Synthesis of desmosine-containing cyclic peptide for the possible elucidation of elastin crosslinking structure

Article abstract of DOI:10.1016/j.tet.2017.05.045

Elastin is a vital extracellular matrix protein, which is known for providing elasticity in numerous tissues. It is formed by the self-assembly and subsequent crosslinking of elastin precursor, tropoelastin. Two tetrafunctional, pyridinium-based amino acids desmosine and isodesmosine are exclusively found in elastin and play an important role as crosslinkers. Structural elucidation of elastin has eluded scientists to date, owing to the highly cross-linked structure and insoluble nature. Therefore, in this study, we aimed to synthesize a desmosine-containing cyclic peptide as a partial elastin mimic, in order to eventually facilitate the elucidation of the crosslinking pattern of elastin by mass spectrometric analysis.

Full text of DOI:10.1016/j.tet.2017.05.045

Accepted Manuscript
Synthesis of desmosine-containing cyclic peptide for the possible elucidation of
elastin crosslinking structure
Keita Ogawa, Takahiro Hayashi, Yong Y. Lin, Toyonobu Usuki
PII:
S0040-4020(17)30520-3
10.1016/j.tet.2017.05.045
TET 28711
DOI:
Reference:
To appear in: Tetrahedron
Received Date: 20 April 2017
Revised Date: 9 May 2017
Accepted Date: 11 May 2017
Please cite this article as: Ogawa K, Hayashi T, Lin YY, Usuki T, Synthesis of desmosine-containing
cyclic peptide for the possible elucidation of elastin crosslinking structure, Tetrahedron (2017), doi:
10.1016/j.tet.2017.05.045.
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ACCEPTED MANUSCRIPT
Synthesis of desmosine-containing cyclic peptide for the possible
elucidation of elastin crosslinking structure
Keita Ogawaa, Takahiro Hayashia, Yong Y. Linb, Toyonobu Usukia,*
a Department of Materials and Life Sciences, Faculty of Science and Technology, Sophia
University, 7-1 Kioicho, Chiyoda-ku, Tokyo 102-8554, Japan
b Mount Sinai St. Luke’s Hospital Center, 1111 Amsterdam Ave., New York, New York 10025,
USA
Keywords: desmosine, cyclic peptide, Sonogashira cross-coupling, Negishi cross-coupling,
intramolecular condensation
* Corresponding author. Tel.; +81 3 3238 3446; fax: +81 3 3238 3361
E-mail address: t-usuki@sophia.ac.jp (T. Usuki).
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Abstract:
O
O
O
BocHN
H
O
H
N
H
N
O
BocHN
CO2tBu
N
N
H
CO2Bn
NHCbz
N
H
CO2Bn
HN
HN
H2N
O
O
CO2H
CO2H
+
NHCbz
CO2tBu
H2N
H2N
I
CO2H
Br
Br
CbzHN
N
I
CO2tBu
N
N
desmosine with cyclic peptide
Elastin is a vital extracellular matrix protein, which is known for providing
elasticity in numerous tissues. It is formed by the self-assembly and subsequent
crosslinking of elastin precursor, tropoelastin. Two tetrafunctional, pyridinium-based amino
acids desmosine and isodesmosine are exclusively found in elastin and play an important
role as crosslinkers. Structural elucidation of elastin has eluded scientists to date, owing to
the highly cross-linked structure and insoluble nature. Therefore, in this study, we aimed to
synthesize a desmosine-containing cyclic peptide as a partial elastin mimic, in order to
eventually facilitate the elucidation of the crosslinking pattern of elastin by mass
spectrometric analysis.
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Introduction:
Elastic fibers in extracellular matrices are significant structural components of skin,
blood vessels, alveolus, ligamentum, and other connective tissues, where they are
responsible for the retention of mechanical strength and elasticity.1 Elastin, a major
structural component of elastic fibers, is a highly crosslinked insoluble protein formed by
oxidative deamination of lysine (Lys) residues in the soluble precursor tropoelastin,
catalyzed by lysyl oxidase.2 Desmosine and isodesmosine (Figure 1) are two major
pyridinium amino acids that serve as important crosslinkers binding the polymeric chains of
amino acids in the 3D network of elastin.3,4
NH2
CO2H
CO2H
CO2H
CO2H
CO2H
NH2
CO2H
NH2
CO2H
H2N
H2N
H2N
H2N
N
N
NH2
CO2H
desmosine
isodesmosine
Figure 1. Structures of desmosine and isodesmosine.
Amino acid sequence analysis has shown that crosslinking occurs within the
hydrophilic (rich in Lys and alanine (Ala)) domains of elastin.5 Mecham and co-workers
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reported that one crosslinking structure involved hydrophilic domains between 19 and 25,
which are joined by desmosine (Figure 2, left). However, the detailed structure remains
unclear.6
24
O
O
17
22
Domain 19
N
H
23
16
HN
H2N
HO2C
K
A
A
K
A
15
27
Y
NH2
25
14
21C' O2H
13
26
O
HN
20
18'
18
4
20'
desmosine
H2N
CO2H
5
6
3
2
19'
19
21
12CO2H
1 N
A
K
S
A
A
K
A
H2N
CO2H
11
9
7
H2N
Domain 25
10
8
desmosine with cyclic peptide (1)
Figure 2. Proposed schematic model of elastin crosslinking peptide (left, A: alanine, K:
lysine, S: serine, Y: tyrosine) and structure of desmosine-containing cyclic peptide (1,
right).
Structural identification of crosslinking moieties involving desmosine is difficult
using available proteomic mass spectrometric techniques. Recently, a new software, Poly
Lynx, was developed for the analysis of crosslinking peptides;7 however, the
characterization of crosslinked peptides from mature elastin digests remains a considerable
challenge. The degradation of elastin-containing tissues is irreversible and occurs in several
widely prevalent diseases, such as atherosclerosis,8 aortic aneurysms,8 cystic fibrosis,9 and
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chronic obstructive pulmonary disease (COPD).10 In order to understand the relationship
between these diseases and elastin, the detailed crosslinking structure should be elucidated.
