Total Synthesis of Isodesmosine by Stepwise, Regioselective Negishi and Sonogashira Cross-Coupling Reactions

Article abstract of DOI:10.1002/ejoc.201500449

Isodesmosine is a crosslinking pyridinium amino acid of elastin and is an attractive biomarker for the diagnosis of chronic obstructive pulmonary disease (COPD). In this paper, we describe the total synthesis of isodesmosine. The key features of this synt

Full text of DOI:10.1002/ejoc.201500449

FULL PAPER
DOI: 10.1002/ejoc.201500449
Total Synthesis of Isodesmosine by Stepwise, Regioselective Negishi and
Sonogashira Cross-Coupling Reactions
Yohei Koseki,[a] Takanori Sugimura,[a] Keita Ogawa,[a] Rina Suzuki,[a] Haruka Yamada,[a]
Noriyuki Suzuki,[a] Yoshiro Masuyama,[a] Yong Y. Lin,[b] and Toyonobu Usuki*[a]
Dedicated to Professor Koji Nakanishi on the occasion of his 90th birthday
Keywords: Isodesmosine / Cross-coupling / Negishi reaction / Sonogashira reaction / Total synthesis
Isodesmosine is a crosslinking pyridinium amino acid of elas-
tin and is an attractive biomarker for the diagnosis of chronic
obstructive pulmonary disease (COPD). In this paper, we de-
scribe the total synthesis of isodesmosine. The key features
of this synthesis are stepwise and regioselective palladium-
catalyzed Negishi and Sonogashira cross-coupling reactions
for efficient introduction of amino acid segments on the pyr-
idine ring.
Introduction
Isodesmosine (1) and desmosine (2) are pyridinium com-
pounds bearing amino acid moieties at the 1,2,3,5- and
1,3,4,5-positions, respectively, and are found only in the
elastin matrix (Figure 1).[1,2] Elastic fibers exist in vertebrate
tissues, such as those found in the lung, skin, blood vessels,
and heart, and play an important role in providing their
elasticity.[3,4] The unique functions of elastin are mainly
derived from its ability to self-assemble and form a
crosslinked structure. Elastin, the main component of elas-
tic fibers, is an extremely insoluble extracellular matrix pro-
tein that consists of soluble precursor tropoelastin mono-
mers crosslinked by amino acids. Hydrophobic domains in
the rubber-like protein make elastin easily stretchable when
an external tensile force is applied, while the stretching can
be released without external action. Therefore, the elastin
crosslinkers are significant amino acids with respect to their
elasticity.
Figure 1. Structures of isodesmosine (1) and desmosine (2).
The degradation of elastin-containing tissues that occurs
in several widely prevalent diseases, such as atherosclero-
sis,[5] aortic aneurysms,[5] cystic fibrosis,[6,7] and chronic ob-
structive pulmonary disease (COPD),[8] has been associated
with increased excretion in the urine, plasma, and other
clinical peptide samples of these two pyridinium com-
pounds. In the lungs, elastin degradation occurs with the
progression of COPD. COPD is a respiratory disease that
occurs mainly as a consequence of smoking, but also due
to α1-antitrypsin deficiency (AATD).[9] According to the
World Health Organization (WHO), COPD was the third
leading cause of death worldwide in 2012.[10] Despite the
fact that an increase in the number of COPD patients is
likely, investigation of therapeutic agents that prevent the
progression of this disease is just beginning. The develop-
ment of biomarkers that indicate the severity of COPD and
the therapeutic response of patients would aid this effort.[11]
The irreversible degradation of lung elastin that occurs
in COPD is known to give rise to amino acids, particularly
[a] Department of Materials and Life Sciences, Faculty of Science
and Technology, Sophia University,
7-1 Kioicho, Chiyoda-ku, Tokyo 102-8554, Japan
E-mail: t-usuki@sophia.ac.jp
http://www.mls.sophia.ac.jp/~usuki/index.html
[b] St. Luke’s-Roosevelt Hospistal Center, Mount Sinai School of
Medicine,
432 W 58th St, New York, NY 10019, USA
Supporting information for this article is available on the
WWW under http://dx.doi.org/10.1002/ejoc.201500449.
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Total Synthesis of Isodesmosine
isodesmosine (1) and desmosine (2), which can be measured
specifically and sensitively in clinical urine and plasma sam-
ples by liquid chromatography–mass spectrometry (LC-MS
or LC-MS/MS) analysis.[12,13] Therefore, the elastin
crosslinking amino acids 1 and 2 are attractive biomarkers
for both drug discovery efforts and the rapid diagnosis of
COPD.
Recently, the total synthesis of desmosine (2) starting
from 4-hydroxypyridine or 3,5-dibromopyridine was
achieved in our laboratory.[14] The synthesis relied on step-
wise palladium-catalyzed cross-coupling reactions between
the pyridine cores and the corresponding segments as key
steps. The biomimetic total synthesis by a lanthanoid-pro-
moted Chichibabin pyridine synthesis has also been re-
ported.[15] Herein, the total synthesis of isodesmosine (1)
through stepwise and regioselective Sonogashira and Negi-
shi cross-coupling reactions between 2,3,5-trihalogenated
pyridines and the corresponding amino acids is described.
Results and Discussion
As illustrated in Scheme 1, the quaternary pyridinium
salt would be formed at the last stage by N-alkylation of
precursors 3 or 4 with iodo amino acid segment 5.[16] Syn-
thesis of 3 or 4 would involve palladium-catalyzed Sonoga-
shira and/or Negishi cross-coupling reactions of trihalogen-
ated pyridines 6 or 7 and the corresponding iodoalkylated
l-glycines 8[16] and 9[16] or terminal alkyne 10.[14a,14c] Be-
cause target 1 has substituents with different carbon chain
lengths at different positions of the pyridine ring, it is an
asymmetric molecule, and thus stepwise, regioselective reac-
tions are necessary. The key strategy of the present synthesis
was to utilize the different reactivities of each compound as
determined by the types of halogen substituents and their
positions on the pyridine ring.
