Total synthesis and biological activity of dolastatin 16

Article abstract of DOI:10.1039/c6ob02657e

The total synthesis of dolastatin 16, a macrocyclic depsipeptide first isolated from the sea hare Dolabella auricularia as a potential antineoplastic metabolite by Pettit et al., was achieved in a convergent manner. Dolastatin 16 was reported by Tan to ex

Full text of DOI:10.1039/c6ob02657e

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Biomol. Chem., 2017, DOI: 10.1039/C6OB02657E.
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DOI: 10.1039/C6OB02657E
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Total Synthesis and Biological Activity of Dolastatin 16
Loida O. Casalme,a Arisa Yamauchi,a Akinori Sato,a Julie G. Petitbois,a Yasuyuki Nogata,b Erina
Yoshimura,c Tatsufumi Okino,a Taiki Umezawa,*a and Fuyuhiko Matsuda*a
Received 00th January 20xx,
Accepted 00th January 20xx
The total synthesis of dolastatin 16, a macrocyclic depsipeptide first isolated from the sea hare Dolabella auricularia as a
potential antineoplastic metabolite by Pettit et al., was achieved in a convergent manner. Dolastatin 16 was reported by Tan
to exhibit strong antifouling activity, and thus shows promise for inhibiting the attachment of marine benthic organisms such
as Amphibalanus amphitrite to ships and submerged artificial structures. Therefore, dolastatin 16 is a potential compound
for a new, environmentally friendly antifouling material to replace banned tributyltin‐based antifouling paints. The synthesis
of dolastatin 16 involved the use of prolinol to prevent formation of a diketopiperazine composed of L‐proline and N‐methyl‐
D‐valine during peptide coupling. This strategy for the elongation of peptide chains allowed the efficient and scalable
synthesis of one segment, which was subsequently coupled with a second segment and cyclized to form the macrocyclic
framework of dolastatin 16. The synthetic dolastatin 16 exhibited potent antifouling activity similar to that of natural
dolastatin 16 toward cypris larvae of Amphibalanus amphitrite.
DOI: 10.1039/x0xx00000x
www.rsc.org/
copper‐based antifouling agents, but these require a high
concentration of copper and a co‐biocide to achieve the same
efficacy. Concerns about copper toxicity have led several
countries to review their existing copper environmental risk
assessments in coastal waters, and a number of countries have
already banned copper‐based antifouling paints in areas with a
high density of boats.9 Thus, the development of antifouling
agents without heavy metals is highly desired.
Marine organisms prevent fouling of their outer surfaces
through the use of natural chemical defense substances with
antifouling properties without causing serious environmental
problems.10 Therefore, natural antifouling products, especially
those with potent settlement‐inhibiting activities but without
biocidal properties, are potential candidates as non‐biocide‐
based and environmentally friendly antifouling agents. Several
marine antifouling natural products have been reported over
the past decade, resulting from the search for nontoxic and
environmentally benign active components for antifouling
paints.11,12
Introduction
Biofouling is the accumulation of organisms on immersed
artificial structures such as ship hulls, jetty pilings, aquaculture
net cages, and seawater intake pipes, and results in significant
economic and environmental problems. For example, the
settlement of marine benthic organisms on a ship’s surface
increases fuel consumption by as much as 40% due to friction.1
In addition, frequent dry‐docking and maintenance to remove
biofouling organisms is an expense. Antifouling paints have
been used to address this problem and minimize the associated
economic costs. Tributyltin (TBT)‐based antifouling paint was
developed during the 1960s and was so efficient against a broad
range of fouling organisms that it became the leading solution,
adopted by approximately 70% of the world’s shipping fleets.2
However, harmful effects of TBT on marine organisms such as
fish,3 crustaceans,4 and especially molluscs5 were subsequently
reported. Nanogram per liter concentrations of TBT induce
masculinization of female gastropods and have resulted in the
extinction of certain species.6 As of 2004, approximately 150
gastropod species worldwide have been affected.7
Consequently, the International Maritime Organization (IMO)
prohibited the use of TBT‐based antifouling paints on ships in
2008.8 Currently, TBT‐based paints have been replaced by
Dolastatin 16 (1), a macrocyclic depsipeptide, was first
isolated in 1997 from the sea hare Dolabella auricularia as a
potential anticancer compound by Pettit and co‐workers.13 This
unique depsipeptide proved to strongly inhibit the growth of a
variety of human cancer cell lines and thus was a candidate for
further development as an anticancer drug. Gerwick et al. also
described the isolation of
Lyngbya majuscula, in 2002.14 The unique structural feature of
is the presence of the unusual amino acids dolamethylleuine
) and dolaphenvaline ( ). The stereostructures of and were
not assigned in the first report, but subsequent X‐ray
crystallographic studies of the natural product showed that
1 from a Madagascan cyanobacterium,
a. Graduate School of Environmental Science, Hokkaido University, N10W5 Sapporo
060‐0810, Japan. E‐mail: umezawa@ees.hokudai.ac.jp,
1
(
2
3
2
3
fmatsuda@ees.hokudai.ac.jp
b. Environmental Science Research Laboratory, Central Research Institute of Electric
Power Industry, 1646 Abiko, Abiko, Chiba 270‐1194, Japan.
c. CERES, Inc., 1‐4‐5 Midori, Abiko, Chiba 270‐1153, Japan.
†Electronic Supplementary Information (ESI) available: Preparation of
7,
8,
10, 17
and 20, optimizations for 18
,
22 and 23, and 1H and 13C NMR spectra for all
compounds. See DOI: 10.1039/x0xx00000x
This journal is © The Royal Society of Chemistry 20xx
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Organic & Biomolecular Chemistry
asymmetric Mannich reactions.18 A notable feature of these
View Article Online
DOI: 10.1039/C6OB02657E
syntheses is construction of the contiguous stereogenic centers
of
2
and
3
with almost complete diastereo‐ and
enantioselectivity by employing chiral organocatalysts. With
adequate amounts of the unusual amino acids synthesized,
attention was focused on the assembly of the macrocyclic
framework of
synthesis of
activities of synthetic
1
. Herein, the synthetic details of the total
are described and the significant biological
are reported.
1
1
Results and discussion
A retrosynthetic analysis for the synthesis of dolastatin 16 using
a build‐up approach is presented in Scheme 1. The synthesis of
1
was envisioned via macrolactonization between the hydroxy
group in lactate and the carboxylic acid in dolamethylleuine of
. Synthesis of was designed by condensation of O‐benzyl‐
lactic acid ( ) and peptide fragments and . Fragment would
be prepared from carboxylic acid 10 and southern segment 11
The southern segment 11 was traced back from ‐proline benzyl
ester hydrochloride (12) and the two unusual amino acid units
and
Preparation of 11 is shown in Scheme 2. Dolamethylleuine
benzyl ester (13) was obtained from through a Mitsunobu
Figure 1. Structure of dolastatin 16 (1) and the unusual amino acids 2 and 3.
6
6
L‐
the absolute configurations of the contiguous stereocenters of
and
were (2R,3R) and (2S,3R), respectively.15
In 2010, Tan’s group reported that effectively inhibited the
larval settlement and metamorphosis of the barnacle
Amphibalanus amphitrite with an EC50 value of 0.003
g/mL.16
The LC50/EC50 ratio of is 6000, and, therefore, was expected
7
8
9
9
2
3
.
1
L

