Syntheses of natural and deuterated desmosines via palladium-catalyzed cross-coupling reactions

Tetrahedron 71 (2015) 1851e1862

Contents lists available at ScienceDirect

Tetrahedron journal homepage: www.elsevier.com/locate/tet

Syntheses of natural and deuterated desmosines via palladium-catalyzed cross-coupling reactions Rina Suzuki, Hiroto Yanuma, Takahiro Hayashi, Haruka Yamada, Toyonobu Usuki * Department of Materials and Life Sciences, Faculty of Science and Technology, Sophia University, 7-1 Kioicho, Chiyoda-ku, Tokyo 102-8554, Japan

a r t i c l e i n f o

a b s t r a c t

Article history: Received 6 January 2015 Received in revised form 28 January 2015 Accepted 30 January 2015 Available online 7 February 2015

Desmosine is a crosslinking pyridinium amino acid of elastin and is an attractive biomarker for the diagnosis of chronic obstructive pulmonary disease (COPD). Herein, we describe the total synthesis of natural desmosine and its deuterated derivatives. The synthesized deuterated derivatives could be used as internal standards for the development of an isotope-dilution liquid chromatographyemass spectrometry (LCeMS/MS) analytical method for the accurate determination of desmosine in clinical samples. The key features of our synthesis are stepwise chemo- and regioselective palladium-catalyzed Sonogashira and Negishi cross-coupling reactions for the efficient introduction of the amino acid side chains onto the pyridine core. Ó 2015 Elsevier Ltd. All rights reserved.

“This paper is dedicated to Professor Koji Nakanishi on the occasion of his 90th birthday.”

Keywords: Desmosine Biomarker Total synthesis Sonogashira cross-coupling Negishi cross-coupling Deuterium

1. Introduction Elastic fibers exist in vertebrate tissues, including the lungs, skin, blood vessels, and heart, and play an important role in providing their elasticity and stretchiness (Fig. 1).1,2 These unique functions are mainly derived from their self-assembling nature and crosslinking structure. Elastin, the main component of elastic fibers, is an extremely insoluble extracellular matrix protein that consists of soluble precursor tropoelastin monomers connected in a three-

dimensional (3-D) crosslinking network by amino acids. Although model structures of elastin have been proposed,3 elucidation of the entire 3-D structure of elastin utilizing spectroscopic methods, such as nuclear magnetic resonance (NMR),4 X-ray diffraction,5 circular dichroism (CD),6 scanning electron microscopy (SEM),7 etc., has been limited to date due to its insoluble nature. On the other hand, the chemical structures of the amino acid crosslinkers have been largely elucidated. Desmosine (1) and isodesmosine (2), which exist only in the elastin matrix, are major

Fig. 1. Depiction of elastic fibers.

* Corresponding author. Tel.: þ81 3 3238 3446; fax: þ81 3 3238 3361; e-mail address: [email protected] (T. Usuki). http://dx.doi.org/10.1016/j.tet.2015.01.064 0040-4020/Ó 2015 Elsevier Ltd. All rights reserved.

pyridinium amino acids that serve as the crosslinking molecules that bind the polymeric chains in elastin into a sophisticated 3-D network (Fig. 2).8,9 Desmopyridine (3) is an elastin crosslinker

1852

R. Suzuki et al. / Tetrahedron 71 (2015) 1851e1862

Fig. 2. Structures of desmosine (1), isodesmosine (2), desmopyridine (3), and neodesmosine (4).

isolated from acid hydrolyzates of bovine ligamentum nuchae,10 while neodesmosine (4) was isolated from bovine ligamentum nuchae.11 The total syntheses of 3 and 4 have been achieved by our group.12 The quantities of these pyridinium amino acids in human aorta elastin have been found to gradually increase with age. The irreversible degradation of elastin-containing tissues also occurs in several widely prevalent diseases, such as atherosclerosis,13 aortic aneurysm,13 cystic fibrosis,14 and chronic obstructive pulmonary disease (COPD),15 resulting in the excretion of pyridinium amino acids 1e4 in bodily fluids. Therefore, the crosslinkers 1e4 are expected to be useful biomarkers for elastin damage. Of the diseases that involve elastin damage, the respiratory disorder COPD is a leading cause of morbidity and mortality. According to the World Health Organization (WHO), COPD affects over 60 million people; it is currently the third leading cause of death worldwide.16 The risks for COPD are related to the interactions between many environmental factors, such as tobacco smoking, and the genetic factor known as a1-antitrypsin deficiency (AATD).17 At present, there is a lack of effective medicines that can prevent progression of the disease, and thus survival rates cannot be improved. As a result, intensive drug discovery activities are underway to find effective treatments. The development of biomarkers that indicate the severity of COPD and the therapeutic response of patients would thus aid in these efforts.18

The irreversible degradation of lung elastin that occurs with COPD is known to give rise to these amino acids, particularly desmosine 1 and isodesmosine 2, which can be measured specifically and sensitively in clinical samples, such as plasma, urine, and sputum, via liquid chromatographyemass spectrometry (LCeMS or LCeMS/MS) analysis.19,20 Therefore, the elastin crosslinking amino acids 1 and 2 are attractive biomarkers for both drug discovery and the rapid diagnosis of COPD. However, the methodologies for the precise analysis of desmosine biomarkers using LCeMS or LCeMS/ MS techniques that have been developed by pharmaceutical companies and academic researchers19,20 require the use of internal standards, which must be analogs of the target molecules. Thus, a stable isotope-labeled internal standard is needed for the precise LCeMS/MS analysis of desmosines.20 Recently, we reported the synthesis of deuterated desmosine with high stability and a definite structure, which contributed to the precise quantitative analysis of desmosines.20d The total synthesis of 1 has been previously achieved in 11% yield over 13 steps in our laboratory.21,22 The synthesis relied on stepwise and regioselective Sonogashira cross-coupling reactions between the pyridine core and corresponding alkynes. After preparation of the trisubstituted pyridine, however, several steps were required to convert the oxazolidine rings to the corresponding amino acid moieties, which contributed to degradation of the overall yield. An attempt was also made to apply this route to the synthesis of deuterated derivatives, but it was not possible to prepare a key intermediate compound. Thus, there is significant demand for an efficient chemical synthesis of desmosines that is applicable for the preparation of isotope-labeled derivatives. Herein, we describe in detail the total synthesis of COPD biomarker desmosine (1) utilizing Sonagashira and Negishi cross-coupling reactions as key steps in order to avoid the need for further conversion of the side chains.23 The shortened and improved total synthesis of 1 was also successfully applied to the synthesis of several deuterated derivatives. Therefore, this synthetic route should enable the continuous supply of these compounds as COPD biomarkers. 2. Results and discussion 2.1. Synthetic study via Negishi cross-coupling reactions As illustrated in Scheme 1, 1 would be retrosynthetically derived from trisubstituted pyridine 5 and iodo amino acid (u-iodobutyl Lglycine) derivative 6 through late-stage formation of the

Scheme 1. Retrosynthesis of desmosine 1 via stepwise Negishi cross-coupling reactions.

R. Suzuki et al. / Tetrahedron 71 (2015) 1851e1862

pyridinium salt, as was previously reported.21 Compound 6 can be prepared from 5-benzoxyl-(S)-4-[(tert-butoxycarbonyl)amino]-5oxopentanoic acid.24 Synthesis of trialkylated pyridine 5 would then involve stepwise and regioselective Negishi cross-coupling reactions between trihalogenated pyridine 7, 8, or 9 and the iodo amino acid segments 10 and 11. The gram-scale synthesis of 3,5-dibromo-4-iodopyridine 7 was accomplished via the dibromination and subsequent Sandmeyer reaction of 4-aminopyridine.12a The preparation of 8 and 9 was achieved starting from 4-hydroxypyridine via 3,5-regioselective introduction of iodine, replacement of the hydroxy group with bromine, and then substitution of the bromine with iodine.21 The iodo amino acids 10 and 11 were prepared according to the literature procedure.24 The Negishi cross-coupling reaction between 3,5-dibromo-4-iodopyridine 7 and amino acid segment 11 was first attempted using 10 mol % Pd2(dba)3 and 40 mol % P(2-furyl)325,26 in N,N-dimethylformamide (DMF) (Scheme 2). However, due to the high reactivity of the iodine group, the chemo- and regioselective introduction of 11 at 4 position of 7 to afford product 12 did not occur and a complex mixture was obtained. It should be noted that in this Negishi reaction, the modified protocol involving the use of an extra Zn equivalent that was removed from the organozinc reagent via centrifugation was employed.25

Scheme 2. Attempted Negishi cross-coupling reaction of 7 and 11.