Our synthesis commenced with the preparation of tri-
halogenated pyridines 6 and 7 starting from 2-hydroxypyr-
idine (11) (Scheme 2). The 3,5-regioselective iodination of
11 with I2 under reflux conditions gave 2-hydroxy-3,5-di-
iodopyridine (12) in 95% yield.[17] The hydroxy group of
Scheme 1. Retrosynthesis of 1.
12 was then converted into the bromide using phosphorus cies, palladium-catalyzed cross-coupling reactions with 6
tribromide (PBr3) at 145 °C to afford 2-bromo-3,5-diiodo- were investigated. First, the Negishi cross-coupling reaction
pyridine (6) in 95% yield. Interestingly, the bromination did between 2-bromo-3,5-diiodopyridine (6) and amino acid
not proceed with N-bromosuccinimide (NBS). Replacement segment 8, which was prepared from 5-benzyloxyl-(S)-4-
of the bromide of 6·HCl with iodide then proceeded using [(tert-butoxycarbonyl)amino]-5-oxopentanoic acid,[16] was
sodium iodide (NaI) to produce 2,3,5-triiodopyridine (7) in performed (Scheme 3). After insertion of zinc into the pro-
59% yield.[18] The structures of 6 and 7 were determined tected γ-iodoalkylated l-glycine 8, the excess zinc was re-
unambiguously by single-crystal X-ray analysis.
moved from the organozinc reagent by centrifugation.[19]
In order to achieve high regioselectivity and chemoselec- The cross-coupling reaction was then conducted by
tivity based on the different reactivities of the halogen spe- using 4 mol-% tris(dibenzylideneacetone)dipalladium(0)
Scheme 2. Synthesis of trihalogenated pyridines 6 and 7.
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Scheme 3. First attempts at stepwise cross-coupling reactions using 6.
[Pd2(dba)3] and 16 mol-% tri(2-furyl)phosphine [P(2- (2D-NMR) spectroscopy, including heteronuclear multiple
furyl)3][20] at room temperature in N,N-dimethylformamide bond correlation (HMBC), and mass spectrometry (MS).
(DMF) for 1 h to afford the desired 3,5-dicoupled product
Next, to obtain the trialkylated pyridine 3, the second
13 in 47% yield. Chemo- and regioselective incorporation Negishi cross-coupling reaction between 13 and δ-iodoalk-
of amino acid segment 8 into the 3- and 5-positions was ylated l-glycine 9, which was prepared from (S)-4-benzyl-
confirmed by two-dimensional nuclear magnetic resonance oxy-3-[(tert-butoxycarbonyl)amino]-4-oxobutanoic acid,[16]
Table 1. First Negishi cross-coupling reaction of 7 and 8.
Entry
Pd cat. (mol-%)
Ligand (mol-%)
Yield of 14 [%]
Yield of 15 [%]
Yield of 16 [%]
1
2
3
4
5
Pd-PEPPSI-IPr (4)
Pd(PPh3)4 (8)
Pd2(dba)3 (4)
Pd2(dba)3 (4)
Pd2(dba)3 (4)
–
–
trace
35
16
46
12
0
0
9
11
0
0
0
8
5
4
P(2-furyl)3 (16)
P(2-furyl)3 (32)
P(o-tol)3 (32)
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Total Synthesis of Isodesmosine
was attempted under the conditions of 10 mol-% Pd2(dba)3 (Table 1). First, the Negishi cross-coupling reaction be-
and 40 mol-% P(2-furyl)3 (Scheme 3). However, the reaction tween 7 and 8 was investigated. The reaction of 2,3,5-tri-
did not proceed, and starting material 13 was recovered in iodopyridine (7) with the pure organozinc reagent derived
53% yield. Therefore, the use of alkynyl amino acid seg- from γ-iodoalkylated l-glycine 8[16] was performed by
ment 10, an amino acid with less steric hindrance, was con- using 4 mol-% Pd-PEPPSI-IPr[21] from 0 °C to room tem-
sidered. Thus, the Sonogashira cross-coupling reaction of perature for 5 h to afford a trace amount of the desired
compound 13 and alkyne 10, which was prepared from l- product 14 (Entry 1). When the reaction was conducted by
serine,[14a,14c] was attempted by using 10 mol-% Pd2(dba)3, using 8 mol-% tetrakis(triphenylphosphine)palladium(0)
40 mol-% P(2-furyl)3, and 40 mol-% copper iodide (CuI) in [Pd(PPh3)4], the monocoupled product 14 was obtained in
DMF and diisopropylethylamine (iPr2NEt) at 50 °C for 1 h 35% yield (Entry 2). Investigation of different catalyst li-
with the hope of obtaining 2,3,5-tricoupled pyridine 4 gands was then performed by using 4 mol-% Pd2(dba)3 as
[20]
(Scheme 3). Instead, however, the homocoupling of 10 oc- the catalyst (Entries 3–5). With 16 mol-% P(2-furyl)3
as
curred quantitatively, and 100% of the starting material 13 the ligand, the desired product 14 was obtained in 16%
was recovered. These results suggested that oxidative ad- yield, along with 2,5-dicoupled product 15 and 3,5-dicou-
dition of palladium to the C–Br bond at the 2-position of pled product 16 in 9% and 8% yield, respectively (Entry 3).
13 may be prevented due to the lower reactivity of the The obtained products 14, 15, and 16 were separated by
bromide and the steric hindrance of the alkyl chain includ- silica gel column chromatography, and the structures of 14
ing the sp3 carbon atom at the 3-position. This synthetic and 15 were confirmed by spectroscopic analysis (HMBC
route was thus ruled out.
and MS). Both compounds 14 and 16 were found to be
An alternative, non-chemoselective strategy was then useful for the total synthesis of isodesmosine (1) (see be-
pursued by using 2,3,5-triiodopyridine 7 instead of 6 low). When the amount of P(2-furyl)3 was increased to
Scheme 4. Attempted Negishi cross-coupling reaction of 14 and 9.
Table 2. Sonogashira cross-coupling reaction of 10 and 14.
Entry
10 [equiv.]