4
5.
1
1
to be a promising lead compound alternative to the heavy‐
metal‐based antifouling agents currently used. Pettit’s group
4
reaction with benzyl alcohol, followed by deprotection of the
Boc group with TFA. The carboxylic acid 14 was prepared in 74%
completed the first total synthesis of
isolated from natural sources exhibited impressive activity
against several human cancer cell lines, whereas synthetic did
not possess significant activity. This discrepancy raises the
question of whether the antifouling properties of synthetic
are also much lower than those of isolated from natural
sources. Concise and scalable syntheses of N‐Boc‐
dolamethylleuine ( ) and N‐Boc‐dolaphenvaline ( ), the N‐Boc‐
protected unusual amino acids in , have been developed using
1 1
in 2015.17 Surprisingly,
yield (over two steps) by condensation between
5 and 12 in the
1
presence of 4‐(4,6‐dimethoxy‐1,3,5‐triazin‐2‐yl)‐4‐
methylmorpholinium chloride (DMTMM)19 and subsequent
hydrogenolysis to remove the benzyl ester. Amide formation
with 13 and 14 in a similar manner in the presence of Et3N
afforded 11 in 94% yield.
1
1
4
5
1
Scheme 1. Retrosynthetic analysis of dolastatin 16 (1).
2 | Org. Biomol. Chem., 2016, 00, 1‐3
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Next, the peptide backbone of
1
was completed as shown in
View Article Online
Scheme 3. After removal of the Boc prDoOteI:c1t0in.1g03g9/rCo6uOpB0o2f61571E
,
coupling reaction with 1020 in the presence of DMTMM
furnished
9 in high yield (89% over two steps). Using the same
protocol, amide 15 was synthesized from
(over two steps). Finally, all fragments of
9
1
and 820 in 45% yield
were assembled by
removal of the Boc group of 15 with TFA, followed by coupling
with 720 in 54% yield (over two steps). Further optimizations
resulted in moderate yields of 15 and 6. Obviously, the modest
yields afforded in the last two coupling reactions (45% and 54%,
respectively) were not acceptable in this advanced stage of the
synthesis.
To develop a more efficient total synthesis, an alternative
convergent route was strategized. The improved retrosynthetic
analysis for the convergent route is illustrated in Scheme 4. A
coupling reaction between the northern segment 16 and the
southern segment 11 would access 1 via 6, because the above‐
mentioned peptide formation reaction between 10 and 11
proceeded in high yields (89% over two steps) without
epimezation21 (Scheme 3), although this concept is the same as
Pettit’s synthesis. This strategy was considered best for the total
synthesis of dolastatin 16 because 11, which contains the
unusual amino acid units, could be used as the most advanced
Scheme 2. Synthesis of southern segment 11.
intermediate for the preparation of 6. The northern segment 16
would be obtained from
condensations.
7
,
8
, and 10 by stepwise fragment
O
O
N
N
N
O
O
O
O
HN
1
O
O
BnO
O
N
BnO
N
H
6
O
O
N
N
N
7 + 8 + 10
O
O
O
O
HO
BnO
16
+
NHBoc
O
O
O
N
BnO
N
H
11
Scheme 4. Improved retrosynthetic analysis of dolastatin 16 (1).
Scheme 3. Synthesis of 6.
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In a preliminary study to construct 16, coupling reactions
between
and TFA salt 1720 were conducted (Scheme 5).
However, extensive attempts under various reaction conditions
provided only low yields of the desired amide 18 22
In the
bromotripyrrolidinophosphonium
View Article Online
DOI: 10.1039/C6OB02657E
8
.
presence
of
hexafluorophosphate (PyBroP),23 the yield of 18 was 28%, but
the main product of this reaction was the diketopiperazine 19
composed of L‐proline and N‐methyl‐D‐valine. Formation of
diketopiperazine is a well‐known side reaction in the synthesis
of dipeptide esters containing N‐methyl or prolyl‐type amide
linkages.24 To minimize the tendency of dipeptide 17 to cyclize
into diketopiperazine, prolinol was used in place of proline ester
as the C‐terminal amino acid.
Scheme 5. Diketopiperazine formation.
The synthesis of 16 commenced with a condensation
reaction between N‐Boc‐N‐methyl‐
21) to afford amide 22 (Scheme 6). Careful optimization22
D‐valine (20) L‐prolinol
20 and
(
allowed a high yield and scale‐up (up to 2.6 mmol of 21) for this
reaction with the EDCI/HOAt system. After TFA‐promoted
cleavage of the Boc group of 22, coupling reaction of the
resulting TFA salt with
9 produced amide 23. After extensive
investigations22 with coupling reagents, such as triphosgene,25
HATU,26 DECP,27 or EDCI, for synthesis of 23, PyBroP and iPr2NEt
were found to provide 23 in 76% yield without epimerization.
The effectiveness of PyBroP in facilitating the coupling reactions
of N‐methylated amino acids is well recognized.23 To complete
the components of the northern segment, the Boc group of 23
was deprotected using TFA, and the resulting TFA salt was
condensed with O‐benzyl‐L‐lactic acid (7)
20 to give amide 24 in
82% yield (two steps). Finally, successive Dess‐Martin and
Pinnick oxidation of 24 afforded 16 with an overall yield of
95%.28 The synthetic steps leading to 16 demonstrated high
yields for amide bond formation with various secondary amines
in the presence of the unprotected primary alcohol, resulting
from the judicious choice of coupling reagents.29 In addition, the
conversion of the primary hydroxy group to a carboxylic acid
was efficient. These results demonstrate that the use of
aminoalcohol instead of the corresponding ‐amino acid ester
is an effective strategy for chain elongation of peptide
frameworks.
With segments 11 and 16 synthesized, the total synthesis of
1
was completed as shown in Scheme 7. Cleavage of the Boc
group from 11 with TFA, followed by a coupling reaction with 16
using DMTMM furnished the linear precursor in 92% yield
(over two steps). Global deprotection of the benzyl groups of
afforded the desired seco acid. Lastly, the Shiina protocol30 was
used for macrolactonization to afford in 31% overall yield (two
6
6
1
Scheme 6. Synthesis of northern segment 16.
steps), because other procedures such as the Yamaguchi
lactonization31 did not provide the desired product. All data (1H
and 13C NMR, HRMS, and optical properties) for synthetic
1
were identical to those reported by Pettit and co‐workers13,17
for the natural and synthetic samples.
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Table 1. Biological activity of synthetic dolastatin 16 (1) and segments 16 and 25
View Article Online
DOI: 10.1039/C6OB02657E
Cytotoxicity
compound
EC50 (g/mL)d
LC50 (g/mL)d
LC50 (g/mL)e
> 30
Synthetic 1
Natural 1a
Synthetic 1b
16
< 0.03
0.003
–
> 10
20
–
–
> 10f
> 10
1.17
0.10
> 10
> 10
> 10
> 100
10—30
8.6
25
CuSO4
c
aobtained by Tan, see ref. 16. bobtained by Pettit, see ref. 17. creference. dagainst cypris
larvae of Amphibalanus amphitrite. eagainst MCF‐7 cell (breast cancer cell) fdescribed as
GI50 (g/mL)
Scheme 7. Total synthesis of dolastatin 16 (1).
Next, the biological activities of
1 and of synthetic
intermediates 16 and 25, the latter being the amine derivative
of 11, were evaluated. Antifouling activity was evaluated as 50%
effective concentration (EC50) of each compound against the
settlement of the cypris larvae of Amphibalanus amphitrite
after a 48h incubation period (Table 1). Despite the concerns
for 11 and 16 provided subgram amounts for overall yields of
56% (7 steps) and 69% (3 steps), respectively. The synthesis of
16 was characterized using prolinol to prevent formation of
that synthetic
1 would possess much lower antifouling activity
diketopiperazine containing
L‐proline and N‐methyl‐D‐valine.
compared to its natural counterpart (as shown by Pettit), the
synthetic sample was found to be highly potent (EC50 <0.03
The product, , obtained from this synthesis also allowed
1
confirmation of its significant antifouling activity, yet low
toxicity. Detailed investigation of the structure‐activity relations