Based on the study by Mecham and co-workers, we envisioned a crosslinking
structure containing desmosine with Ala-Ala into the 3,4-positions, desmosine with cyclic
peptide (1, Figure 2, right). Utilizing our synthetic strategy for the total synthesis of
desmosine,11-13 we aimed to synthesize 1, which contains a macrocycle with two alanine
residues in the 3 and 4 positions, which would be structurally similar to part of elastin.
Synthetic studies of cyclic peptides that contain a pyridine ring are unusual,14 and thus are
of considerable interest. Furthermore, we hoped to elucidate the crosslinking structure of
elastin by comparative analysis of 1 and degradated elastin peptides. By employing MS/MS
as an analytical technique, if we could find a degradated product with the same fragment
peak as 1, a partial structure of elastin could be elucidated. Therefore, in order to contribute
to the elucidation of the elastin crosslinking structure by an innovative chemical synthetic
approach, we synthesized 1 via stepwise Sonogashira and Negishi cross-coupling reactions
and intramolecular condensation as key steps.
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Results and discussion:
According to the retrosynthetic analysis shown in Scheme 1, 1 could be derived
from cyclic peptide 2 or 3, which contain a pyridine ring, and iodo amino acid (ω-iodobutyl
L-glycine) derivative 4 through late stage formation of the pyridinium salt. The macrocycle
of 2 or 3 could be constructed by hydrogenation and intramolecular condensation of 5 or 6.
Synthesis of 5 or 6 would involve chemo- and regioselective palladium-catalyzed
Sonogashira and Negishi cross-coupling reactions between trihalogenated pyridine 7,11c,11d
which can be prepared from commercially available 4-aminopyridine,15 terminal alkyne 8,
and iodo amino acid 9 or 10. The protected alkyne 8 could be formed by intermolecular
condensation between 11 and 12.
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Scheme 1. Retrosynthesis of 1.
O
O
I
N
H
HN
HN
BocHN
1
+
O
CO2R
tBuO2C
BocHN
CO2R
N(Boc)2
N
4
2: R = Me
3: R = tBu
hydrogenation and
intramolecular condensation
I
O
H
N
NHCbz
CO2R
Br
Br
O
BocHN
CO2R
N
H
CO2Bn
I
Sonogashira
and Negishi
cross-couplings
N
9: R = Me
10: R = tBu
7
+
NHCbz
O
BocHN
H
N
CbzHN
CO2R
N
H
CO2Bn
N
O
5: R = Me
6: R = tBu
8
intermolecular
condensation
O
NHBoc
CO2H
11
H2N
+
N
H
CO2Bn
12
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We commenced the synthesis of 1 by preparing terminal alkyne 8 (Scheme 2).
Carboxylic acid 11 was prepared by hydrolysis of 13 quantitatively while amine 12 was
synthesized by removal of tert-butyl group of 14 quantitatively.16 The intermolecular
condensation between 13 and 14 using 1-ethyl-3-(3-dimethylaminopropyl) carbodiimide
hydrochloride (EDCꢀHCl) as a condensation agent and 1-hydroxybenzotriazole
monohydrate (HOBtꢀH2O) then provided alkyne with Ala-Ala 8 in 92% yield.
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Scheme 2. Synthesis of 8: (a) 2N NaOH, MeOH, rt, 0.5 h, quant; (b) TFA, CH2Cl2, rt, 1 h,
quant; (c) EDCꢀHCl, HOBtꢀH2O, iPr2NEt, CH2Cl2/DMF (2:1), rt, 5 h, 92%.
O
NHBoc
BocHN
N
H
CO2Bn
CO2Bn
13
14
a
b
O
NHBoc
H2N
·
TFA
N
H
CO2Bn
CO2H
12
11
c
O
BocHN
H
N
N
H
CO2Bn
O
8
For the synthesis of iodo amino acid 9, the amino group and carboxylic acid of
-homoserine were protected with carbobenzoxy (Cbz) and Me groups to afford 15 in 65%
L
yield over two steps (Scheme 3).17 Finally, replacement of the hydroxyl group of 15 with
iodine via the Appel reaction gave 9 in 94% yield.
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Scheme 3. Synthesis of 9: (a) CbzCl, 1N NaHCO3, 40 °C, 24 h; (b) TMS-diazomethane,
hexane, MeOH, rt, 6 h, 65% (2 steps); (c) I2, PPh3, imidazole, CH2Cl2, 0 °C, 1 h, 94%.
NH2
NHCbz
CO2Me
NHCbz
CO2Me
a, b
c
HO
CO2H
-homoserine
HO
I
L
15
9
For the synthesis of 10, treatment of -aspartic acid with thionyl chloride (SOCl2)
L
in MeOH gave the β-methylated carboxylic acid; subsequent treatment with carbobenzoxy
chloride (CbzCl) and potassium carbonate (K2CO3) gave 16 in 76% yield over two steps
(Scheme 4). Protection of 16 with a tBu group using tert-butyl bromide (tBuBr), K2CO3,
and benzyltriethylammonium chloride (BnEt3NCl) in acetonitrile (MeCN) afforded 17 in
80% yield.18 Next, 17 was hydrolyzed to afford β-carboxylic acid 18 in 97% yield. The
reduction of 18 via an activated ester afforded alcohol 19 in 90% yield over two steps.19
25
Specific rotation of the obtained 19 was [α]D = -16.7 (c 0.75, EtOAc), which was good
agreement with literature value ([α]D25 = -14.0 (c 0.75, EtOAc)).20 Finally, 19 was iodinated
via the Appel reaction to give 10 in 85% yield.21
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Scheme 4. Synthesis of 10: (a) MeOH, SOCl2, rt, 25 min; (b) CbzCl, K2CO3, H2O, ether, rt,
4 h, 76% (2 steps); (c) tBuBr, K2CO3, BnEt3NCl, MeCN, rt, 5 h, then 50 °C, 26 h, 80%; (d)
2N NaOH, MeOH, rt, 3 h, 97%; (e) NMM, EtOCOCl, THF, -15 °C, 1 h; (f) NaBH4, THF,
H2O, 0 °C, 0.5 h, 90% (2 steps); (g) I2, PPh3, imidazole, CH2Cl2, 0 °C, 1 h, 85%.