Yield of 18 [%]
Yield of 19 [%]
Yield of 20 [%]
1
2
2.4
1.4
31
43
4
16
44
34
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T. Usuki et al.
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32 mol-%, the yield of 14 was improved to 46%, with 15
and 16 obtained in 11% and 5% yield, respectively (En-
try 4). With tri(o-tolyl)phosphine [P(o-tol)3][22] as the li-
gand, the yield decreased to 12% (Entry 5). The preferential
formation of the desired compound 14 may occur, because
the reactivity at the 5-position in 2,3,5-triiodoyridine (7) is
probably higher than that at the other ring positions.
Next, because the 2-position of the pyridine ring was
considered to be more reactive than the 3-position,[23] the
cross-coupling reaction of 14 at the 2-position with sub-
strates of appropriate carbon chain lengths was investi-
gated. The regioselective Negishi cross-coupling reaction
between 2-position of 14 and iodo amino acid 9 was consid-
ered first (Scheme 4). When the reaction was run by using
8 mol-% Pd2(dba)3 and 16 mol-% P(2-furyl)3 from 0 °C to
50 °C for 48 h, the desired product 17 was not observed,
and a complex mixture was obtained with no recovery of
starting materials. This result suggested that the additional
CH2 group in the alkyl chain may have prevented the cross-
coupling reaction.
Sonogashira cross-coupling at the 2-position of 14 with
less hindered alkyne 10 was then investigated (Table 2).
When 2.4 equimolar amounts of 10 were used in the pres-
ence of 20 mol-% Pd(PPh3)4 and 40 mol-% CuI in iPr2NEt
and DMF at room temperature, the desired product 18 was
obtained in 31% yield (Entry 1). In addition, the undesired
dicoupled product 19 and tricoupled product 20 were also
obtained in 4% and 44% yield, respectively. In order to
avoid the generation of 20, a smaller excess of 10 was used
(1.4 equimolar; Entry 2), and the yield of 18 was improved
to 43%, although undesired 20 was still formed in 34%
yield. However, as expected, the desired product 18 was ob-
tained in higher yield than the undesired compound 19.
Scheme 5. Synthesis of 4.
Incorporation of γ-iodoalkylated l-glycine 8 into the 3- the dicoupled product 16 (a byproduct in the first Negishi
positon of 18 was then attempted by a Negishi cross-cou- cross-coupling reaction, see Table 1) was performed by
pling reaction using 20 mol-% Pd-PEPPSI-IPr (Scheme 5). using 8 mol-% Pd2(dba)3, 32 mol-% P(2-furyl)3, and
The desired tricoupled pyridine 4 was obtained in 54% 32 mol-% CuI at room temperature. In this manner, by con-
yield. Compound 4 was also obtained in 65% yield when trolling the reactivities of the trihalogenated pyridines with
the Sonogashira cross-coupling reaction of alkyne 10 and the corresponding coupling substrates, the desired pyridine
Scheme 6. Total synthesis of isodesmosine 1.
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Total Synthesis of Isodesmosine
data are reported in terms of chemical shift (δ, ppm). The carbon
numberings for the compound names and the assignment of the
NMR peaks due to compound 1 were adapted from ref.[15] Electron
ionization (EI) mass spectra were recorded with a JEOL JMS-700
instrument. Electrospray ionization (ESI) mass spectra were re-
corded with a JEOL JMS-T100LC instrument.
4 bearing amino acid substituents with the appropriate
carbon chain lengths at the correct positions was success-
fully prepared.
Formation of the pyridinium salt of 4 with ω-iodoalkyl-
ated l-glycine 5, which was obtained from (S)-5-benzoxy-4-
[(tert-butoxycarbonyl)amino]-5-oxopentanoic acid,[16] was
then conducted in nitromethane (MeNO2) at 70 °C to af-
ford 21 in 45% yield (Scheme 6).[24] After reduction of the
benzyl (Bn) and alkyne groups with H2 and Pd/C, the tert-
butoxycarbonyl (Boc) protecting groups were removed by
using trifluoroacetic acid (TFA) to give crude 1. Purifica-
tion of the crude product by reverse-phase high-perform-
ance liquid chromatography (RP-HPLC) afforded pure iso-
desmosine (1) in 47% yield over two steps.
2-Hydroxy-3,5-diiodopyridine (12): To a solution of 2-hydroxypyr-
idine (11) (2.47 g, 26.0 mmol, 1.0 equiv.) in H2O (33 mL) was added
a solution of NaOH (6.55 g, 164 mmol, 6.3 equiv.) and NaOAc
(19.9 g, 242 mmol, 9.3 equiv.) in H2O (82 mL). The solution was
stirred and refluxed, and then powdered I2 (23.0 g, 90.6 mmol,
3.5 equiv.) was added. The solution was subsequently acidified with
50% AcOH and then neutralized with 40% NaOH. This acidifica-
tion-neutralization procedure was performed under reflux condi-
tions and repeated three times within 20 min. In the third acidifica-
tion step, 50% AcOH was added until free iodine was generated.
After removal of excess I2 by boiling, the residue was filtered,
washed with boiling water, and dried to give product 12 as a color-
less powder (8.6 g, 24.8 mmol, 95%); m.p. 197–215 °C. IR (KBr):
Conclusions
ν = 3443, 2683, 1632, 1523, 865, 622, 553 cm–1. 1H NMR
The total synthesis of isodesmosine (1), a crosslinking
amino acid of elastin and a COPD biomarker, was achieved
in 1.2% yield over nine steps via 18, which was prepared
from 2-hydroxypyridine (11). The key transformations were
the stepwise, regioselective Sonogashira and Negishi cross-
coupling reactions of triiodopyridine 7 and the correspond-
ing coupling substrates. This achievement should contribute
to the elucidation of the crosslinking structure of elastin
and the development of COPD biomarkers. The synthetic
strategy described above may also be useful for the prepara-
tion of various other substituted pyridines in a regioselec-
tive fashion.