g/mL) and more effective than CuSO4 as a fouling inhibitor.
The EC50 values of 16 and 25 indicated moderate to weak
activity. These results demonstrate that all components of
of
1 and the preparation of molecular probes for elucidating a
1
mechanism of action are currently underway.
and/or the cyclic structure are essential for strong antifouling
activity. The 50% lethal concentrations toward the same larvae
(LC50 >10

g/mL) as well as MCF‐7 cells (LC50 >30

1
g/mL)32 were
is a novel
Acknowledgements
much greater than the EC50 value, and thus
This work was financially supported by the Sasagawa
Foundation and Grant‐in‐Aid for Young Scientists (B)
(15K16551) to TU. We thank Dr. L. T. Tan for the gift of natural
candidate for an environmentally friendly antifouling material.
In addition, the MCF‐7 cell results agreed with those in the
report by Pettit (GI50 >10 
g/mL).17 The LC50 value of 16 on MCF‐
1.
7 cells (LC50 > 100 µg/mL) was much greater than the value
produced by 25 (LC50 between 10 and 30 µg/mL) containing
unusual amino acids.
Experimental
General Methods. Tetrahydrofuran (THF), methanol (CH3OH),
and acetonitrile (CH3CN) were purchased from Kanto Chemical
Co. Inc. Dichloromethane (CH2Cl2) and triethylamine (Et3N)
were distilled from CaH2. All commercially obtained reagents
were used as received.
Conclusions
In summary, the total synthesis of dolastatin 16 (1) was
achieved using a convergent process. The synthesis involved
scalable and concise preparation of the southern and northern
segments 11 and 16, and efficient assembly of the two
Analytical TLC was carried out using pre‐coated silica gel
plates (Merck TLC silica gel 60F254). Wakogel 60N 63‐212 m
segments to construct the macrocyclic skeleton of
1 after
was used for column chromatography. IR spectra were recorded
on a JASCO FTIR‐4100 Type A spectrometer using a NaCl cell. 1H
and 13C NMR spectra were recorded using a JNM‐EX 400 (400
MHz and 100 MHz) spectrometer. Chemical shifts are reported
considering unsuccessful results. The synthetic sequences
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1
in ppm relative to CHCl3 (δ = 7.26) in CDCl3 for H NMR, and 2.02‐2.08 (2H, m), 2.09‐2.18 (1H, m), 2.39 (1H, ddV,ieJw=A6rti.c8le,O1n3lin.4e
DOI: 10.1039/C6OB02657E
CDCl3 (δ = 77.0) for 13C NMR. Splitting patterns are designated Hz), 2.74 (1H, dd, J = 7.3, 13.4 Hz), 3.13‐3.19 (1H, m), 3.28‐3.36
as s, d, t, q, and m, indicating singlet, doublet, triplet, quartet, (1H, m), 4.38‐4.44 (1H, m), 4.52 (1H, dd, J = 4.4, 8.3 Hz), 5.31
and multiplet, respectively.
TFA∙H‐Dml‐OBn (13). To a solution of N‐Boc‐dolamethylleuine δ 14.3, 24.9, 27.9, 28.3, 38.4, 39.9, 46.9, 54.0, 59.3, 79.8, 126.2,
) (Boc‐Dml‐OH) (116 mg, 0.473 mmol) in THF (2.4 mL) were 128.3, 129.4, 140.1, 155.9, 172.7, 174.0; HRMS (ESI) m/z: [M +
added BnOH (53.9
L, 0.520 mmol), PPh3 (186 mg, 0.710 mmol), Na]+ Calcd for C21H30O5N2Na 413.2047; Found 413.2047.
and DIAD (0.373 mL, 0.710 mmol) at 0 C under Ar atmosphere. Boc‐Dpv‐Pro‐Dml‐OBn (11). To a solution of crude TFA∙H‐Dml‐
(1H, d, J = 9.3 Hz), 7.15‐7.28 (5H, m); 13C NMR (CDCl3, 100 MHz)
(
4