Since the coupling reaction between 7 and 11 was unsuccessful, the 3,5-chemo- and regioselective Negishi cross-coupling reaction was planned. Thus, the Negishi cross-coupling reaction of 4-bromo3,5-diiodopyridine 8 and g-iodoalkylated L-glycine 10 was investigated (Table 1). Reaction of 8 with 10 using 10 mol % Pd2(dba)3 and 40 mol % P(2-furyl)325,26 in DMF at 50  C for 20 h afforded the desired di-coupled product 13 in 44% yield (entry 1). When the reaction time was reduced to 5 h under the same conditions, the yield was slightly improved to 49% (entry 2). Replacing the P(2furyl)3 ligand with AsPh3, which is known to be more effective in palladium-catalyzed reactions,26 led to a decrease in the yield to Table 1 Negishi cross-coupling reaction of 8 and 10

Entry

Ligand (40 mol %)

Time (h)

Yield (%)

1 2 3 4

P(2-furyl)3 P(2-furyl)3 AsPh3 AsPh3

20 5 20 5

44 49 33 71

1853

33% when the reaction was run for 20 h, but a significant increase in the yield to 71% with a reaction time of just 5 h (entries 3 and 4, respectively). With AsPh3, the longer reaction time led to a lower yield, suggesting that the ligand may cause the decomposition of the substrates and/or product. However, when the Negishi crosscoupling reaction of 3,4,5-triiodopyridine 9 and the iodo amino acid 10 was performed using 10 mol % Pd2(dba)3 and 40 mol % P(2furyl)3 at 50  C for 54 h, no coupling products, such as 14, were observed (Scheme 3), and a complex mixture was obtained.

Scheme 3. Negishi cross-coupling reaction of 9 and 10.

In order to obtain the trialkylated pyridine 5, the second Negishi cross-coupling reaction between 13 and d-iodoalkylated L-glycine 1124 was attempted, but this reaction also did not proceed (Scheme 4). Instead, starting material 13 was recovered in 71% yield. Therefore, the use of the less hindered alkynyl amino acid segment 15, which can be prepared from L-serine21 was considered. The Sonogashira cross-coupling reaction of 13 and alkyne 15 was then attempted using 10 mol % Pd2(dba)3, 40 mol % P(2-furyl)3, and 40 mol % CuI in DMF and i-Pr2NEt at 50  C with the hope of obtaining 3,4,5-trisubstituted pyridine 16 (Scheme 4). However, the starting material was once again recovered, this time in 92% yield, and the desired coupling product 16 was not observed. These results are probably due to the steric repulsion of the 18,180 -di-sp3carbon atoms during the oxidative addition of palladium to the 4bromo-position on the pyridine ring, while the Sonogashira cross-coupling reaction to the 4-bromo-3,5-dialkynylpyridine (i.e., 18,180 -di-sp carbons) was successful.21 This synthetic route was thus ruled out. 2.2. Synthesis via Sonogashira and Negishi cross-coupling reactions Since the introduction of the side chain at the 4 position of the 3,5-disubstituted pyridine resulted in failure so far, an alternative strategy, the side chain insertion order, was taken into account. As illustrated in Scheme 5, in the new approach, the quaternary pyridinium salt would be ultimately formed via alkylation of precursor 16 with terminal iodo amino acid segment 6.24 Synthesis of 16 would involve stepwise, chemo- and regioselective palladiumcatalyzed Sonogashira and Negishi cross-coupling reactions of trihalogenated pyridine 7 or 17 with the corresponding terminal alkyne 15 and then the g-iodoalkylated L-glycine 10,24 respectively. The key strategy was to utilize the different reactivities of each compound as determined by the substituent positions and the halogen species on the pyridine ring. In addition, to avoid steric hindrance, the terminal alkyne 15 was incorporated at the 4 position of the pyridine core first, and then, the amino acid segment was introduced at the 3 and 5 positions. The chemo- and regioselective Sonogashira cross-coupling reaction of 3,5-dibromo-4-iodopyridine 7 with alkyne 15 at the 4 position was performed using 10 mol % Pd2(dba)3, 40 mol % P(2furyl)3, and i-Pr2NEt in DMF for 15 h to afford the corresponding monoalkyne 18 in 58% yield (Scheme 6). Incorporation of g-iodoalkylated L-glycine 10 into the 3,5-positons of 18 was then attempted

1854

R. Suzuki et al. / Tetrahedron 71 (2015) 1851e1862

Scheme 4. Attempted Negishi cross-coupling reaction of 13 and 11 and Sonogashira cross-coupling reaction of 13 and 15.

Scheme 5. Retrosynthesis of desmosine 1 via stepwise Sonogashira and Negishi cross-coupling reactions.

via a Negishi cross-coupling reaction using Pd-PEPPSI-IPr, which was developed by Organ and co-workers.27 When the reaction was conducted with 3,5-dibromopyridine 18, the desired trisubstituted pyridine 16 was obtained in 60% yield, along with the di-coupled product 19 in 10% yield. Interestingly, the Negishi cross-coupling reaction of the di-coupled product 19 with 10 using Pd-PEPPSI-IPr only gave a trace amount of 16. This result suggests that the reaction mechanisms for the formation of the trisubstituted and disubstituted products differ somehow. The chemo- and regioselective Sonogashira cross-coupling reaction of the 4 position of 3,5dichloro-4-iodopyridine 17, which is commercially available, and alkyne 15 was also performed under the same conditions as those used for the coupling reaction with 7 (Scheme 6). The corresponding monoalkyne 20 was obtained in 80% yield, which was greater than that obtained with 7. The different reactivities may be due to the difference in the steric hindrance of the neighboring iodine groups, with increased hindrance preventing the oxidative addition to the palladium. However, the Negishi cross-coupling of 10 and

dichloropyridne 20 using Pd-PEPPSI-IPr did not proceed, and the starting material was recovered in 44% yield. This result may suggest that the reactivity of the chloropyridine is reduced compared to that of the bromopyridine and iodopyridine.25 Formation of the pyridinium salt of 16 with u-iodoalkylated Lglycine 6 was then achieved in nitromethane (MeNO2) at 60e80  C, affording 21 in 99% yield (Scheme 7).28 After reduction of the benzyl (Bn) and alkyne groups with H2 and Pd/C, the tert-butoxycarbonyl (Boc) protecting groups were successfully removed using trifluoroacetic acid (TFA) to give crude 1. Purification via C18 (reversed-phase) column chromatography afforded pure desmosine 1 in 63% yield over two steps. Spectroscopic data for synthetic 1, including the optical rotation, were in good agreement with those obtained for natural 1. Thus, the total synthesis of (þ)-desmosine 1, a crosslinking pyridinium amino acid of elastin, was achieved via chemo- and regioselective Sonogashira and Negishi cross-coupling reactions as key transformations in 22% yield over five steps starting from 3,5-

R. Suzuki et al. / Tetrahedron 71 (2015) 1851e1862

Scheme 6. Synthesis of trisubstituted pyridine 16.

Scheme 7. Total synthesis of desmosine 1.

1855

1856

R. Suzuki et al. / Tetrahedron 71 (2015) 1851e1862

dibromo-4-iodopyridine 7. The straightforward synthetic route is greatly shortened compared to the previous synthesis of 1 via stepwise Sonogashira cross-coupling reactions starting from 4hydroxypyridine (11% yield over 13 steps).21 Another elastin crosslinker desmopyridine 3 was also synthesized using this route based on regioselective Sonogashira and Negishi cross-coupling reactions. Starting from compound 16, the reduction of the Bn and alkyne groups with H2 and Pd/C was conducted. The Boc groups were then successfully removed using TFA to give crude 3 (Scheme 8). Purification via C18 chromatography afforded the pure desmopyridine 3 quantitatively in two steps. The spectroscopic data for 3, including NMR spectra, were in good agreement with those for natural 3.10,12a

Catalytic deuterogenation with methanol-d4 was performed on pyridinium salt 21, which was synthesized via Sonogashira and Negishi cross-coupling reactions (Scheme 9). This deuterogenation gave 22, a compound with four deuterium atoms incorporated in the side chain located at the 4 position. After deprotection of the Boc groups, the synthesis of 1-d4 was assessed by electrospray ionization (ESI)-MS [high resolution MS (HRMS) (m/z) calcd for C241H362H4N5O8 [M]þ: 530.3124, found: 530.3143]. The deuterated ratio of the end product was d4¼51%, d3¼39%, and d2¼10% as determined by MS. The presence of protic residues in the porous configuration of the Pd/C is thought to have inhibited 100% deuterium insertion in the side chain located at the 4 position. However, the synthesis of this desmosine-d4

Scheme 8. Synthesis of desmopyridine 3.

Scheme 9. Synthesis of desmosine-d4.