˜
(300 MHz, [D6]DMSO): δ = 12.12 (s, 1 H, OH), 8.21 (d, J = 2.4 Hz,
1 H, H6), 7.70 (d, J = 2.4 Hz, 1 H, H4) ppm. 13C NMR (125 MHz,
[D6]DMSO): δ = 158.8, 155.0, 141.4, 94.6, 65.1 ppm. ESI-HRMS:
calcd. for C5H2ONI2 [M – H]– 345.8231, found 345.8230.
2-Bromo-3,5-diiodopyridine (6): Compound 12 (70.4 mg, 0.2 mmol,
1.0 equiv.) was added to a solution of PBr3 (1.5 mL, 16.0 mmol,
80 equiv.), and the mixture was heated to 145 °C and stirred at
this temperature for 4.5 h. The solution was then cooled to room
temperature, diluted with ethyl acetate (EtOAc), and neutralized
with an aqueous NaOH solution at 0 °C. The aqueous layer was
then extracted with EtOAc. The combined organic layers were
washed with brine, dried with Na2SO4, and concentrated in vacuo
to afford 6 as an orange powder (78.8 mg, 0.19 mmol, 95%); Rf =
0.80 (hexane/EtOAc = 2:1); m.p. 83 °C. IR (KBr): ν = 1636, 1523,
˜
1405, 1385, 1344, 1181, 1126, 1004, 889, 755, 707, 637, 511 cm–1.
1H NMR (300 MHz, CDCl3): δ = 8.52 (d, J = 2.1 Hz, 1 H, H6),
8.39 (d, J = 2.1 Hz, 1 H, H4) ppm. 13C NMR (75 MHz, CDCl3):
δ = 155.2, 154.8, 147.6, 100.9, 91.3 ppm. EI-HRMS: calcd. for
C5H2BrI2N [M]+ 408.7460, found 408.7455. CCDC-1057744 (for
6) contains the supplementary crystallographic data for this paper.
These data can be obtained free of charge from The Cambridge
Crystallographic Data Centre via www.ccdc.cam.ac.uk/data_
request/cif.
Experimental Section
General Procedures: All non-aqueous reactions were conducted un-
der nitrogen with magnetic stirring by using freshly distilled sol-
vents unless otherwise indicated. Tetrahydrofuran (THF) was dis-
tilled from sodium/benzophenone ketyl and/or stored over acti-
vated molecular sieves. iPr2NEt, acetonitrile (MeCN), and MeNO2
were distilled and/or stored over activated molecular sieves. DMF
was distilled from MgSO4 and stored over activated molecular
sieves. All reagents were obtained from commercial suppliers and
used without further purification unless otherwise stated. The en-
antiomerically pure (Ͼ99.5%) amino acids used as starting materi-
als were purchased from Watanabe Chemical Industries, Ltd (Hiro-
shima, Japan). Analytical thin layer chromatography (TLC) was
performed on Silica gel 60 F254 plates produced by Merck KGaA
(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 Chemical, Ltd
(Tokyo, Japan). Melting points were determined with an AS ONE
ATM-01 apparatus. Optical rotations were determined with a JA-
SCO P-2200 digital polarimeter at the sodium lamp D line (λ =
589 nm) and are reported as follows: [α]TD (c g/100 mL, solvent).
Infrared (IR) spectra were recorded with a JASCO FT-IR 4100
2,3,5-Triiodopyridine (7): To a solution of 6 (1.43 g, 3.49 mmol,
1.0 equiv.) in THF (17.5 mL) was added 4 m HCl in Et2O
(1.05 mL). After stirring at room temperature for 5 min, the mix-
ture was concentrated in vacuo. NaI (10.46 g, 69.80 mmol,
20 equiv.) in MeCN (35 mL) was then added to the mixture. After
stirring under reflux conditions for 30 h, 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 dried with Na2SO4 and concentrated
in vacuo. Purification by silica gel column chromatography (hex-
ane/EtOAc = 10:1) followed by recrystallization from hexane af-
forded 7 as a colorless powder (945.5 mg, 2.07 mmol, 59%); Rf =
0.80 (hexane/EtOAc = 2:1); m.p. 100 °C. IR (KBr): ν = 1515, 1378,
˜
spectrometer and are reported in wavenumbers (cm–1). H and 13C
1338, 1214, 1111, 995, 887, 743, 701, 634, 501 cm–1. 1H NMR
(300 MHz, CDCl3): δ = 8.51 (d, J = 2.1 Hz, 1 H, H6), 8.29 (d, J =
1.8 Hz, 1 H, H4) ppm. 13C NMR (75 MHz, CDCl3): δ = 154.7,
153.4, 128.7, 109.4, 92.4 ppm. EI-HRMS: calcd. for C5H2I3N
[M]+ 456.7322, found 456.7325. CCDC-1057745 (for 7) contains
the supplementary crystallographic data for this paper. These data
1
NMR spectra were recorded with a JEOL JNM-EXC 300 or a
JEOL JNM-ECA 500 spectrometer. 1H NMR spectroscopic data
are reported as follows: chemical shift (δ, ppm), integration, multi-
plicity (s, singlet; d, doublet; t, triplet; q, quartet; m, multiplet),
coupling constants (J) in Hz, assignments. 13C NMR spectroscopic
Eur. J. Org. Chem. 2015, 4024–4032
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T. Usuki et al.
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can be obtained free of charge from The Cambridge Crystallo-
graphic Data Centre via www.ccdc.cam.ac.uk/data_request/cif.
903, 862, 751, 698, 603, 496 cm–1. H NMR (300 MHz, CDCl3): δ
= 8.08 (d, J = 2.4 Hz, 1 H, H6), 7.74 (d, J = 2.1 Hz, 1 H, H4),
7.42–7.31 (m, 5 H, Bn), 5.18 (dd, J = 36.0, 12.3 Hz, 2 H, Bn), 5.15–
5.06 (m, 1 H, NH), 4.43–4.33 (m, 1 H, H24), 2.59–2.35 (m, 2 H,
H22), 2.16–2.03 (m, 2 H, H23), 1.95–1.81 (m, 1 H, H23), 1.44 (s,
9 H, tBu) ppm. 13C NMR (75 MHz, CDCl3): δ = 172.0, 155.3,
149.0, 146.6, 137.1, 135.2, 128.9, 128.7, 128.6, 128.5, 127.0, 107.8,
80.4, 67.5, 53.1, 33.9, 28.4, 27.7 ppm.