a, b
NHCbz
CO2H
NHCbz
CO2tBu
NH2
CO2H
c
MeO2C
MeO2C
HO2C
L
-aspartic acid
16
17
O
NHCbz
CO2tBu
O
NHCbz
CO2tBu
d
e
HO2C
O
O
18
NHCbz
NHCbz
CO2tBu
g
f
HO
CO2tBu
I
10
19
With coupling substrates 8, 9, and 10 in hand, we attempted the C-C bond
formation between 3,5-dibromo-4-iodopyridine 7 and the corresponding alkyne and iodo
amino acids utilizing palladium-catalyzed cross-coupling reactions for the synthesis of 5
(Scheme 5). Based on established synthetic methods,11-13 the Sonogashira cross-coupling
reaction between 7 and 8 was performed using 10 mol% Pd2(dba)3, 40 mol% P(2-furyl)3,
and 40 mol% CuI in N,N-dimethylformaide (DMF) and N,N-diisopropylethylamine
(iPr2NEt) at room temperature to give monocoupled pyridine 20 in 80% yield. The
introduction of iodoalkylated -glycine at the 3,5-positions of 20 was then attempted via
L
Negishi cross-coupling using PEPPSI-IPr, as reported by Organ and co-workers.22 The
modified reaction23 was also performed using 20 mol% PEPPSI-IPr at room temperature
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for 12 h to afford tricoupled pyridine 5 in 55% yield with a trace amount of the dicoupled
product. Tricoupled compound 5 was hydrogenated with H2 and Pd/C in tert-butyl alcohol
(tBuOH) to reduce the alkyne and remove the Bn and Cbz groups; product 21 was obtained
in 61% yield. Intramolecular condensation of 21 using condensation agent
N-[1-(cyano-2-ethoxy-2-oxoethylideneaminooxy)dimethylamino(morpholino)]uranium
hexafluorophosphate (COMU)24 afforded macrocycle 22. While the use of
O-(7-aza-1H-benzotriazol-1-yl)-N,N,N’,N’-tetramethyluronium
hexafluorophosphate)
(HATU) afforded a trace amount of 22, COMU gave the better yield and the macrocycle
was successfully obtained in 26% yield.
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Scheme 5. Sonogashira and Negishi cross-coupling reactions and intramolecular
condensation: (a) Pd2(dba)3 (10 mol%), P(2-furyl)3 (40 mol%), CuI (40 mol%), iPr2NEt,
DMF, rt, 2 h, 80%; (b) Zn, TMSCl, DMF, rt, 1 h, then PEPPSI-IPr (20 mol%), rt, 12 h,
55%; (c) H2, Pd/C, tBuOH, rt, 21 h, 61%; (d) COMU, iPr2NEt, CH2Cl2/DMF (2:1), rt, 24 h,
26%.
O
BocHN
H
N
O
N
H
CO2Bn
H
N
O
O
N
H
CO2Bn
8 (1 eq)
BocHN
NHCbz
CO2Me
a
+
I
+
I
Br
Br
Br
Br
9 (5 eq)
N
N
20
7 (1 eq)
O
O
H
N
H
N
O
O
N
H
CO2Bn
N
H
CO2H
NH2
BocHN
CO2Me
b
BocHN
CO2Me
c
NHCbz
CO2Me
H2N
CO2Me
CbzHN
N
N
21
5
O
O
N
H
HN
HN
BocHN
CO2Me
d
O
H2N
CO2Me
N
22
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Next, the amino group of 22 was protected with Boc (Scheme 6).25 Then, the
reaction of pyridinium salt 2 with ω-iodoalkylated -glycine 4, which was prepared from
L
known alcohol 2312a via the Appel reaction, in nitromethane (MeNO2) at 50 °C to 60 °C for
48 h afforded 24 in 69% yield. We tried to remove the Boc, tBu, and Me groups
simultaneously using trifluoroacetic acid (TFA) at once to obtain the target compound 1.
However, residual Me groups, such as in 25, were confirmed by matrix assisted laser
desorption/ionization time-of-flight mass spectrometry (MALDI-TOFMS), indicating that
the reaction did not proceed completely. This synthetic route was thus ruled out, since
selective ester hydrolysis is difficult in the presence of amides.
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Scheme 6. Synthesis of precursors of 1: (a) (Boc)2O, NaHCO3, EtOH, rt, 1 h, quant; (b) I2,
PPh3, imidazole, CH2Cl2, 0 °C, 1 h, 52%; (c) 4, MeNO2, 50 °C, 24 h, then 60 °C, 24 h,
69%; (d) TFA, H2O, 50 °C, 12 h.
O
O
O
O
N
H
N
H
HN
HN
HN
HN
BocHN
CO2Me
BocHN
CO2Me
R
N(Boc)2
CO2tBu
a
+
O
O
4 (3 eq): R = I
23: R = OH
H2N
CO2Me
BocHN
CO2Me
b
N
22
N
2
O
O
O
O
N
H
N
H
HN
HN
HN
HN
H2N
BocHN
c
d
O
O
CO2Me
CO2Me
CO2H
BocHN
CO2Me
H2N
H2N
CO2Me
CO2tBu
N
N
(Boc)2N
diMe-protected bicyclic desmosine (25)
24
(suggested by MALDI-TOFMS)
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As an alternative strategy, introduction of iodoalkylated -glycine 10 at the
L
3,5-positions of monocoupled 20 was attempted via Negishi cross-coupling reaction
(Scheme 7). The desired tricoupled compound, 6 was obtained in 50% yield along with a
trace amount of the dicoupled compound; the reduction of 6 led to cyclization precursor 26
in 51% yield. 26 was subjected to intramolecular condensation using COMU, affording
cyclic peptide 27 in 21% yield. Protection by Boc of 27 afforded 3-protected 27 was
obtained in 56% yield; subsequent alkylation with 4 gave 28, which contains a pyridinium
moiety, in 41% yield. Finally, the Boc and tBu groups were removed using TFA to give
crude 1. Purification via C18 column chromatography afforded the desired
desmosine-containing peptide 1 quantitatively.
