Total synthesis of the COPD biomarker desmosine via stepwise Sonogashira cross-coupling reactions

Article abstract of DOI:10.1246/bcsj.20140394

Desmosine, a COPD biomarker and elastin crosslinker, is a tetrasubstituted pyridinium amino acid. In this paper, the total synthesis of desmosine is described utilizing stepwise and regioselective palladium-catalyzed Sonogashira cross-coupling reactions o

Full text of DOI:10.1246/bcsj.20140394

Total Synthesis of the COPD Biomarker Desmosine
via Stepwise Sonogashira Cross-Coupling Reactions
Haruka Yamada, Takahiro Hayashi, and Toyonobu Usuki*
Department of Materials and Life Sciences, Faculty of Science and Technology, Sophia University,
7-1 Kioicho, Chiyoda-ku, Tokyo 102-8554
E-mail: t-usuki@sophia.ac.jp
Received: December 17, 2014; Accepted: January 21, 2015; Web Released: January 27, 2015
Desmosine, a COPD biomarker and elastin crosslinker, is a tetrasubstituted pyridinium amino acid. In this paper,
the total synthesis of desmosine is described utilizing stepwise and regioselective palladium-catalyzed Sonogashira cross-
coupling reactions of trihalopyridines and terminal alkynes as key steps. The 13-step synthesis starting from 4-
hydroxypyridine was achieved in 11% overall yield.
Chronic obstructive pulmonary disease (COPD), which is a
respiratory disorder, is reported to be the third leading cause
of death worldwide in 2012 by the World Health Organi-
zation (WHO).1 The risk of developing COPD is related to
the interactions among many environmental factors including
tobacco smoking, air pollution, and possession of the gene for
α1-antitrypsin deficiency (AATD).2 Due to a lack of effective
medicines for the prevention of the progression of COPD,
survival rates have not been improved to date. Thus, intensive
drug discovery activities with the goal of identifying a cure
for the disease are underway in the pharmaceutical industry
and academia. The development of biomarkers that can act as
indicators of the severity of COPD and the level of patient
response to medicines would aid these efforts.3
Elastin fibers are found in the extracellular matrix and are
an essential structural component of the lungs, ligaments, skin,
blood vessels, etc. Desmosine (1, Figure 1) and isodesmosine
(2) exist only in the elastin matrix and are tetrasubstituted
pyridinium amino acids that serve as crosslinkers of elastin,
leading to the formation of a sophisticated three-dimensional
network.4,5 It has been proposed that the biosynthetic formation
of crosslinkers 1 and 2 occurs via Mannich reactions and aldol
condensations after oxidative transformation of the lysine of
elastin by lysyl oxidase.6 Recently, the irreversible degradation
of lung elastin that occurs in COPD has been shown to give rise
to 1 and 2, which can be measured specifically and sensitively in
clinical samples such as plasma, urine, sputum, and broncho-
alveolar lavage fluid (BALF) via liquid chromatography-mass
spectrometry (LC-MS or LC-MS/MS) analysis.7,8 Therefore,
the elastin crosslinking amino acids 1 and 2 are attractive bio-
markers for both drug discovery efforts and the rapid diagnosis
of COPD.
Recently, we reported the isotope-dilution LC-MS/MS
analysis of these biomarkers using isotopically labeled
desmosine (desmosine-d4) as an internal standard.8d The syn-
thesis of desmosine-d4 was achieved using stepwise palladium-
catalyzed Negishi and Sonogashira cross-coupling reactions.8d,9
The lanthanide-promoted Chichibabin pyridine synthesis of 1
and 2 was also achieved in our laboratory.10 Total syntheses of
the crosslinking amino acids desmopyridine and neodesmosine
have been also reported.11 In order to expand the potential
application of desmosine 1 as a COPD biomarker and elastin
crosslinker, investigation of alternative synthetic routes for
the preparation of 1 has been attempted. Herein, we report
in detail12 the total synthesis of desmosine 1 via stepwise and
regioselective palladium-catalyzed Sonogashira cross-coupling
reactions as key steps for C-C bond formation between the
pyridine core and relevant alkynes.
Results and Discussion
The target molecule 1 consists of a pyridine core and four
amino acids that have carbon chains with different lengths (C4
to C6). The Sonogashira cross-coupling13 is well known as a
useful catalytic reaction for C-C bond formation between
arylhalides and alkynes. As illustrated in the retrosynthetic
analysis in Scheme 1, it was thus envisioned that desmosine 1
would be derived from 3,4,5-tri-serial-alkynyl pyridine 3 and
ω-iodoalkylated L-glycine 414 through late-stage formation of
the pyridinium salt. Synthesis of 3 would then involve step-
wise and regioselective palladium-catalyzed Sonogashira cross-
coupling reactions between trihalogenated pyridine 5 or 6 and
the corresponding terminal alkynes 7 and 8. Trihalopyridines
5 and 6 would be obtained from commercially available 4-
hydroxypyridine (9), and protected alkynes 7 and 8 could be
Figure 1. Structures of desmosine (1) and isodesmosine (2).
Bull. Chem. Soc. Jpn. 2015, 88, 673–683 | doi:10.1246/bcsj.20140394
© 2015 The Chemical Society of Japan | 673

Scheme 1. Retrosynthetic analysis of desmosine (1).
substituent using PBr3 at 145 °C without solvent to afford 4-
bromo-3,5-diiodopyridine (5) in 98% yield. In order to increase
the reactivity of the bromine substituent, the pyridinium salt
with HCl was formed before iodination. Replacement of the
bromine substituent of 5-HCl with iodide then proceeded using
NaI in MeCN to afford 3,4,5-triiodopyridine (6) in 95% yield.16
The structures of 5 and 6 were determined unambiguously via
single-crystal X-ray analysis.
The syntheses of terminal alkynes 7 and 8 are shown in
Schemes 3 and 4, respectively. Because protected ethynyl
glycine was reported to be extremely labile,17 (R)-Garner’s
aldehyde 10 was selected as the precursor for one of the cross-
coupling substrates. Compound 10 was prepared from D-serine
according to a literature procedure in 73% yield over five steps
(Scheme 3).18 Formation of terminal alkyne 7 from aldehyde
10 via the dibromoolefin using Corey-Fuchs methodology19
did not afford the desired product. On the other hand, the
Ohira-Bestmann reagent,20 dimethyl-(1-diazo-2-oxopropyl)-
phosphonate, was found to react with (R)-Garner’s aldehyde
10 in the presence of K2CO3 in MeOH under milder conditions
to give terminal alkyne 7 in 83% yield.21
Scheme 2. Synthesis of trihalopyridines.
formed from (R)-Gernar’s aldehyde 10 and β-iodoalkylated
L-glycine 11, respectively.
