An enantioselective total synthesis of tubulysin V

Article abstract of DOI:10.1016/j.tet.2016.08.038

Tubulysin V has been enantioselectively synthesized from the units of dipeptide 23, Tuv and Tup. The features of this synthetic strategy is included three portions, the Tuv fragment 17 was diastereoselectively synthesized from the D-malic acid, the stereocenters of the Tup unit was constructed by the asymmetric reduction as well as methylation, and the epimerization for several known methods was successfully avoided by condensation of fragment 19 with 24, and by deprotection with hydrogenation.

Full text of DOI:10.1016/j.tet.2016.08.038

Accepted Manuscript
An enantioselective total synthesis of tubulysin V
Wei Tao, Wen Zhou, Zhu Zhou, Chang-Mei Si, Xun Sun, Bang-Guo Wei
PII:
S0040-4020(16)30814-6
10.1016/j.tet.2016.08.038
TET 28019
DOI:
Reference:
To appear in: Tetrahedron
Received Date: 17 July 2016
Revised Date: 9 August 2016
Accepted Date: 12 August 2016
Please cite this article as: Tao W, Zhou W, Zhou Z, Si C-M, Sun X, Wei B-G, An enantioselective total
synthesis of tubulysin V, Tetrahedron (2016), doi: 10.1016/j.tet.2016.08.038.
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An enantioselective total synthesis of
tubulysin V
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Wei Tao,a,b Wen Zhou,b Zhu Zhou,b Chang-Mei Si,b,* Xun Sunb and Bang-Guo Weia,b,*
aInstitutes of Biomedical Sciences, Fudan University, 130 Dongan Road, Shanghai 200032, China
bSchool of Pharmacy, Fudan University, 826 Zhangheng Road, Shanghai 201203, China
Final Condensation
Amidation
Ph
O
H
OH
O
N
N
Asymmetric Reduction
N
N
N
H
H
S
O
CO2H
Asymmetric Addition
Evans' Methylation
Tubulysin V 1e

ACCEPTED MANUSCRIPT
1
Tetrahedron
journal homepage: www.elsevier.com
An enantioselective total synthesis of tubulysin V
Wei Tao,a,b Wen Zhou,b Zhu Zhou,b Chang-Mei Si,b,* Xun Sun b and Bang-Guo Weia,b,*
aInstitutes of Biomedical Sciences, Fudan University, 130 Dongan Road, Shanghai 200032, China
bDepartment of Natural Products Chemistry, School of Pharmacy, Fudan University, 826 Zhangheng Road, Shanghai, 201203, China
ARTICLE INFO
ABSTRACT
Article history:
Received
Received in revised form
Accepted
Tubulysin V has been enantioselectively synthesized from the units of dipeptide 23, Tuv and
Tup. The features of this synthetic strategy is included three portions, the Tuv fragment 17 was
disasteroselectively synthesized from the D-malic acid, the stereocenters of the Tup unit was
constructed by the asymmetric reduction as well as methylation, and the epimerization for
several known methods was successfully avoided by condensation of fragment 19 with 24, and
by deprotection with hydrogenation.
Available online
keywords:
Natural product
Anti-cancer
Asymmetric synthesis
Tubulysin
2009 Elsevier Ltd. All rights reserved.
Malic acid
1. Introduction
and non-proteinogenic N-methyl picolinic acid (D-Mep). One of
the challenging for enantioselective synthesis of these natural
Tubulysins, an interesting family of peptidic secondary
metabolites of myxobacteria, was firstly isolated by Höfle and
co-workers in 2000.1 These natural products show exceptionally
potent cell growth inhibitors that act by disturbing the
microtubule construction and consequently inhibiting tubulin
polymerization with IC50 values ranging from 0.01 to 10 nM.2
The mode of action differs from those of paclitaxel, vinblastine
and the epothilones, and the activity exhibit more significant than
that of these known drugs for a variety of cancers by 20- to
10000-fold.2a,2b,3 As shown in Figure 1, although the structure of
tubulysins has been classified as a straight tetrapeptide, which
includes four subunits: terminal amino acid with phenol (Tut,
tubutyrosine) or phenyl (Tup, tubuphenylalanine), non-
proteinogenic tubuvaline (Tuv), proteinogenic isoleucine (L-Ile)
products is a asymmetric method for Tuv, Tut or Tup fragments.
The other is the condensation for the N-terminal residue of Tuv
and N,O-acetal substituent ester groups. Moreover, epimerization
could easily occur during the peptide formation. Due to its
limitation from natural sources, challengingly asymmetric
strategy, intriguing efficacy for cancer cell growth inhibition and
high demand in preclinical as well as clinical chemotherapeutics
studies, during the past ten years or so tremendous efforts have
been devoted to the asymmetric synthesis of tubulysins and their
analogous as well as these mechanisms of action.4-8
The first challenging part for the synthesis of tubulysins is the
generation of the stereocenters in the Tuv 17, Tut or Tup 13
fragment. To our best knowledge, almost all of these studies
focused on the asymmetric synthesis of hydroxyl in Tuv
fragment and methyl center in Tut or Tup fragment. But these
known results showed that not all of the stereoselectivity for
hydroxyl or methyl is satisfied.4 Interestingly, chiral N-tert-
butanesulfinamide, as pioneered by Ellman and Davis9, was
selected as an effective auxiliary to build the stereochemistry of
tubulysins by several group, and sometimes suffered from
unsatisfied stereoselectivity5e,7b,c,8l,n, despite its robust chemical
transformation. Recently, one of our interests is to explore the
utility of chiral N-tert-butanesulfinyl imines, and we have
successfully accomplished several bioactive natural products.10 It
is worth mentioning that an interesting stereoselective
migration11a and an efficient diastereoselective approach to
access trans-5-hydroxy-6-substituted-2-piperidinones11b,c or
trans-4-hydroxy-6-substituted-2-2-pyrrolidinones11d have been
successfully achieved. As one of our continuous interests in
R3
O
OR2
S
O
H
N
N
N
R1
N
N
H
O
CO2H
Tup
Mep
Ile
Tuv
Tubulysin A 1a R1= CH2OCOCH2CH(CH3); R2=Ac; R3=OH
Tubulysin B 1b R1= CH2OCOCH2CH2CH3); R2=Ac; R3=OH
Tubulysin D 1c R1= CH2OCOCH2CH(CH3)2; R2=Ac; R3=H
Tubulysin U 1d R1= H; R2=Ac; R3=H
Tubulysin V 1e R1= H; R2= H; R3=H
Figure 1. The structures of tubulysins 1a~1e.

ACCEPTED MANUSCRIPT
2
Tetrahedron
utility of the chiral N-tert-butanesulfinamide used in the
To obtain optically pure 13, as shown in Scheme 2, we
decided to investigate the way of asymmetric reduction. Weinreb
amide 7 was conveniently achieved from 3, then covered to
ketone 8. The condensation of ketone 8 with chiral N-tert-
butanesulfinamide in the presence of titanium tetrachloride gave
imine. Treatment of imine in situ with sodium borohydride
produced desired compound 9 in 55% yield9e. Although several
known conditions for condensation were investigated, the results
turned out to be fruitless. The minor diastereoisomer could not be
thoroughly separated by flash silica gel chromatography.