2.3. Synthetic study of isotopic labeled desmosines Having accomplished a more efficient total synthesis of desmosine, the next aim was to synthesize a desmosine internal standard for LCeMS or LCeMS/MS analysis. We estimated that the isotopic labeling of the amino acid substituent of the pyridinium moiety would be unsuitable for MS analysis, because it would likely be degraded by collision energy. Therefore, desmosine-d4, a desmosine with four deuterium atoms incorporated into the side chain at the 4 position of the pyridine core, was initially designed. This synthesis of desmosine-d4 takes advantage of the above-described route to desmosine, with introduction of the deuterium substituents achieved via catalytic hydrogenation.

should contribute to the establishment of a method for the precise quantitative analysis of desmosines in clinical COPD samples.20d Next, because the deuterated ratio proved to be unsatisfactory, the synthesis of isotope-labeled desmosine with an improved deuterated ratio was considered. In the alternative approach, rather than deuterogenation, deuterium was introduced on one of the amino acid segments, and particularly the substrate for the Negishi cross-coupling reaction. Using sodium borodeuteride-d4 (99% pure) as a reagent for reduction, commercially available 4-benzoxyl-(S)3-[(tert-butoxycarbonyl)amino]-4-oxobutanoic acid 23 was reduced to give deuterated alcohol 24 with d2¼87%. The Appel reaction was then performed to afford 10-d2 in 82% yield over two

R. Suzuki et al. / Tetrahedron 71 (2015) 1851e1862

steps (Scheme 10).24 At this point, the deuterated ratio of 10-d2 was d2¼87%, which was likely due to incorporation of protons from the H2O used in the first step (23/24).

1857

conditions required for the final step, these last two steps were each performed in a deuterium environment to afford isotopically pure desmosine-d8 (1-d8). Thus, synthesis of 1-d8 from 21-d(2þ2)

Scheme 10. Synthesis of 10-d2.

The deuterated iodo amino acid 10-d2 was then incorporated into the 3 and 5 positions of the pyridine core 18 via the Negishi cross-coupling reaction (Scheme 11). The temperature of this Negishi cross-coupling reaction was increased to 60  C in order to reduce the reaction time, because the progress of the reaction was relatively slow under conventional conditions due to the isotopic effect. According to the MS data for 16-d(2þ2), the deuterated ratio of 10-d2 was retained, thus providing 16-d(2þ2) with d4¼76%. This ratio was also retained in the pyridinium salt 21-d(2þ2).

was achieved via catalytic deuterogenation with methanol-d4 and deprotection using TFA/D2O in 45% yield over the two steps. However, the deuterated ratio was d8¼55%, d7¼33%, and d6¼12%. Because there was no proton source in the reaction, intramolecular DeH exchange is conceivable. It is known that the deuterogenation of alkynes with unprotected carboxylic acid substituents results in moderate deuterated ratios.29 In this route, the cleavage of the Bn group occurs simultaneously with reduction of the alkyne. The generated free carboxyl groups may thus be the cause of this DeH

Scheme 11. Alternative synthetic approach to deuterated desmosines.

Subsequently, following the desmosine route, the synthesis of desmosine with two deuterium atoms in each of the amino acids at the 3 and 5 positions (desmosine-d(2þ2), 1-d(2þ2)) was achieved (Scheme 12). Specifically, compound 1-d(2þ2) was obtained from 21-d(2þ2) in 76% yield over two steps (hydrogenation and deprotection of the Boc groups using TFA). The synthesis of 1-d(2þ2) was confirmed by MS [ESI-HRMS (m/z) calcd for C241H362H4N5O8 [M]þ: 530.3124, found: 530.3128]. Initially, the hydrogenation step was performed using a balloon at 1 atm. This desmosine-d(2þ2) contained mostly d3 (d4¼26%, d3¼74%) due to DeH exchange with protons of the methanol solvent, because the hydrogenation required more than 6 days, which was sufficient time to allow for degradation of the isotopic ratio. Therefore, the hydrogenation was performed at high pressure (2e5 atm) using a hydrogen cylinder in order to reduce the reaction time to 6 h. In this case, the deuterated ratio for desmosine-d(2þ2) was improved to d4¼56%, d3¼33%, and d2¼11%, but some distribution in the isotopic purity remained. In order to prevent the decrease in the deuterated ratio at the 18 and 180 positions during the hydrogenation and under the acidic

exchange. The deuterated yield of 1-d4, 1-d(2þ2), and 1-d8 remained somewhere around 55%. A compound with a high isotopic purity would be a more effective internal standard. Thus there is still room for improvement in the isotopic ratio of isotopically labeled desmosines. 3. Conclusion In summary, an efficient total synthesis of the COPD biomarker desmosine 1, a crosslinking amino acid of elastin, has been achieved via stepwise chemo- and regioselective Sonogashira and Negishi cross-coupling reactions as key transformations in 22% yield over five steps starting from 3,5-dibromo-4-iodopyridine 7. The method was also applied to the elastin crosslinker desmopyridine 3 and the deuterated desmosines 1-d4, 1-d(2þ2), and 1-d8, which could serve as internal standards for LCeMS/MS analysis. Therefore, this route should enable the establishment of a method for the precise quantitative analysis of desmosine.20d The synthetic strategy described above is currently being applied to the preparation of various other crosslinking amino acids to elucidate

1858

R. Suzuki et al. / Tetrahedron 71 (2015) 1851e1862

Scheme 12. Synthesis of desmosine-d(2þ2) and desmosine-d8.

the 3-D structure of elastic fiber, including degraded elastin peptides that are generated as a result of COPD and can indicate the severity of the disease. 4. Experimental section

shift (d, ppm). ESI-MS spectra were recorded on a JEOL JMS-T100LC instrument. Measurements for X-ray crystallographic analysis were made on a Rigaku Mercury CCD area detector with graphite monochromated Mo Ka radiation at 93 K. The carbon numbering on 1H NMR of all compounds is corresponding with desmosine 1 and desmopyridine 3 (Fig. 2).

4.1. General All non-aqueous reactions were conducted under an atmosphere of nitrogen with magnetic stirring using freshly distilled solvents unless otherwise indicated. Tetrahydrofuran (THF), and DMF were dried by distillation and stored over activated molecular sieves. Trimethylsilyl chloride (TMSCl), and diisopropylethylamine (iPr2NEt) were dried by distillation before use. Dehydrated methanol (MeOH) was purchased from Kanto Chemicals (Tokyo, Japan). Protected aspartic acid 23 (>99.5% purity) was purchased from Watanabe Chemicals (Hiroshima, Japan). 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 KGaA. Column chromatography was performed with acidic Silica gel 60 (spherical, 40e50 mm) or neutral Silica gel 60N (spherical, 40e50 mm) produced by Kanto Chemicals. Natural desmosine 1 was purchased from Elastin Products (St. Louis, MO). 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 (l¼589 nm) D line and are reported as follows: [a]TD (c g/100 mL, solvent). UV spectra were recorded on a JASCO V-560 UV/VIS spectrophotometer. Infrared (IR) spectra were recorded on a JASCO FT-IR 4100 spectrometer and are reported in wavenumbers (cm1). 1H and 13C NMR spectra were recorded on a JEOL JNM-EXC 300 spectrometer or on a JEOL JNMECA 500 spectrometer. 1H NMR data are reported as follows: chemical shift (d, 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

4.2. (S)-Benzyl (3-(5-(R)-21-(benzyloxy)-20-(tert-butoxycarbonylamino)-5-oxobutyl)-4-bromopyridin-3-yl)-210 -(tertbutoxycarbonylamino)butanoate (13) Zinc dust (200 mg, 3.0 mmol) was placed in a nitrogen-purged 1.5 mL Eppendorf microtube. DMF (150 mL) and TMSCl (60.0 mL, 0.47 mmol) were added, and the resulting mixture was stirred vigorously for 15 min at room temperature. 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 cooled to room temperature. A solution of benzyl 2-(S)-((tertbutoxycarbonyl)amino)-4-iodobutanoate 1024 (210 mg, 0.5 mmol, 5.4 equiv), which was recrystallized in pentene, in DMF (150 mL) was added to the activated zinc and rinsed with 100 mL DMF. The reaction mixture was then stirred at room temperature for 1 h. The insertion of zinc was monitored by TLC analysis (hexane/ethyl acetate (EtOAc)¼5:1). After completion of the insertion, the zinc solution was allowed to settle using a centrifuge for 1 min at room temperature. The organozinc solution was collected via a microsyringe and added to a 10 mL flask containing Pd2(dba)3 (12.2 mg, 0.01 mmol), AsPh3 (12.8 mg, 0.04 mmol), and 4-bromo-3,5-diiodopyridine 8 (38.9 mg, 94.9 mmol, 1.0 equiv). After stirring for 5 h at 50  C, the reaction 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 then concentrated in vacuo to give the crude product as a yellow oil. Purification by flash silica gel column chromatography (hexane/Et2O¼1:2) afforded the pure 13 (49.8 mg, 67.2 mmol, 71%) as a yellow oil; Rf 0.66 (hexane/EtOAc¼1:2); [a]25 D