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Scheme 7. Synthesis of 1: (a) Zn, TMSCl, DMF, rt, 1 h, then PEPPSI-IPr (20 mol%), rt, 12
h, 50%; (b) H2, Pd/C, tBuOH, rt, 21 h, 51%; (c) COMU, iPr2NEt, CH2Cl2/DMF (2:1), rt, 24
h, 21%; (d) (Boc)2O, NaHCO3, EtOH, rt, 1 h, 56%; (e) 4 (3 eq), MeNO2, 50 °C, 24 h, then
60 °C, 24 h, 41%; (fd) TFA, H2O, rt, 2 h, quant.
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O
O
H
N
H
N
O
BocHN
CO2tBu
O
N
H
CO2Bn
N
H
CO2Bn
BocHN
NHCbz
CO2tBu
a
+
I
NHCbz
Br
Br
10 (5 eq)
CbzHN
CO2tBu
N
N
20
6
O
H
N
O
O
O
N
H
N
H
CO2H
NH2
HN
BocHN
b
BocHN
CO2tBu
c
O
CO2tBu
HN
H2N
CO2tBu
H2N
CO2tBu
N
27
N
26
O
O
O
O
N
H
N
H
HN
HN
BocHN
CO2tBu
HN
HN
BocHN
CO2tBu
d
e
O
O
BocHN
CO2tBu
BocHN
CO2tBu
CO2tBu
N
N
(Boc)2N
3
28
O
O
N
H
HN
HN
H2N
O
CO2H
CO2H
f
H2N
CO2H
N
H2N
desmosine with cyclic peptide (1)
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MALDI-TOFMS/MS analysis of the obtained 1 was then carried out. The product
ion of 1 at m/z 582584 was observed in addition to precursor ion [1]+ at m/z 651 in the
MS/MS spectra. This product ion was assumed to be [1 – Ala]+, which would be derived
from the cleavage of the peptide bonds.26 This result is expected to facilitate the elucidation
of the crosslinking structure of elastin.
Conclusion:
In summary, the synthesis of desmosine-containing cyclic peptide 1 was achieved
via chemo- and regioselective Sonogashira and Negishi cross-coupling reactions and
intramolecular condensation as key steps in 0.8% yield over 11 steps from -alanine. This is
L
the first synthesis of a cyclic peptide containing desmosine, which may resemble part of
elastin. MALDI-TOFMS/MS analysis of 1 was examined and confirmed the expected
product ion. This work is anticipated to shed light on the crosslinking structure of elastin
via MS/MS comparative analysis, which will be carried out in due course.
Experimental section:
General procedures
All non-aqueous reactions were conducted under an atmosphere of nitrogen with
magnetic stirring unless otherwise indicated. tBuOH was dried by distillation by CaH2 and
stored over activated molecular sieves. DMF was dried by distillation by MgSO4 and stored
over activated molecular sieves. iPr2NEt, N-methylmorphiline (NMM) and trimethylsilyl
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chloride (TMSCl) were distilled over CaH2. Dehydrated methanol (MeOH) and ethanol
(EtOH) were purchased from Kanto Chemicals (Tokyo, Japan) and Wako Pure Chemical
Industries (Osaka, Japan), respectively. Other solvents, such as dichloromethane (CH2Cl2),
MeCN, diethyl ether (Et2O), and tetrahydrofuran (THF) were purchased from commercial
suppliers and stored over activated molecular sieves. All reagents were obtained from
commercial suppliers and used without further purification unless otherwise stated.
N-Bromosuccinimide (NBS) was recrystallized by water. Triphenylphosphine (PPh3) was
recrystallized by EtOH. The enantiomerically pure amino acids,
L-alanine and
L-homoserine,
were purchased from Watanabe Chemical Industries, Ltd (Hiroshima, Japan) and
L-aspartic
acid was purchased from Wako Pure Chemical Industries (Osaka, Japan). Analytical thin
layer chromatography (TLC) was performed on Silica gel 60 F254 plates produced by Merck
(Darmstadt, Germany). Column chromatography was performed with acidic Silica gel 60
(spherical, 40-50 µm) or neutral Silica gel 60N (spherical, 40-50 µm) produced by Kanto
Chemicals (Tokyo, Japan). Removal of small amount of solvent was performed by Smart
Evaporator CEV1-SQ-P2 (Kanagawa, Japan).
Melting points were measured by an AS one ATM-01 apparatus. Optical rotations
were measured on a JASCO P-2200 digital polarimeter at the sodium lamp (λ = 589 nm) D
T
line and are reported as follows: [α]D (c g/100 mL, solvent). Infrared (IR) spectra were
recorded on a JASCO FT-IR 4100 spectrometer and are reported in wavenumbers (cm-1).
1H and 13C nuclear magnetic resonance (NMR) spectra were recorded on a JEOL
JNM-EXC 300 spectrometer (300 MHz) or on a JEOL JNM-ECA 500 spectrometer (500
1
MHz). H NMR data are reported as follows: chemical shift (
δ
, ppm), integration,
multiplicity (s, singlet; d, doublet; t, triplet; q, quartet; m, multiplet), coupling constants (J)
in Hz, assignments. 13C NMR data are reported in terms of chemical shift (
δ
, ppm). EI-MS
spectra were recorded on a JEOL JMS-700 instrument. FAB-MS spectra were also
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observed on the JEOL JMS-700. Electrospray ionization-mass spectrometer (ESI-MS)
spectra were recorded on a JEOL JMS-T100LC instrument. MALDI-TOFMS and
MALDI-TOFMS/MS spectra were observed on a Shimadzu AXIMA Performance
instrument on CID of 651.40 (gate: 600.00-700.00) with Reflectron Mode, Power of 70.
16-(S)-tert-Butoxycarbonylamino-pent-15-ynoic acid (11):
To a solution of 16 (523.2 mg, 1.72 mmol) in MeOH (5.0 mL) was stirred at 0 °C.