However, the crude lactam was converted to alcohol 10 by
treatment with HCl/Dioxane and subsequent with Boc2O, which
was easily separated by flash silica gel chromatography in 49%
overall yield with excellent diastereoselectivity (dr > 99:1).
Treatment of alcohol 10 with PDC produced the cyclized product
11 in 80% yield13. The ring-opening of lactam 11 with lithium
hydroxide monohydrate in the presence of H2O2,15 smoothly gave
acid in quantitative yield without purification, which was directly
treated with BnBr in the presence of NaHCO3 to afford ester 12
in 64% overall yield. Finally, the compound 12 was converted to
free amide 13 by treating with TFA in quantitative crude yield.
synthesis of natural products and bioactive secondary
metabolites,12 we decided to investigate an enantioselective
approach for the asymmetric synthesis of tubulysin V induced by
this popular auxiliary. Herein, we present this highly
enantioselective method for the asymmetric synthesis of
tubulysin V.
2. Results and discussion
The retrosynthetic analysis of tubulysin V (1e), as showed in
Figure 2, was divided into three domains, Tup 13, Tuv 17 and
peptide fragment 23. The Tuv fragment 17 is easily synthesized
by our recent method.13 The peptide fragment 23 was accessible
by condensation of protected Mep and Lle according to our
previous works12. In this paper, we focus on the asymmetric
syntheses of Tup 13 fragment through the stereocontrolled
reduction induced by chiral N-tert-butanesulfinamide. Moreover,
the epimerization was avoided by the different sequence of
condensation during the peptide formation and non-alkaline
conditions for the deprotection of amino acid.
Amidation
Final Condensation
O
Ph
O
O
H
OH
O
b
a
TBSO
TBSO
4
N
N
N
Bn
N
N
N
OCH3
H
H
7
O
S
8
S
O
CO2H
Tubulysin V 1e
NHBoc
Bn
d
HN
e
c
HO
TBSO
Ph
O
H
Bn
NH2
(S)
OTBS
N
O
N
9
10
BnO
OH
Ph
OBn
N
(S)
BocHN
NH2TFA
Bn
NHBoc
O
f
O
g
S
BnO
O
BnO
N
Bn
Tup 13
Tuv 17
Peptide fragments 23
Boc
11
O
O
Evans' Methylation
O
S
RMgBr
13
O
S
OTBS
12
Ref. 13
OTBS
OH
N
N
Scheme 2. Reagents and conditions: a. i) NaClO2, NaH2PO4, 2-methylbut-
2-ene, t-BuOH/H2O, rt, overnight; ii) N,O-Dimethylhydroxylamine
hydrochloride, HATU, DIPEA, overnight, 63% (from 3); b. BnMgBr, THF, 0
oC to rt, 8 h, 38%; c. i) Ti(OEt)4, (R)-2-methylpropane-2-sulfinamide, THF,
BocHN
BnO
OTBS
β-chiral aldimines 26
Our previous work.
14 (dr > 98:2)
O
Ph
Asymmetric Reduction
refluxed 8 h,; ii) NaBH4, -20oC to rt, dr > 99:1; d. HCl/Dioxane, 0 C, 2 h,
o
Figure 2. Retrosynthetic analysis of tubulysin V 1e.
then Boc2O, TEA, rt, overnight, 49% (4 steps); e. i) PDC, DMF, rt, overnight,
80%; f. i) LiOH·H2O, H2O2, THF/H2O, 0oC to rt, overnight, ii) BnBr,
NaHCO3, DMSO, 24 h, 64% (2 steps); g. TFA, 0 oC, 2 h, quantitative crude
yield.
The synthesis of Tup 13 is demonstrated in Scheme 1 and
Scheme 2. Firstly, we plan to construct the stereocenter by the
asymmetric addition of imine 5 with Grignard reagent. Thus the
primary alcohol 3 was easily prepared from the commercial
lactone 2 by our similar synthetic route12b. Then the alcohol 3
was converted to chiral imine 5 by oxidation with Dess-Martin
periodinane14 and subsequent condensation with chiral N-tert-
butanesulfinamide in the presence of anhydrous cupric sulphate
in 69% overall yield. Then when the imine 5 was treated with
BnMgBr, the desired product 6 was produced in 79% combined
Next, we focused on the preparation of the fragment 17 of
tubulysin V. Scheme 3 illustrates the preparation of Tuv 17.
Chiral fragment 14, derived from the D-malic acid by our
previous procedure13, was oxidized to give the 15, which was
reacted with H-Cys-OH and subsequent continuously treated
with MnO2 to afford 16 in 63% overall yield5c. Treatment of
compound 16 with lithium hydroxide monohydrate (LiOH·H2O)
gave acid 17 in quantitative yield.
yield.
To
our
disappointment,
there
was
slight
diastereoselectivity for the addition of compound 5 (dr = 60:40).
ref. 12b
a
TBSO
TBSO
O
O
OTBS
o
OH
c
O
4
2
3
Ph
O
O
b
S
N
H
TBSO
S
N
5
6 dr = 60:40
Scheme 1. Reagents and conditions: a. (COCl)2, DMSO, DCM, -78 C;
Scheme 3. Reagents and conditions: a. (COCl)2, DMSO, DCM, -78 oC; b. i)
(b) CuSO4, (R)-2-methylpropane-2-sulfinamide, PPTS, DCM, 30 h, 69%; (c)
BnMgBr, THF, -78 oC to rt, 79%.
H-Cys-OMe·HCl, EtOH, rt, 3 h, 74% for two steps; ii) activated MnO2,

ACCEPTED MANUSCRIPT
3
CH3CN, 60oC, 24 h, 63%; c. LiOH·H2O, THF/H2O, 0oC, 5 h, quantitative
3. Conclusions
crude yield.
In summary, we established a convenient method for highly
enantioselective synthesis of tubulysin V 1e. In this strategy, the
Tuv unit 17 was disasterselectively prepared from the D-malic
acid, and the stereocenters of Tup unit was constructed by the
asymmetric reduction as well as methylation. Moreover, the
epimerization was avoided by condensation of fragment 19 with
24, and by deprotection with hydrogenation. Further diversity-
oriented syntheses of tubulysins by this strategy are now in
progress in our laboratory.
With fragments Tup and Tuv in hand, we focused on the
synthesis of tubulysin V. The crude Tuv uint 17 was coupled
with Tup unit 13 under HATU/HOAt/DIPEA condition to give
compound 18 in 75% yield (Scheme 4). Treatment of 18 with
HCl/dioxane afforded crude amine without further purification,
which was treated with N-Boc isoleucine in the presence of
HATU/DIPEA16 to give tripeptide 20 in 76% yield. To our
disappointment, during this condensation, the α-carbon chiral
center of Ile unit was partial epimerization (dr = 86:14).
4. Experimental section
General: THF was distilled from sodium/benzophenone.
Reactions were monitored by thin layer chromatography (TLC)
on glass plates coated with silica gel with fluorescent indicator.