R. Suzuki et al. / Tetrahedron 71 (2015) 1851e1862

8.1 (c 1.0, MeOH); IR (neat, cm1) 3354, 2975, 2928, 1713, 1500, 1454, 1366, 1254, 1164, 1052, 1026, 911, 736, 698; 1H NMR (300 MHz, CDCl3) d 8.15 (2H, s, H2/6), 7.36 (10H, s, Bn), 5.24e5.12 (6H, m, Bn/20NH/200 NH), 4.42 (2H, d, J¼7.8 Hz, H20/200 ), 2.84e2.64 (4H, m, H18/180 ), 2.19e2.07 (2H, m, H19/190 ), 1.96e1.84 (2H, m, H19/190 ), 1.45 (18H, s, tBu); 13C NMR (75 MHz, CDCl3) d 172.07, 155.40, 135.28, 128.82, 128.75, 128.62, 101.22, 80.27, 67.43, 53.34, 32.45, 29.89, 29.81, 28.43, 28.38; ESI-HRMS (m/z) calcd for C37H46BrN3NaO8 [MþNa]þ 764.2345, found 762.2344. 4.3. (S)-Benzyl 16-(tert-butoxycarbonylamino)-13-(3,5dibromopyridin-14-yl)pent-14-ynoate (18) A solution of 3,5-dibromo-4-iodopyridine 712a,23 (CCDC1021115, 15.8 mg, 44.0 mmol, 1.0 equiv), benzyl 16-(S)-((tertbutoxycarbonyl)amino)pent-14-ynoate 1521 (26.7 mg, 68 mmol, 1.5 equiv), Pd2(dba)3 (5.5 mg, 4.5 mmol, 10 mol %), P(2-furyl)3 (4.2 mg, 18 mmol, 40 mol %), and CuI (3.4 mg, 18 mmol, 40 mol %) in DMF (2.3 mL) was degassed by freeze/pump/thaw techniques. iPr2NEt (0.45 mL) was then added to the resulting solution. After stirring at 40  C for 15 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 then concentrated in vacuo. Purification on silica gel column chromatography (hexane/EtOAc¼6:1) afforded 18 (13.6 mg, 25.3 mmol, 58%); Rf 0.34 (hexane/EtOAc¼5:1); [a]20 D þ22.9 (c 0.1, CHCl3); IR (neat, cm1) 3629, 3423, 3234, 3033, 2976, 2931, 2410, 2235, 1715, 1500, 1445, 1367, 1343, 1165, 1062, 1021, 993, 889, 856, 760, 731, 698, 573, 461; 1H NMR (300 MHz, CDCl3) d 8.65 (2H, s, H2/6), 7.36e7.28 (5H, m, Bn), 5.54 (1H, d, J¼7.5 Hz, 16NH), 5.28e5.18 (2H, m, Bn), 4.66e4.64 (1H, m, H16), 3.15 (2H, d, J¼4.8 Hz, H15), 1.45 (9H, s, tBu); 13C NMR (CDCl3, 75 MHz) d 170.3, 115.2, 155.1, 149.7, 135.1, 134.2, 128.6, 128.4, 99.8, 80.4, 79.7, 67.7, 52.0, 28.4, 24.3; EIMS (m/z) calcd for C22H22Br2N2O4 [M]þ 535.99, found 535.85; ESI-HRMS (m/z) calcd for C22H22Br2N2NaO4 [MþNa]þ 558.9844, found 558.9829. 4.4. ((21S,210 S)-Benzyl 18,180 -(16-((S)-21-(benzyloxy)-21-(tertbutoxycarbonyl amino)-17-oxopent-13-ynyl)pyridine-3,5-diyl) bis(20-(tert-butoxycarbonylamino)butanoate)) (16), and ((21S)-benzyl 18-(16-((S)-21-(benzyloxy)-21-(tert-butoxycarbonyl amino)-17-oxopent-13-ynyl)-5-bromopyridine-3diyl)-(20-(tert-butoxycarbonylamino)butanoate)) (19) Zinc dust (200 mg, 3.0 mmol) was placed in a nitrogen-purged 1.5 mL Eppendorf microtube. DMF (150 mL) and TMSCl (60.0 mL, 0.47 mmol) were added to the microtube, and the resulting mixture was stirred vigorously for 15 min at room temperature. 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 cooled to room temperature. A solution of benzyl 20-(S)-((tert-butoxycarbonyl)amino)-18-iodobutanoate 1024 (211 mg, 0.5 mmol, 5.0 equiv), which was recrystallized in pentene, in DMF (150 mL) was added to the activated zinc and rinsed with 100 mL DMF. The reaction mixture was then stirred at room temperature for 1 h. The insertion of zinc was monitored by TLC (hexane/EtOAc¼5:1). 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 added to a 10 mL flask, containing Pd-PEPPSI-IPr (13.6 mg, 20 mol %) and 18 (72.6 mg, 0.13 mmol, 1.0 equiv). After stirring for 6 h at 40  C and 18 h at 50  C, 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

1859

washed with brine, dried over Na2SO4, and then concentrated in vacuo to give the crude product as a yellow oil. Purification by flash silica gel column chromatography (hexane/EtOAc¼3:1/1:1) afforded 16 (77.4 mg, 80.4 mmol, 60%) and 19 (7.2 mg, 9.6 mmol, 10%). Compound 16: a yellow oil; Rf 0.28 (hexane/EtOAc¼1:1); [a]20 D þ18.3 (c 0.1, CHCl3); IR (neat, cm1) 3365, 3033, 2976, 2932, 2231, 1714, 1584, 1503, 1455, 1421, 1390, 1366, 1252, 1217, 1165, 1055, 1026, 1000, 913, 861, 778, 752, 698, 584, 530, 485, 466, 420; 1H NMR (300 MHz, CDCl3) d 8.18 (2H, s, H2/6), 7.33e7.20 (15H, m, Bn), 5.98 (1H, s, 16NH), 5.43 (2H, m, 20NH/200 NH), 5.25e5.10 (6H, m, Bn), 4.60 (1H, m, H16), 4.43 (2H, m, H20/200 ), 3.04 (2H, m, H15), 2.63 (4H, m, H18/180 ), 2.12e2.02 and 1.94e1.84 (4H, m, H19/190 ), 1.43 (27H, s, tBu); 13C NMR (CDCl3, 75 MHz) d 172.3, 170.6, 155.5, 147.6, 137.2, 135.4, 135.1, 128.7, 128.6, 128.5, 128.4, 80.4, 80.1, 67.6, 67.2, 60.4, 53.5, 52.5, 33.0, 28.4, 28.3, 28.0, 24.0; ESI-MS calcd for C54H66N4NaO12 [MþNa]þ 985.46, found 985.47; ESI-HRMS (m/z) calcd for C54H66N4NaO12 [MþNa]þ 985.4575, found 985.4573. Compound 19: a yellow oil; Rf 0.60 (hexane/EtOAc¼1:1); 1H NMR (300 MHz, CDCl3) d 8.57 (1H, s, H2/6), 8.22 (1H, s, H2/6), 7.33e7.20 (10H, m, Bn), 5.77 (1H, s, 16NH), 5.43 (1H, s, 20NH), 5.26e5.11 (4H, m, Bn), 4.62 (1H, m, H16), 4.46 (1H, m, H20), 3.08 (2H, m, H15), 2.66 (2H, m, H18), 2.12e1.84 (2H, m, H19), 1.43 (27H, s, tBu); ESI-HRMS (m/z) calcd for C38H44BrN3NaO8 [MþNa]þ 772.2209, found 772.2176. 4.5. (S)-Benzyl 16-(tert-butoxycarbonylamino)-13-(3,5dichloropyridin-14-yl)pent-14-ynoate (20) A solution of 3,5-dichloro-4-iodopyridine 17 (100.5 mg, 0.367 mmol, 1.0 equiv), benzyl 16-(S)-((tert-butoxycarbonyl)amino) pent-14-ynoate 1524 (166.2 mg, 54.8 mmol, 1.5 equiv), Pd2(dba)3 (33.4 mg, 36.5 mmol, 10 mol %), P(2-furyl)3 (33.8 mg, 0.146 mmol, 40 mol %), and CuI (27.8 mg, 0.146 mmol, 40 mol %) in DMF (18.3 mL) was degassed by freeze/pump/thaw techniques. iPr2NEt (3.6 mL) was then added to the resulting solution. After stirring at 40  C for 17 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 then concentrated in vacuo. Purification on silica gel column chromatography (hexane/ EtOAc¼10:1) afforded 20 (131.5 mg, 0.293 mmol, 80%) as a yellow oil; Rf 0.31 (hexane/EtOAc¼5:1); 1H NMR (300 MHz, CDCl3) d 8.48 (2H, s, H2/6), 7.32e7.28 (5H, m, Bn), 5.52 (1H, d, J¼7.8 Hz, 16NH), 5.28e5.17 (2H, m, Bn), 4.68e4.62 (1H, m, H16), 3.16 (2H, d, J¼4.8 Hz, H15), 1.45 (9H, s, tBu); 13C NMR (CDCl3, 75 MHz) d 170.2, 155.1, 146.8, 135.1, 133.6, 130.3, 128.6, 128.5, 128.4, 101.2, 80.4, 67.7, 52.1, 28.4, 28.2, 24.3; EIMS (m/z) calcd for C22H22Cl2N2O4 [M]þ 448.10, found 448.10; EI-HRMS (m/z) calcd for C22H22Cl2N2O4 [M]þ 448.0957, found 448.0954. 4.6. (3,5-Bis((S)-21-(benzyloxy)-20-(tert-butoxycarbonylamino)-21-oxobutyl)-4-((S)-17-(benzyloxy)-16-(tertbutoxycarbonylamino)-17-oxopent-13-ynyl)-1-((S)-12-(benzyloxy)-11-(tert-butoxycarbonylamino)-12-oxohexyl)pyridinium iodide) (21) A mixture of 16 (34.7 mg, 36.0 mmol, 1.0 equiv) and benzyl 2(S)-((tert-butoxycarbonyl)amino)-6-iodohexanoate 624 (32.2 mg, 72.0 mmol, 2.0 equiv) in MeNO2 (1.0 mL) was heated at 60  C for 22 h, and at 80  C for 26.5 h. The reaction mixture was then concentrated in vacuo. Purification on silica gel column chromatography (hexane/EtOAc¼1:1/CH2Cl2/MeOH¼10:1) afforded 21 (50.4 mg, 35.7 mmol, 99%) as a yellow solid; Rf 0.50 (CH2Cl2/  MeOH¼10:1); [a]25 D þ8.1 (c 0.1, CHCl3); mp 83e86 C; IR (KBr, cm1) 3384, 2976, 2228, 1712, 1632, 1500, 1455, 1366, 1252, 1165, 1054, 752, 699, 602, 408; 1H NMR (300 MHz, CDCl3) d 8.93 (2H, s,