The mixture was stirred at room temperature for 16 h, OBn (13) and 14 (253 mg, 0.648 mmol) in CH3CN (3.2 mL) were
quenched with saturated NaHCO3, extracted with EtOAc, added Et3N (0.542 mL, 3.89 mmol) and DMTMM (179 mg, 0.648
washed with brine, dried over Na2SO4, and concentrated in mmol) under Ar atmosphere. After 16 h of stirring at room
vacuo. The crude product was purified using column temperature, the mixture was concentrated in vacuo. The
chromatography (5% EtOAc in hexane) to afford Boc‐Dml‐OBn residue was purified using column chromatography (10% EtOAc
as a colorless oil (120 mg, 0.358 mmol, 76%): [α]23D = +15.4 (c in hexane) to afford 11 as a transparent solid (372 mg, 0.612
0.23, CHCl3); IR (neat) 3750, 2974, 2876, 2360, 2341, 1716, 1507, mmol, 94%): [α]23D = +17.6 (c 2.05, CHCl3); IR (neat) 3734, 3417,
1
1166, 772, 669 cm–1; H NMR (CDCl3, 400 MHz) δ 0.86‐0.90 (6H, 3311, 2973, 2876, 2360, 2341, 1715, 1507, 1245, 1171, 752, 700
1
m), 1.20 (3H, d, J = 7.3 Hz), 1.40 (9H, s), 1.57‐1.64 (1H, m), 2.78‐ cm–1; H NMR (CDCl3, 400 MHz) δ 0.76‐0.86 (9H, m), 1.13 (3H,
2.85 (1H, m), 3.35‐3.41 (1H, m), 5.05‐5.12 (2H, m), 5.23 (1H, d, J d, J = 7.3 Hz), 1.37‐1.45 (10H, m), 1.78‐1.90 (2H, m), 1.92‐2.10
= 10.8 Hz), 7.31‐7.37 (5H, m); 13C NMR (CDCl3, 100 MHz) δ 15.7, (2H, m), 2.14‐2.26 (1H, m), 2.40 (1H, dd, J = 6.8, 13.4 Hz), 2.73
19.2, 19.9, 28.4, 31.8, 40.5, 58.6, 66.3, 78.8, 128.1, 128.3, 128.6, (1H, dd, J = 7.3, 13.2 Hz), 2.77‐2.85 (1H, m), 3.18‐3.30 (2H, m),
135.7, 156.4, 175.6; HRMS (ESI) m/z: [M + Na]+ Calcd for 3.64 (1H, dt, J = 3.4, 9.8 Hz), 4.45‐4.50 (2H, m), 4.90‐5.00 (2H,
C19H29NO4Na 358.1989; Found 358.1992.
m), 5.31 (1H, d, J = 9.2 Hz), 6.79 (1H, d, J = 10.2 Hz), 7.08‐7.15
To Boc‐Dml‐OBn (255 mg, 0.760 mmol) was added (1H, m), 7.18‐7.34 (9H, m); 13C NMR (CDCl3, 100 MHz) δ 14.0,
TFA/CH2Cl2 (1:4 v/v, 25 mL). After 1 h of stirring at room 15.9, 19.5, 19.8, 24.9, 28.3, 29.1, 31.9, 38.3, 39.7, 40.5, 46.7,
temperature, the solution was concentrated in vacuo to afford 53.9, 57.0, 60.6, 66.3, 79.5, 126.1, 128.0, 128.2, 128.3, 128.6,
crude 13, which was used in the next step without further 129.5, 135.6, 140.3, 155.9, 171.5, 171.9, 175.9; HRMS (ESI) m/z:
purification.
[M + Na]+ Calcd for C35H49N3O6Na 630.3514; Found 630.3509.
Boc‐ ‐MeVal‐Pro‐Dpv‐Pro‐Dml‐OBn (9). To Boc‐Dpv‐Pro‐Dml‐
OBn (11) (70.5 mg, 0.116 mmol) was added TFA/CH2Cl2 (1:4 v/v,
Boc‐Dpv‐Pro‐OH (14). To a solution of N‐Boc‐dolaphenvaline (
(Boc‐Dpv‐OH) (208 mg, 0.709 mmol) and HCl∙H‐Pro‐OBn (12
5
)
)
D
(257 mg, 1.06 mmol) in CH3CN (3.5 mL) was added DMTMM 3.9 mL). After 1 h of stirring at room temperature, the solution
(294 mg, 1.06 mmol) under Ar atmosphere. After 16 h of stirring was concentrated in vacuo to afford crude TFA∙H‐Dpv‐Pro‐Dml‐
at room temperature, the mixture was concentrated in vacuo. OBn, which was used in the next step without further
The residue was purified using column chromatography (10% purification.
EtOAc in hexane) to afford Boc‐Dpv‐Pro‐OBn as a transparent
To a solution of the crude TFA salt and 10 (38.1 mg, 0.116
solid (259 mg, 0.539 mmol, 76%): [α]23D = –31.8 (c 2.21, CHCl3); mmol) in CH3CN (1.5 mL) were added DMTMM (32.1 mg, 0.116
IR (neat) 3734, 3308, 2976, 2360, 2341, 1746, 1709, 1647, 1497, mmol) under Ar atmosphere. After 16 h of stirring at room
1433, 1169, 752, 700 cm–1; 1H NMR (CDCl3, 400 MHz) δ 0.79 (3H, temperature, the mixture was concentrated in vacuo. The
d, J = 6.8 Hz), 1.37 (9H, s), 1.77‐1.86 (3H, m), 1.96‐2.02 (1H, m), residue was purified using column chromatography (20%
2.04‐2.14 (1H, m), 2.34 (1H, dd, J = 6.8, 13.4 Hz), 2.72 (1H, dd, J acetone in hexane) to afford 9 as a colorless oil (84.3 mg, 0.103
= 6.8, 13.4 Hz), 3.10‐3.20 (1H, m), 3.24‐3.31 (1H, m), 3.34‐4.40 mmol, 89% for 2 steps): [α]23D = +13.8 (c 0.43, CHCl3); IR (neat)
(1H, m), 4.46‐4.50 (1H, dd, J = 6.3, 8.8 Hz), 5.06 (2H, s), 5.22 (1H, 3317, 2967, 2875, 1685, 1649, 1518, 1454, 1152, 754, 701 cm–
1
d, J = 9.3 Hz), 7.10‐7.28 (10H, m); 13C NMR (CDCl3, 100 MHz) δ
;
1H NMR (CDCl3, 400 MHz, mixture of rotamers) δ 0.70‐0.90
14.1, 24.9, 28.3, 28.9, 38.4, 40.0, 46.4, 54.0, 58.8, 66.8, 79.5, (15H, m), 1.15 (3H, d, J = 7.3 Hz), 1.30‐1.50 (10H, m), 1.79‐2.43
126.0, 128.1, 128.2, 128.3, 128.49, 128.50, 128.54, 129.4, 135.5, (12H, m), 2.58‐2.80 (4H, m), 3.10‐3.29 (2H, m), 3.60‐3.77 (4H,
140.4, 155.9, 170.9, 171.8; HRMS (ESI) m/z: [M + Na]+ Calcd for m), 4.26‐4.80 (3H, m), 4.90‐5.00 (2H, m), 6.80 (1H, d, J = 10.2
C28H36N2O5Na 503.2516; Found 503.2515.
Hz), 6.92‐7.30 (10H, m); 13C NMR (CDCl3, 100 MHz, mixture of
To a solution of Boc‐Dpv‐Pro‐OBn (259 mg, 0.539 mmol) in rotamers) δ 14.3, 15.86, 15.93, 17.86, 17.92, 18.3, 19.5, 19.80,
CH3OH (2.7 mL) was carefully added 20% Pd(OH)2/C (25.9 mg, 19.82, 20.1, 24.8, 25.1, 26.8, 27.0, 28.1, 28.2, 28.3, 28.35, 28.4,
10 wt%) under Ar atmosphere. The solution was purged with H2 29.1, 29.3, 29.5, 31.8, 31.9, 38.5, 39.6, 39.7, 40.4, 40.6, 46.6,
gas and stirring was continued under H2 at room temperature 46.7, 47.4, 52.4, 52.5, 57.0, 59.3, 60.1, 60.2, 60.6, 60.7, 61.2,
for 16 h. The solution was filtered through celite and 61.6, 62.6, 66.3, 76.6, 79.8, 80.1, 80.2, 126.03, 126.09, 128.0,
concentrated in vacuo. The crude product was purified using 128.07, 128.08, 128.16, 128.22, 128.3, 128.56, 128.58, 129.50,
column chromatography (30% EtOAc in hexane) to afford 14 as 129.53, 129.6, 135.5, 135.6, 140.26, 140.33, 155.2, 156.3, 156.8,
a transparent solid (205 mg, 0.525 mmol, 97%): [α]23D =–40.5 (c 169.0, 169.7, 170.2, 170.5, 171.1, 171.2, 171.3, 171.47, 171.50,
1.10, CHCl3); IR (neat) 3734, 3302, 2977, 2360, 2341, 1715, 1647, 171.54, 172.5, 175.87, 175.92; HRMS (ESI) m/z: [M + Na]+ Calcd
1615, 1507, 1455, 1168, 754, 702 cm–1; 1H NMR (CDCl3, 400 for C46H67N5O8Na 840.4882; Found 840.4883.
MHz) δ 0.89 (3H, d, J = 6.8 Hz), 1.40 (9H, s), 1.83‐1.91 (2H, m),
6 | Org. Biomol. Chem., 2016, 00, 1‐3
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Organic & Biomolecular Chemistry
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Organic & Biomolecular Chemistry
PAPER
Boc‐Pro‐O‐Hiv‐
mg, 17.0
mol) was added TFA/CH2Cl2 (1:4 v/v, 0.60 mL). After 175.9; HRMS (ESI) m/z: [M + Na]+ CalDcdOI:f1o0r.10C3691/HC864ONB60O21615N7aE
1 h of stirring at room temperature, the solution was 1099.6090; Found 1099.6082.
concentrated in vacuo to afford crude TFA∙H‐ ‐MeVal‐Pro‐Dpv‐ Boc‐Pro‐O‐Hiv‐ ‐MeVal‐Pro‐OBn (18) and cyclo(‐
Pro‐Dml‐OBn, which was used in the next step without further Pro‐) (19). To a solution of crude 17 and Boc‐Pro‐O‐Hiv‐OH (
purification.
(41.0 mg, 0.130 mmol) in CH3CN (0.65 mL) were added iPr2NEt
To a solution of the crude TFA salt and Boc‐Pro‐O‐Hiv‐OH ( (136 L, 0.780 mmol) and PyBroP (60.6 mg, 0.130 mmol) under
(5.36 mg, 17.0 mol) in CH3CN (1.7 mL) was added DMTMM Ar atmosphere. After 16 h of stirring at room temperature, the
(9.41 mg, 34.0 mol) under Ar atmosphere. After 48 h of stirring mixture was concentrated in vacuo. The residue was purified
D‐MeVal‐Pro‐Dpv‐Pro‐Dml‐OBn (15). To 9 (13.9 169.9, 171.0, 171.1, 171.2, 171.3, 171.5, 171.6, 171.7, 172.5,
View Article Online