Synthesis of 3,4,5-trihalopyridines 5 and 6 began with
4-hydroxypyridine (9) (Scheme 2). The 3,5-regioselective
introduction of iodide into 9 using N-iodosuccinimide (NIS)
in CCl4 gave 4-hydroxy-3,5-diiodopyridine (12) in just 21%
yield. Alternatively, treatment of 9 with I2 under acidification-
neutralization and reflux conditions gave 12 in 94% yield.15
The hydroxy group of 12 was then substituted with a bromine
For the synthesis of alkynyl amino acid 8, the intermediate
iodo amino acid 11 was prepared from L-serine in 50% yield
674 | Bull. Chem. Soc. Jpn. 2015, 88, 673–683 | doi:10.1246/bcsj.20140394
© 2015 The Chemical Society of Japan

Scheme 3. Synthesis of terminal alkyne 7.
iodo-position in 6 was considered to be more reactive than the
3-, and 5-iodo-positions, the cross-coupling reaction between
6 and 2.1 equivalent of alkyne 8 was attempted using 5 mol %
[PdCl2(PPh3)2] in the presence of 10 mol % CuI in N,N-
dimethylformamide (DMF) and i-Pr2NEt at room temperature
(Scheme 5). Unexpectedly, the 4-monoalkynylpyridine was not
obtained at all. Instead, 3-monoalkynyl pyridine 14 and 3,5-
dialkynylpyridine 15 were obtained in 52% and 22% yields,
respectively. We thus concluded that the reactivity of the
4-iodo-position in 6 was actually less than that of the 3- and/or
5-iodo-positions due to the steric hindrance of the neighbor-
ing iodine groups, which prevented oxidative addition of the
palladium catalyst.
As an alternative strategy, the Sonogashira cross-coupling
reaction between 5 or 6 and alkyne 7 was investigated
(Table 1). As shown in Entry 1, the reaction of 3,4,5-
triiodopyridine (6) and 8 equivalents of 7 in DMF and i-Pr2NEt
in the presence of a catalytic amount of [PdCl2(PPh3)2] and CuI
at room temperature for 40 h gave the desired 4-iodo-3,5-
dialkynyl product 17 in 68% yield. When 20 mol % [Pd(PPh3)4]
was used as the catalyst, the yield was improved to 80% or
better (Entries 2-5). Replacement of the catalyst alone led to an
increase in the yield to 80% (Entry 2). Increasing the reaction
temperature to 45 °C from room temperature further improved
the yield to 85% (Entry 3). Changing the ratio of DMF and
Hünig’s base from 57/1 to 5/1 enabled a reduction in the
equivalents of alkyne 7 from 8 to 3.8 and also led to a further
increase in the yield to 90% (Entries 3-5). As a side note,
when catalytic Pd(OAc)2 with phosphine ligands was used,
an inseparable tri-substituted pyridine was observed (data not
shown). Interestingly, when 4-bromo-3,5-diiodopyridine (5)
was employed in the coupling reaction with the same reagents,
the reaction time for production of 4-bromo-3,5-dialkynyl
product 16 was greatly shortened to 8 h, but the yield remained
fairly high (Entry 6), possibly resulting from the reduced steric
hindrance and/or the higher electronegativity of the 4-bromo-
group compared to that of the 4-iodo-substituent.
Scheme 4. Synthesis of terminal alkyne 8.
over three steps (Scheme 4).14 The coupling reaction of freshly
prepared organocopper compound RCu(CN)ZnI22 was accom-
plished with protected halogenated ethynes obtained from
trimethylsilylethyne.23 Use of trimethylsilyl (TMS)-protected
iodoethyne as a coupling substrate afforded the product 13
in 35% yield along with the exo-olefin side product (via β-
elimination). Using the less reactive TMS-protected bromo-
ethyne avoided the side reaction and provided 13 in 72% yield.
Treatment with tetrabutylammonium fluoride (TBAF) and
EtOH in tetrahydrofuran (THF) at 0 °C then led to the desired
terminal alkynyl amino acid 8 in 92% yield.24 When the same
reaction was run with MeOH in THF, the methyl ester by-
product resulting from ester exchange was observed along with
removal of the benzyl group.
With trihalopyridines 5 and 6 and terminal alkynes 7 and
8 in hand, the next task for achieving the total synthesis of
desmosine 1 was to conduct a studies of C-C bond formation
reactions between the pyridine core and the corresponding
alkynes via palladium-catalyzed Sonogashira cross-coupling
in a regio- and chemoselective fashion.25 First, because the 4-
Scheme 5. Initial attempt at the first Sonogashira cross-coupling reaction for the synthesis of desmosine 1.
Bull. Chem. Soc. Jpn. 2015, 88, 673–683 | doi:10.1246/bcsj.20140394
© 2015 The Chemical Society of Japan | 675

Table 1. First Sonogashira Cross-Coupling Reaction in the Synthesis of Desmosine 1
Entry
Substrate
7/equiv
Conditions
Yield/%
1
2
3
4
5
6
6
6
6
6
6
5
8.0
8.0
8.0
6.0
3.8
3.8
[PdCl2(PPh3)2] (15 mol %), DMF/iPr2NEt (57/1), rt, 40 h
[Pd(PPh3)4] (20 mol %), DMF/iPr2NEt (57/1), rt, 38 h
[Pd(PPh3)4] (20 mol %), DMF/iPr2NEt (57/1), 45 °C, 54 h
[Pd(PPh3)4] (20 mol %), DMF/iPr2NEt (5/1), 45 °C, 30 h
[Pd(PPh3)4] (20 mol %), DMF/iPr2NEt (5/1), 45 °C, 30 h
[Pd(PPh3)4] (20 mol %), DMF/iPr2NEt (5/1), 45 °C, 8 h
68 (17)
80 (17)
85 (17)
89 (17)
90 (17)
86 (16)
Table 2. Second Sonogashira Cross-Coupling Reactions in the Synthesis of Desmosine 1
Entry
Substrate
8/equiv
Conditions
Yield/%
1
2
3
4
5
6
17
17
17
17
17
16
4.0
4.0
4.0
4.0
3.0
3.0
[PdCl2(PPh3)2] (20 mol %), rt, 21 h
42
69
72
72
86
77
Pd(OAc)2 (20 mol %), P(o-tol)3 (40 mol %), rt, 22 h
Pd(OAc)2 (20 mol %), P(2-furyl)3 (40 mol %), rt, 12 h
[Pd2(dba)3] (10 mol %), P(o-tol)3 (40 mol %), rt, 26 h
[Pd2(dba)3] (10 mol %), P(2-furyl)3 (40 mol %), rt, 8 h
[Pd2(dba)3] (10 mol %), P(2-furyl)3 (40 mol %), 50 °C, 72 h
The second Sonogashira cross-coupling reaction between the
obtained dialkynyl pyridines 16 or 17 and alkynyl amino acid
8 was then investigated (Table 2). The reaction of 4-iodo-
3,5-dialkynylpyridine 17 with 20 mol % [PdCl2(PPh3)2] in the
presence of 40 mol % CuI at room temperature for 21 h gave
the desired coupling product 3 in 42% yield (Entry 1). Use
of 20 mol % Pd(OAc)2 with tri(o-tolyl)phosphine [P(o-tol)3]
and tri(2-furyl)phosphine [P(2-furyl)3] afforded 3 in 69% and
72% yields, respectively (Entries 2 and 3).26 Using 10 mol %
and deprotection of the benzyl ester, giving 18 (Scheme 6).
When the reaction was run at 50 °C for 5 days, however, the
product was completely decomposed. Re-benzylation of car-
boxylic acid 18 with benzyl bromide (BnBr) in the presence of
27
Cs2CO3 and tetrabutylammonium iodide (TBAI) afforded 19
in 65% yield. Formation of the pyridinium salt with iodo amino
acid 4, which was obtained from protected glutamic acid,14
in MeNO2 at 50 °C was then accomplished to produce 20 in
82% yield.28 Conversion of the oxazolidine rings in 20 to
the corresponding amino alcohols by treatment with p-toluene-
sulfonic acid (PTSA),29 followed by reaction with di-tert-butyl
dicarbonate [(Boc)2O] gave the diol 21 in 25% yield. How-
ever, a mixture of by-product methyl esters formed via ester
exchange was observed. Because the improvement of the yield
for this reaction was difficult, an alternative synthetic route
was investigated.