1860

R. Suzuki et al. / Tetrahedron 71 (2015) 1851e1862

H2/6), 7.48e7.30 (20H, m, Bn), 6.05 (1H, s, 16NH), 5.63 (2H, m, 20NH/200 NH), 5.24e5.12 (4H, m, Bn), 4.72e4.70 (2H, m, H7), 4.58 (1H, m, H16), 4.35e4.27 (3H, m, H11/20/200 ), 3.16 (2H, t, J¼5.1 Hz, H15), 2.87 (4H, m, H18/180 ), 2.22e2.02 (6H, m, H8/19/190 ), 1.76e1.62 (4H, m, H9/10), 1.45e1.42 (36H, s, tBu); 13C NMR (CDCl3, 75 MHz) d 170.1, 155.7, 143.0, 135.4, 135.3. 134.9, 128.7, 128.6, 128.5, 128.4, 80.2, 67.8, 67.4, 67.2, 31.5, 28.4, 28.3; ESIHRMS (m/z) calcd for C72H92N5O16 [M]þ 1282.6539, found 1282.6534. 4.7. 4-(16-(S)-Amino-16-carboxy-butyl)-1-(11-(S)-amino-11carboxy-pentyl)-3,5-bis-(20-(S)-amino-20-carboxy-propyl)pyridinium, desmosine (1) A solution of 21 (46.5 mg, 33.0 mmol, 1.0 equiv) in MeOH (1.2 mL) was treated with 10% Pd/C (192.5 mg, 0.18 mmol, 6.0 equiv) and under H2 atmosphere hydrogenated using a balloon at room temperature. After stirring for 4 days at room temperature, the reaction mixture was separated by filtration through a Celite pad on neutral silica gel eluting with MeOH and the filtrate was then concentrated in vacuo to afford crude mixture of 210 as yellow solid (31.8 mg, 30.2 mmol); 1H NMR (D2O, 300 MHz) d 8.52 (2H, s, H2/6), 4.50 (2H, m, H7), 3.93 and 3.797 (3H, m, H11/16/20/200 ), 2.91 (6H, m, H13/18/ 180 ), 2.14e1.63 (14H, m, H8/9/10/14/15/19/190 ), 1.46 (36H, m, tBu); ESI-MS (m/z) calcd for C44H72N5O6 [M]þ 926.50, found 926.51. The obtained product was used to the next reaction without further purification. A mixture of TFA and distilled H2O (2.0 mL, TFA/H2O¼95:5) was added to the crude 210 (10.8 mg, 10.2 mmol, 1.0 equiv) at room temperature and stirred for 3 h. The solvent was removed in vacuo. Purification on C18 column chromatography (0.1% TFA in distilled H2O) afforded desmosine 1 as a yellow solid (8.9 mg, 13.9 mmol, 63% (2 steps)); Rf 0.22 [MeOH (0.1% TFA)/H2O 1 (0.1% TFA)¼1:9]; [a]20 D þ9.8 (c 0.10, H2O); H NMR (D2O, 500 MHz) d 8.53 (2H, s, H2/6), 4.50 (2H, t, J¼7.2 Hz, H7), 3.87e3.84 (1H, m, H20), 3.79e3.78 (1H, m, H16), 3.76e3.72 (1H, m, H11), 3.05e2.99 (2H, m, H13), 2.94e2.86 (4H, m, H18/180 ), 2.18e2.13 (4H, m, H19/ 190 ), 2.07e2.00 (4H, m, H8/15), 1.90e1.88 (2H, m, H10), 1.67e1.33 (4H, m, H9/14); 13C NMR (125 MHz, D2O) d 175.1, 174.6 (C12/17/21), 159.2 (C4), 142.3 (C2/6), 140.8 (C3/5), 63.8 (C7), 55.0, 54.9, 54.8 (C11/16/20/200 ), 31.7, 31.1 (C8/10/15/19/190 ), 30.6 (C8), 28.9 (C13), 25.3 (C9/14); ESI-HRMS (m/z) calcd for C24H40N5O8 [M]þ 526.2877, found 526.2877. These data were in good agreement with natural 1. 4.8. Desmopyridine (3) A solution of 16 (46.2 mg, 478.0 mmol, 1.0 equiv) in MeOH (1.0 mL) was treated with 10% Pd/C (255.2 mg, 0.24 mmol, 5.0 equiv) and hydrogenated at balloon pressure at room temperature. After stirring for 6 days at room temperature, the insoluble was separated by filtration through a Celite pad on neutral silica gel eluting with MeOH and the filtrate was concentrated in vacuo. Concentration of the filtrate yielded crude product. The product was used to the next reaction without further purification. A mixture of TFA and distilled H2O (2.0 mL, TFA/H2O¼95:5) was added to the crude product (49.7 mg) at room temperature and stirred for 2 h. The solvent was removed in vacuo. Purification on C18 silica gel column chromatography (0.1% TFA in distilled H2O) yielded desmopyridine 3 as a white solid (71.9 mg, 0.18 mmol, quant); Rf 0.30 [MeOH (0.1% TFA)/H2O (0.1% TFA)¼1:9]; [a]20 D þ2.5 (c 0.10, H2O); 1H NMR (D2O, 300 MHz) d 8.52 (2H, s, H2/6), 4.11 (2H, t, J¼5.5 Hz, H9/90 ), 4.03 (1H, t, J¼5.4 Hz, H14), 3.14e2.90 (4H, m, H7/ 70 /11), 2.33 (2H, d, J¼5.1 Hz, H13), 2.25 (4H, d, J¼6.3 Hz, H8/80 ), 1.80e1.60 (2H, m, H12); 13C NMR (75 MHz, D2O) d 172.6 (C10/100 / 15), 160.6 (C4), 139.5 (C3/5), 139.4 (C2/6), 54.3 (C9/90 /14), 43.8 (C11), 30.9 (C8/80 ), 30.7 (C13), 26.2 (C7/70 ), 25.2 (C12). These data were in good agreement with those described for natural 3.10,12a