Flash chromatography was performed on silica gel (300–400
mesh) with Petroleum ether /EtOAc as eluent. Optical rotations
were measured on a polarimeter with a sodium lamp. HRMS
were measured on a LCMS-IT-TOF apparatus. IR spectra were
OP2
S
O
a
+
13
17
N
P1HN
b
N
H
CO2Bn
18 (P1=Boc; P2=TBS)
19 (P1=P2=H)
recorded using film on
a
Fourier Transform Infrared
O
OH
O
Spectrometer. NMR spectra were recorded at 400 MHz, and
chemical shifts are reported in δ (ppm) referenced to an internal
TMS standard for 1H NMR and CDCl3 (77.0 ppm) for 13C NMR.
BocHN
N
c
N
N
H
H
S
CO2Bn
4.1. (S)-4-(tert-Butyldimethylsilyloxy)-3-methylbutan-1-ol 3
20
The compound 3 was prepared by our previous method.12b
[α]D25= -7.89 (c 0.68, CHCl3); IR (film) : νmax 3431, 2955, 2924,
2851, 1460, 1377, 1257, 1061, 836 cm-1;1H NMR (400 MHz,
CDCl3) δ 3.74-3.68 (m, 1H), 3.66-3.60 (m, 1H), 3.53 (dd, J = 9.8,
4.6 Hz, 1H), 3.44 (dd, J = 9.8, 7.0 Hz, 1H), 2.91 (brs, 1H), 1.83-
1.76 (m, 1H), 1.66-1.51 (m, 2H), 0.92 (s, 9H), 0.91 (m, 3H), 0.07
(s, 6H) ppm; 13C NMR (100 MHz, CDCl3) δ 68.7, 61.1, 37.8,
33.8, 25.9, 18.3, 17.2, -5.4, -5.5 ppm; HRMS (ESI) calcd for
(C11H26O2Si+H)+ : 219.1780, found 219.1773.
Scheme 4. Reagents and conditions: a. HOAt, HATU, Et3N, DMF, 0oC to
rt, overnight, 75%; b. HCl/dioxane, 0oC,
h; c. (2S,3S)-2-(tert-
4
butoxycarbonyl)-3-methylpentanoic acid, HATU, DIPEA, CH2Cl2, 76%
combined yield, dr = 86:14.
In order to avoid partial racemization of lle, we decided to
prepare the peptide fragment 23. Protected 21, prepared from H-
Ile-OH, was directly amidated with acid 22 in the presence of
HATU/DIPEA to afford compound 23 in 57% yield. Then the
acid 24 was easily achieved by hydrogenation (Pd/C, H2) in
quantitative yield (Scheme 5).
4.2. (R,E)-N-((S)-4-(tert-Butyldimethylsilyloxy)-3-
methylbutylidene)-2-methylpropane-2-sulfinamide 5
O
O
To a cooled solution (-78oC) of oxalyl chloride (8.7 mL, 100
mmol) in dry DCM (100 mL) was slowly dropped a solution of
DMSO (14.3 mL, 200 mmol) in DCM (20 mL). After being
stirred for 1 h at -78oC, a solution of 3 (10.9 g, 50 mmol) in DCM
(20 mL) was dropped and the resulted mixture was stirred for 3 h.
Then the reaction was quenched with TEA (41.9 mL, 300 mmol)
and allowed to warm to room temperature. The mixture was
diluted with an aqueous solution of NH4Cl and separated. The
aqueous layer was extracted with DCM (70 mL × 3). The
combined organic layers were washed with brine (50 mL × 3).
Dried, filtrated and concentrated to give the crude 4 without
further purification, which was dissolved in dry DCM (200 mL).
R-(+)-tert-Butylsulfinamide (6.05 g, 50 mmol) and PPTS (0.63 g,
2.5 mmol) and CuSO4 (15.96 g, 100 mmol) was added in one
portion. After being stirred for 24 h, the mixture was filtrated and
concentrated. The residue was purified by flash chromatography
on silica gel (EtOAc/PE = 1:20) to give 5 (11.0 g) as a yellow oil
in 69% overall yield. [α]D23= -155.1 (c 1.0, CHCl3) IR (film) :
νmax 3445, 2953, 2921, 2860, 1624, 1474, 1359, 1260, 1085 cm-1;
1H NMR (400 MHz, CDCl3): δ 8.06-8.02 (m, 1H), 3.48 (dd, J =
10.0, 5.6 Hz, 1H), 3.40 (dd, J = 9.6, 6.4 Hz, 1H), 2.67-2.60 (m,
1H), 2.34-2.25 (m, 1H), 2.11-2.01 (m, 1H), 1.16 (s, 9H), 0.94-
0.89 (m, 3H), 0.86-0.84 (m, 9H), 0.01 (s, 3H), 0.00 (s, 3H). 13C
NMR (100 MHz, CDCl3) δ 169.3, 67.5, 56.6, 39.8, 33.7, 25.9,
22.4, 16.6, -5.4, -5.5 ppm; HRMS (ESI) calcd for
(C15H33NO2SSi+H)+ : 320.2080, found 320.2074.
H
a
H2N
N
OBn
N
b
OP
+
OH
N
O
O
23 P = Bn
24 P = H
22
21
Scheme 5 Reagents and Conditions: a. HATU, DIPEA, DCM, 57%; b.
Pd/C, H2, MeOH, 3 h, quantitative crude yield.
Finally, the free acid 24 was directly treated with 19 in the
presence of HATU/ DIPEA to afford protected tubulysin V 25
(Scheme 6). We are pleased that there was no epimerization of
25 during this assembling sequence. Then the 25 was treated
under hydrogen in the presence of Pd/C to obtain the desired
tubulysin V 1e {[]D -9.7 (c 0.37, MeOH), lit5c -11.4 (c 0.7,
22
MeOH); lit5e -11.9 (c 0.73, MeOH)} in 31% yield. The spectral
data of the synthetic 1e were identical to the reported data.5c,e
O
OH
O
H
a
N
N
+
24
19
N
N
N
H
H
S
O
CO2P
25 (P=Bn)
Tubulysin V 1e (P=H)
b
Scheme 6. Reagents and conditions: a. HATU, DIPEA, CH2Cl2; b. Pd/C, H2,
MeOH, 2 h, 31% yield of preparative HPLC for two steps.

ACCEPTED MANUSCRIPT
4
Tetrahedron
4.3. tert-Butyl (2R,4S)-5-(tert-butyldimethylsilyloxy)-4-methyl-
The crude aldehyde 4 (6.1 g, 23.3 mmol) and 2,3-Dimethyl-2-
o
1-phenylpentan-2-ylcarbamate 10
butene (20 mL) was stirred in t-BuOH (30 mL) at 0 C. Then an
aqueous solution (30 mL) of NaClO2 (6.3 g, 70 mmol) and
NaH2PO4·2H2O (10.9 g, 70 mmol) was dropped and stirred for 9
h. The mixture was diluted with water and extracted with EtOAc
(80 mL × 3) and the combined organic layers were washed with
brine (40 mL × 3). Dried, filtrated and concentrated gave the
crude acid without further purification. The above crude acid was
Method A: To a solution of compound 5 (5.2 g, 16.55 mmol)
in anhydrous THF (130 mL) was treated with a solution of
o
BnMgBr (33.1 mL, 33.1 mmol, 1 M in THF) at -78 C to rt for
overnight. The mixture was quenched with a saturated aqueous
solution of NH4Cl and separated. The aqueous layer was
extracted with DCM (50 mL × 3). The combined organic layers
were washed with brine, dried, filtrated and concentrated to give
crude mixture, which was dissolved in EtOH (30 mL) and cooled
to 0oC. Then, a solution of HCl/dioxane (15 mL) was dropped.