D
D
D‐MeVal‐
8)
8)



at room temperature, the mixture was concentrated in vacuo. using column chromatography (20% acetone in hexane) to
The residue was purified using column chromatography (20% afford 18 (22.4 mg, 0.036 mmol, 28% for 2 steps) and 19 (12.4
acetone in hexane) to afford 15 as a colorless oil (7.82 mg, 7.70 mg, 0.059 mmol, 45% for 2 steps).

mol, 45% for 2 steps): [α]23D = +15.2 (c 0.52, CHCl3); IR (neat) Amide 18. Colorless oil; [α]23D = +6.5 (c 0.62, CHCl3); IR (neat)
3800, 2969, 2876, 2318, 1746, 1684, 1647, 1541, 1508, 1456, 2971, 2876, 1744, 1700, 1649, 1397, 1254, 1169, 1088, 999, 752,
1396, 1171, 1088, 1011, 754, 701 cm–1; 1H NMR (CDCl3, 400 699 cm–1; 1H NMR (CDCl3, 400 MHz, mixture of rotamers) δ
MHz, mixture of rotamers) δ 0.73‐1.20 (24H, m), 1.37‐1.44 (10H, 0.65‐1.00 (12H, m), 1.31‐1.40 (9H, m), 1.70‐2.30 (10H, m), 2.61
m), 1.74‐2.60 (16H, m), 2.70‐2.86 (2H, m), 2.99 (3H, s), 3.31‐3.65 (0.82H, s), 2.67 (1.50H, s), 2.85 (0.24H, s), 2.88 (0.44H, s), 3.24‐
(8H, m), 4.28‐4.32 (0.4H, m), 4.39‐4.48 (2.6H, m), 4.74‐5.12 (4H, 3.51 (4H, m), 4.20‐4.71 (3H, m), 4.83‐5.14 (3H, m), 7.18‐7.31 (5H,
m), 6.76‐6.82 (1.6H, m), 6.89 (0.4H, d, J = 8.3 Hz), 7.12‐7.40 (10H, m); 13C NMR (CDCl3, 100 MHz, mixture of rotamers) δ 15.9, 16.0,
m); 13C NMR (CDCl3, 100 MHz, mixture of rotamers) 15.9, 16.06, 16.2, 17.9, 19.4, 19.7, 19.9, 20.1, 22.1, 23.1, 23.2, 23.8, 24.0,
16.11, 16.2, 18.0, 18.1, 19.49, 19.53, 19.58, 19.7, 19.80, 19.82, 24.9, 25.0, 26.2, 26.4, 28.4, 28.5, 28.7, 28.9, 28.97, 29.02, 29.6,
24.8, 24.9, 25.0, 26.4, 26.5, 28.3, 28.35, 28.43, 28.5, 28.88, 29.8, 29.9, 30.4, 30.5, 30.6, 30.7, 46.1, 46.2, 46.3, 46.4, 46.5,
28.93, 29.2, 30.2, 31.9, 38.5, 39.7, 40.3, 46.7, 46.9, 47.3, 52.8, 46.7, 58.3, 58.5, 58.7, 58.7, 59.1, 59.2, 59.8, 60.0, 60.1, 66.8,
57.0, 58.5, 60.0, 60.7, 60.8, 66.26, 66.31, 75.2, 76.6, 77.2, 79.69, 67.1, 75.0, 75.5, 76.7, 77.0, 77.3, 79.6, 79.8, 128.08, 128.14,
79.73, 126.0, 126.1, 127.9, 127.98, 128.0, 128.16, 128.17, 128.3, 128.4, 128.5, 135.57, 135.59, 135.6, 135.7, 153.9, 167.76,
128.25, 128.34, 128.6, 129.5, 129.6, 135.5, 140.2, 140.5, 153.8, 167.83, 168.1, 168.9, 169.2, 169.5, 171.8, 172.2, 172.4, 172.7,
168.6, 169.2, 169.5, 171.1, 171.5, 171.57, 171.62, 172.4, 172.8, 173.4; HRMS (ESI) m/z: [M + Na]+ Calcd for C33H49N3O8Na
173.1, 175.9 δ ; HRMS (ESI) m/z: [M + Na]+ Calcd for 638.3412; Found 638.3409.
C56H82N6O11Na 1037.5934; Found 1037.5926.
BnO‐Lac‐Pro‐O‐Hiv‐
‐MeVal‐Pro‐Dpv‐Pro‐Dml‐OBn (6). To 15 IR (neat) 3734, 3479, 2965, 1651, 1456, 1403, 1296, 669 cm–1;
(8.2 mg, 8.10
mol) was added TFA/CH2Cl2 (1:4 v/v, 0.30 mL). 1H NMR (CDCl3, 400 MHz) δ 1.02 (3H, d, J = 6.8 Hz), 1.09 (3H, d,
After 1 h of stirring at room temperature, the solution was J = 6.8 Hz), 1.80‐2.00 (3H, m), 2.15‐2.22 (1H, m), 2.38‐2.44 (1H,
concentrated in vacuo to afford crude TFA∙H‐Pro‐O‐Hiv‐ m), 3.00 (3H, s), 3.45‐3.52 (2H, m), 3.59‐3.66 (2H, m), 4.06‐4.12
Diketopiperazine 19. Colorless oil; [α]23D =+3.90 (c 0.19, CHCl3);
D

D
‐
MeVal‐Pro‐Dpv‐Pro‐Dml‐OBn, which was used in the next step (1H, m); 13C NMR (CDCl3, 100 MHz) δ 18.8, 19.7, 22.5, 29.7, 32.1,
without further purification.
34.6, 45.7, 58.9, 71.2, 165.0, 167.7; HRMS (ESI) m/z: [M + Na]+
7) (1.6 Calcd for C11H18N2O2Na 233.1261; Found 233.1262.
To a solution of the crude TFA salt and BnO‐Lac‐OH (
mg, 8.90

mol) in CH2Cl2 (0.20 mL) were added Et3N (1.13

L, Boc‐
D
‐MeVal‐Pro‐CH2OH (22). To a solution of Boc‐
D‐MeVal‐OH
8.10 mol) and DECP (1.23


L, 8.10 mol) under Ar atmosphere.