Thus, 19 was treated with PTSA to convert the oxazolidine
rings to amino alcohols, which were subsequently reacted with
(Boc)2O to afford diol 22 in 80% yield (Scheme 7). The diol
22 was oxidized under Dess-Martin periodinane (DMP)30 and
Kraus-Pinnick oxidation31 conditions to afford the carboxylic
acid 23 in 99% overall yield. Unfortunately, the dibenzylation
of 23 did not succeed, but gave a complex mixture (data not
26
[Pd2(dba)3] with 40 mol % of P(o-tol)3 and P(2-furyl)3 at
room temperature for 26 and 8 h, the desired trialkynylpyridine
3 was obtained in 72% and 86% yields, respectively (Entries 4
and 5). Interestingly again, the reactivity of the bromide was
different. When 4-bromo-3,5-dialkynylpyridine 16 was sub-
jected to this Sonogashira cross-coupling at 50 °C, the reaction
took much longer (72 h), and the product 3 was obtained in
lower yield (77%, Entry 6). This difference in reactivity is
likely due to the fact that palladium inserts more readily into
the C-I bond than into the C-Br bond during the oxidative
addition stage.
Next, hydrogenation of the obtained trialkynyl pyridine 3
using H2 and Pd/C in MeOH at room temperature for 4 days
led to quantitative conversion of the alkyne groups to alkanes
676 | Bull. Chem. Soc. Jpn. 2015, 88, 673–683 | doi:10.1246/bcsj.20140394
© 2015 The Chemical Society of Japan

Scheme 6. Synthesis of diol 21.
Scheme 7. Total synthesis of desmosine (1).
shown). Therefore, direct formation of the pyridinium salt with
hydrogenation with Pd/C and trifluoroacetic acid (TFA),
respectively, to provide crude desmosine. Purification via
reversed-phase high-performance liquid chromatography (RP-
HPLC) afforded pure 1 in 40% yield from 24. Thus, the total
iodo amino acid 4 in MeNO2 at 50 to 60 °C was accomplished,
providing 24 in 76% yield.28 The benzyl and t-butoxycarbonyl
protecting groups were then removed in a stepwise fashion via
Bull. Chem. Soc. Jpn. 2015, 88, 673–683 | doi:10.1246/bcsj.20140394
© 2015 The Chemical Society of Japan | 677

synthesis of COPD biomarker desmosine 1, a crosslinking
amino acid of elastin, was achieved in 13 steps starting from
4-hydroxypyridine (9) with an overall yield of 11%.
ment or on a JEOL JMS-700 instrument. Fast atom bombard-
ment (FAB)-MS spectra were also observed on the JEOL JMS-
700. Electrospray ionization-mass spectrometer (ESI-MS) spec-
tra were recorded on a JEOL JMS-T100LC instrument or on
a Thermo Exactive spectrometer. MS data are reported in mass-
to-charge ratio (m/z). JASCO HPLC systems PU-2085, MD-
2010, and CO-2060 were used for the purification of 1.
20
The optical rotation of synthetic 1 was observed to be ½ꢀꢀD
+7.6 (c 0.1, H2O), whereas that of natural 1 was found to be
20
½ꢀꢀD +10.0 (c 0.1, H2O). These results suggest that natural
desmosine 1 is a chiral molecule derived from the L-amino
acid, as expected. Other spectroscopic data, including NMR
spectra, were also in good agreement with those obtained for
natural 1.
1
The carbon numbering on H NMR of all compounds corre-
sponds with desmosine 1 in ref 12.
4-Hydroxy-3,5-diiodopyridine (12). To a solution of start-
ing material 4-hydroxypyridine (9) (5.01 g, 52.6 mmol, 1.0
equiv) in H2O (67 mL) was added a solution of NaOH (13.2 g,
330 mmol, 6.3 equiv) and NaOAc (40.1 g, 489 mmol, 9.3 equiv)
in H2O (167 mL). After the solution was stirred and refluxed,
powdered I2 (46.9 g, 184 mmol, 3.5 equiv) was added to the
solution. The solution was then acidified with 50% AcOH, and
neutralized with 40% NaOH. This acidification-neutralization
procedure was conducted under reflux and repeated three times
in 20 min. The forth acidification was made until free iodine
was precipitated. After sublimation of the I2 by boiling, the
residue was filtered, washed with boiling water, and then dried
to give the product 12 as a colorless powder (17.18 g, 49.5
mmol, 94%); mp 280 °C with decomposition; IR (KBr, cm¹1):
3166, 2952, 2880, 2805, 2360, 1605, 1525, 1334, 1270, 1038,
Conclusion
The total synthesis of the COPD biomarker (+)-desmosine
1, a crosslinking amino acid of elastin, has been achieved in
13 steps in 11% overall yield starting from 4-hydroxypyri-
dine (9) via stepwise and regioselective palladium-catalyzed
Sonogashira cross-coupling reactions as key steps. This
achievement will contribute to the development of method-
ology for the precise LC-MS/MS detection of desmosines as
important biomarkers for the globally prevalent disease COPD.
The synthetic strategy described above is currently being
applied to the preparation of various other amino acid cross-
linkers in order to elucidate the three-dimensional structure
of elastin fiber, including degraded elastin peptides that are
generated as a result of COPD and can be indicative of the
severity of the disease.
1
846, 746, 712, 597; H NMR (300 MHz, DMSO-d6): ¤ 11.95
(1H, s, OH), 8.27 (2H, s, H2/6); 13C NMR (75 MHz, DMSO-
d6): ¤ 170.52, 143.04, 86.59; ESI-HRMS (m/z) calcd for
Experimental
¹
C5H2I2NO [M ¹ H] 345.8231, found 345.8231.
General Procedures.
All nonaqueous reactions were
4-Bromo-3,5-diiodopyridine (5). To a solution of 12 (34.7
mg, 0.10 mmol, 1.0 equiv) was added PBr3 (1.0 mL, 10.6 mmol,
106 equiv). The solution was heated to 145 °C, and then stirred
for 4.5 h at this temperature. The mixture was cooled to room
temperature, diluted with EtOAc, and then quenched with a
NaOH aqueous solution to pH 9 at 0 °C. 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 5 as a yellow powder (40.0
mg, 0.098 mmol, 98%); Rf 0.57 (hexane/EtOAc = 5:1); mp
158-161 °C; IR (KBr, cm¹1): 3066, 1815, 1684, 1524, 1496,
conducted under an atmosphere of nitrogen with magnetic
stirring unless otherwise indicated. tert-Butanol (t-BuOH) was
dried by distillation over CaH2 and stored over activated
molecular sieves. BF3¢OEt2 was distilled over CaH2. Other sol-
vents, such as acetonitrile (MeCN), dichloromethane (CH2Cl2),
diethyl ether (Et2O), methanol (MeOH), tetrahydrofuran (THF),
and triethylamine (Et3N) 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. Analytical
thin layer chromatography (TLC) was performed on Silica gel
60 F254 plates produced by Merck. Column chromatography
was performed with acidic Silica gel 60 (spherical, 40-50
¯m) or neutral Silica gel 60N (spherical, 40-50 ¯m) produced
by Kanto Chemicals (Tokyo, Japan). Natural desmosine 1 was
purchased from Elastin Products Company, Inc (Missouri,
USA).