Then 1N NaOH (5.0 mL) was added dropwise at 0 °C. The whole mixture was left stirring
for 2 h at room temperature. The resulting solution was acidified with 1N HCl and
extracted with ether. The combined extracts were washed with brine, and dried over
Na2SO4. The solvent was removed in vacuo. Purification on silica gel column
chromatography (hexane/EtOAc = 3:1) yielded the product 11 as a colorless oil (368.4 mg,
20
20
1.73 mmol, quant); [α]D +45.3 (c 1.0, CHCl3) (lit value: [α]D +58.2 (c 1.0, CHCl3)27);
1H NMR (300 MHz, CDCl3)
δ
4.27 (1H, t, J = 6.0 Hz, H16), 2.76-2.60 (2H, m, H15),
13
2.36-2.35 (1H, m, H13), 1.46 (9H, s, tBu); C NMR (75 MHz, CD3OD)
δ 173.8, 157.5,
80.7, 80.1, 72.1, 53.6, 28.7, 22.8; EI-MS (m/z) calcd for C10H15NO4 [M]+ 213.10, found
212.85.
L
-Alanyl-
L
-alanine benzyl ester trifluoroacetate (12):
To a solution of 15 (3.51 g, 10 mmol) in CH2Cl2 (48 mL) was stirred at 0 °C. Then
TFA (23 mL) was added dropwise at 0 °C. The whole mixture was left stirring for 2 h at
room temperature. The solvent was removed in vacuo. Purification on silica gel column
chromatography (CH2Cl2/MeOH = 10/1) yielded the product 12 as a yellow oil (4.00 g, 10
mmol, quant); Rf 0.22 (CH2Cl2/MeOH = 10/1); [α]D25 -30.7 (c 1.0, CHCl3); IR (neat, cm-1)
1
3071, 1742, 1666, 1556, 1455, 1386, 1344, 1198, 1133, 1050, 947, 837, 799, 722, 697; H
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ACCEPTED MANUSCRIPT
NMR (300 MHz, CD3OD)
δ
7.36-7.30 (5H, m, Bn), 5.18 (1H, d, J = 12.3 Hz, Bn) , 5.12
(1H, d, J = 12.3 Hz, Bn), 4.51 (1H, q, J = 7.2 Hz, H22/25), 3.95-3.92 (1H, m, H22/25),
1.45-1.42 (3H, d, J = 8.1 Hz, H24/27), 1.42 (3H, d, J = 7.5 Hz, H24/27); 13C NMR (75
MHz, CD3OD)
δ 173.6, 171.2, 137.1, 129.7, 129.5, 129.4, 68.1, 50.1, 17.7, 17.3;
FAB-HRMS (m/z) calcd for C13H19N2O3 [M+H]+ 251.1396, found 251.1378.
26-[23-(16-tert-Butoxycarbonylamino-pent-14-ynoylamino)-propionylamino]-propioni
c acid benzyl ester (8):
To a solution of 11 (456.3 mg, 2.1 mmol, 1.0 eq) in CH2Cl2/DMF (20 mL/2 mL),
EDC·HCl (492.7 mg, 2.6 mmol, 1.2 eq) followed by HOBt·H2O (396.1 mg, 2.6 mmol, 1.2
eq) were stirred at 0 °C for 15 mins. A solution of 12 (743.2 mg, 2.1 mmol, 1.0 eq) in DMF
(8 mL) followed by iPr2NEt (1.1 mL, 6.4 mmol, 3.0 eq) were then added dropwise at 0 °C.
The whole mixture was left stirring for 5 h at room temperature. After partitioning between
CH2Cl2 and water, the organic layer was washed first with saturated NaHCO3 solution, then
0.1N HCl, brine and finally dried over Na2SO4 and evaporated. Purification on silica gel
column chromatography (hexane/EtOAc = 1:1) yielded 8 as a colorless solid (886.7 mg, 2.0
mmol, 93%); Rf 0.50 (hexane/EtOAc = 1/2); mp = 115-117 °C; [α]D 25 -32.2 (c 1.0, CHCl3);
IR (neat, cm-1) 3308, 2980, 1743, 1682, 1644, 1519, 1456, 1392, 1368, 1254, 1202, 1166,
1061, 635; 1H NMR (300 MHz, CD3OD)
δ 7.39-7.30 (5H, m, Bn), 6.82 (1H, d, J = 6.9 Hz,
NH), 6.63 (1H, br, NH), 5.26 (1H, m, NH), 5.20 (1H, d, J = 12.0 Hz, Bn), 4.59 (1H, m,
H16/22/25), 4.50 (1H, m, H16/22/25), 4.27-4.26 (1H, m, H16/22/25), 2.84-2.54 (2H, m,
H15), 2.08 (1H, t, J = 2.7 Hz, H13), 1.46 (9H, s, tBu), 1.41 (3H, d, J = 7.2 Hz, H24/27),
1.39 (3H, d, J = 7.5 Hz, H24/27); 13C NMR (75 MHz, CDCl3)
δ 172.1, 171.0, 170.9, 169.7,
135.0, 128.3, 128.1, 127.9, 78.8, 71.6, 66.9, 48.7, 48.5, 47.9, 47.8, 28.0, 22.2, 18.0, 17.8;
ESI-HRMS (m/z) calcd for C12H31N3NaO6 [M+Na]+ 468.2111, found 468.2120.
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ACCEPTED MANUSCRIPT
Methyl 20-(S)-[(benzyloxycarbonyl)amino]-18-hydroxybutyrate (17):
To a solution of -homoserine (2.40 g, 20.2 mmol, 1.0 eq) in 1N NaHCO3 (96 mL)
L
was added CbzCl (3.5 mL, 24.3 mmol, 1.2 eq). After stirring for 24 h at 40 °C, the reaction
mixture was cooled to 0 °C and acidified to pH 3 with 1N HCl. The aqueous layer was then
extracted with EtOAc. The aqueous phase was readjusted to pH 3 after each extraction.
After the combined organic layers were washed with brine, dried over Na2SO4, and
concentrated in vacuo, crude product was obtained as a colorless solid, and was used to the
next reaction without further purification.