(
20) (608 mg, 2.63 mmol) and L‐prolinol (21) (H‐Pro‐CH2OH)
After 36 h of stirring at room temperature, the mixture was (266 mg, 2.63 mmol) in THF (13 mL) were added NaHCO3 (221
concentrated in vacuo. The residue was purified using column mg, 2.63 mmol), HOAt (358 mg, 2.63 mmol), and EDCI (504 mg,
chromatography (20% acetone in hexane) to afford
6 (4.7 mg, 2.63 mmol) under Ar atmosphere. After 16 h of stirring at room
4.40

mol, 54% for 2 steps): [α]23D = +10.0 (c 1.74, CHCl3); IR temperature, the mixture was concentrated in vacuo. The
(neat) 3734, 3413, 3309, 2969, 2876, 1735, 1646, 1508, 1428, residue was purified using column chromatography (10%
1183, 1101, 752 cm–1; 1H NMR (CDCl3, 400 MHz, mixture of acetone in hexane) to afford 22 as a colorless oil (773 mg, 2.46
rotamers) δ 0.69‐1.10 (24H, m), 1.30‐1.45 (4H, m), 1.60‐2.85 mmol, 94%): [α]23D = +78.2 (c 1.28, CHCl3); IR (neat) 3734, 3445,
(20H, m), 2.94 (3H, s), 3.10‐3.70 (5H, m), 4.05‐5.10 (11H, m), 2966, 2874, 2360, 2341, 1626, 1541, 1472, 1391, 1257, 1150,
6.74 (1.5 H, d, J = 10.2 Hz), 6.80 (0.5 H, d, J = 9.2 Hz), 7.00‐7.20 1051, 930, 882, 769, 669 cm–1; 1H NMR (CDCl3, 400 MHz,
(1H, m), 7.24‐7.29 (14H, m); 13C NMR (CDCl3, 100 MHz, mixture mixture of rotamers) δ 0.83 (3H, d, J = 6.8 Hz), 0.89 (3H, d, J =
of rotamers) δ 13.8, 14.3, 15.89, 15.92, 16.2, 16.7, 17.1, 17.3, 6.4 Hz), 1.43‐1.47 (9H, m), 1.50‐1.60 (1H, m), 1.76‐1.93 (2H, m),
18.0, 18.1, 19.4, 19.5, 19.7, 19.77, 19.82, 20.2. 22.4, 24.8, 24.9, 1.98‐2.07 (1H, m), 2.20‐2.35 (1H, m), 2.75 (3H, s), 3.38‐3.56 (2H,
24.99, 25.01, 25.7, 26.5, 28.1, 28.3, 28.7, 29.0, 29.1, 29.2, 29.9, m), 3.57‐3.70 (2H, m), 4.20‐4.29 (1.3H, m), 4.53 (0.7 H, d, J =
30.2, 31.6, 31.86, 31.93, 38.4, 38.6, 39.6, 39.7, 40.4, 40.6, 46.5, 10.7 Hz), 4.79 (0.3H, d, J = 7.8 Hz), 4.96 (0.7 H, d, J = 7.3 Hz); 13
C
46.7, 46.8, 46.9, 47.2, 52.5, 53.2, 56.97, 56.99, 58.7, 59.1, 59.2, NMR (CDCl3, 100 MHz, mixture of rotamers) δ 18.0, 18.3, 19.7,
60.1, 60.3, 60.67, 60.70, 66.2, 66.3, 70.9, 71.2, 75.0, 75.3, 75.6, 20.1, 24.4, 24.5, 26.8, 26.9, 28.2, 28.3, 28.4, 28.9, 29.4, 47.5,
75.8, 126.00, 126.04, 127.66, 127.71, 127.9, 127.95, 127.99, 47.9, 58.1, 61.2, 61.5, 61.6, 63.1, 67.5, 67.7, 80.0, 80.3, 155.3,
128.17, 128.22, 128.3, 128.4, 128.58, 128.59, 129.5, 129.6, 156.3, 171.2, 172.0; HRMS (ESI) m/z: [M + Na]+ Calcd for C16H30
135.5, 135.6, 137.76, 137.81, 140.4, 140.5, 167.7, 168.8, 169.4, N2O4Na 337.2098; Found 337.2101.
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Page 8 of 10
PAPER
Organic & Biomolecular Chemistry
Boc‐Pro‐O‐Hiv‐
D
‐MeVal‐Pro‐CH2OH (23). To 22 (50.6 mg, 0.161 under Ar atmosphere. The mixture was stirred for 16 h at 0 °C,
View Article Online
DOI: 10.1039/C6OB02657E
mmol) was added TFA/CH2Cl2 (1:4 v/v, 5.4 mL). After 1 h of quenched with copious amount of saturated Na2S2O3 solution,
stirring at room temperature, the solution was concentrated in then saturated NaHCO3, extracted with Et2O, washed with brine,
vacuo to afford crude TFA∙H‐D‐MeVal‐Pro‐CH2OH, which was dried over Na2SO4, and concentrated in vacuo. The crude
used in the next step without further purification.
aldehyde was used in the next step without further purification.
To a solution of the crude TFA salt and Boc‐Pro‐O‐Hiv‐OH (
8)
To a solution of the crude aldehyde in tBuOH/H2O (3:1 v/v,
i
(50.8 mg, 0.161 mmol) in CH3CN (1.0 mL) were added Pr2NEt 14 mL) at 0 °C were added 2‐methyl‐2‐butene (3.5 mL, 33.0
(0.280 mL, 1.61 mmol) and PyBroP (113 mg, 0.242 mmol) under mmol), NaH2PO4 (266 mg, 2.22 mmol), and NaClO2 (301 mg,
Ar atmosphere. After 16 h of stirring at room temperature, the 3.33 mmol). The mixture was stirred for 16 h at 0 °C, quenched
mixture was concentrated in vacuo. The residue was purified with saturated NH4Cl, extracted with EtOAc, washed with brine,
using column chromatography (20% acetone in hexane) to dried over Na2SO4, and concentrated in vacuo. The crude
afford 23 as a colorless foam (63.0 mg, 0.123 mmol, 76% for 2 product was purified using column chromatography (20%
steps): [α]23D = +32.8 (c 4.20, CHCl3); IR (neat) 3446, 2971, 2877, acetone in hexane) to afford 16 as a colorless foam (624 mg,
2360, 2341, 1747, 1699, 1637, 1399, 1366, 1167, 1121, 1088, 1.06 mmol, 95% for 2 steps): [α]23D = +12.2 (c 1.25, CHCl3); IR
1011, 754, 666 cm–1; 1H NMR (CDCl3, 400 MHz, mixture of (neat) 3734, 2970, 2877, 2360, 2341, 1743, 1647, 1456, 1187,
rotamers) δ 0.76‐1.05 (12H, m), 1.35‐1.41 (9H, m), 1.65‐2.40 1114, 1013, 751 cm–1; 1H NMR (CDCl3, 400 MHz, mixture of
(10H, m), 2.99 (3H, s), 3.22‐3.80 (6H, m), 4.10‐4.25 (1H, m), 4.26‐ rotamers) δ 0.68‐1.01 (12H, m), 1.33‐1.42 (3H, m), 1.70‐2.32
4.40 (1H, m), 4.92‐5.00 (2H, m); 13C NMR (CDCl3, 100 MHz, (10H, m), 2.83 (0.1H, s), 2.92 (1.9H, s), 2.97 (1.0H, s), 3.30‐3.72
mixture of rotamers) δ 16.2, 16.7, 16.9, 17.9, 18.1, 18.2, 19.1, (4H, m), 4.10‐4.35 (2H, m), 4.40‐4.75 (3H, m), 4.90‐5.10 (2H, m),
19.3, 19.6, 19.65, 19.72, 21.6, 23.2 24.0, 24.46, 24.50, 26.3, 26.5, 7.19‐7.31 (5H, m); 13C NMR (CDCl3, 100 MHz, mixture of
27.88, 27.89, 28.1, 28.2, 28.3, 28.39, 28.42, 28.7, 29.1, 29.4, rotamers) δ 16.6, 17.1, 17.4, 17.8, 18.1, 18.2, 18.8, 19.4, 19.5,
29.5, 29.95, 29.99, 30.04, 30.07, 30.4, 46.0, 46.2, 46.4, 47.8, 19.8, 22.1, 24.9, 25.1, 25.7, 26.5, 26.6, 27.8, 28.07, 28.13, 29.3,
47.9, 57.8, 58.2, 58.37, 58.42, 59.8, 60.0, 60.1, 60.5, 60.7, 65.1, 29.6, 29.9, 30.4, 31.4, 46.6, 46.8, 47.0, 47.4, 58.8, 60.0, 60.1,
65.3, 66.0, 66.5, 75.3, 75.5, 79.7, 79.8, 153.7, 154.4, 167.8, 60.5, 70.9, 71.5, 75.0, 75.1, 75.35, 75.41, 127.7, 127.8, 127.9,
169.5, 169.6, 169.8, 169.9, 173.1, 173.4; HRMS (ESI) m/z: [M + 128.38, 128.40, 137.5, 137.7, 167.8, 169.1, 169.9, 171.5, 171.9,
Na]+ Calcd for C26H45N3O7Na 534.3150; Found 534.3144.
BnO‐Lac‐Pro‐O‐Hiv‐ ‐MeVal‐Pro‐CH2OH (24). To Boc‐Pro‐O‐ 586.3134; Found 586.3136.
Hiv‐ ‐MeVal‐Pro‐CH2OH (23) (696 mg, 1.36 mmol) was added BnO‐Lac‐Pro‐O‐Hiv‐ ‐MeVal‐Pro‐Dpv‐Pro‐Dml‐OBn (6). To 11
173.6, 173.7; HRMS (ESI) m/z: [M – H]– Calcd for C31H44N3O8
D
D
D
TFA/CH2Cl2 (1:4 v/v, 45 mL). After 1 h of stirring at room (90.0 mg, 0.148 mmol) was added TFA/CH2Cl2 (1:4 v/v, 5.0 mL).
temperature, the solution was concentrated in vacuo to afford After 1 h of stirring at room temperature, the solution was
crude TFA∙H‐Pro‐O‐Hiv‐D‐MeVal‐Pro‐CH2OH, which was used in concentrated in vacuo to afford crude TFA∙H‐Dpv‐Pro‐Dml‐OBn,
the next step without further purification.
To a solution of the crude TFA salt and
which was used in the next step without further purification.
To a solution of the crude TFA salt and BnO‐Lac‐Pro‐O‐Hiv‐
7
(245 mg, 1.36
mmol) in CH3CN (6.8 mL) were added Et3N (1.14 mL, 8.16 mmol)
D‐MeVal‐Pro‐OH (16) (87.0 mg, 0.148 mmol) in CH3CN (1.5 mL)
and DMTMM (376 mg, 1.36 mmol) under Ar atmosphere. After were added Et3N (0.124 mL, 0.888 mmol) and DMTMM (41.0
16 h of stirring at room temperature, the mixture was mg, 0.148 mmol) under Ar atmosphere. After 16 h of stirring at
concentrated in vacuo. The residue was purified using column room temperature, the solution was concentrated in vacuo. The
chromatography (20% acetone in hexane) to afford 24 as a residue was purified using column chromatography (40%
colorless foam (635 mg, 1.11 mmol, 82% for 2 steps): [α]23D = – acetone in hexane) to afford
2.60 (c 1.05, CHCl3); IR (neat) 3734, 3446, 2966, 2875, 2360, 0.136 mmol, 92% for 2 steps).
2341, 1744, 1636, 1456, 1188, 1112, 1013, 750, 699 cm–1; 1H Dolastatin 16 (1). To a solution of
6
as a transparent solid (146 mg,
6
(13.0 mg, 12.0 mol) in