1
1406, 1205, 1161, 1086, 1003, 885, 725, 692, 517; H NMR
1
(300 MHz, DMSO-d6): ¤ 8.85 (2H, s, H2/6); H NMR (300
MHz, CDCl3): ¤ 8.81 (2H, s, H2/6); 13C NMR (75 MHz,
CDCl3): ¤ 156.6, 145.8, 100.7; EI-MS (m/z) calcd for
C5H2BrI2N [M]+ 408.75, found 408.85; ESI-HRMS (m/z)
calcd for C5H3BrI2N [M + H]+ 409.7538, found 409.7532.
The structure of 5 was also confirmed by single-crystal X-ray
analysis (CCDC-853164).
Optical rotations were measured on a JASCO P-2200 digital
polarimeter at the sodium lamp (- = 589 nm) D line and are
T
reported as follows: ½ꢀꢀD (c g/100 mL, solvent). Infrared (IR)
3,4,5-Triiodopyridine (6). To a solution of 5 (39.9 mg,
0.097 mmol, 1.0 equiv) in THF (0.5 mL) was added a 3 M HCl
solution, which was diluted with Et2O. After stirring for 5 min
at room temperature, the mixture was evaporated in vacuo. To
the mixture was added NaI (307.9 mg, 2.05 mmol, 21.2 equiv)
in MeCN (1.5 mL). After stirring for 24 h under reflux con-
dition, the mixture was diluted with CH2Cl2, and quenched
with 10% K2CO3 and 5% NaHSO3 solutions. The aqueous
layer was then extracted with CH2Cl2. The combined organic
layers were washed with brine, dried over Na2SO4, and concen-
trated in vacuo. Purification on silica gel column chromatog-
spectra were recorded on a JASCO FT-IR 4100 spectrometer
and are reported in wavenumbers (cm¹1). H and 13C nuclear
1
magnetic resonance (NMR) spectra were recorded on a JEOL
JNM-EXC 300 spectrometer (300 MHz) or on a JEOL JNM-
ECA 500 spectrometer (500 MHz). H NMR data are reported
as follows: chemical shift (¤, 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 (¤, ppm). Electron ionization (EI)-MS
spectra were recorded on a Shimadzu GCMS QP-5050 instru-
1
678 | Bull. Chem. Soc. Jpn. 2015, 88, 673–683 | doi:10.1246/bcsj.20140394
© 2015 The Chemical Society of Japan

raphy (hexane/EtOAc = 5:1) afforded 6 as a colorless powder
(42.0 mg, 0.092 mmol, 95%). Recrystallization of the product
from benzene or hexane gave 6 as colorless needles; Rf 0.60
(hexane/EtOAc = 5:1); mp 150-152 °C; IR (KBr, cm¹1):
3434, 3060, 1817, 1520, 1485, 1376, 1198, 1157, 1072, 885,
722, 695, 676, 504; 1H NMR (300 MHz, CDCl3): ¤ 8.79 (2H, s,
H2/6); 13C NMR (75 MHz, CDCl3): ¤ 154.3, 107.7; EI-MS
(m/z) calcd for C5H2I3N [M]+ 456.73, found 456.73; ESI-
HRMS (m/z) calcd for C5H3I3N [M + H]+ 457.7400, found
457.7391. The structure of 6 was also confirmed by single-
crystal X-ray analysis (CCDC-853163).
gel column chromatography (hexane/EtOAc = 10:1) afforded
13 as a yellow oil (1.40 g, 3.74 mmol, 72%); Rf 0.47 (hexane/
25
EtOAc = 5:1); ½ꢀꢀD +6.8 (c 0.1, CHCl3); IR (neat, cm¹1):
3442, 3373, 3091, 3066, 3034, 2960, 2927, 2856, 2179, 1949,
1748, 1717, 1500, 1456, 1367, 1344, 1251, 1215, 1165, 1051,
1029, 1008, 842, 760, 698, 641; 1H NMR (300 MHz, CDCl3): ¤
7.40-7.30 (5H, m, Bn), 5.31 (1H, d, J = 8.1 Hz, NH), 5.24 (1H,
d, J = 12.3 Hz, Bn), 5.15 (1H, d, J = 12.3 Hz, Bn), 4.54-4.48
(1H, m, H16), 2.82 (1H, dd, J = 16.8, 4.8 Hz, H15), 2.72 (1H,
dd, J = 16.8, 4.8 Hz, H15), 1.45 (9H, s, t-Bu), 0.13 (9H, s,
TMS); 13C NMR (75 MHz, CDCl3): ¤ 170.7, 155.2, 135.4,
128.7, 128.5, 128.3, 100.9, 88.6, 80.2, 67.4, 52.3, 28.4, 24.3,
0.1; EI-MS (m/z) calcd for C16H21NO4Si [M ¹ (CH3)3]+
319.12, found 319.20; FAB-MS (m/z) calcd for C20H30NO4Si
[M + H]+ 376.19, found 376.22.
2,2-(S)-Dimethyl-3-(tert-butoxycarbonyl)-4-ethynyl-
oxazolidine (7). To a solution of Garner’s aldehyde 1018
(1.28 g, 5.6 mmol, 1.0 equiv) and dimethyl-1-diazo-2-oxoprop-
yl phosphonate (1.61 g, 8.4 mmol, 1.5 equiv) in MeOH (20 mL)
was added K2CO3 (1.55 mg, 11.2 mmol, 2.0 equiv) in one por-
tion at 0 °C. After stirring for 1 h at 0 °C, then for 2 h at room
temperature, the 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 concentrated
in vacuo. Purification on silica gel column chromatography
(hexane/EtOAc = 20:1) afforded 7 as a colorless oil (1.05 g,
Benzyl (S)-2-(tert-Butoxycarbonyl)aminopent-4-ynoate
(8). To a solution of 13 (462.3 mg, 1.23 mmol, 1.0 equiv) in
THF (16.4 mL) and EtOH (358 ¯L, 6.16 mmol, 5.0 equiv)
cooled to 0 °C was added TBAF (607 ¯L, 616 ¯mol, 1.0 M
solution in THF, 0.5 equiv). After stirring at 0 °C for 1.5 h, the
reaction mixture was diluted with EtOAc, and quenched with
saturated 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. Purifica-
tion on silica gel column chromatography (hexane/EtOAc =
5:1) afforded 8 as a yellow oil (358.1 mg, 1.18 mmol, 92%);
20
4.65 mmol, 83%); Rf 0.50 (hexane/EtOAc = 5:1); ½ꢀꢀD +90.9
20
(c 0.1, CHCl3) [lit value21 of the enantiomer: ½ꢀꢀD ¹99 (c 0.96,
CHCl3)]; IR (neat, cm¹1): 3293, 3261, 2981, 2935, 2879, 2116,
20
1702, 1477, 1458, 1377, 1265, 1245, 1210, 1180, 1150, 1095,
Rf 0.43 (hexane/EtOAc = 5:1); ½ꢀꢀD +8.1 (c 0.1, CHCl3); IR
1
1067, 1055, 855, 806, 770, 676; H NMR (300 MHz, CDCl3):
(neat, cm¹1): 3390, 3298, 3090, 3066, 3034, 2978, 2933, 2123,
1955, 1455, 1428, 1155, 1696, 1506, 1062, 1022, 988, 753, 698,
¤ 4.58-4.48 (1H, m, H20), 4.05-3.97 (2H, m, CH2), 2.26 (1H,
s, H19), 1.61-1.47 (15H, m, Me/tBu); 13C NMR (75 MHz,
CDCl3): ¤ 151.5, 151.3, 94.3, 93.9, 82.7, 82.3, 80.7, 80.2, 70.5,
70.1, 68.6, 48.3, 28.3, 26.8, 25.8, 25.1, 24.3; EI-MS (m/z)
calcd for C11H16NO3 [M ¹ CH3]+ 210.11, found 210.10.