To a solution of crude product in hexane (102 mL) and MeOH (43 mL) was added
TMS-diazomethane (2.0 M n-hexane solution; 13.2 mL, 26.3 mmol, 1.3 eq). After stirring
for 6 h at room temperature, the reaction mixture was concentrated in vacuo. Purification
on silica gel column chromatography (hexane/EtOAc = 2:1) afforded the product 17 as a
colorless oil (3.52 g, 13.2 mmol, 65% (2 steps)); Rf 0.47 (hexane/EtOAc = 1:1); IR (neat,
1
cm-1) 2949, 1737, 1694, 1540, 1437, 1349, 1271, 1227, 1056, 748, 699; H NMR (300
MHz, CDCl3)
δ 7.40-7.28 (5H, m, Bn), 5.71 (1H, d, NH), 5.16-5.07 (2H, t, J = 12.3 Hz,
Bn), 4.55 (1H, m, H20), 3.75-3.61 (5H, m, H18/Me), 2.20-2.09 (1H, m, H19), 1.78-1.68
(1H, m, H19); 13C NMR (75 MHz, CDCl3)
δ
173.1, 156.8, 136.2, 128.6, 128.3, 128.2, 67.3,
58.5, 52.6, 51.4, 35.4; ESI-HRMS (m/z) calcd for C13H17NNaO5 [M+Na]+ 290.1004, found
290.0981.
20-(S)-20-(Benzyloxycarbonyl)amino]-18-iodobutyric acid methyl ester (9):
PPh3 (4.04 g, 15.4 mmol, 2.0 eq) and imidazole (1.05 g, 15.4 mmol, 2.0 eq) were
dissolved in CH2Cl2 (26 mL) with stirring. Iodine (I2) (3.91 g, 15.4 mmol, 2.0 eq) was
added to the solution at 0 °C. Then, the solution was warmed to room temperature with
-23-

ACCEPTED MANUSCRIPT
stirring for 10 min, and was cooled to 0 °C. A solution of 17 (2.06 g, 7.71 mmol, 1.0 eq) in
CH2Cl2 (15 mL) was also added to the cooled solution. After stirring at 0 °C for 1 h, the
reaction mixture filtered through a short column of neutral silica gel eluting with
hexane/Et2O (=1:1, ca. 500 mL) and the filtrate was concentrated in vacuo. Purification on
silica gel column chromatography (hexane/EtOAc = 5:1) afforded 9 as a colorless solid
(2.72 g, 7.2 mmol, 94%); Rf 0.30 (hexane/EtOAc = 5:1); [α]D25 +15.0 (c 0.98, CHCl3) (lit28
25
value: [α]D +15.2 (c 0.99, CHCl3)); IR (neat, cm-1) 3300, 2914, 2380, 1737, 1690, 1540,
1
1435, 1349, 1296, 1271, 1226, 1165, 1142, 1052, 1019, 994, 784, 747, 697, 644; H NMR
(300 MHz, CDCl3)
δ 7.41-7.29 (5H, m, Bn), 5.31 (1H, d, NH), 5.12 (2H, s, Bn), 4.47-4.40
(1H, m, H20), 3.77 (3H, s, Me), 3.20-3.15 (2H, t, J = 7.3 Hz, H18), 2.46-2.42 (1H, m, H19),
13
2.28-2.16 (1H, m, H19); C NMR (75 MHz, CDCl3)
δ 171.8, 156.0, 136.0, 128.6, 128.4,
128.2, 67.3, 54.7, 52.8, 36.9, -0.65; EI-HRMS (m/z) calcd for C13H16N1O4I1 [M]+ 377.0124,
found 377.0118.
(S)-20-[(Benzyloxycarbonyl)amino]-19-(methoxycarbonyl)propionic acid (16):
L-Aspartic acid (10.0 g, 75.1 mmol) was added to MeOH (50 mL) and cooled to
-30 °C. SOCl2 (7.5 mL) was added dropwise to the mixture, the cooling bath was removed
and the solution was slowly warmed to room temperature. After stirring for 25 minutes,
Et2O (1.0 mL) was added to the mixture and upon cooling and shaking, the product was
precipitated as a colorless solid which was filtered immediately, washed with ice cold
diethyl ether and collected. The amine hydrochloride was obtained as a colorless solid, and
was used to the next reaction without further purification.
To the amine hydrochloride dissolved in water (310 mL) was added Et2O (120 mL),
K2CO3 (14.5 g, 105.1 mmol) and CbzCl (15 mL, 105.1 mmol). The solution was stirred at
room temperature for 4 h, the layers were separated, the aqueous layer was washed with
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ACCEPTED MANUSCRIPT
Et2O and acidified to pH 1 with 1N HCl. The resulting solution was extracted with Et2O.
The combined organic phases were dried over Na2SO4 and the solvents evaporated.
Purification on silica gel column chromatography (CH2Cl2/MeOH = 10/1) yielded the
product 16 as a colorless solid (16.0 g, 57.0 mmol, 76% (2 steps)); IR (neat, cm-1) 2945,
1
1690, 1538, 1264, 1179, 1035, 873, 747, 636; H NMR (300 MHz, CD3OD)
δ 7.36-7.28
(5H, m, Bn), 5.10 (2H, s, Bn), 4.54 (1H, dd, J = 7.2, 5.2 Hz, H20), 3.67 (3H, s, Me),
13
2.94-2.75 (2H, m, H19); C NMR (75 MHz, CDCl3)
δ 174.9, 171.6, 156.3, 136.4, 128.6,
128.3, 128.2, 67.4, 52.3, 50.2, 36.4; ESI-HRMS (m/z) calcd for C13H15NNaO6 [M+Na]+
304.0797, found 304.0772.
β-Methyl α-tert-but (20S)-N-(benzyloxycarbonyl)aspartate (17):
To a solution of 16 (980.0 mg, 3.49 mmol, 1.0 eq) in MeCN (12 mL) was added
K2CO3 (3.2 g, 23.2 mmol, 6.5 eq) and BnEt3NꢀHCl (0.81 g, 3.56 mmol, 1.0 eq). After
stirring at room temperature for 5 h, the reaction mixture was added tBuBr (4.1 mL, 36.3
mmol, 10 eq). After stirring at 50 °C for 26 h, the reaction mixture was cooled to room
temperature and added H2O and EtOAc. The aqueous layer was then extracted with EtOAc.