15: Rf = 0.30 (hexane/EtOAc = 2:1). [α]2D5 = –6.2 (c = 1.0 in MeOH).
1H NMR (300 MHz, CDCl3): δ = 8.20 (s, 1 H, H6), 7.78 (s, 1 H,
H4), 7.41–7.30 (m, 10 H, Bnϫ2), 5.41–5.28 (m, 1 H, NH), 5.26–
5.07 (m, 5 H, Bnϫ2/NH), 4.51–4.40 (m, 1 H, H16), 4.43–4.33 (m,
1 H, H24), 3.04–2.95 (m, 2 H, H13), 2.60–2.44 (m, 2 H, H22), 2.33–
2.19 (m, 1 H, H15), 2.16–2.00 (m, 2 H, H15/23), 1.95–1.81 (m, 1
H, H23), 1.44 (s, 18 H, tBuϫ2) ppm. 13C NMR (75 MHz, CDCl3):
δ = 172.5, 172.1, 159.2, 155.6, 155.4, 149.1, 146.9, 135.8, 135.5,
135.3, 128.9, 128.8, 128.7, 128.6, 128.5, 128.4, 128.2, 96.3, 80.4,
80.0, 67.4, 67.2, 53.5, 53.2, 36.6, 34.1, 31.2, 28.5, 28.4, 27.8 ppm.
ESI-HRMS: calcd. for C37H46IN3NaO8 [M + Na]+ 810.2227,
found 810.2215.
16: Rf = 0.44 (hexane/EtOAc = 2:1). 1H NMR (300 MHz, CDCl3):
δ = 7.94 (s, 1 H, H6), 7.42–7.28 (m, 10 H, Bnϫ2), 7.06 (s, 1 H, H4),
5.41–5.28 (m, 1 H, NH), 5.26–5.05 (m, 5 H, Bnϫ2/NH), 4.47–4.29
(m, 1 H, H20/24), 2.73–2.39 (m, 2 H, H19/23), 2.19–1.99 (m, 2 H,
H18/22), 1.95–1.80 (m, 1 H, H18/22), 1.45, 1.44 (s, 18 H, tBuϫ2)
ppm. 13C NMR (75 MHz, CDCl3): δ = 172.1, 155.4, 148.7, 140.7,
137.0, 136.2, 135.3, 135.3, 128.9, 128.8, 128.7, 128.6, 121.8, 80.3,
67.4, 53.2, 34.8, 32.9, 31.7, 28.5, 28.4, 28.0 ppm. ESI-HRMS: calcd.
for C37H46IN3NaO8 [M + Na]+ 810.2227, found 810.2203.
1
Benzyl (S)-18-[5-{(S)-24-Benzyloxycarbonyl-24-(tert-butoxycarb-
onyl)aminopropyl}-2-bromopyridin-1-yl]-20-[(tert-butoxycarbonyl)-
amino]butanoate (13): Zinc dust (200.3 mg, 3.1 mmol, 33 equiv.)
was placed in a nitrogen-purged 1.5 mL microtube, and then DMF
(150 μL) and trimethylsilyl chloride (60 μL, 0.47 mmol, 5.0 equiv.)
were added to the tube. After stirring vigorously at room tempera-
ture for 15 min, the liquid was removed by using a microsyringe,
and the remaining solid was dried by using a hot-air gun under
reduced pressure. The activated zinc was then cooled to room tem-
perature, and a solution of benzyl (S)-2-[(tert-butoxycarbonyl)-
amino]-3-iodobutanoate (8) (210.6 mg, 0.5 mmol, 5.3 equiv.) in
DMF (150 μL and rinsed with 50 μL ϫ2) was added. After stirring
at room temperature for 1 h, the zinc duct was settled by using a
centrifuge separator. The liquid was then removed from the acti-
vated zinc by using a microsyringe with DMF (200 μL) and added
to a 10 mL flask containing Pd2(dba)3 (4.8 mg, 3.9 μmol, 4 mol-
%), P(2-furyl)3 (4.0 mg, 17.2 μmol, 18 mol-%), and 2-bromo-3,5-
diiodopyridine (6) (38.3 mg, 93.5 μmol, 1.0 equiv.). After stirring at
0 °C for 2 h and then at room temperature for 1 h, the reaction
mixture was diluted with EtOAc and quenched with brine. The
aqueous layer was extracted with EtOAc, and then the combined
organic layers were dried with Na2SO4 and concentrated in vacuo.
Purification by silica gel column chromatography (hexane/EtOAc
= 2:1 Ǟ 1:1) afforded 13 as a yellow oil (32.2 mg, 43.5 μmol, 47%);
1
Rf = 0.35 (hexane/EtOAc = 1:2). H NMR (300 MHz, CDCl3): δ
= 7.95 (d, J = 2.4 Hz, 1 H, H6), 7.16 (d, J = 2.3 Hz, 1 H, H4),
7.41–7.32 (m, 10 H, Bnϫ2), 5.24–5.08 (m, 6 H, Bnϫ2/NH), 4.39
(m, 2 H, H20/24), 2.71–2.59 (m, 2 H, H18), 2.59–2.46 (m, 2 H,
H22), 2.01–2.00 (m, 2 H, H19/23), 1.98–1.81 (m, 2 H, H19/23), 1.45
(s, 9 H, tBu), 1.44 (s, 9 H, tBu) ppm. 13C NMR (75 MHz, CDCl3):
δ = 172.2, 155.5, 147.9, 141.8, 138.9, 137.1, 136.0, 135.4, 135.3,
128.9, 128.8, 128.7, 80.3, 67.4, 53.3, 53.2, 34.1, 32.4, 31.2, 28.5,
28.0 ppm. ESI-MS: calcd. for C37H46BrN3NaO8 [M + Na]+ 762.24,
found 762.24.