After being stirred for 0.5h, the reaction was concentrated at 0 oC
for 0.5 h. The resulted mixture was diluted with DCM (90 mL)
and added Boc2O (5.98 g, 27.4 mmol) and TEA (15.9 mL, 114
mmol). After stirring for 12h, the mixture was quenched with a
aqueous solution of NH4Cl and separated. The aqueous layer was
extracted with DCM (50 mL × 3) and the combined organic
layers were washed with brine (50 mL × 2). Dried, filtrated and
concentrated, the residue was purified by flash chromatography
on silica gel (PE/EA = 3:1) to give major 10 (1.70 g, 42%) as a
colorless oil and a minor compound (1.13 g, 28%) as a colorless
oil.
o
dissolved in dry DCM (100 mL) and cooled to 0 C. Then N,O-
DimethylhydroxylamineHydrochloride (2.7 g, 28.0 mmol),
HATU (13.3 g, 35.0 mmol) and DIPEA (8.1 mL, 46.6 mmol)
was added successively and the mixture was stirred for overnight
o
at 0 C to room temperature. The mixture was quenched with an
aqueous solution of NH4Cl and the resulted mixture was
separated. The aqueous layer was extracted with DCM (50 mL ×
3) and the combined organic layers were washed with brine (40
mL × 3). Dried, filtrated and concentrated, the residue was
purified by flash chromatography on silica gel (EtOAc/PE = 1:20)
25
to give 7 (6.80 g) as a colorless oil in 63% overall yield. [α]D
=
-10.2 (c 0.90, CHCl3); IR (film) : νmax 3031, 2931, 2862, 1709,
1
1454, 1414, 1253, 1209, 1105, 1028 cm-1; H NMR (400 MHz,
CDCl3) δ 3.69 (s, 3H), 3.53 (dd, J = 10.0, 4.8 Hz, 1H), 3.46 (dd,
J = 10.0, 6.0 Hz, 1H), 3.19 (s, 3H), 2.61 (dd, J = 18.2, 9.0 Hz,
1H), 2.31-2.12 (m, 2H), 0.96 (d, J = 6.0 Hz, 3H), 0.90 (s, 9H),
0.05 (s, 6H) ppm; 13C NMR (100 MHz, CDCl3) δ 174.1, 67.6,
61.1, 35.3, 32.5, 25.9, 18.3, 16.8, -5.2, -5.5 ppm; HRMS (ESI)
calcd for [C13H29NO3Si+Na+]: 298.1814, found: 298.1805.
Method B: Compound 8 (4.48 g, 15 mmol), R-(+)-tert-
Butylsulfinamide (1.82 g, 1.5 mmol) and Ti(OEt)4 (5.05 g, 2.8
o
mmol) was stirred in dry THF (100 mL) at 0 C for 1h, and then
the reaction was heated to reflux until the compound was
disappeared by TLC (about for another 20 h). After being cooled
4.5 (S)-5-(tert-Butyldimethylsilyloxy)-4-methyl-1-phenylpentan-
2-one 8
o
to room temperature, the mixture was cooled to -60 C. The
suspension of NaBH4 (570 mg, 1.5 mmol) in THF (10 mL) was
dropped and stirred for 1 h at this temperature. Then the mixture
was allowed to warm to room temperature and stirred for 4 h.
The reaction mixture was carefully quenched with MeOH, and
then the reaction was diluted with an aqueous solution of
NaHCO3 and brine. The resulted mixture was extracted with
EtOAc (30mL × 4) and the combined organic layers were washed
with an aqueous solution of NaHCO3 and brine. Dried, filtrated
and concentrated to give crude mixture, which was dissolved in
Compound 7 (1.1 g, 4.1 mmol) was stirred in dry THF (20 mL)
and cooled to 0 C, and then a solution of BnMgBr (6.2 mL, 6.2
o
mmol, 1M in THF) was slowly dropped. After being stirred for 8
o
h at 0 C to room temperature, the reaction was quenched with a
aqueous solution of NH4Cl. The resulted mixture was extracted
with EtOAc (50 mL × 3) and the combined organic layers were
washed with brine (30 mL × 3). Dried, filtrated and concentrated,
the residue was purified by flash chromatography on silica gel
(EtOAc/PE = 1:40) to give 8 (477 mg) as a colorless oil in 38%
yield. [α]D25 = + 3.3 (c 2.21, CHCl3); IR (film) : νmax 2956, 2929,
2885, 2857, 1714, 1472, 1462, 1455, 1256, 1095, 837, 777, 700
o
HCl/dioxane (15 mL) and stirred for 2 h at 0 C. After being
concentrated, the resulted residue was dissolved in dry DCM (10
mL). Boc2O (3.27 g, 15.0 mmol) and TEA (12.5 mL, 90 mmol)
was added and stirred for overnight. The mixture was quenched
with an aqueous solution of NH4Cl and separated. The aqueous
layer was extracted with DCM (50 mL × 3) and the combined
organic layers were washed with brine (50 mL × 2). Dried,
filtrated and concentrated, the residue was purified by flash
chromatography on silica gel (PE/EA = 3:1) to give 10 (2.1 g,
49%) as a colorless oil.
The major product: [α]D23= +12.55 (c 2.0, CHCl3); IR (film) :
νmax 3427, 2969, 1684, 1506, 1452, 1386, 1359, 1249, 1171, 1041,
1002, 700 cm-1; 1H NMR (400 MHz, CDCl3) δ 7.33 -7.17 (m, 5H),
4.49 (d, J = 9.2 Hz, 1H), 4.07 (s, 1H), 3.49 (s, 2H), 2.85-2.65 (m,
2H), 2.47 (s, 1H), 1.80-1.72 (m, 1H), 1.59-1.53 (m, 1H), 1.41 (s,
9H), 1.30-1.21 (m, 1H), 0.96-0.93 (m, 3H). 13C NMR (100 MHz,
CDCl3) δ 155.8, 138.0, 129.5, 128.3, 126.3, 79.3, 67.9, 49.6, 41.7,
39.3, 32.5, 28.4, 17.8 ppm; HRMS (ESI) calcd for
(C17H27NO3+H)+ : 294.2069, found 294.2064.
1
cm-1; H NMR (400 MHz, CDCl3) : δ 7.37-7.28 (m, 2H), 7.28-
7.23 (m, 1H), 7.21-7.16 (m, 2H), 3.67 (s, 2H), 3.44 (dd, J = 9.8,
5.0 Hz, 1H), 3.32 (dd, J = 10.0, 6.4 Hz, 1H), 2.67-2.57 (m, 1H),
2.25-2.12 (m, 2H), 0.90-0.85 (m, 9H), 0.85-0.80 (m, 3H), 0.05- -
0.05 (m, 6H) ppm; 13C NMR (100 MHz, CDCl3) : δ 208.1, 134.4,
129.4, 128.7, 126.9, 67.4, 50.6, 45.6, 32.0, 25.9, 18.3, 16.7, -5.4,
-5.5 ppm; HRMS (ESI) calcd for [C18H30O2Si +Na+]: 329.1913,
found: 329.1922.