1
653; H NMR (300 MHz, CDCl3): ¤ 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,
t-Bu); 13C NMR (75 MHz, CDCl3): ¤ 170.7, 155.2, 135.3,
128.7, 128.6, 128.4, 80.3, 78.6, 71.8, 67.6, 52.2, 28.4, 23.0;
FAB-MS (m/z) calcd for C17H22NO4Si [M + H]+ 304.15,
found 304.24.
Benzyl
(S)-2-(tert-Butoxycarbonyl)amino-5-trimethyl-
silylpent-4-ynoate (13). CuCN (465.6 mg, 5.20 mmol, 1.0
equiv) and LiCl (437.4 mg, 10.3 mmol, 2.0 equiv) were dried
together by heating under vacuum at 150 °C for 2 h in one flask.
While the CuCN and LiCl were drying, zinc powder (2.09 g,
32.0 mmol, 6.2 equiv) was dried under vacuum with heating
using a heat gun over 5 min. To the flask containing the zinc
powder were added DMF (2.6 mL) and TMSCl (198 ¯L, 1.56
mmol, 5 mol % to Zn) at room temperature. After stirring for
30 min at room temperature, a solution of benzyl (S)-2-(tert-
butoxycarbonyl)amino-3-iodopropanoate (11)14 (2.10 g, 5.17
mmol, 1.0 equiv) in DMF (2.6 mL, washed with 0.3 mL © 2)
was slowly added to this suspension. After confirming the
insertion of zinc into 11 by TLC, to another flask containing
the CuCN and LiCl in DMF (10.0 mL) cooled to ¹20 °C was
added a solution of Zn-inserted 11 over 30 min to produce
Cu-11.
After stirring for 15 min at ¹20 °C, freshly prepared (2-
bromoethynyl)trimethylsilane (1.14 g, 6.44 mmol, 1.25 equiv)
was added dropwise to the reaction mixture of Cu-11 over
5 min. The mixture was then allowed to slowly warm to room
temperature and continue stirring for 16 h. The mixture was
diluted with EtOAc, and 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 concentrated in vacuo. Purification on silica
Benzyl 14-(S)-2-(tert-Butoxycarbonyl)amino-5-(4,5-di-
iodopyridin-3-yl)pent-4-ynoate (14), and Dibenzyl 5,5¤-(4-
Iodopyridin-3,5-diyl)di[(S)-2-(tert-butoxycarbonyl)amino-
pent-4-ynoate] (15). A solution of 6 (16.2 mg, 0.035 mmol,
1.0 equiv) and 8 (22.5 mg, 0.074 mmol, 2.1 equiv) in DMF (0.4
mL) and iPr2NEt (61.7 ¯L) was added CuI (0.6 mg, 3.15 ¯mol,
10 mol %) and [PdCl2(PPh3)2] (1.2 mg, 1.71 ¯mol, 5 mol %).
After stirring at room temperature for 16 h, the reaction mixture
was diluted with EtOAc, and quenched with saturated 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 on silica
gel column chromatography (hexane/Et2O = 4:1) afforded 14
(11.7 mg, 0.019 mmol, 52%) and 15 (6.4 mg, 0.008 mg, 22%),
1
respectively. 14: a yellow oil; H NMR (300 MHz, CDCl3): ¤
8.73 (1H, s, H6), 8.27 (1H, s, H2), 7.37-7.27 (5H, m, Bn), 5.51
(1H, d, J = 8.1 Hz, NH), 5.27 (1H, d, J = 12.3 Hz, Bn), 5.19
(1H, d, J = 12.0 Hz, Bn), 4.64 (1H, dt, J = 8.1, 4.8 Hz, CH),
3.08 (2H, d, J = 4.8 Hz, CH2), 1.45 (9H, s, tBu); EI-MS (m/z):
calcd for C22H22I2N2O4 [M]+ 631.97, found 632.10. 15: a
1
yellow oil; H NMR (300 MHz, CDCl3): ¤ 8.21 (2H, s, H2/6),
7.38-7.28 (10H, m, Bn), 5.53 (2H, d, J = 8.4 Hz, NH), 5.27
Bull. Chem. Soc. Jpn. 2015, 88, 673–683 | doi:10.1246/bcsj.20140394
© 2015 The Chemical Society of Japan | 679

25
(2H, d, J = 12.0 Hz, Bn), 5.19 (2H, d, J = 12.0 Hz, Bn), 4.64
(2H, dt, J = 8.1, 4.2 Hz, CH), 3.07 (4H, d, J = 4.2 Hz, CH2),
1.45 (18H, s, tBu).
as a colorless oil; Rf 0.30 (hexane/EtOAc = 1:2); ½ꢀꢀD +148.6
(c 0.1, CHCl3); IR (neat, cm¹1): 3431, 3345, 2979, 2934,
2876, 2359, 2251, 2230, 1746, 1705, 1499, 1454, 1379, 1261,
1171, 1103, 1054, 914, 844, 735, 698, 580, 542; 1H NMR
(300 MHz, CDCl3): ¤ 8.49 (1H, s, H2/6), 7.44-7.30 (5H, m,
Bn), 5.22 (1H, d, J = 12.3 Hz, Bn), 5.15 (2H, d, J = 12.3
Hz, Bn/NH), 4.95-4.81 (2H, m, H20/20¤), 4.57 (1H, m, H16),
4.11 (4H, m, H21/21¤), 3.15-3.00 (2H, m, H15), 1.66 (6H, s,
Me), 1.53 (6H, s, Me), 1.48 (18H, s, tBu), 1.44 (9H, s, tBu);
13C NMR (75 MHz, CDCl3): ¤ 170.4, 155.2, 151.4, 150.9,
135.3, 128.3, 121.4, 97.9, 95.5, 94.3, 80.9, 80.4, 78.8, 68.7,
67.3, 65.9, 52.2, 49.3, 28.4, 27.1, 25.9, 25.2, 24.5; ESI-HRMS
(m/z) calcd for C46H58N4NaO10 [M + Na]+ 849.4051, found
849.4053.
(S)-2-(tert-Butoxycarbonyl)amino-5-[3,5-bis(2-{[(S)-2,2-
dimethyl-3-tert-butoxycarbonyl-1,3-oxazolidin-4-yl]ethyl})-
pyridin-4-yl]pentanoic Acid (18). A solution of 3 (66.7 mg,
80.7 ¯mol, 1.0 equiv) in MeOH (2.9 mL) was treated with 10%
Pd/C (53.8 mg, 50.5 ¯mol, 63 mol %) and hydrogenated at
balloon pressure at room temperature. After stirring for 4 days
at room temperature, the insoluble material was separated by
filtration through neutral silica gel and Celite eluting with
MeOH. The filtrate was then concentrated in vacuo. Concen-
tration of the filtrate yielded carboxylic acid 18 (62.9 mg, 84.0
¯mol, quant) as a colorless solid; 1H NMR (500 MHz,
CD3OD): ¤ 8.16 (2H, s, H2/6), 4.05-3.90 (8H, m, NH, H16/
20/20¤), 2.84-2.63 (6H, m, H13/18/18¤), 1.97-1.95 (2H, m,
H14), 1.86-1.79 (4H, m, H19/19¤), 1.57-1.43 (41H, m, H15/
Me/tBu); ESI-MS (m/z): calcd for C39H65N4O10 [M + H]+
749.47, found 749.48.