NMR (CDCl3, 400 MHz, mixture of rotamers) δ 0.77‐1.09 (12H, CH3OH (1.5 mL) was carefully added 20% Pd(OH)2/C (2.60 mg,
m), 1.38‐1.43 (3H, m), 1.61‐2.50 (10H, m), 2.88 (0.3H, s), 2.95 20 wt%) under Ar atmosphere at room temperature. The
(0.1 H, s), 3.02 (2.6 H, s), 3.34‐3.80 (6H, m), 4.03‐4.16 (1H, m), reaction mixture was stirred under H2 atmosphere (3 atm) for
4.19 (1H, q, J = 6.3 Hz), 4.30‐4.69 (3H, m), 4.94‐5.09 (2H, m), 16 h. The solution was filtered through celite and concentrated
7.24‐7.32 (5H, m); 13C NMR (CDCl3, 100 MHz, mixture of in vacuo to afford the crude seco acid, which was used in the
rotamers) δ 16.4, 17.1, 17.26, 17.30, 18.0, 18.1, 18.3, 19.0, 19.2, next step without further purification.
19.6, 19.7, 19.8, 21.4, 24.5, 24.7, 25.16, 25.24, 26.4, 26.5, 27.9,
28.0, 28.1, 29.0, 29.5, 30.0, 30.2, 31.4, 46.2, 46.6, 47.8, 48.0, mg, 12.0
58.3, 58.8, 59.1, 59.9, 60.0, 60.2, 60.8, 65.1, 65.6, 66.8, 69.7, crude seco acid and Et3N (1.7
To a solution of MNBA (20.7 mg, 60.0
mol) in toluene (6.4 mL) was added a solution of the
L, 12.0 mol) in toluene (1.3 mL)
mol) and DMAP (14.7