4.6 (3S,5R)-tert-Butyl 5-benzyl-3-methyl-2-oxopyrrolidine-1-
carboxylate 11
Compound 10 (2 g, 6.8 mmol) and PDC (12.82 g, 34 mmol) were
stirred in dry DMF (30 mL) for 24 h at room temperature. The
reaction was diluted with water and extracted with EtOAc (50
mL × 4). The combined organic layers were washed with bine,
dried, filtrated and concentrated. The residue was purified by
flash chromatography on silica gel (EtOAc/PE = 1:7) to give 11
(1.58 g) as a colorless oil in 80% yield. [α]D23= +18.95 (c 2.0,
CHCl3); IR (film): νmax 3450, 2976, 2932, 1784, 1748, 1710, 1455,
1
The minor product: H NMR (400 MHz, CDCl3) δ 7.30 -7.24
(m, 2H), 7.22 -7.15 (m, 3H), 4.43 (d, J = 6.8 Hz, 1H), 3.91 (s,
1H), 3.51-3.45 (m, 1H), 3.36 (dd, J = 10.4, 6.4 Hz, 1H), 2.82-
2.70 (m, 2H), 2.06-1.94 (m, 1H), 1.74-1.65 (m, 1H), 1.52-1.44
(m, 1H), 1.39 (s, 9H), 1.24-1.20 (m, 1H), 0.89-0.86 (m, 3H).
1
1368, 1308, 1150, 975, 740, 696 cm-1; H NMR (400 MHz,
CDCl3) δ 7.38-7.16 (m, 5H), 4.33-4.26 (m, 1H), 3.15 (dd, J =
13.2, 3.6 Hz, 1H), 2.72 (dd, J = 13.2, 9.2 Hz, 1H), 2.49-2.37 (m,
1H), 2.05 (dd, J = 12.8, 8.4 Hz, 1H), 1.60 (s, 9H), 1.18-1.14 (m,
3H). 13C NMR (100 MHz, CDCl3) δ 176.6, 150.1, 137.4, 129.4,
4.4. (S)-4-(tert-Butyldimethylsilyloxy)-N-methoxy-N,3-
dimethylbutanamide 7

ACCEPTED MANUSCRIPT
5
128.7, 126.8, 82.9, 56.9, 39.2, 36.2, 30.8, 28.1, 15.4 ppm; HRMS
(ESI) calcd for (C17H27NO3+H)+ 290.1756, found 290.1750.
4.9 (2S,4R)-Benzyl 4-(2-((1R,3R)-3-(tert-butoxycarbonyl)-1-
(tert-butyldimethylsilyloxy)-4-methylpentyl)thiazole-4-
carboxamido)-2-methyl-5-phenylpentanoate 18
4.7 (2S,4R)-Benzyl 4-(tert-butoxycarbonyl)-2-methyl-5-
phenylpentanoate 12
Compound 17 (201 mg, 0.438 mmol) was dissolved in THF (6
mL) and was treated with a solution of LiOH·H2O (37 mg, 0.876
mmol, H2O, 2 mL) at room temperature for 5 h. The reaction was
acidified with HCl (1 M) and extracted with EtOAc (20 mL × 3).
The combined organic layers were dried over Na2SO4 and
concentrated to give crude acid without further purification. The
compound 12 (192 mg, 0.484 mmol) was treated with TFA/DCM
(5 mL, v/v = 1:1). Then the reaction was concentrated to give salt
without further purification. The above crude acid, salt, HOAt
(65 mg, 0.484 mmol), HATU (184 mg, 0.484 mmol) and Et3N
To a solution of compound 11 (1.52 g, 5.25 mmol) in THF (25
mL) was treated with an aqueous solution of LiOH·H2O (1.25 g,
29.7 mmol, 8 mL) at room temperature for overnight. The
mixture was acidified with an aqueous solution of 5% KHSO4
and extracted with EtOAc (30 mL × 4). The combined organic
layers were washed with brine. Dried, filtrated and concentrated
to give crude acid without further purification. The above crude
acid was dissolved in dry DMSO (35 mL), then NaHCO3 (441
mg, 5.25 mmol) and BnBr (0.93 mL, 7.88 mmol) was added in
one portion. After stirring for 24 h, the mixture was quenched
with water and the resulted was extracted with EtOAc (30 mL ×
4). The combined organic layers were washed with brine (20 mL
× 4). Dried, filtrated and concentrated, the residue was purified
by flash chromatography on silica gel (EtOAc/PE = 1:10) to give
12 (1.33 g) as a colorless oil in 64% yield. [α]D23=+22.25 (c 2.0,
CHCl3); IR (film) : νmax 3485, 2977, 2926, 1635, 1498, 1455,
o
(0.123 mL, 0.88 mmol) were stirred in dry DMF (5 mL) at 0 C
to room temperature for 15h. The reaction was diluted with water
and extracted with EtOAc (20mL × 3). The combined organic
layers were washed with bine (10mL × 3), dried, filtrated and
concentrated. The residue was purified by flash chromatography
on silica gel (EtOAc/PE= 1:10) to give 18 (244 mg) as a colorless
23
oil in 75% yield. [α]D = +35.58 (c 2.0, CHCl3); IR (film): vmax
3392, 3025, 2958, 2929, 2856, 1716, 1666, 1540, 1496, 1390,
1365, 1253, 1170, 1087, 1006, 838, 780, 685 cm-1; 1H NMR (400
MHz, CDCl3) δ 7.83 (s,1H), 7.24-7.02 (m, 10H), 6.99-6.92
(m,1H), 4.99-4.89 (m, 2H), 4.89-4.83 (m, 1H), 4.50-4.40 (m, 1H),
4.38-4.22 (m, 1H), 3.60-3.34 (m, 1H), 2.88-2.70 (m, 2H), 2.60-
2.48 (m, 1H), 1.96-1.85 (m, 1H), 1.80-1.68 (m, 2H),1.58-1.45 (m,
2H), 1.36-1.29 (m, 9H),1.08-1.04 (m, 3H), 0.80 (s, 9H),0.77-0.73
(m, 6H), 0.00 (s, 3H), -0.21 (s, 3H). 13C NMR (100 MHz, CDCl3)
δ 176.9, 175.9, 160.7, 155.4, 149.7, 137.6, 136.1, 129.6, 128.5,
128.3, 128.2, 128.1, 126.5, 122.8, 79.0, 70.2, 66.4, 52.0, 48.4,
41.1, 41.0, 37.7, 36.6, 28.4, 25.8, 18.1, 18.0, 17.9, 17.8, -4.7, -5.2
ppm; HRMS (ESI) calcd for (C40H59N3O6SSi+H)+: 738.3972,
found 738.3966.