4.9. 4-(16-(S)-Amino-16-carboxy-butyl)-1-(11-(S)-amino-11carboxy-pentyl)-3,5-bis-(20-(S)-amino-20-carboxy-[D4]propyl)-pyridinium, desmosine-d4 (1-d4) A solution of 21 (35.2 mg, 27.4 mmol, 1.0 equiv) in CD3OD (0.9 mL) was treated with 10% Pd/C (145.6 mg, 0.14 mmol, 5.0 equiv) and under D2 atmosphere deuterated using a balloon at room temperature. After stirring for 6 days at room temperature, the reaction mixture was separated by filtration through a Celite pad on neutral silica gel eluting with MeOH and the filtrate was then concentrated in vacuo to afford crude mixture of 22 as yellow solid (17.4 mg, 18.7 mmol); ESI-MS (m/z) calcd for C44D4H68N5O6 [M]þ 930.52, found 930.39. The obtained product was used to the next reaction without further purification. A mixture of TFA and distilled H2O (7.0 mL, TFA/H2O¼95:5) was added to the crude 22 (17.4 mg, 18.7 mmol, 1.0 equiv) at room temperature and stirred for 2 h. The solvent was removed in vacuo. Purification on C18 column chromatography (0.1% TFA in distilled H2O) afforded desmosine-d4 1-d4 as a yellow solid (28.9 mg, 44.9 mmol, quant (2 steps)); Rf 0.22 [MeOH (0.1% TFA)/H2O (0.1% TFA)¼1:9]; 1H NMR (D2O, 300 MHz) d 8.56 (2H, s, H2/6), 4.52 (2H, t, J¼6.9 Hz, H7), 4.14e4.12 (2H, m, H20/200 ), 4.05e3.96 (1H, m, H11/16), 3.08e2.91 (4H, m, H18/180 ), 2.24e2.22 (4H, m, H19/190 ), 2.10 (2H, m, H15), 2.04e1.97 (4H, m, H8/10), 1.41 (2H, m, H9); ESIMS (m/z) calcd for C24H36D4N5O8 [M]þ 530.3128, found 530.3115. 4.10. Benzyl 20-(S)-((tert-butoxycarbonyl)amino)-18-hydroxy18-(D2)-butanoate (24) 4-Benzoxyl-(S)-[(tert-butoxycarbonyl)amino]-5-oxobutanoic acid 23 (200.4 mg, 0.62 mmol, 1.0 equiv) was dissolved in THF (3.0 mL) and stirred. The solution was cooled to 15  C, and Nmethylmorpholine (76.0 mL, 0.68 mmol, 1.1 equiv) and EtOCOCl (65.0 mL, 0.68 mmol, 1.1 equiv) 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 NaBD4 (39.0 mg, 0.94 mmol, 1.5 equiv) was added, followed by the dropwise addition of H2O (0.6 mL). The reaction mixture was stirred at 0  C for 40 min. 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/ EtOAc¼1:1) yielded benzyl 20-(S)-[(tert-butoxycarbonyl)amino]-4hydroxy-3-(2D)-butanoate (24) as a colorless oil (175.6 mg, 0.56 mmol, 91%); Rf 0.43 (hexane/EtOAc¼1:1); [a]27 D 41.7 (c 0.1, MeOH); IR (ATR, cm1) 3381, 2979, 1710, 1511, 1369, 1244, 1167, 1053, 966, 912, 858, 744, 699, 592; 1H NMR (300 MHz, CDCl3) d 7.40e7.30 (5H, m, Bn), 5.39 (1H, d, J¼6.7 Hz, NH), 5.19 (2H, dd, J¼12.7, 12.1 Hz, Bn), 4.58e4.48 (1H, m, CH), 2.21e2.10 (1H, m, CH2), 1.65e1.55 (1H, m, CH2), 1.44 (9H, s, tBu); 13C NMR (75 MHz, CDCl3) d 173.3, 156.9, 135.7, 129.3, 129.0, 128.8, 81.0, 67.8, 55.5, 51.1, 36.4, 28.7; ESI-HRMS (m/z) calcd for C16H21D2N1O5 [MþNa]þ 334.1600, found 334.1620. 4.11. Benzyl 20-(S)-((tert-butoxycarbonyl)amino)-18-iodo-18(D2)-butanoate (10-d2) Triphenylphosphine (209.7 mg, 0.80 mmol, 2.0 equiv) and imidazole (53.9 mg, 0.79 mmol, 2.0 equiv) were dissolved in CH2Cl2 (1.2 mL) with stirring. The solution was cooled to 0  C and I2 (202.0 mg, 0.80 mmol, 2.0 equiv) was added. The solution was warmed to room temperature and cooled to 0  C. A solution of benzyl 20-(S)-((tert-butoxycarbonyl)amino)-18-hydroxy-18-(2D)butanoate 24 (122.5 mg, 0.39 mmol, 1.0 equiv), which was recrystallized in pentane, in CH2Cl2 (0.7 mL) was also added. After stirring

R. Suzuki et al. / Tetrahedron 71 (2015) 1851e1862

for 2 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/ EtOAc¼5:1) yielded benzyl 2-(S)-[(tert-butoxycarbonyl)amino]-4iodo-3-(2D)-butanoate (10-d2) as a yellow oil (136.3 mg, 0.32 mmol, 82%); Rf 0.66 (hexane/EtOAc¼2:1); [a]27 D 32.5 (c 0.1, MeOH); IR (ATR, cm1) 3350, 2980, 1756, 1684, 1511, 1452, 1368, 1293, 1249, 1222, 1180, 1092, 1041, 946, 856, 786, 756, 697, 601; 1H NMR (300 MHz, CDCl3) d 7.41e7.31 (5H, m, Bn), 5.18 (2H, dd, J¼12.6, 12.0 Hz, Bn), 5.07 (1H, m, NH), 4.37 (1H, m, CH), 2.45e2.36 (1H, m, CH2), 2.23e2.12 (1H, m, CH2), 1.44 (9H, s, tBu); 13C NMR (75 MHz, CDCl3) d 171.5, 155.4, 135.2, 128.8, 128.7, 128.4, 80.4, 67.5, 54.5, 36.8, 28.4, 8.1; ESI-HRMS (m/z) calcd for C16H20D2I1N1O5 [MþNa]þ 444.0617, found 444.0609. 4.12. ((21S,210 S)-Benzyl 18,180 -(4-((S)-17-(benzyloxy)-16-(tertbutoxycarbonylamino)-17-oxopent-13-ynyl)pyridine-3,5-diyl) bis(21-(tert-butoxycarbonylamino)-18-(D2)-butanoate)) (16d(2D2)) Zinc dust (200 mg, 3.0 mmol) was placed in a nitrogen-purged 1.5 mL microtube. Dry DMF (150 mL) and trimethylsilyl chloride (60.0 mL, 0.47 mmol) were added, and the resulting mixture was stirred vigorously for 15 min at room temperature. Stirring was stopped, and the solution was removed by microsyringe. The remaining solid was dried using a hot air gun at reduced pressure. The activated zinc was cooled to room temperature, and a solution of benzyl 20-(S)-((tert-butoxycarbonyl)amino)-18-iodo-18-(2D)butanoate (10-d2) (211 mg, 0.5 mmol, 5.0 equiv), which was recrystallized in pentene, in dry DMF (150 mL and rinsed with 100 mL DMF) was added to the activated zinc. The reaction mixture was stirred at room temperature for 1 h, after which time TLC analysis (hexane/EtOAc¼5:1) revealed that no starting material remained. Stirring was stopped and the zinc duct was allowed to settle using a centrifuge separator. The solution was removed from the activated zinc via microsyringe with 200 mL DMF and add to a 10 mL flask, containing Pd-PEPPSI-IPr (13.6 mg, 20 mol %) and compound 18 (54.2 mg, 0.10 mmol, 1.0 equiv). Stirring was continued for 1.5 h at 60  C, the reaction 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 to give the crude product as yellow oil. Purification by flash column chromatography (hexane/EtOAc¼3:1/1:1) afforded the pure 16-d(2þ2) (56.0 mg, 58.4 mmol, 58%) as a brown oil and 19-d2 (2.2 mg, 2.9 mmol, 3%) as a yellow oil, respectively. 16-d(2þ2): Rf 0.31 1 (hexane/EtOAc¼1:1); [a]20 D þ18.0 (c 0.1, CHCl3); IR (ATR, cm ) 3364, 3064, 2976, 2938, 2839, 2362, 2332, 1718, 1603, 1512, 1451, 1413, 1367, 1290, 1247, 1167, 1053, 856, 757, 702; 1H NMR (300 MHz, CDCl3) d 8.17 (2H, s, H2/6), 7.36e7.21(15H, m, Bn), 5.99 (H, s, 16NH), 5.41 (2H, m, 20NH/200 NH), 5.25e5.10 (6H, m, Bn), 4.60 (1H, m, H16), 4.43 (2H, m, H20/200 ), 3.05 (2H, m, H15), 2.08e2.02 and 1.90e1.83 (4H, m, H19/190 ), 1.43 (27H, s, tBu); 13C NMR (CDCl3, 75 MHz) d 172.7, 171.0, 155.8, 147.4, 138.0, 135.5, 129.1, 129.0, 128.9, 80.9, 80.5, 68.1, 67.6, 60.9, 53.8, 52.9, 33.3, 28.9, 28.8, 24.5; ESIHRMS (m/z) calcd for C54H62D4N4NaO4 [MþNa]þ 989.4822, found 989.4774. 4.13. (3,5-Bis((S)-21-(benzyloxy)-20-(tert-butoxycarbonylamino)-21-oxo-18-(D2)-butyl)-4-((S)-17-(benzyloxy)16-(tert-butoxycarbonylamino)-17-oxopent-1-ynyl)-1-((S)-12(benzyloxy)-11-(tert-butoxycarbonylamino)-12-oxohexyl)pyridinium iodide) (21-d(2D2)) A mixture of 16-d(2þ2) (46.7 mg, 47.8 mmol, 1.0 equiv) and benzyl 2-(S)-((tert-butoxycarbonyl)amino)-6-iodohexanoate 624