Benzyl (S)-13-[5-{(S)-24-Benzyloxycarbonyl-24-(tert-butoxy-
carbonyl)aminopropyl}-3-iodopyridin-1-yl]-16-[(tert-butoxycarbon-
yl)amino]pent-14-ynoate (18): A solution of 14 (15.6 mg, 25.1 μmol,
1.0 equiv.), 10 (10.9 mg, 35.9 μmol, 1.4 equiv.), Pd(PPh3)4 (5.8 mg,
5.02 μmol, 20 mol-%), and CuI (1.8 mg, 9.45 μmol, 38 mol-%) in
DMF (1.2 mL, 20 μm) was degassed by using the freeze/pump/thaw
technique. Next, iPr2NEt (250 μL) was added, and the resulting
solution was stirred at room temperature for 2.5 h. The reaction
mixture was then diluted with EtOAc and quenched with a satu-
rated NH4Cl solution. The aqueous layer was extracted with
EtOAc, and the combined organic layers were washed with brine,
dried with Na2SO4, and concentrated in vacuo. Purification by sil-
ica gel column chromatography (hexane/EtOAc = 2:1) afforded 18
(8.5 mg, 10.7 μmol, 43 %) as a yellow oil, 19 (3.2 mg, 4.0 μmol,
16%) as a yellow oil, and 20 (8.5 mg, 8.7 μmol, 34%) as a yellow
oil.
18: Rf = 0.26 (hexane/EtOAc = 2:1). [α]2D5 = –4.5 (c = 0.9, MeOH).
1H NMR (300 MHz, CDCl3): δ = 8.24 (s, 1 H, H6), 7.85 (s, 1 H,
H4), 7.39–7.28 (m, 10 H, Bnϫ2), 5.63 (d, J = 9.0 Hz, 1 H, NH),
5.28–5.08 (m, 5 H, Bnϫ2/NH), 4.67–4.58 (m, 1 H, H16), 4.44–
4.34 (m, 1 H, H24), 3.10 (t, J = 5.6 Hz, 2 H, H15), 2.66–2.40 (m,
2 H, H22), 2.19–2.04 (m, 1 H, H23), 1.96–1.84 (m, 1 H, H23), 1.45,
1.44 (s, 18 H, tBu ϫ 2) ppm. 13C NMR (75 MHz, CDCl3): δ =
172.0, 170.5, 155.2, 149.0, 145.7, 145.0, 137.2, 135.3, 132.3, 132.2,
132.1, 128.9, 128.7, 128.6, 128.5, 128.4, 98.4, 84.6, 80.3, 67.7, 67.5,
Benzyl (S)-24-[(tert-Butoxycarbonyl)amino]-22-(2,3-diiodopyridin-1-
yl)butanoate (14): Zinc dust (199.7 mg, 3.1 mmol, 33 equiv.) was
placed in a nitrogen-purged 1.5 mL microtube, and then DMF
(150 μL) and trimethylsilyl chloride (60 μL, 0.47 mmol, 5.0 equiv.)
were added to the tube. After stirring vigorously at room tempera-
ture for 15 min, the liquid was removed by using a microsyringe,
and the remaining solid was dried by using a hot-air gun under
reduced pressure. The activated zinc was then cooled to room tem-
perature, and a solution of benzyl (S)-2-[(tert-butoxycarbonyl)-
amino]-3-iodobutanoate (8) (209.9 mg, 0.5 mmol, 5.3 equiv.) in
DMF (150 μL and rinsed with 50 μL ϫ2) was added. After stirring
at room temperature for 1 h, the zinc dust was settled by using a
centrifuge separator. The liquid was then removed from the acti-
vated zinc by using a microsyringe with DMF (200 μL) and added
to a 10 mL flask at 0 °C containing Pd2(dba)3 (4.8 mg, 3.9 μmol,
4 mol-%), P(2-furyl)3 (7.6 mg, 32.7 μmol, 32 mol-%), and 2,3,5-tri-
iodopyridine (7) (42.5 mg, 93.0 μmol, 1.0 equiv.) in DMF (450 μL).
After stirring at room temperature for 5 h, the reaction mixture
was diluted with EtOAc and quenched with brine. The aqueous
layer was then extracted with EtOAc, and the combined organic
layers were dried with Na2SO4 and concentrated in vacuo. Purifica-
tion by silica gel column chromatography (hexane/EtOAc = 3:1 Ǟ
2:1) afforded 14 as a yellow oil (26.5 mg, 42.6 μmol, 46%), 15 as a
yellow oil (8.3 mg, 10.5 μmol, 11%), and 16 as a yellow oil (3.5 mg,
4.4 μmol, 5%).
53.2, 52.2, 34.0, 29.8, 28.5, 28.4, 28.2, 23.9 ppm. IR (neat): ν =
˜
3372, 2968, 2237, 1714, 1504, 1455, 1367, 1260, 1159, 1023, 909,
864, 799, 733, 679, 451 cm–1. ESI-MS: calcd. for C38H44IN3NaO8
[M + Na]+ 820.21, found 820.27.
19: Rf = 0.39 (hexane/EtOAc = 2:1). 1H NMR (300 MHz, CDCl3):
δ = 8.01 (d, J = 2.1 Hz, 1 H, H6), 7.39–7.25 (m, 10 H, Bnϫ2),
7.23 (d, J = 2.1 Hz, 1 H, H4), 5.56 (d, J = 9.6 Hz, 1 H, NH), 5.29–
5.05 (m, 5 H, Bnϫ2/NH), 4.67–4.58 (m, 1 H, H3), 4.44–4.34 (m,
14: Rf = 0.59 (hexane/EtOAc = 2:1). [α]2D5 = –6.3 (c = 1.0, MeOH).