71.0, 74.5, 75.0, 75.3, 75.9, 77.6, 127.4, 127.7, 127.8, 128.2, for a period of 4 hours and 20 minutes under Ar atmosphere.
128.4, 128.9, 137.7, 137.8, 138.1, 167.8, 169.5, 169.8, 170.0, The solution was stirred for 16 h at room temperature,
171.1, 171.4, 171.5, 172.2, 172.7; HRMS (ESI) m/z: [M + Na]+ concentrated in vacuo, extracted with EtOAc, washed
Calcd for C31H47N3O7Na 596.3306; Found 596.3301.
BnO‐Lac‐Pro‐O‐Hiv‐ ‐MeVal‐Pro‐OH (16). To a solution of 24 over Na2SO4, and concentrated in vacuo. The crude product was
(635 mg, 1.11 mmol) in CH2Cl2 (22 mL) at 0 °C were added purified using column chromatography (20% acetone in hexane)
NaHCO3 (336 mg, 4.00 mmol) and DMP (1.22 g, 2.89 mmol) to afford dolastatin 16 ( ) as a transparent solid (3.30 mg, 3.70
sequentially with 1 N HCl, saturated NaHCO3 and brine, dried
D
1
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The test compounds were dissolved in ethanol and aliquots

mol, 31% for 2 steps): [α]23D = +11.8 (c 0.38, CH3OH); IR (neat)
View Article Online
3394, 3326, 2965, 2876, 2360, 2341, 1748, 1733, 1652, 1506, of the solution were transferred toDOwI:e1l0ls.10o39f/Ca6O2B042‐6w5e7Ell
1457, 1424, 1388, 1299, 1184, 1091, 1015, 752, 702 cm–1; 1H polystyrene culture plates and air‐dried. Four wells were used
NMR (CDCl3, 400 MHz) δ 0.78‐0.91 (15H, m), 0.99‐1.06 (9H, m), for each concentration. To each well were added filtered
1.43 (3H, d, J = 6.8 Hz), 1.45‐1.60 (2H, m), 1.65‐2.44 (15H, m), seawater (2.0 mL, salinity 28) and six 2‐day‐old cyprids. The
2.45‐2.55 (2H, m), 2.78‐2.90 (2H, m), 3.08 (3H, s), 3.35‐3.50 (2H, plates were kept in the dark at 25 °C for 48 h. The numbers of
m), 3.60‐3.70 (2H, m), 3.85‐3.92 (1H, m), 4.44 (1H, d, J = 6.8 Hz), cyprids that attached, metamorphosed, died, or did not settle
4.54 (1H, d, J = 7.8 Hz), 4.60‐4.64 (1H, m), 4.94 (1H, d, J = 8.8 Hz), were counted under a microscope. Three or four trials were
5.12‐5.20 (2H, m), 5.41 (1H, d, J = 2.9 Hz), 6.72 (1H, d, J = 8.8 Hz), done for each concentration. Probit analysis was used to
7.12‐7.19 (1H, m), 7.20‐7.30 (2H, m), 7.34 (2H, d, J = 7.3 Hz) 7.68 calculate the EC50 values.
(1H, d, J = 10.2 Hz); 13C NMR (CDCl3, 100 MHz) δ 15.0, 15.3. 16.2, Cytotoxicity Assay.33 The cytotoxicity of dolastatin 16 (
1) was
17.4, 17.9, 19.86, 19.88, 19.93, 20.5, 21.9, 24.9, 25.2, 25.6, 25.8, determined by a standard MTT assay. First, MCF‐7 breast cancer
28.4, 29.8, 30.9, 31.0, 32.5, 38.8, 41.0, 41.1, 46.1, 46.6, 47.7, cells (Culture Collections, Public Health England) were
50.7, 56.5, 58.0, 59.0, 59.6, 61.5, 66.8, 76.5, 126.3, 128.5, 129.7, maintained in RPMI‐1640 medium with 10% fetal bovine serum
140.7, 169.2, 169.5, 169.7, 171.1, 171.19, 171.21, 172.4, 174.8; (FBS). Afterwards, the cells were seeded in wells of a 96‐well
HRMS (ESI) m/z: [M + Na]+ Calcd for C47H70N6O10Na 901.5046; plate at a density of 1.0 x 104 cells per well. After 24 h at 37 °C
Found 901.5042. Spectroscopic data were identical with natural with 5% CO2, the cells were incubated with increasing
dolastatin 16 (
H‐Dpv‐Pro‐Dml‐OBn (25). To a solution of Boc‐Dpv‐Pro‐Dml‐ was removed after 72 h and replaced with a 100 μL solution of
OBn (11) (23.1 mg, 38.0 mol) was added TFA/CH2Cl2 (1:4 v/v, MTT in RPMI‐1640 with 10% FBS (0.5 mg/mL) and incubated for
1
) ([α]23D = +15.5 (c 0.20, CH3OH)).13
concentrations of 1 under the same conditions. The medium

1.3 mL). After 1 h of stirring at room temperature, the solution 3 h at 37 °C with 5% CO2. The MTT solution was aspirated and
was concentrated in vacuo to afford crude TFA∙H‐Dpv‐Pro‐Dml‐ replaced by 100 μL of DMSO. After 15 min of incubation, the
OBn, which was used in the next step without further optical density was measured at 570 nm using a microplate
purification.
To a solution of the crude TFA salt in CH2Cl2 (0.190 mL) was cytotoxicity relative to untreated cells. Each sample
added 4 M NaOH (0.190 mL, 0.760 mmol). After 1 h of stirring concentration of and negative control (1% EtOH in medium)
reader. The results are expressed as the mean percentage of
1
at room temperature, the solution was quenched with H2O (5.0 was tested four times, except for the positive control (cisplatin),
mL), extracted with CH2Cl2, dried over Na2SO4, and which was tested twice.
concentrated in vacuo. The crude product was purified using
column chromatography (30% EtOAc in hexane) to afford 25 as
a colorless oil (14.2 mg, 28.0 
mol, 74%): [α]23D = –74.5 (c 1.00,
Notes and references
CHCl3); IR (neat) 3410, 2964, 2876, 1717, 1653, 1508, 1362,
1173, 753, 701 cm–1; 1H NMR (CDCl3, 400 MHz) δ 0.75‐0.88 (9H,
m), 1.16 (3H, d, J = 7.3 Hz), 1.45‐1.51 (1H, m), 1.70‐1.78 (1H, m),
1.80‐1.88 (2H, m), 1.89‐2.08 (1H, m), 2.62 (1H, dd, J = 6.3, 13.1
Hz), 2.10‐2.18 (1H, m), 2.76 (1H, dd, J = 8.3, 13.4 Hz), 2.83‐2.86
(1H, m), 3.03 (1H, dd, J = 7.8, 16.8 Hz), 3.20‐3.26 (1H, m), 3.36‐
3.48 (1H, m), 3.68 (1H, dt, J = 3.4, 9.8 Hz), 4.53 (1H, dd, J = 3.4,
8.3 Hz), 5.00 (1H, d, J = 12.2 Hz), 5.04 (1H, d, J = 12.2 Hz), 6.84
(1H, d, J = 10.2 Hz), 7.10‐7.19 (1H, m), 7.20‐7.40 (9H, m); 13C
NMR (CDCl3, 100 MHz) δ 12.9, 13.2, 13.4, 14.0, 15.8, 19.5, 19.7,
19.8, 22.0, 24.9, 25.1, 29.0, 31.7, 31.8, 38.1, 39.7, 40.3, 40.6,
46.3, 52.5, 53.9, 57.0, 57.2, 60.7, 66.2, 66.5, 126.0, 127.9,
128.17, 128.24, 128.3, 128.40, 128.44, 128.5, 128.9, 129.26,
129.29, 135.3, 135.6, 140.6, 171.7, 175.1, 175.8; HRMS (ESI)
m/z: [M + Na]+ Calcd for C30H42N3O4 508.3170; Found 508.3166.
Antifouling Assay.12a,12d The adult barnacles, Amphibalanus
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as I‐II stage nauplii upon immersion in seawater after drying
overnight. The nauplii thus obtained were cultured in filtered
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cells/mL. Larvae reached the cyprid stage in 5 days. The cyprids
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