1861

(47.8 mg, 95.7 mmol, 2.0 equiv) in MeNO2 (1.3 mL) was heated at 80  C for 50 h. The reaction mixture was concentrated in vacuo. Purification on silica gel column chromatography (hexane/EtOAc¼1:1/CH2Cl2/MeOH¼10:1) yielded 21-d(2þ2) (65.7 mg, 51.0 mmol, quant) as a brown solid; Rf 0.50 (CH2Cl2/ 1 MeOH¼10:1); [a]25 D þ10.6 (c 0.1, CHCl3); IR (ATR, cm ) 3359, 2979, 2230, 1733, 1631, 1512, 1452, 1371, 1247, 1169, 1050, 744, 699, 553; 1H NMR (300 MHz, CDCl3) d 8.95 (2H, s, H2/6), 7.40e7.26 (20H, m, Bn), 6.08 (1H, s, 16NH), 5.63 (2H, m, 20NH/ 200 NH), 5.23e5.10 (4H, m, Bn), 4.73e4.71 (2H, m, H7), 4.58 (1H, m, H16), 4.38e4.27 (3H, m, H11/20/200 ), 3.16 (2H, t, J¼5.0 Hz, H15), 2.22e2.00 (6H, m, H8/19/190 ), 1.88e1.66 (4H, m, H9/10), 1.41e1.38 (36H, s, tBu); 13C NMR (CDCl3, 75 MHz) d 170.6, 155.9, 143.4, 135.8, 135.6. 135.3, 129.3, 129.1, 128.8, 128.6, 68.2, 67.8, 67.6, 31.8, 28.7, 28.6; ESI-HRMS (m/z) calcd for C72H88D4N5O16 [M]þ 1286.6790, found 1286.6804. 4.14. 4-(16-(S)-Amino-16-carboxy-butyl)-1-(11-(S)-amino-11carboxy-pentyl)-3,5-bis-(20-(S)-amino-20-carboxy-18-(D2)propyl)-pyridinium, desmosine (1-d(2D2)) A solution of 21-d(2þ2) (26.6 mg, 20.6 mmol, 1.0 equiv) in MeOH (0.8 mL) was treated with 10% Pd/C (131.4 mg, 0.12 mmol, 6.0 equiv) and hydrogenated under the pressure using hydrogen cylinder. The solution was stirred at 5 atm for 6 h, then at 2 atm for 3 h at room temperature. After stirring, the insoluble was separated by filtration through a Celite pad on neutral silica gel eluting with MeOH and the filtrate was concentrated in vacuo. Concentration of the filtrate yielded 210 -d(2þ2) as yellow solid (12.2 mg, 13.1 mmol, 63%). The product was used in the next reaction without further purification; 1H NMR (CD3OD, 300 MHz) d 8.53 (2H, s, H2/6), 4.53 (2H, m, H7), 3.83 and 3.80 (3H, m, H11/16/20/200 ), 2.91 (2H, m, H13), 2.16e1.91 (14H, m, H8/9/10/14/15/19/190 ), 1.46 (36H, m, tBu); ESIMS (m/z) calcd for C44H68D4N5O6 [M]þ 930.52, found 930.32. A mixture of TFA and distilled H2O (2.0 mL, TFA/H2O¼95:5) was added to the crude 210 -d(2þ2) (14.5 mg, 15.6 mmol, 1.0 equiv) at room temperature and stirred for 3 h. The solvent was removed in vacuo. Purification on C18 silica gel column chromatography (0.1% TFA in distilled H2O) yielded desmosine-d(2þ2) 1-d(2þ2) as a colorless solid (6.8 mg, 12.8 mmol, 82%.); Rf 0.31 [MeOH (0.1% TFA)/H2O (0.1% TFA)¼ 1:9]; 1H NMR (D2O, 500 MHz) d 8.54 (2H, s, H2/6), 4.50 (2H, t, J¼7.2 Hz, H7), 4.01e3.99 (1H, m, H20), 3.94e3.92 (1H, m, H16), 3.90e3.88 (1H, m, H11), 3.06e2.85 (2H, m, H13), 2.14e2.06 (4H, m, H19/190 ), 2.06e1.98 (4H, m, H8/15), 1.96e1.88 (2H, m, H10), 1.70e1.35 (4H, m, H9/14); ESI-HRMS (m/z) calcd for C24H36D4N5O8 [M]þ 530.3124, found 530.3128. 4.15. 4-(16-(S)-Amino-16-carboxy-13,14-(D4)-butyl)-1-(11-(S)amino-11-carboxy-pentyl)-3,5-bis-(20-(S)-amino-20-carboxy18-(D2)-propyl)-pyridinium, desmosine (1-d8) A solution of 21-d(2þ2) (49.5 mg, 38.4 mmol, 1.0 equiv) in CD3OD (0.8 mL) was treated with 10% Pd/C (234.6 mg, 0.23 mmol, 6.0 equiv) and deuterogenated using a balloon at room temperature. After stirring for 7 days, the reaction mixture was separated by filtration through a Celite pad on neutral silica gel eluting with MeOH and the filtrate was concentrated in vacuo. Concentration of the filtrate yielded crude 210 -d8 as yellow solid (18.3 mg, 19.6 mmol, 51%). The product was used to the next reaction without further purification; 1H NMR (CD3OD, 300 MHz) d 8.66 (2H, s, H2/6), 4.51 (2H, m, H7), 4.18e3.93 (3H, m, H11/16/20/200 ), 2.16e1.62 (12H, m, H8/9/10/15/19/190 ), 1.45 (36H, m, tBu); ESI-MS (m/z) calcd for C44H64D8N5O6 [M]þ 934.55, found 934.45. A mixture of TFA and distilled H2O (2.0 mL, TFA/H2O¼95:5) was added to the crude 210 -d8 (18.3 mg, 19.6 mmol, 1.0 equiv) at room temperature and stirred for 3 h. The solvent was removed in vacuo.

1862

R. Suzuki et al. / Tetrahedron 71 (2015) 1851e1862

Purification on C18 silica gel column chromatography (0.1% TFA in distilled H2O) yielded desmosine-d8 1-d8 as a colorless solid (9.3 mg, 17.4 mmol, 89%.); Rf 0.31 [MeOH (0.1% TFA)/H2O (0.1% TFA)¼ 1:9]; 1H NMR (D2O, 500 MHz) d 8.56 (2H, s, H2/6), 4.52 (2H, t, J¼7.2 Hz, H7), 4.04e3.98 (1H, m, H20), 3.96e3.91 (1H, m, H16), 3.90e3.86 (1H, m, H11), 2.23e2.20 (4H, m, H19/190 ), 2.06e2.01 (4H, m, H8/15), 1.98e1.90 (2H, m, H10), 1.57e1.31 (2H, m, H9); ESI-HRMS (m/z) calcd for C241H322H8N516O8 [M]þ 534.3379, found 534.3382. Acknowledgements We thank Dr. Yong Y. Lin (Mount Sinai School of Medicine), Prof. Gerard M. Turino (Mount Sinai School of Medicine), Prof. Yoshiro Masuyama (Sophia University) for many suggestions, and Prof. Noriyuki Suzuki (Sophia University) for measuring X-ray analysis. This work was supported by a Grant-in-Aid for Young Scientists (B) from the Japan Society for the Promotion of Science (JSPS; KAKENHI Grant Nos. 22710224 and 25750388), the Sophia University Collaborative Research Grant, the Shimadzu Science Foundation, the SEI Group CSR Foundation, and the Naito Science Foundation. Supplementary data 1 H and 13C NMR spectra, CIF data of compound 7. Supplementary data associated with this article can be found in the online version, at http://dx.doi.org/10.1016/j.tet.2015.01.064.

