-28), 68.6 (C-49),
58.7 (C-2), 56.6 (C-8), 55.9 (C-38), 54.2 (C-44), 53.5 (C-29), 52.2 (C-6),
51.7 (C-21), 46.5 (C-5), 45.9 (C-18), 41.1 (C-27), 40.6 (C-22), 37.8
(C-30), 34.7 (C-9), 31.2 (C-16), 31.0 (C-40), 30.9 (C-42), 30.2 (C-45),
28.8(C-3), 24.9(C-4), 24.8(C-23), 23.1(C-39), 23.0(C-24), 21.8(C-25),
20.7 (C-50), 19.5 (C-47), 17.8 (C-46), 17.4 (C-19); HRESIMS calcd for
C50H74N8O12Na [M + Na]+ 1001.5324, found 1001.5283.
5 Erlanger BF, Sachs H, Brand E. The synthesis of peptides related to
gramicidin S J. Am. Chem. Soc. 1954; 76: 1806–1810.
6 McDermott JR, Benoiton NL. N-Methylamino acids in peptide
synthesis. II.
A
new synthesis of N-benzyloxycarbonyl,
N-methylamino acids. Can. J. Chem. 1973; 51: 1915–1919.
¨
7 Heinz G,Schroder E.Synthesenvonarginin-haltigenpeptiden.Justus
Liebigs Ann. Chem. 1961; 642: 145–162.
8 Zhang W, Ma Z-H, Mei D, Li C-X, Zhang X-L, Li Y-X. Total synthesis
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Cytotoxic Evaluation In Vitro
9 Miller SC, Scanlan TS. Site-selective N-methylation of peptides on
solid support. J. Am. Chem. Soc. 1997; 119: 2301–2302.
The prepared compounds (1a, 1b, 1c, and 1) were submitted to
ShanghaiInstituteofMateriaMedicaChineseAcademyofSciences
to test their cytotoxicities. The cytotoxic effects were examined us-
ing sulforhodamine B (SRB) assay against A549, LoVo, KB, BEL-7402
and MTT tetrazolium dye assay against P388 and HL-60 cell lines.
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(+)-jasplakinolide. J. Org. Chem. 1991; 56: 5196–5202.
11 Aurelio L, Box JS, Brownlee RTC, Hughes AB, Sleebs MM. An efficient
synthesis of N-methyl amino acids by way of intermediate
5-oxazolidinones. J. Org. Chem. 2003; 68: 2652–2667.
12 Myers AG, Gleason JL, Yoon T, Kung DW. Highly practical
methodology for the synthesis of D- and L-α-amino acids,
N-protected α-amino acids, and N-methyl-α-amino acids J. Am.
Chem. Soc. 1997; 119: 656–673.
Acknowledgements
This work was financially supported by Hi-Tech Research and
Development Program of China (2006AA09Z409), and School of
Pharmacy, Fudan University.
13 Freidinger RM, Hinkle JS, Perlow DS, Arison BH. Synthesis of
9-fluorenylmethyloxycarbonyl-protected N-alkyl amino acids by
reduction of oxazolidinones. J. Org. Chem. 1983; 48: 77–81.
14 Andres JM, Pedrosa R. Diastereoselective synthesis of enantiopure
γ -amino-β-hydroxy acids by Reformatsky reaction of chiral
α-dibenzylamino aldehydes. Tetrahedron 2001; 57: 8521–8530.
15 Kondekar NB, Kandula SV, Kumar P. Application of the asymmetric
aminohydroxylation reaction for the syntheses of HIV-protease
inhibitor, hydroxyethylene dipeptide isostere and γ -amino acid
derivative. Tetrahedron Lett. 2004; 45: 5477–5479.
Supporting information
Supporting information may be found in the online version of this
article.
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and statine analogs via 4-(N,N-dibenzylamino)-3-keto esters. J. Org.
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c
www.interscience.com/journal/psc Copyright ꢀ 2010 European Peptide Society and John Wiley & Sons, Ltd. J. Pept. Sci. 2010; 16: 364–374