The combined organic layers were washed with brine, dried over Na2SO4, and concentrated
in vacuo. Purification on silica gel column chromatography (hexane/EtOAc = 5:1) afforded
the product 16 as a yellow oil (944.9 mg, 2.80 mmol, 80%); Rf 0.53 (hexane/EtOAc = 2:1);
1H NMR (300 MHz, CDCl3)
δ 7.40-7.30 (5H, m, Bn), 5.37 (1H, d, J = 7.8 Hz, NH), 5.24
(1H, d, J = 12.3 Hz, Bn), 5.18 (1H, d, J = 12.0 Hz, Bn), 4.54-4.49 (1H, m, H16), 2.83-2.67
(2H, m, H15), 2.01 (1H, t, J = 2.7 Hz, H13), 1.44 (9H, s, tBu); 13C NMR (75 MHz, CDCl3)
δ
171.1, 169.5, 155.9, 136.3, 128.5, 128.1, 128.0, 82.5, 66.9, 51.8, 50.9, 36.8, 28.3, 27.8;
EI-MS (m/z) calcd for C17H23NO6 [M]+ 337.15, found 337.10.
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ACCEPTED MANUSCRIPT
20-tert-Butyl hydrogen (S)-N-benzyloxycarbonylaspartate (18):
To a solution of 17 (2.31 g, 6.85 mmol) in MeOH (4.9 mL) was added 2N NaOH
(4.4 mL). After stirring at room temperature for 3 h, MeOH was removed at reduced
pressure and the remaining aqueous layer was washed with ether. The aqueous layer was
then acidified to pH 2 with 3N HCl and extracted with EtOAc. The combined organic
layers were washed with brine, dried over Na2SO4, and concentrated in vacuo. Purification
on silica gel column chromatography (hexane/EtOAc = 2/1) afforded the product 18 as
colorless oil (2.13 g, 6.59 mmol, 97%); IR (neat, cm-1) 1739, 1555, 1371, 1230, 1155, 1061,
1
750, 699; H NMR (300 MHz, CDCl3)
δ 7.36-7.31 (5H, m, Bn), 5.72 (1H, d, J = 7.8 Hz,
NH), 5.12 (2H, s, Bn), 4.53-4.51 (1H, m, H20), 3.06-2.83 (2H, m, H19), 1.44 (9H, s, tBu);
13C NMR (75 MHz, CDCl3)
δ
176.6, 169.6, 156.2, 136.2, 128.6, 128.2, 83.0, 67.3, 50.8,
36.8, 27.9; EI-MS (m/z) calcd for C16H21NO6 [M]+ 323.14, found 323.05.
tert-Butyl 20-(S)-[(benzyloxycarbonyl)amino]-18-hydroxybutyrate (19):
18 (10.6 g, 32.9 mmol, 1.0 eq) was dissolved in THF (150 mL) and stirred. The
solution was cooled to -15 °C, and NMM (4.0 mL, 36.2 mmol, 1.1 eq) and ethyl
chloroformate (EtOCOCl) (3.4 mL, 36.2 mmol, 1.1 eq) were added. The reaction mixture
was then stirred for 1 h. The precipitated N-methylmorpholine hydrochloride was removed
by filtration and washed with THF (10 mL). The filtrate was cooled to 0 °C and sodium
borohydride (NaBH4) (1.86 g, 49.4 mmol, 1.5 eq) was added, followed by the dropwise
addition of water (32 mL). The reaction mixture was stirred at 0 °C for 0.5 h. The resulting
solution was quenched with saturated aqueous NH4Cl, stirred for 10 min, and extracted
with EtOAc. The combined extracts were washed with brine, and dried over Na2SO4.
Purification on silica gel column chromatography (hexane/EtOAc = 2:1) yielded the
product 19 as a colorless oil (9.09 g, 29.4 mmol, 90%); Rf 0.43 (hexane/EtOAc = 1/1);
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ACCEPTED MANUSCRIPT
[α]D25 -16.7 (c 0.75, EtOAc) (lit value: [α]D25 -14.0 (c 0.75, EtOAc)29) 1H NMR (300 MHz,
CDCl3) δ 7.37-7.32 (5H, m, Bn), 5.58 (1H, m, NH), 5.15 (1H, d, J = 12.0 Hz, Bn), 5.10 (1H,
d, J = 12.3 Hz, Bn), 4.42-4.40 (1H, m, H20), 3.72-3.62 (2H, m, H18), 2.15 (1H, m, H19),
1.65-1.62 (1H, m, H19), 1.47 (9H, s, tBu); 13C NMR (75 MHz, CDCl3)
δ
171.6, 156.8,
136.1, 82.2, 67.0, 58.3, 51.8, 35.6, 27.9; ESI-HRMS (m/z) calcd for C16H23NNaO5
[M+Na]+ 332.1474, found 332.1453.
20-(S)-20-(Benzyloxycarbonylamino)-18-iodobutyric acid tert-butyl ester (10):
PPh3 (15.4 g, 58.8 mmol, 2.0 eq) and imidazole (4.0 g, 58.8 mmol, 2.0 eq) were
dissolved in CH2Cl2 (20 mL) with stirring. The solution was cooled to 0 °C and I2 (14.9 g,
58.8 mmol, 2.0 eq) was added. The solution was warmed to room temperature and cooled
to 0 °C. A solution of 19 (9.09 g, 29.4 mmol, 1.0 eq) in CH2Cl2 (30 mL) was also added.
After stirring for 1 h, the mixture was diluted with Et2O. The residue was filtered through a
short column of neutral silica gel eluting with Et2O. Purification on silica gel column
chromatography (hexane/EtOAc = 6:1) yielded the product 10 as a colorless oil (10.5 g,
20
25.0 mmol, 85%); Rf 0.61 (hexane/EtOAc = 2/1); [α]D +14.1 (c 1.0, CHCl3); IR (neat,
cm-1) 3734, 3649, 3336, 2978, 2361, 1716, 1522, 1455, 1393, 1368, 1224, 1151, 1043, 845,
1
738, 697; H NMR (300 MHz, CDCl3)
δ 7.38-7.31 (5H, m, Bn), 5.32 (1H, m, NH), 5.11
(2H, s, Bn), 4.28 (1H, m, H20), 3.18-3.14 (2H, m, H18), 2.41 (1H, m, H19), 2.18 (1H, m,
13
H19), 1.47 (9H, s, tBu); C NMR (75 MHz, CDCl3)
δ 170.4, 156.0, 136.2, 128.6, 128.3,
128.3, 82.9, 67.2, 55.4, 37.6, 20.1; EI-HRMS (m/z) calcd for C16H22INO4 [M]+ 419.0594,
found 419.0596.