1390, 1365, 1253, 1171, 1005, 698 cm-1; H NMR (CDCl3, 400
1
MHz) δ 7.43-7.13 (m, 10H), 5.20-5.10 (m, 2H), 4.35(d, J = 8.8
Hz, 1 H), 4.00-3.85 (m, 1H), 2.80 (m, 2H), 2.75-2.63 (m, 1H),
2.00-1.91 (m, 1H), 1.55-1.45 (m, 1H), 1.42(s, 9H), 1.23-1.19 (m,
3H); 13CNMR (CDCl3, 100 MHz) δ 176.1, 155.3, 137.9, 136.1,
129.5, 128.6, 128.4, 128.1, 126.4, 79.1, 66.3, 49.8, 41.4, 38.0,
36.5, 28.4, 17.7 ppm; HRMS (ESI) calcd for (C24H31NO4+H)+:
398.2331, found 398.2326.
4.8
Methyl
2-((1R,3R)-3-(tert-butoxycarbonyl)-1-(tert-
butyldimethylsilyloxy)-4-methylpentyl)thiazole-4-carboxylate 16
To a cooled solution (-78oC) of oxalyl chloride (0.60 mL, 6.6
mmol) in dry DCM (10 mL) was slowly dropped a solution of
DMSO (1 mL, 13.2 mmol) in DCM (3 mL). After being stirred
for 1 h at -78oC, a solution of 14 (1.30 g, 3.3 mmol) in DCM (5
mL) was dropped and the resulted mixture was stirred for 3h. The
reaction was quenched with TEA (2.75 mL, 19.8 mmol) and
allowed to warm to room temperature. Then a solution of L-
cysteine methyl ester hydrochloride (1.49 g, 8.7 mmol, 5 mL
EtOH) was added in one portion and the reaction was stirred for
anther 3h at the room temperature. The mixture was concentrated
and the residue was dissolved in EtOAc (50 mL) and washed
with brine (20 mL × 3). Dried, filtrated and concentrated, the
residue was purified by flash chromatography on silica gel
(EtOAc/PE = 1:6) to give a mixture (1.32 g) as a colorless oil.
The above mixture was dissolved in dry MeCN (15 mL), then
activated MnO2 (2.44 g, 28 mmol) was added in one portion and
the reaction was stirred at 60 oC for 9 h. Another portion
activated MnO2 (2.44 g, 28 mmol) was added. After being stirred
for another 15 h, the reaction was cooled to room temperature
and filtrated. The filtrate was concentrated and the residue was
purified by flash chromatography on silica gel (EtOAc/PE = 1:5)
to give 16 (0.63 g) as a colorless oil in 63% yield. [α]D23 = + 29.4
(c 1.0, CHCl3); IR (film): νmax 3449, 2958, 2929, 2860, 1712,
4.10 (2S,3S)-Benzyl 3-methyl-2-((R)-1-methylpiperidine-6-
carboxamido)pentanoate 23
Acid 22 (0.45 g, 3.15 mmol) and amine 21 (0.45 g, 2 mmol) were
stirred in cooled (-10 oC) DCM (20 mL), then HATU (1.18 g, 3.1
mmol) and DIPEA (2.5 mL, 14 mmol) were added respectively.
After being stirred for overnight, the mixture was quenched with
an aqueous solution of NaHCO3 and extracted with DCM (20 mL
× 3). The combined organic layers were washed with bine (10
mL × 3), dried, filtrated and concentrated. The residue was
purified by flash chromatography on silica gel (MeOH/DCM =
23
1:50) to give 23 (395 mg) as a colorless oil in 57% yield. [α]D
=
+48.6 (c 1.0, CHCl3); IR (film): vmax 3445, 2959, 2937, 2844,
2784, 1737, 1652, 1501, 1452, 1189, 1145, 1036, 756, 697 cm-1,
1H NMR (400 MHz, CDCl3) δ 7.40-7.30 (m, 5H), 7.10-7.02
(m,1H), 5.25-5.07 (m, 2H), 4.63 (dd, J=9.2, 4.8 Hz, 1H), 2.94-
2.86 (m, 1H), 2.53-2.45 (m, 1H), 2.22 (s, 3H), 2.07-2.01 (m, 1H),
2.00-1.95 (m, 1H), 1.91-1.83 (m, 1H), 1.73-1.59 (m, 2H), 1.56-
1.47 (m, 1H), 1.47-1.36 (m, 2H),1.24-1.08 (m, 2H), 0.95-0.85 (m,
6H); 13C NMR (100 MHz, CDCl3) δ 173.4, 170.7, 134.4, 127.5,
127.4, 68.6, 65.9, 55.0, 54.3, 43.9, 36.6, 29.6, 24.1, 24.0, 22.2,
14.9, 10.5 ppm; HRMS (ESI) calcd for (C20H30N2O3+H)+:
347.2335, found 347.2329.
1
1637, 1506, 1353, 1249, 1172, 1090, 1002, 898, 772; H NMR
(400 MHz, CDCl3) δ 8.15 (s, 1H), 5.20 (dd, J = 9.6, 2.0 Hz, 1H),
4.63 (d, J = 9.2 Hz, 1H), 3.98 (s, 3H), 3.74-3.65(m, 1H), 1.91-
1.81 (m, 2H), 1.67-1.62 (m, 1H), 1.46(s, 10H), 0.96 (s, 9H), 0.90-
0.83 (m, 6H), 0.17 (s, 3H), 0.07 (s, 3H). 13C NMR (100 MHz,
CDCl3) δ 178.5, 161.9, 155.4, 146.3, 127.5, 78.8, 70.4, 52.5, 51.9,
41.5, 32.6, 28.4, 25.8, 18.1, 18.0, 17.7, 5.3, 4.7 ppm; HRMS (ESI)
calcd for (C22H40N2O5SSi)+ : 473.2505, found 473.2500.
4.11
(2S,4R)-4-(2-((1R,3R)-1-Hydroxy-4-methyl-3-((2S,3S)-3-
methyl-2-((R)-1-methylpiperidine-2-
carboxamido)pentanamido)pentyl)thiazole-4-carboxamido)-2-
methyl-5-phenylpentanoic acid 1e
Compound 18 (85 mg, 0.115 mmol) was treated with
HCl/dioxane (5 ml) for 2 h，then the mixture was concentrated
to give crude salt without further purification. Compound 23 (40
mg, 0.115 mmol) and Pd/C (4 mg,) were stirred in MeOH (30

ACCEPTED MANUSCRIPT
6
Tetrahedron
4425; (b) Braig, S.; Wiedmann, R. M.; Liebl, J.; Singer, M.;
mL) under hydrogen atmosphere for 3 h. The suspension was
Kubisch, R.; Schreiner, L.; Wagner, E.; Vollmar, A. M.; Abhari,
B. A.; Fulda, S.; Kazmaier, U. Cell Death Dis 2014, 5, e1001; (c)
Hoffmann, J.; Gorges, J.; Junk, L.; Kazmaier, U. Org. Biomol.
Chem. 2015, 13, 6010-6020; (d) Rath, S.; Liebl, J.; Fuerst, R.;
Ullrich, A.; Burkhart, J. L.; Kazmaier, U.; Herrmann, J.; Mueller,
R.; Guenther, M.; Schreiner, L.; Wagner, E.; Vollmar, A. M.;
Zahler, S. Br. J. Pharmacol. 2012, 167, 1048-1061; (e) Eirich, J.;
Burkhart, J. L.; Ullrich, A.; Rudolf, G. C.; Vollmar, A.; Zahler, S.;
Kazmaier, U.; Sieber, S. A. Mol. BioSyst. 2012, 8, 2067-2075; (f)
Burkhart, J. L.; Mueller, R.; Kazmaier, U. Eur. J. Org. Chem.