IR (neat): ν = 3354, 2975, 1709, 1500, 1454, 1397, 1161, 1107, 1022,
˜
4030
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© 2015 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
Eur. J. Org. Chem. 2015, 4024–4032

Total Synthesis of Isodesmosine
1 H, H24), 3.06 (t, J = 4.5 Hz, 2 H, H3), 2.61–2.41 (m, 2 H, H22),
2.18–1.96 (m, 1 H, H23), 1.95–1.79 (m, 1 H, H23), 1.45 (s, 18 H,
tBu ϫ 2) ppm. ESI-MS: calcd. for C38H44IN3NaO8 [M + Na]+
820.21, found 820.18.
20: Rf = 0.17 (hexane/EtOAc = 2:1). 1H NMR (300 MHz, CDCl3):
δ = 8.19 (s, 1 H, H6), 7.41–7.25 (m, 16 H, H4/Bnϫ3), 5.69–5.56
(m, 2 H, NH), 5.28–5.05 (m, 7 H, Bnϫ2/NH), 4.68–4.54 (m, 2 H,
H16/3), 4.47–4.34 (m, 1 H, H24), 3.20–2.92 (m, 4 H, H15/3), 2.68–
2.42 (m, 2 H, H22), 2.18–1.98 (m, 1 H, H23), 1.95–1.81 (m, 1 H,
H23), 1.45, 1.41 (s, 27 H, tBu ϫ 3) ppm. ESI-MS: calcd. for
C55H64N4NaO12 [M + Na]+ 995.44, found 995.38.
2-[(S)-16-Benzyloxycarbonyl-16-(tert-butoxycarbonyl)aminobut-13-
ynyl]-1-[(S)-11-benzyloxycarbonyl-11-(tert-butoxycarbonyl)amino-
pentyl]-3,5-bis[(S)-20/24-benzyloxycarbonyl-20/24-(tert-butoxycarb-
onyl)aminopropyl]pyridinium (21): A mixture of 4 (31.9 mg,
33.1 μmol, 1.0 equiv.) and benzyl 2-(S)-[(tert-butoxycarbonyl)-
amino]-6-iodohexanoate (5) (59.3 mg, 132.5 μmol, 4.0 equiv.) in
MeNO2 (1.3 mL) was heated at 70 °C for 24 h. The reaction mix-
ture was then concentrated in vacuo. Purification by silica gel col-
umn chromatography (CH2Cl2/MeOH = 30:1) afforded 21
(21.0 mg, 14.9 μmol, 45%) as a yellow oil; Rf = 0.50–0.63 (CH2Cl2/
MeOH = 10:1). [α]2D5 = –10.7 (c = 0.9, MeOH). IR (neat): ν = 3354,
˜
2976, 2928, 2232, 1711, 1500, 1455, 1366, 1253, 1164, 1052, 1026,
751, 698, 493, 463, 406 cm–1. 1H NMR (300 MHz, CDCl3): δ =
9.78 (s, 1 H, H6), 7.85 (s, 1 H, H4), 7.42–7.29 (m, 20 H, Bnϫ4),
6.08–5.94 (m, 1 H, NH), 5.76–5.57 (m, 2 H, NH), 5.31–5.07 (m, 9
H, Bnϫ4/NH), 4.81–4.68 (m, 2 H, H7), 4.68–4.11 (m, 4 H, H11/
16/20/24), 3.33–2.64, 2.33–1.55 (m, 16 H, H8/9/10/15/18/19/22/23),
1.43, 1.41 (s, 36 H, tBuϫ4) ppm. 13C NMR (75 MHz, CDCl3): δ
= 172.3, 171.8, 171.7, 171.3, 170.0, 155.8, 155.5, 145.3, 144.5, 141.1,
135.4, 135.2, 134.9, 133.8, 128.8, 128.7, 128.6, 128.5, 81.0, 80.2,
80.1, 68.1, 67.6, 67.3, 60.5, 60.3, 52.8, 32.8, 32.1, 31.7, 29.8, 29.5,
29.1, 28.5, 28.4, 24.5, 22.8, 22.3, 21.2, 14.3 ppm. ESI-MS: calcd.
for C72H92N5O16 [M]+ 1282.65, found 1282.61.
Benzyl (S)-13-[3,5-Bis-{(S)-20/24-benzyloxycarbonyl-20/24-(tert-
butoxycarbonyl)aminopropyl}pyridin-1-yl]-16-[(tert-butoxycarbon-
yl)amino]pent-14-ynoate (4): Zinc dust (206.6 mg, 3.2 mmol,
33 equiv.) was placed in a nitrogen-purged 1.5 mL microtube, and
then DMF (150 μL) and trimethylsilyl chloride (65 μL, 0.51 mmol,
5.0 equiv.) were added to the tube. After stirring the mixture vigor-
ously at room temperature for 15 min, the solution was removed
by using a microsyringe, and the remaining solid was dried by using
a hot-air gun under reduced pressure. The activated zinc was then
cooled to room temperature, and a solution of benzyl (S)-2-[(tert-
butoxycarbonyl)amino]-3-iodobutanoate (8) (225.4 mg, 0.5 mmol,
5.3 equiv.) in DMF (150 μL and rinsed with 50 μL ϫ2) was added.
After stirring of this mixture at room temperature for 1 h, the zinc
duct was settled by using a centrifuge separator. The liquid was
then removed from the activated zinc by using a microsyringe with
DMF (200 μL) and added to a 10 mL flask at 0 °C containing Pd-
PEPPSI-IPr (13.6 mg, 20.0 μmol, 4 mol-%) and 18 (80.4 mg,
100 μmol, 1.0 equiv.). After stirring at room temperature for 6 h,
the reaction mixture was diluted with EtOAc and quenched with
brine. The aqueous layer was then extracted with EtOAc, and the
combined organic layers were dried with Na2SO4 and concentrated
in vacuo. Purification by silica gel column chromatography (hex-
ane/EtOAc = 1:1) afforded 4 as a colorless solid (52.8 mg,
54.8 μmol, 54%); Rf = 0.48 (hexane/EtOAc = 1:1). [α]2D5 = –3.3 (c
2-[(S)-16-Amino-16-carboxybutyl]-1-[(S)-11-amino-11-carboxy-
pentyl]-3,5-bis[(S)-20/24-amino-20/24-carboxypropyl]pyridinium,
Isodesmosine (1): A solution of 21 (9.9 mg, 7.0 μmol, 1.0 equiv.) in
MeOH (0.14 mL) was treated with 10% Pd/C (37.3 mg, 35.1 μmol,
500 mol-%) and hydrogenated at balloon pressure and room tem-
perature with stirring for 3 d. The insoluble material was then sepa-
rated by filtration through neutral silica gel and Celite with MeOH
as the eluent. The filtrate (5.7 mg) in MeOH (0.08 mL) was treated
again with 10% Pd/C (21.5 mg) and hydrogenated at balloon pres-
sure and room temperature with stirring for 1 d. The insoluble ma-
terial was then separated by filtration through neutral silica gel and
Celite by using MeOH as the eluent. The filtrate was concentrated
in vacuo (5.5 mg), and the resulting residue was used in the next
reaction without further purification.