3,5-Bis{[(S)-2,2-dimethyl-3-tert-buthoxycarbonyl-1,3-
oxazolidin-4-yl]ethynyl}-4-iodopyridine (17).
A solution
of 6 (14.8 mg, 32.4 ¯mol, 1.0 equiv), 7 (27.6 mg, 122.5 ¯mol,
3.8 equiv), [Pd(PPh3)4] (7.4 mg, 6.40 ¯mol, 20 mol %), and CuI
(2.6 mg, 13.7 ¯mol, 42 mol %) in DMF (1.6 mL, 20 ¯M to 6)
was degassed by freeze/pump/thaw techniques. iPr2NEt (320
¯L) was added to the resulting solution. After stirring at 45 °C
for 30 h, the reaction mixture was diluted with EtOAc, and
quenched with saturated 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 on silica gel column chromatography
(hexane/Et2O = 1:1) afforded 17 (19.1 mg, 29.3 ¯mol, 90%) as
25
a colorless solid; Rf 0.40 (hexane/EtOAc = 1:2); ½ꢀꢀD +148.6
(c 0.1, CHCl3); IR (neat, cm¹1): 3530, 3385, 2980, 2925, 2873,
2234, 1698, 1541, 1475, 1457, 1373, 1262, 1208, 1169, 1095,
1
1057, 945, 845, 806, 768, 672, 538, 513; H NMR (300 MHz,
CDCl3): ¤ 8.34 (1H, s, H2/6), 4.89-4.78 (2H, m, H20/20¤),
4.19-4.11 (4H, m, H21/21¤), 1.70 (6H, s, Me), 1.54 (6H,
s, Me), 1.43 (18H, s, tBu); 13C NMR (75 MHz, CDCl3): ¤
151.1, 150.0, 127.4, 95.5, 94.6, 94.1, 80.6, 68.7, 65.9, 49.2,
28.6, 27.2, 26.1, 25.3, 24.4; ESI-HRMS (m/z) calcd for
C29H38IN3NaO6 [M + Na]+ 674.1703, found 674.1693.
4-Bromo-3,5-bis{[(S)-2,2-dimethyl-3-tert-butoxycarbon-
yl-1,3-oxazolidin-4-yl]ethynyl}pyridine (16). A solution of
5 (13.6 mg, 33.1 ¯mol, 1.0 equiv), 7 (28.6 mg, 126.9 ¯mol, 3.8
equiv), [Pd(PPh3)4] (7.5 mg, 6.49 ¯mol, 20 mol %), and CuI
(2.4 mg, 12.6 ¯mol, 38 mol %) in DMF (1.6 mL, 20 ¯M to 5)
was degassed by freeze/pump/thaw techniques. iPr2NEt (320
¯L) was added to the resulting solution. After stirring at 45 °C
for 8 h, the reaction mixture was diluted with EtOAc, and
quenched with saturated 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 on silica gel column chromatography
(hexane/Et2O = 1:1) afforded 16 (17.3 mg, 28.6 ¯mol, 86%) as
Benzyl (S)-2-(tert-Butoxycarbonyl)amino-5-[3,5-bis(2-
{[(S)-2,2-dimethyl-3-tert-butoxycarbonyl-1,3-oxazolidin-4-
yl]ethyl})pyridin-4-yl]pentanoate (19). To a solution of the
obtained 18 (20.2 mg, 27.0 ¯mol, 1.0 equiv) and Cs2CO3 (29.2
mg, 8.94 mmol, 3.3 equiv) in DMF (0.27 mL) was added TBAI
(11.6 mg, 31.4 ¯mol, 1.2 equiv) and BnBr (3.4 ¯L, 28.6 ¯mol,
1.06 equiv) at ¹20 °C. After stirring for 4 h at ¹20 °C followed
by 12 h at room temperature, the mixture was diluted with
EtOAc, and quenched with saturated 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 on silica gel column chro-
matography (hexane/EtOAc = 1:3) afforded 19 (14.6 mg, 17.4
¯mol, 65%) as a colorless oil; Rf 0.48 (hexane/EtOAc = 1:5);
1
a colorless solid; H NMR (300 MHz, CDCl3): ¤ 8.44 (1H, s,
H2/6), 4.88-4.78 (2H, m, H20/20¤), 4.14 (4H, m, H21/21¤),
1.68 (6H, s, Me), 1.54 (6H, s, Me), 1.42 (18H, s, tBu); ESI-MS
(m/z) calcd for C29H38BrN3NaO6 [M + Na]+ 626.16, found
626.24.
25
Benzyl (S)-2-(tert-Butoxycarbonyl)amino-5-(3,5-bis{[(S)-
2,2-dimethyl-3-tert-butoxycarbonyl-1,3-oxazolidin-4-yl]-
ethynyl}pyridin-4-yl)pent-4-ynoate (3). A solution of 17
(16.8 mg, 25.8 ¯mol, 1.0 equiv), 8 (23.7 mg, 78.1 ¯mol, 3.0
equiv), [Pd2(dba)3] (3.2 mg, 2.62 ¯mol, 10 mol %), P(2-furyl)3
(2.5 mg, 10.8 ¯mol, 42 mol %) and CuI (2.0 mg, 10.5 ¯mol,
41 mol %) in DMF (1.29 mL, 20 ¯M to 17) was degassed by
freeze/pump/thaw techniques. iPr2NEt (22.6 ¯L) was added
to the resulting solution. After stirring at room temperature
for 8.5 h, the reaction mixture was diluted with EtOAc, and
quenched with saturated 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 silica gel column chromatography
(hexane/Et2O = 1:1) afforded 3 (18.3 mg, 22.1 ¯mol, 86%)
½ꢀꢀD ¹28.5 (c 0.07, CHCl3); IR (neat, cm¹1): 2979, 2933,
2873, 2357, 1739, 1696, 1389, 1365, 1253, 1166, 1094, 858,
1
681; H NMR (500 MHz, CDCl3): ¤ 8.20 (2H, s, H2/6), 7.33-
7.29 (5H, m, Bn), 5.25 (1H, s, NH), 5.18 (1H, d, J = 12.0 Hz,
Bn), 5.19 (1H, d, J = 12.5 Hz, Bn), 4.33 (1H, m, H16), 3.98-
3.78 (6H, H20/20¤/21/21¤), 2.54 (6H, H13, H18/18¤), 2.00-
1.94 (2H, m, H14), 1.79 (4H, m, H19/19¤), 1.59-1.44 (41H,
H15/Me/tBu); ESI-HRMS (m/z): calcd for C46H71N4O10
[M + H]+ 839.5170, found 839.5151.