2011, 3050-3059.
filtrated and concentrated to give crude acid without further
purification. The above crude salt, acid and HATU (66 mg, 0.173
mmol) were stirred in dry DCM (2 mL) at 0 C, then DIPEA
o
(0.14 mL, 0.801 mmol) was dropped and the mixture was stirred
for 24h at the temperature. The result mixture was quenched with
a saturated solution of NaHCO3. The resulted mixture was
extracted with DCM (15 mL × 4). The combined organic layers
were washed with brine one time, dried and concentrated to give
crude 25. The crude 25, Pd/C (20 mg) was stirred in MeOH (30
mL) under hydrogen atmosphere for 4 h. After being filtrated and
concentrated, the residue was purified by prepative HPLC
(MeOH/H2O = from 60:1 to 99:1, tR= 8.4min) to provide pure
tubulysin V 1e as a white foam (24 mg) in 31% yield. [α]D24= -
9.7 (c 0.37, MeOH); 1H NMR (600 MHz, CD3OD) δ 8.06 (s, 1H),
7.27-7.15 (m, 5H), 4.42-4.36 (m, 1H), 4.24 (d, J = 12.0 Hz, 1H),
4.12-4.07 (m, 1H), 3.80-3.75 (m, 1H), 3.52-3.47 (m, 1H), 3.12-
3.06 (m, 1H), 2.93-2.90 (m, 2H), 2.79 (s, 3H), 2.59-2.54 (m, 1H),
2.20-2.15 (m, 1H), 2.15-2.09(m, 1H), 2.07-1.72 (m, 9H), 1.68-
1.58 (m, 3H), 1.34-1.22 (m, 2H), 1.20-1.17 (m, 3H), 1.04-0.91
(m, 12H); 13C NMR (CD3OD, 150 MHz) δ: 177.8, 177.2, 171.5,
167.2, 161.2, 148.7, 137.4, 128.4, 127.3, 125.4, 122.3, 67.8, 66.3,
58.0, 54.2, 50.7, 48.6, 40.9, 40.2, 38.7, 37.2, 35.8, 35.5, 31.8,
28.2, 23.9, 22.0, 20.4, 17.5, 16.7, 16.5, 14.1, 9.2 ppm; HRMS
(ESI) calcd for (C35H54N5O6S+H)+: 672.3795, found 672.3789.
7. For synthesis of other natural tubulysins see: (a) Pando, O.;
Dorner, S.; Preusentanz, R.; Denkert, A.; Porzel, A.; Richter, W.;
Wessjohann, L. Org. Lett. 2009, 11, 5567-5569; (b) Peltier, H. M.;
McMahon, J. P.; Patterson, A. W.; Ellman, J. A. J. Am. Chem.
Soc. 2006, 128, 16018-16019; (c) Yang, X.-D.; Dong, C.-M.;
Chen, J.; Ding, Y.-H.; Liu, Q.; Ma, X.-Y.; Zhang, Q.; Chen, Y.
Chem. - Asian J. 2013, 8, 1213-1222; (d) Nicolaou, K. C.; Yin, J.;
Mandal, D.; Erande, R. D.; Klahn, P.; Jin, M.; Aujay, M.;
Sandoval, J.; Gavrilyuk, J.; Vourloumis, D. J. Am. Chem. Soc.
2016, 138, 1698-1708.
8. For synthesis of analogues of tubulysins see: (a) Wipf, P.; Wang,
Z. Org. Lett. 2007, 9, 1605-1607; (b) Raghavan, B.;
Balasubramanian, R.; Steele, J. C.; Sackett, D. L.; Fecik, R. A. J.
Med. Chem. 2008, 51, 1530-1533; (c) Vlahov, I. R.; Wang, Y.;
Vetzel, M.; Hahn, S.; Kleindl, P. J.; Reddy, J. A.; Leamon, C. P.
Bioorg. Med. Chem. Lett. 2011, 21, 6778-6781; (d) Floyd, W. C.,
III; Datta, G. K.; Imamura, S.; Kieler-Ferguson, H. M.; Jerger, K.;
Patterson, A. W.; Fox, M. E.; Szoka, F. C.; Frechet, J. M. J.;
Ellman, J. A. ChemMedChem 2011, 6, 49-53; (e) Patterson, A.
W.; Peltier, H. M.; Sasse, F.; Ellman, J. A. Chem. - Eur. J. 2007,
13, 9534-9541; (f) Shibue, T.; Okamoto, I.; Morita, N.; Morita,
H.; Hirasawa, Y.; Hosoya, T.; Tamura, O.; Bioorg. Med. Chem.
Lett. 2011, 21, 431-434; (g) Pando, O.; Stark, S.; Denkert, A.;
Porzel, A.; Preusentanz, R.; Wessjohann, L. A. J. Am. Chem. Soc.
2011, 133, 7692-7705; (h) Sreejith, S. P.; Sani, M.; Saunders, F.
R.; Wallace, H. M.; Zanda, M. Synlett 2011, 1673-1676; (i) Wang,
Z.; McPherson, P. A.; Raccor, B. S.; Balachandran, R.; Zhu, G.;
Day, B. W.; Vogt, A.; Wipf, P. Chem. Biol. Drug Des. 2007, 70,
75-86; (j) Shankar, S. P.; Jagodzinska, M.; Malpezzi, L.; Lazzari,
P.; Manca, I.; Greig, I. R.; Sani, M.; Zanda, M. Org. Biomol.
Chem. 2013, 11, 2273-2287; (k) Burkhart, J. L.; Kazmaier, U.
RSC Adv. 2012, 2, 3785-3790; (l) Yang, X.; Dong, C.; Chen, J.;
Liu, Q.; Han, B.; Zhang, Q.; Chen, Y. Tetrahedron Lett. 2013, 54,
2986-2988; (m) Balasubramanian, R.; Raghavan, B.; Steele, J. C. ;
Sackett, D. L.; Fecik, R. A. Bioorg. Med. Chem. Lett. 2008, 18,
2996-2999; (n) Patterson, A. W.; Peltier, H. M.; Ellman, J. A. J.
Org. Chem. 2008, 73, 4362-4369; (o) Park, Y.; Lee, J. K.; Ryu, J.-
S. Synlett 2015, 1063-1068.
9. For reviews on chiral N-tert-butanesulfinamide, see: (a) Ellman, J.
A.; Owens, T. D.; Tang, T. P. Acc. Chem. Res. 2002, 35, 984-995;
(b) Lin, G.-Q.; Xu, M.-H.; Zhong, Y.-W.; Sun, X.-W. Acc. Chem.