References and notes 1. Debelle, L.; Tamburro, A. M. Int. J. Biochem. Cell Biol. 1999, 31, 261e272. 2. Rosenbloom, J.; Abrams, W. R.; Mecham, R. P. FASEB J. 1993, 7, 1208e1218. 3. (a) Hoeve, C. A. J.; Flory, P. J. Biopolym. 1974, 13, 677e686; (b) Urry, D. W. Adv. Exp. Med. Biol. 1974, 43, 211e243; (c) Weis-Fogh, T.; Andersen, S. O. Nature 1970, 227, 718e721; (d) Urry, D. W. Ultrastruct. Pathol. 1983, 4, 227e251. 4. Bochicchio, B.; Floquet, N.; Pepe, A.; Alix, A. J. P.; Tamburro, A. M. Chem.dEur. J. 2004, 10, 3166e3176. 5. Srokowski, E. M.; Woodhouse, K. A. J. Mater. Med. 2013, 24, 71e84. 6. Bochicchio, B.; Pepe, A. Chirality 2011, 23, 694e702. €der, C. U.; Wichapong, K.; Sippl, W.; 7. Heinz, A.; Ruttkies, C. K. H.; Jahreis, G.; Schra Keeley, F. W.; Neubert, R. H. H.; Schmelzer, C. E. H. Biochim. Biophys. Acta 2013, 1830, 2994e3004. 8. (a) Partridge, S. M.; Elsden, D. F.; Thomas, J. Nature 1963, 197, 1297e1298; (b) Thomas, J.; Elsden, D. F.; Partridge, S. M. Nature 1963, 200, 651e652. 9. Akagawa, M.; Suyama, K. Connect. Tissue Res. 2000, 41, 131e141. 10. Umeda, H.; Takeuchi, M.; Suyama, K. J. Biol. Chem. 2001, 276, 12579e12587. 11. (a) Nagai, Y. Connect. Tissue 1983, 14, 112e113; (b) Nakamura, F.; Suyama, K. Agric. Biol. Chem. 1991, 55, 547e554.

12. (a) Total synthesis of desmopyridine 3: Murakami, Y.; Yanuma, H.; Usuki, T. Tetrahedron: Asymmetry 2012, 23, 1557e1563; (b) Total synthesis of neodesmosine 4: Yanuma, H.; Usuki, T. Heterocycles 2013, 87, 55e63. 13. Watanabe, M.; Sawai, T. Tohoku J. Exp. Med. 1999, 187, 291e303. 14. (a) Bode, D. C.; Pagani, E. D.; Cumiskey, W. R.; von Roemeling, R.; Hamel, L.; Silver, P. J. Pulm. Pharmacol. Ther. 2000, 13, 175e180; (b) Schwartz, E.; Cruickshank, F. A.; Lebwohl, M. Exp. Mol. Pathol. 1990, 52, 63e68. 15. Viglio, S.; Iadarola, P.; Lupi, A.; Trisolini, R.; Tinelli, C.; Balbi, B.; Grassi, V.; €ring, G.; Meloni, F.; Meyer, K. C.; Dowson, L.; Hill, S. L.; Worlitzsch, D.; Do Stockley, R. A.; Luisetti, M. Eur. Respir. J. 2000, 15, 1039e1045. 16. WHO. WHO “The Top 10 Causes of Death”. http://www.who.int/mediacentre/ factsheets/fs310/en/ [accessed 05.01.15]. 17. (a) Yoshida, T.; Tuder, R. M. Physiol. Rev. 2007, 87, 1047e1082; (b) Mannino, D. M.; Buist, A. S. Lancet 2007, 370, 765e773. 18. (a) Turino, G. M.; Ma, S.; Lin, Y. Y.; Cantor, J. O.; Luisetti, M. Am. J. Respir. Crit. Care Med. 2011, 184, 637e641; (b) Martinez, F. J.; Donohue, J. F.; Rennard, S. I. Lancet 2011, 378, 1027e1037. 19. (a) Kaga, N.; Soma, S.; Fujimura, T.; Seyama, K.; Fukuchi, Y.; Murayama, K. Anal. Biochem. 2003, 318, 25e29; (b) Boutin, M.; Berthelette, C.; Gervais, F. G.; Scholand, M.-B.; Hoidal, J.; Leppert, M. F.; Bateman, K. P.; Thibault, P. Anal. Chem. 2009, 81, 1881e1887; (c) Shiraishi, K.; Matsuzaki, K.; Matsumoto, A.; Hashimoto, Y.; Iba, K. J. Oleo Sci. 2010, 59, 431e439; (d) Miliotis, T.; Lindberg, C.; € m, S. J. Chromatogr., A 2013, 1308, 73e78; (e) Semb, K. F.; van Geest, M.; Kjellstro Albarbarawi, O.; Barton, A.; Lin, Z.; Takahashi, E.; Buddharaju, A.; Brady, J.; Miller, D.; Palmer, C. N. A.; Huang, J. T. J. Anal. Chem. 2010, 82, 3745e3750; (f) € beli, H. Anal. Biochem. 2013, 436, Lamerz, J.; Friedlein, A.; Soder, N.; Cutler, P.; Do 127e136; (g) Ongay, S.; Hendriks, G.; Hermans, J.; van den Berge, M.; ten Hacken, N. H. T.; van de Merbel, N. C.; Bischoff, R. J. Chromatogr., A 2014, 1326, 13e19. 20. (a) Ma, S.; Lieberman, S.; Turino, G. M.; Lin, Y. Y. Proc. Natl. Acad. Sci. U.S.A. 2003, 100, 12941e12943; (b) Ma, S.; Lin, Y. Y.; Turino, G. M. Chest 2007, 131, 1363e1371; (c) Ma, S.; Turino, G. M.; Lin, Y. Y. J. Chromatogr., B 2011, 879, 1893e1898; (d) Ma, S.; Turino, G. M.; Hayashi, T.; Yanuma, H.; Usuki, T.; Lin, Y. Y. Anal. Biochem. 2013, 440, 158e165; (e) Murakami, Y.; Suzuki, R.; Yanuma, H.; He, J.; Ma, S.; Turino, G. M.; Lin, Y. Y.; Usuki, T. Org. Biomol. Chem. 2014, 12, 9887e9894. 21. (a) Usuki, T.; Yamada, H.; Hayashi, T.; Yanuma, H.; Koseki, Y.; Suzuki, N.; Masuyama, Y.; Lin, Y. Y. Chem. Commun. 2012, 48, 3233e3235; (b) Yamada, H.; Hayashi, T.; Usuki, T. Bull. Chem. Soc. Jpn. 2015, http://dx.doi.org/10.1246/bcsj. 20140394 22. Biomimetic total synthesis of 1 and 2 was also reported: (a) Usuki, T.; Sugimura, T.; Komatsu, A.; Koseki, Y. Org. Lett. 2014, 16, 1672e1675; (b) Sugimura, T.; Komatsu, A.; Koseki, Y.; Usuki, T. Tetrahedron Lett. 2014, 55, 6343e6346. 23. For preliminary communication of our research, see: Yanuma, H.; Usuki, T. Tetrahedron Lett. 2012, 53, 5920e5922. 24. Koseki, Y.; Yamada, H.; Usuki, T. Tetrahedron: Asymmetry 2011, 22, 580e586. 25. Usuki, T.; Yanuma, H.; Hayashi, T.; Yamada, H.; Suzuki, N.; Masuyama, Y. J. Heterocycl. Chem. 2014, 51, 269e273. 26. Farina, V.; Krishnan, B. J. Am. Chem. Soc. 1991, 113, 9585e9595. 27. Valente, C.; Belowich, M. E.; Hadei, N.; Organ, M. G. Eur. J. Org. Chem. 2010, 23, 4343e4354. 28. (a) Ren, R. X.-F.; Sakai, N.; Nakanishi, K. J. Am. Chem. Soc. 1997, 119, 3619e3620; (b) Sicre, C.; Cid, M. M. Org. Lett. 2005, 7, 5737e5739. 29. (a) Kurita, T.; Aoki, F.; Mizumoto, T.; Maejima, T.; Esaki, H.; Maegawa, T.; Monguchi, Y.; Sajiki, H. Chem.dEur. J. 2008, 14, 3371e3379; (b) Maegawa, T.; Fujiwara, Y.; Inagaki, Y.; Esaki, H.; Monguchi, Y.; Sajiki, H. Angew. Chem., Int. Ed. 2008, 47, 5394e5397.