4-[(S)-4-Benzyloxycarbonyl-4-(tert-butoxycarbonyl)amino-
butyl]-1-[(S)-5-benzyloxycarbonyl-5-(tert-butoxycarbonyl)-
aminopentyl]-3,5-bis(2-{[(S)-2,2-dimethyl-3-tert-butoxycar-
bonyl-1,3-oxazolidin-4-yl]ethyl})pyridinium Iodide (20).
mixture of 19 (14.6 mg, 17.4 ¯mol, 1.0 equiv) and benzyl (S)-2-
(tert-butoxycarbonyl)amino)-6-iodohexanoate (4)14 (15.6 mg,
A
680 | Bull. Chem. Soc. Jpn. 2015, 88, 673–683 | doi:10.1246/bcsj.20140394
© 2015 The Chemical Society of Japan

34.8 ¯mol, 2.0 equiv) in MeNO2 (0.6 mL) was heated at 50 °C
for 34 h. The reaction mixture was concentrated under reduced
pressure. Purification by silica gel column chromatography
(CH2Cl2/MeOH = 30:1 ¼ 20:1) yielded 20 (16.5 mg, 14.2
¯mol, 82%) as a yellowish oil; 1H NMR (CDCl3, 300 MHz): ¤
9.22 (2H, s, H2/6), 7.35 (10H, m, Bn), 5.31-5.29 (2H, m, NH),
5.16 (3H, m, H7, Bn), 4.61 (1H, m, H16), 4.28 (1H, m, H11),
3.97-3.93 (6H, m, H20/20¤/21/21¤), 2.81-2.77 (6H, m, H13/
18), 2.10-2.07 (2H, m, H19/19¤), 2.04-2.00 (4H, m, H8/19/
19¤), 1.88-1.86 (2H, m, H9), 1.77-1.72 (2H, m, H10), 1.56-
4,4¤-{4-[(S)-4-Benzyloxycarbonyl-4-(tert-butoxycarbonyl)-
aminobutyl]pyridin-3,5-diyl}di[(S)-2-(tert-butoxycarbonyl)-
aminobutanoic Acid] (23). To a solution of Dess-Martin
periodinane (79.7 mg, 188 ¯mol, 8.3 equiv) in CH2Cl2 (0.5 mL)
was slowly added a solution of 22 (17.2 mg, 22.7 ¯mol, 1.0
equiv) in CH2Cl2 (0.3 mL, washed with 0.1 mL © 2) at 0 °C.
After stirring for 2 h at room temperature, the mixture was
diluted with Et2O, quenched with an ice-cold saturated Na2S2O3
solution and an ice-cold saturated NaHCO3 solution. The
aqueous layer was then extracted with Et2O. The combined
organic layers were washed with brine, dried over Na2SO4,
and concentrated in vacuo. The dialdehyde was obtained as a
colorless oil, and was used to the next reaction without further
purification.
+
1.41 (52H, H14/15, Me, tBu); ESI-MS calcd for C64H96N5O14
[M]+ 1158.70, found 1158.76.
4-[(S)-4-Benzyloxycarbonyl-4-(tert-butoxycarbonyl)amino-
butyl]-1-[(S)-5-benzyloxycarbonyl-5-(tert-butoxycarbonyl)-
aminopentyl]-3,5-bis[(S)-3-(tert-butoxycarbonyl)amino-4-
hydroxybutyl]pyridinium Iodide (21). To a solution of 20
(16.5 mg, 14.2 ¯mol, 1.0 equiv) in MeOH (0.6 mL) was added
PTSA monohydrate (6.2 mg, 32.6 ¯mol, 2.3 equiv) at room tem-
perature. After stirring for 19 h at room temperature, Et3N (24.0
¯L, 172 ¯mol, 12 equiv) was added to the resulting solution, and
then, cooling to 0 °C, a solution of (Boc)2O (26.6 mg, 122 ¯mol,
8.6 equiv) in MeOH (0.30 mL, washed with 0.2 mL © 2) was
slowly added. After stirring for an additional 20 h at room tem-
perature, the mixture was diluted with CH2Cl2, quenched with
saturated NaHCO3 solution, extracted with CH2Cl2, washed
with brine, dried over Na2SO4, and evaporated in vacuo. Purifi-
cation on silica gel column chromatography (CH2Cl2/MeOH =
20:1 ¼ 5:1) yielded 21 as a yellow oil; ESI-MS calcd for
To a solution of the dialdehyde in tBuOH (1.0 mL) and cold
β-isoamylene (98 ¯L, 876 ¯mol, 39 equiv) was added a solu-
tion of NaH2PO4 dihydrate (50.3 mg, 322 ¯mol, 14 equiv) and
NaClO2 (47.5 mg, 415 ¯mol, 18 equiv) in deionized water (0.4
mL, washed with 0.2 mL © 2) with using a plastic disposable
syringe (not using a steel needle) at room temperature. After
stirring for 1 h at room temperature, the mixture was diluted
with EtOAc, quenched with saturated 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 on silica gel column chro-
matography (CH2Cl2/MeOH = 20:1 ¼ 1:1) afforded 23 (17.7
mg, 22.5 ¯mol, 99% over two steps) as a colorless solid;
20
Rf 0.39 (CH2Cl2/MeOH = 1:1); ½ꢀꢀD +3.1 (c 0.1, MeOH);
+
C50H88N5O14 [M]+ 1078.68, found 1078.68.
IR (neat, cm¹1): 3408, 2979, 2931, 2803, 1698, 1525, 1456,
1
Benzyl (S)-2-(tert-Butoxycarbonyl)amino-5-{3,5-bis[(S)-
3-(tert-butoxycarbonyl)amino-4-hydroxybutyl]pyridin-4-
yl}pentanoate (22). To a solution of 19 (15.3 mg, 18.2 ¯mol,
1.0 equiv) in MeOH (0.7 mL) was added PTSA monohydrate
(8.0 mg, 42.1 ¯mol, 2.3 equiv) at room temperature. After stir-
ring for 2 days at room temperature, Et3N (23.0 ¯L, 165 ¯mol,
9.0 equiv) was added to the resulting solution. The solution was
cooled to 0 °C, and then a solution of (Boc)2O (24.8 mg, 113
¯mol, 6.2 equiv) in MeOH (0.30 mL, washed with 0.15 mL ©
2) was slowly added. After stirring for an additional 17 h at
room temperature, the mixture was diluted with CH2Cl2, and
quenched with saturated NaHCO3 solution. The aqueous layer
was then extracted with CH2Cl2. The combined organic layers
were washed with brine, dried over Na2SO4, and concentrated
in vacuo. Purification by silica gel column chromatography
(CH2Cl2/MeOH = 20:1 ¼ 10:1) afforded 22 (11.1 mg, 14.6
¯mol, 80%) as a colorless oil; Rf 0.74 (CH2Cl2/MeOH = 5:1);
1368, 1252, 1165, 1028, 860, 754, 699; H NMR (300 MHz,
CD3OD): ¤ 8.16 (2H, s, H2/6), 7.35-7.32 (5H, m, Bn), 5.22
(1H, d, J = 12.3 Hz, Bn), 5.12 (1H, d, J = 12.3 Hz, Bn), 4.20
(1H, m, H16), 4.10 (2H, m, H20/20¤), 2.89 (6H, H13, H18/
18¤), 2.25-1.88 (4H, m, H19/19¤), 1.67-1.52 (4H, m, H14/15),
1.46 (18H, s, tBu), 1.45 (9H, s, tBu); 13C NMR (75 MHz,
CD3OD): ¤ 175.6, 173.7, 157.8, 157.8, 147.4, 137.3, 137.0,
129.3, 129.1, 129.0, 80.4, 80.3, 67.6, 54.8, 54.4, 34.2, 32.5,
30.4, 28.5, 28.0, 27.6, 27.0; ESI-HRMS (m/z): calcd for
C40H59N4O12 [M + H]+ 787.4129, found 787.4102.