Res. 2008, 41, 831-840; (c) Ferreira, F.; Botuha, C.; Chemla, F.;
Perez-Luna, A. Chem. Soc. Rev. 2009, 38, 1162-1186; (d) Robak,
M. T.; Herbage, M. A.; Ellman, J. A. Chem. Rev. 2010, 110, 3600-
3740; For selected application of N-tert-butanesulfinamide, see:
(e) Kochi, T.; Ellman, J. A. J. Am. Chem. Soc. 2004, 126, 15652-
15653; (f) Zhong, Y.-W.; Dong, Y.-Z.; Fang, K.; Izumi, K.; Xu,
M.-H.; Lin, G.-Q. J. Am. Chem. Soc. 2005, 127, 11956-11957; (g)
Robak, M. T.; Herbage, M. A.; Ellman, J. A. Tetrahedron 2011,
67, 4412-4416; (h) Chen, H.; Yu, W.; Guo, X. H.; Meng, W. D.;
Huang, Y. G. Chin. Chem. Lett. 2012, 23, 277-280; (i) Ye, J.-L.;
Zhang, Y.-F.; Liu, Y.; Zhang, J.-Y.; Ruan, Y.-P.; Huang, P.-Q.
Org. Chem. Front. 2015, 2, 697-704.
Acknowledgments
We thank the National Natural Science Foundation of China
(21472022, 21272041, 21072034), and Key Laboratory of
Synthetic Chemistry of Natural Substances, SIOC of Chinese
Academy of Sciences for financial support. The authors thank
Ms. Qian-Ru, Zhou and Mr Xiao-Yun, Wei for preparation of
several intermediate compounds.
References and notes
1. Sasse, F.; Steinmetz, H.; Höfle, G.; Reichenbach, H. J. Antibiot.
2000, 53, 879-885.
2. (a) Steinmetz, H.; Glasser, N.; Herdtweck, E.; Sasse, F.;
Reichenbach, H.; Höfle, G.; Angew. Chem. Int. Ed. 2004, 43,
4888-4892；(b) Steinmetz, H.; Glasser, N.; Herdtweck, E.; Sasse,
F.; Reichenbach, H.; Höfle, G. Angew. Chem. 2004, 116, 4996-
5000; (c) Dömling, A.; Richter, W. Diversity 2005, 9, 141-147；
(d) Khalil, M.; Sasse, F.; Lϋnsdorf, H.; Elnakady, Y.;
Reichenbach, H. Chem. Bio. Chem. 2006, 7, 678-683; (e) Kaur,
G.; Hollingshead, M.; Holbeck, S.; Schauer-Vokašinović.,
Camalier, F. R.; Dömling, A.; Agarwal, S. Biochem. J. 2006, 396,
235-242.
3. (a) Howarth, J.; Kenny, P.; McDonnell, S.; O’Connor, S. Bioorg.
Med. Chem. Lett. 2003, 13, 2693-2697; (b) Höfle, G.; Glaser, N.;
Leibold, T.; Karama, U.; Sasse, F. Pure Appl. Chem. 2003, 75,
167-178.
4. For reviews on synthesis of tubulysins see: (a) Neri, D. Fossati,
G.; Zanda, M. ChemMedChem 2006, 1, 175-180; (b) Xu, X.;
Friestad, G. K.; Lei, Y. Mini-Rev. Med. Chem. 2013, 13, 1572-
1578; (c) Murray, B. C.; Peterson, M. T.; Fecik, R. A. Nat. Prod.
Rep. 2015, 32, 654-662.
5. For synthesis of tubulysin V see: (a) Shibue, T.; Hirai, T.;
Okamoto, I.; Morita, N.; Masu, H.; Azumaya, I.; Tamura, O.
Chem. - Eur. J. 2010, 16, 11678-11688; (b) Shankar, P. S.;
Bigotti, S.; Lazzari, P.; Manca, I.; Spiga, M.; Sani, M.; Zanda, M.
Tetrahedron Lett. 2013, 54, 6137-6141; (c) Sani, M.; Fossati, G.;
Huguenot, F.; Zanda, M. Angew. Chem., Int. Ed. 2007, 46, 3526-
3529; (d) Doemling, A.; Beck, B.; Eichelberger, U.; Sakamuri, S.;
Menon, S.; Chen, Q.-Z.; Lu, Y.; Wessjohann, L. A. Angew.
Chem., Int. Ed. 2006, 45, 7235-7239; (e) Wang, R.; Tian, P.; Lin,
G.-Q. Chin. J. Chem. 2013, 31, 40-48.
10. (a) Liu, R.-C.; Wei, J.-H.; Wei, B.-G.; Lin, G.-Q. Tetrahedron:
Asymmetry 2008, 19, 2731-2734; (b) Xarnod, C.; Huang, W.; Ren,
R.-G.; Liu, R.-C.; Wei, B.-G. Tetrahedron 2012, 68, 6688-6695.
11. (a) Huang, W.; Ye, J.-L.; Zheng, W.; Dong, H.-Q.; Wei, B.-G. J.
Org. Chem. 2013, 78, 11229-11237; (b) Si, C.-M.; Huang, W.;
Du, Z.-T.; Wei, B.-G.; Lin, G.-Q. Org. Lett. 2014, 16, 4328-4331;
(c) Si, C.-M.; Mao, Z.-Y.; Dong, H.-Q.; Du, Z.-T.; Wei, B.-G.;
Lin, G.-Q. J. Org. Chem. 2015, 80, 5824-5833; (d) Si, C.-M.;
Mao, Z.-Y.; Liu, Y.-W.; Du,; Wei, B.-G.; Lin, G.-Q. Org. Chem.
Front. 2015, 2, 1485-1499.
12. (a) Ma, J.-Y.; Xu, L.-F.; Huang, W.-F.; Wei, B.-G.; Lin, G.-Q.
synlett 2009, 1307-1310; (b) Ma, J.-Y.; Huang, W.; Wei, B.-G.
Tetrahedron Lett. 2011, 52, 4598-4601; (c) Huang, W.; Ren, R.-
G.; Dong, H.-Q.; Wei, B.-G.; Lin, G.-Q. J. Org. Chem. 2013, 78,
6. For synthesis of Pretubulysin see: (a) Ullrich, A.; Chai, Y.;
Pistorius, D.; Elnakady, Y. A.; Herrmann, J. E.; Weissman, K. J.;
Kazmaier, U.; Mueller, R. Angew. Chem., Int. Ed. 2009, 48, 4422-

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10747-10762; (d) Ren, R.-G.; Ma, J.-Y.; Mao, Z.-Y.; Liu, Y.-W.;
7
Wei, B.-G. Chin. Chem. Lett. 2015, 26, 1209-1215.
13. Zhou, Q.-R.; Wei, X.-Y.; Li, Y.-Q.; Huang, D.-F.; Wei, B.-G.
Tetrahedron 2014, 70, 4799-4808.
14. (a) Dess, D. B.; Martin, J. C. J. Org. Chem. 1983, 48, 4155-4156;
(b) Dess, D. B.; Martin, J. C.; J. Am. Chem. Soc. 1991, 113, 7277-
7287; (c) Frigerio, M.; Santagostino, M.; Sputore, S. J. Org.
Chem. 1999, 64, 4537-4541.
15. Kende, A. S.; Dong, H.-Q.; Mazur, A. W.; Ebetino, F. H.
Tetrahedron Lett. 2001, 42, 6015-6018.
16. Carpion, L. A. J. Am. Chem. Soc. 1993, 115, 4397-4398.