26-[23-(16-tert-Butoxycarbonylamino-pent-14-ynoylamino)-propionylamino]-propioni
c acid benzyl ester (20):
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ACCEPTED MANUSCRIPT
A solution of 7 (79.2 mg, 0.22 mmol, 1.0 eq), 8 (97.3 mg, 0.22 mmol, 1.0 eq),
Pd2(dba)3 (26.3 mg, 21.8 µmol, 10 mol%), P(2-furyl)3 (20.3 mg, 87.4 µmol, 40 mol%), and
CuI (16.6 mg, 87.4 µmol, 40 mol%) in DMF (7.2 mL) was degassed by freeze/pump/thaw
techniques. iPr2NEt (1.4 mL) was added to the resulting solution. After stirring at room
temperature for 2 h, the reaction mixture was diluted with EtOAc and quenched with
saturated ammonium chloride (NH4Cl) solution. The aqueous layer was then extracted with
EtOAc. The combined organic layers were washed with brine, dried over Na2SO4, and
concentrated in vacuo. Purification by filteration and washed with Et2O yielded the product
20 as a colorless solid (119.0 mg, 0.18 mmol, 80%); Rf 0.30 (hexane/EtOAc = 1:1); IR
(neat, cm-1) 3734, 3306, 2979, 2360, 1735, 1691, 1634, 1508, 1451, 1395, 1368, 1158, 1056,
956, 754, 697; 1H NMR (300 MHz, CD3OD)
δ 7.39-7.30 (5H, m, Bn), 6.82 (1H, d, J = 6.9
Hz, NH), 6.63 (1H, br, NH), 5.26 (1H, m, NH), 5.20 (1H, d, J = 12.0 Hz, Bn), 4.59 (1H, m,
H16/22/25), 4.50 (1H, m, H16/22/25), 4.27-4.26 (1H, m, H16/22/25), 2.84-2.54 (2H, m,
H15), 2.08 (1H, t, J = 2.7 Hz, H13), 1.46 (9H, s, tBu), 1.41 (3H, d, J = 7.2 Hz, H24/27),
1.39 (3H, d, J = 7.5 Hz, H24/27); 13C NMR (75 MHz, CDCl3)
δ 172.5, 171.3, 169.8, 149.8,
135.4, 134.2, 128.7, 128.6, 128.3, 124.6, 123.4, 79.9, 79.5, 67.3, 61.8, 49.2, 48.4, 28.4, 23.6,
22.6, 18.6, 18.3; ESI-HRMS (m/z) calcd for C28H32Br2N4NaO6 [M+Na]+ 701.0586, found
701.0558.
20-Benzyloxycarbonylamino-18-(5-(20’-benzyloxycarbonylamino-20’-methoxycarbon
yl-propyl)-4-{17-[23-(26-benzyloxycarbonyl-ethylcarbamoyl)-ethylcarbamoyl]-16-tert-
butoxycarbonylamino-but-14-ynyl}-pyridin-15-yl)-butyric acid methyl ester (5):
Zinc dust (382.5 mg, 5.9 mmol, 30 eq) was placed in a nitrogen-purged 1.5 mL
Eppendorf microtube. DMF (150 µL) and TMSCl (117 ꢁL, 0.92 mmol) were added to the
microtube, and the resulting mixture was stirred vigorously for 15 min at room temperature.
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ACCEPTED MANUSCRIPT
After stirring, the solution was removed by microsyringe. The remaining solid was dried
using a hot air gun at reduced pressure. The activated zinc was then cool to room
temperature. A solution of 9 (377 mg, 1.0 mmol, 5.0 eq) in DMF (260 µL) was added to the
activated zinc and rinsed with 100 µL DMF. The reaction mixture was then stirred at room
temperature for 1 h. The insertion of zinc was monitored by TLC (hexane/EtOAc = 5/1).
After completion of the insertion, the zinc solution was allowed to settle using a centrifuge
for 1 min at room temperature. The solution was collected via a microsyringe and add to a
10 mL flask, containing PEPPSI-IPr (26.5 mg, 39.0 µmol, 20 mol%) and 16 (132.4 mg,
194.6 µmol, 1.0 eq) in DMF (500 µL). After stirring for 12 h at room temperature, the
reaction mixture was diluted with EtOAc, quenched with a saturated NH4Cl solution. The
aqueous layer was then extracted with EtOAc. The combined organic layers were washed
with brine, dried over Na2SO4, and then concentrated in vacuo to give the crude product as
yellow oil. Purification by flash silica gel column chromatography (hexane/EtOAc = 1/2→
1/4) afforded 5 as a yellow oil (108.5 mg, 106.3 µmol, 55%); Rf 0.19 (hexane/EtOAc =
1
1:2); H NMR (300 MHz, CDCl3)
δ 8.28 (2H, s, H2/6), 7.33 (15H, m, Bn), 6.99-6.77 (1H,
m, NH), 6.13-5.83 (2H, m, NH), 5.18-5.07 (6H, m, Bn), 4.50-4.45 (5H, m,
H16/20/20’/22/25), 3.73-3.66 (6H, m, Me), 2.92-2.77 (6H, m, H15/18/18’), 2.18-1.94 (4H,
m, H19/19’), 1.40 (9H, s , tBu), 1.28-1.23 (6H, m, H24/27); 13C NMR (75 MHz, CDCl3)
δ
172.4, 170.0, 162.8, 146.8, 136.4, 135.4, 128.6, 128.3, 67.2, 53.9, 52.6, 49.0, 48.3, 36.6,
32.7, 31.6, 29.8, 28.3, 23.8, 18.2, 17.8; ESI-HRMS (m/z) calcd for C54H64N6NaO14
[M+Na]+ 1043.4378, found 1043.4399.
20-Amino-18-(5-(20’-amino-20’-methoxycarbonyl-propyl)-4-{16-tert-butoxycarbonyla
mino-16-[17-(23-carboxy-ethy
lcarbamoyl)-ethylcarbamoyl]-butyl}-pyridin-15-yl)-butyric acid methyl ester (21):
-29-