4-[(S)-[4-Benzyloxycarbonyl-4-(tert-butoxycarbonyl)-
aminobutyl]-1-[(S)-5-benzyloxycarbonyl-5-(tert-butoxy-
carbonyl)aminopentyl]-3,5-bis[(S)-3-(tert-butoxycarbonyl)-
amino-3-carboxypropyl]pyridinium Iodide (24). A mix-
ture of 23 (7.3 mg, 9.2 ¯mol, 1.0 equiv) and benzyl (S)-2-(tert-
butoxycarbonyl)amino-6-iodohexanoate (4)14 (12.5 mg, 27.9
¯mol, 3.0 equiv) in MeNO2 (1.2 mL) was heated at 50 °C for
81 h, then warmed up to 60 °C for 19 h. The reaction mixture
was concentrated in vacuo. Purification on silica gel column
chromatography (CH2Cl2/MeOH = 20:1 ¼ 3:1) afforded 24
(7.8 mg, 7.0 ¯mol, 76%) as a yellow oil; Rf 0.48 (CH2Cl2/
25
½ꢀꢀD +37.8 (c 0.10, MeOH); IR (oil, cm¹1): 3344, 2929, 2356,
1692, 1517, 1455, 1365, 1248, 1165, 1054, 751, 716, 699, 462,
1
418, 406; H NMR (300 MHz, CD3OD): ¤ 8.16 (2H, s, H2/6),
7.34-7.30 (5H, m, Bn), 7.08 (1H, d, J = 8.7 Hz, NH), 6.55 (1H,
d, J = 7.5 Hz, NH), 5.21 (1H, d, J = 12.3 Hz, Bn), 5.11 (1H, d,
J = 12.3 Hz, Bn), 4.18 (1H, m, H16), 3.56-3.43 (6H, m, H20/
20¤/21/21¤), 2.72-2.67 (6H, m, H13/18/18¤), 1.99-1.93 (2H,
m, H14), 1.82 (2H, m, H15), 1.68-1.51 (4H, m, H19/19¤), 1.47
(27H, s, tBu); 13C NMR (75 MHz, CD3OD): ¤ 174.0, 158.3,
158.1, 148.4, 137.3, 129.6, 129.4, 129.3, 80.7, 80.1, 67.8, 65.2,
55.1, 53.5, 52.7, 34.2, 34.2, 32.9, 30.8, 28.8, 28.7, 28.0, 27.6;
ESI-HRMS (m/z): calcd for C40H63N4O10 [M + H]+ 759.4544,
found 759.4537.
20
MeOH = 1:1); ½ꢀꢀD ¹1.8 (c 0.1, MeOH); IR (neat, cm¹1):
3369, 2978, 2931, 1709, 1518, 1455, 1367, 1252, 1165, 1052,
1027, 861, 752, 699, 584; 1H NMR (300 MHz, CD3OD): ¤ 8.62
(2H, s, H2/6), 7.35-7.32 (5H, m, Bn), 5.24-5.09 (4H, m, Bn),
4.47 (2H, t, J = 6.9 Hz, H7), 4.21 (1H, m, H16), 4.12 (3H, m,
H11/20/20¤), 2.89 (6H, m, H13/18/18¤), 2.15 (2H, m, H19/
19¤), 2.02 (4H, m, H8/19/19¤), 1.76-1.66 (4H, m, H10/15),
1.60-1.57 (2H, m, H14), 1.45-1.42 (20H, m, H9/tBu);
13C NMR (75 MHz, CD3OD): ¤ 175.6, 174.2, 160.7, 158.4,
Bull. Chem. Soc. Jpn. 2015, 88, 673–683 | doi:10.1246/bcsj.20140394
© 2015 The Chemical Society of Japan | 681

143.5, 142.3, 137.5, 130.1, 130.0, 130.0, 129.9, 129.7, 129.7,
129.6, 129.5, 81.1, 81.0, 68.2, 62.2, 55.1, 54.4, 33.8, 32.9,
32.2, 31.8, 29.8, 29.1, 29.0, 28.6, 27.9, 27.6, 23.9; ESI-HRMS
(m/z): calcd for C58H84N5O16 [M]+ 1106.5908, found
1106.5976.
4-[(S)-4-Amino-4-carboxybutyl]-1-[(S)-5-amino-5-car-
boxypentyl]-3,5-bis[(S)-3-amino-3-carboxypropyl]pyridini-
um Iodide, Desmosine (1). A solution of 17 (20.5 mg, 18.5
¯mol, 1.0 equiv) in MeOH (0.3 mL) was treated with 10% Pd/
C (130.9 mg, 124 ¯mol, 6.7 equiv) and hydrogenated at balloon
pressure at room temperature. After stirring for 24 h at room
temperature, the insoluble material was separated by filtration
through a celite pad on neutral silica gel eluting with MeOH.
The filtrate was then concentrated in vacuo. Concentration of
the filtrate yielded crude tetracarboxylic acid as a colorless
solid. The product was used to the next reaction without further
purification.
A mixture of TFA and distilled water (2.0 mL, 95:5 ratio)
was added to the crude tetracarboxylic acid at room temper-
ature and stirred for 2 h. The solvent was removed on a rotary
evaporator. Purification on C18 silica gel column chromatog-
raphy (0.1% TFA in distilled water) afforded the crude product
as a colorless solid (20.2 mg). The crude product was then
purified by reversed phase HPLC. The conditions were as
follows: column, Cosmosil 5C18-AR-II (10 © 250 mm, Nacalai
Tesque, Kyoto); solvent, linear gradient of 10% MeOH and
90% H2O; flow rate, 1.5 mL min¹1; detection, 254 nm; temper-
ature, 40 °C; Rt = 6.8 min (desmosine 1). As a result, 4.7 mg of
pure desmosine 1 was obtained as a colorless amorphous in
40% over two steps.
Medicine), and Prof. Yoshiro Masuyama (Sophia University)
for their suggestions, Prof. Noriyuki Suzuki (Sophia Univer-
sity) for the crystallographic analyses and suggestions, Prof.
Kyozo Suyama (Tohoku University) for fruitful discussions,
and Ms. Rina Suzuki (Sophia University) for manuscript sug-
gestions. This work was supported by a Grant-in-Aid for
Young Scientists (B) from the Japan Society for the Promo-
tion of Science (JSPS; KAKENHI Grant Nos. 22710224 and
25750388), the Sophia University Collaborative Research
Grant, the EBARA Hatakeyama Memorial Fund (EHMF), the
Japan Gasoline Company - Saneyoshi (JGC-S) Scholarship
Foundation, the Shimadzu Science Foundation, the SEI Group
CSR Foundation, and the Naito Science Foundation.
Supporting Information
1H and 13C NMR spectra of synthetic compounds, and X-ray
crystallographic data of 5 and 6. These materials are available
free of charge on J-STAGE.
References
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Desmosine (Synthetic): Rf 0.28 [MeOH (0.1% TFA)/H2O
20
(0.1% TFA) = 1:9]; ½ꢀꢀD +7.6 (c 0.10, H2O); UV -max: 268,
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Desmosine (Natural): Rf 0.28 [MeOH (0.1% TFA)/H2O
20
(0.1% TFA) = 1:9]; ½ꢀꢀD +10.0 (c 0.10, H2O); UV -max: 268,
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Bull. Chem. Soc. Jpn. 2015, 88, 673–683 | doi:10.1246/bcsj.20140394
© 2015 The Chemical Society of Japan | 683