= 1.0, MeOH). IR (KBr): ν = 3377, 2977, 2369, 1713, 1500, 1456,
˜
1366, 1252, 1164, 1053, 1025, 859, 751, 698, 594, 485 cm–1. 1H
NMR (300 MHz, CDCl3): δ = 8.15 (s, 1 H, H6), 8.23 (s, 1 H, H4),
7.43–7.29 (m, 15 H, Bnϫ3), 5.61–5.52 (m, 1 H, NH), 5.29–5.07
(m, 8 H, Bnϫ3/NHϫ2), 4.65–4.53 (m, 1 H, H16), 4.49–4.32 (m,
1 H, H20/24), 3.10–2.96 (m, 2 H, H15), 2.73–2.44 (m, 4 H, H18/
22), 2.18–1.99 (m, 1 H, H23), 1.95–1.79 (m, 1 H, H23), 1.45, 1.43
(s, 27 H, tBuϫ3) ppm. 13C NMR (75 MHz, CDCl3): δ = 172.4,
172.2, 170.8, 155.3, 147.7, 146.1, 136.6, 136.5, 136.3, 135.9, 135.5,
135.3, 128.8, 128.7, 128.6, 128.5, 128.4, 83.9, 80.4, 80.3, 80.1, 67.6,
67.4, 67.2, 64.4, 53.4, 53.2, 52.5, 34.1, 32.7, 31.7, 29.2, 28.7, 28.4,
24.0 ppm. ESI-HRMS: calcd. for C55H66N4NaO12 [M + Na]+
985.4575, found 985.4544.
A mixture of TFA and distilled water (1.0 mL, 95:5 ratio) was
added to the above residue at room temperature and stirred for
2 h. The solvent was then removed in a rotary evaporator, and the
obtained crude product was purified by RP-HPLC. The conditions
were as follows: column, Cosmosil 5C18-AR-II (10ϫ250 mm, Na-
calai Tesque, Kyoto); solvent, linear gradient by using 10% MeOH
and 90 % H2O (0.1 % TFA); flow rate, 1.5 mL/min; detection,
254 nm; temperature, room temp.; tr = 9.5–10.8 min (isodesmosine,
1). As a result, 2.1 mg of pure isodesmosine (1) was obtained as a
yellow oil [2.1 mg, 3.3 μmol, 47% (2 steps)]. Rf = 0.26 [MeOH
1
(0.1% TFA)/H2O (0.1% TFA) = 1:9]. H NMR (500 MHz, D2O):
Benzyl (S)-13-[(S)-3,5-Bis{20/24-benzyloxycarbonyl-20/24-(tert-
butoxycarbonyl)aminopropyl}pyridin-1-yl]-16-[(tert-butoxycarbon-
yl)amino]pent-14-ynoate (4): A solution of 16 (12.3 mg, 15.6 μmol,
1.0 equiv.), 10 (14.1 mg, 46.5 μmol, 3.0 equiv.), Pd2(dba)3 (1.5 mg,
1.2 μmol, 8 mol-%), P(2-furyl)3 (1.1 mg, 4.7 μmol, 30 mol-%), and
CuI (1.3 mg, 6.8 μmol, 44 mol-%) in DMF (0.63 mL, 25 μm) was
degassed by using the freeze/pump/thaw technique. Next, iPr2NEt
(125 μL) was added, and the resulting solution was stirred at room
temperature for 2.5 h. The reaction mixture was then diluted with
EtOAc and quenched with a saturated NH4Cl solution. The aque-
ous layer was extracted with EtOAc, and the combined organic
layers were washed with brine, dried with Na2SO4, and concen-
trated in vacuo. Purification by silica gel column chromatography
(hexane/EtOAc = 1:1) afforded 4 (9.7 mg, 10.1 μmol, 65%) as a
colorless solid.
δ = 8.57 (s, 1 H, H6), 8.27 (s, 1 H, H4), 4.59–4.54 (m, 2 H, H7),
3.89 (t, J = 5.5 Hz, 1 H, H20), 3.81–3.79 (t, J = 5.5 Hz, 1 H, H16/
24), 3.75 (t, J = 5.5 Hz, 1 H, H11), 3.13 (t, J = 8.3 Hz, 2 H, H13),
3.06–2.98 (m, 1 H, H18), 2.98–2.89 (m, 1 H, H22), 2.91–2.81 (m,
2 H, H18/22), 2.24–2.16 (m, 4 H, H19/23), 2.12–2.05 (m, 2 H, H15),
2.05–1.97 (m, 2 H, H8), 1.95–1.87 (m, 2 H, H10), 1.85–1.66 (m, 2
H, H14), 1.62–1.42 (m, 2 H, H9) ppm. ESI-MS: calcd. for
C24H40N5O8 [M]+ 526.29, found 526.24.
Acknowledgments
We thank Prof. Gerard M. Turino (Mount Sinai School of Medi-
cine) for many suggestions. This work was supported by Grants-
in-Aid for Young Scientists (B) from JSPS (KAKENHI Grant Nos.
Eur. J. Org. Chem. 2015, 4024–4032
© 2015 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
www.eurjoc.org
4031

T. Usuki et al.
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Received: April 8, 2015
Published Online: May 15, 2015
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