Synthesis 2025; 57(14): 2252-2260
DOI: 10.1055/s-0043-1775486
paper

Total Synthesis of Deoxypyridinoline and Deoxypyridinoline-13C2,15N1

Hiroaki Ishii
,
Nao Tanaka
,
Ryosuke Shigeta
,
Saki Kondo
,

This work was supported by the KOSÉ Cosmetology Research Foundation.
 


Abstract

Collagen fibers, which make up 30% of all proteins in vertebrates, are extracellular matrix proteins that provide mechanical strength to connective tissues such as skin and bone, forming the structural foundation of the body. Deoxypyridinoline, a collagen cross-linking amino acid first isolated from bovine bone in 1982, is released into the bloodstream during bone metabolism alongside degraded bone tissue. This makes it a promising biomarker for quantitatively assessing diseases associated with abnormal bone resorption. In this study, we achieved the total synthesis of deoxypyridinoline from commercially available amino acids in five steps with an overall yield of 20%. This synthetic method provides a scalable alternative to isolating the compound from natural sources. As part of ongoing research into its use as a biomarker, we also synthesized isotopically labeled deoxypyridinoline-13C2,15N1, which can be used as an internal standard for quantitative analysis.


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Collagen fibers are the most abundant extracellular matrix proteins in vertebrate bodies, and play a crucial role in the strength of animal skin and bones.[2] Since the concept of collagen molecules was established in 1948 through electron microscopy observations,[3a] 29 different types of collagen molecules have been identified.[3b] Deoxypyridinoline (1), a 1,3,4,5-tetrasubstituted pyridinium compound with three amino acid moieties, was first isolated from bovine bone by Ogawa and co-workers in 1982 (Figure [1]).[4] This compound mainly cross-links type I collagen, which constitutes 90% of the collagen in the human body, contributing to the mechanical stability of its fibrous structure.

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Figure 1 Structure of deoxypyridinoline (1) and deoxypyridinoline-13C2,15N1 (1-13C2,15N1 )

In the biosynthetic process, lysyl oxidase catalyzes the conversion of lysine and hydroxylysine in the telopeptide region into allysine. These partially modified amino acids then undergo condensation reactions with surrounding lysine residues, forming immature cross-linking intermediates such as dehydrohydroxylysinonorleucine and dehydrodihydroxylysinonorleucine. After Amadori rearrangement, these intermediates form ketoamine structures that condense to construct the mature cross-link 1.[5] This compound is also abundant in bone,[6] and is released into the bloodstream as a metabolite when bone is resorbed by osteoclasts. Consequently, compound 1 has recently gained attention as a biomarker for assessing bone metabolism disorders, such as osteoporosis.[7]

The natural supply of 1 is typically achieved through acid hydrolysis followed by subsequent purification; however, large-scale supply through this method remains challenging. Although the total synthesis of 1 has been reported by Reddy and co-workers of Abbott Laboratories[5] [7] [8] and Anastasia and co-workers at the University of Milan,[9–11] these reports date back nearly 20 years, and their reproducibility is unclear. Therefore, in this study, we focused on the total synthesis of 1 using inexpensive, commercially available amino acids as starting materials.[12]

For many years, our laboratory has been involved in the chemical synthesis and quantitative analysis of desmosine, a cross-linking amino acid in elastin, an extracellular matrix protein that contributes to the elasticity and stretchability of skin and blood vessels. To precisely quantify desmosine in biological samples, we have employed an isotope dilution LC-MS/MS method. This method uses a compound with the same chemical structure but a different mass as an internal standard, and the target compound is quantified by comparing it to the standard. Since the quantification relies on the area ratio of mass spectra, it is crucial to ensure a mass difference of at least 3 Da. Our laboratory has achieved the total synthesis of desmosine/isodesmosine-13C3,15N1, which incorporates four isotopic elements,[13] [14] and successfully used this internal standard to compare blood desmosine levels between stroke patients and healthy individuals.[15] This allowed us to gain new insights into how blood desmosine levels fluctuate in disease. In this study, we report the synthesis of 1, and its isotopically labeled form deoxypyridinoline-13C2,15N1 (1-13C2,15N1 ), based on the findings from the synthesis of the natural version.

In synthesizing the natural compound 1, we examined the starting materials based on a synthetic strategy involving the cyclization of tertiary amine 3a to construct the pyridinium ring, followed by global deprotection of tBu and Boc groups (Scheme [1]). Compound 3a would be synthesized by N-alkylation of primary amine 5a [16] using α-halo ketone 4a or 4b.[5] [8] [10] [11] The preparation of 4a and 4b was planned by subjecting a commercially available glutamic acid derivative to a series of reactions, including Wittig reaction.

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Scheme 1 Retrosynthetic analysis of deoxypyridinoline (1) and deoxypyridinoline-13C2,15N1 (1-13C2,15N1 )

The synthesis of α-halo ketones 4a and 4b was carried out using Boc-Glu(OBzl)-OH as the starting material (Scheme [2]). Through a two-step protection group introduction and DIBAL-H reduction, aldehyde 8 was synthesized. Wittig reaction[16] of 8 successfully afforded olefin 9 in 60% yield. Next, the Prilezhaev epoxidation[17] of the obtained 9 was carried out using mCPBA, followed by ring opening with NaI or LiBr in THF containing acetic acid. As a result, halohydrins 11 and 12 were obtained in high yields.[11] Finally, IBX oxidation was performed on both compounds to yield the α-halo ketones 4a and 4b, completing the preparation for N-alkylation.

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Scheme 2 Synthesis of α-halo ketones 4a and 4b. Reagents and conditions: a) tert-butyl 2,2,2-trichloroacetimidate, CH2Cl2, r.t., 20 h, 91%; b) (Boc)2O, MeCN, r.t., 16 h, quant; c) DIBAL-H, Et2O, –90 °C, 15 min, 82%; d) MePPh3Br, nBuLi, THF, –90 °C to 0 °C, 1.5 h, 60%; e) mCPBA, CH2Cl2, r.t., 18 h, 91%; f) NaI, AcOH, THF, r.t., 1.5 h, quant; g) IBX, CH2Cl2, 50 °C, 6 h, 83%; h) LiBr, AcOH, THF, r.t., 22 h, 87%; i) IBX, (CH2Cl)2, 65 °C, 12 h, 94%.

The synthesis of primary amine 5a was achieved by introducing a tBu group to the commercially available lysine derivative Boc-Lys(Z)-OH using tert-butyl 2,2,2-trichloroacetimidate with an 84% yield, followed by removal of the Cbz group through a hydrogenation reaction (Table [1]).[16]

N-Alkylation of primary amine 5a with 4a or 4b was attempted by adding potassium carbonate in MeCN (Table [1]).[5] [7] [10] The instability of 4a resulted in a sluggish reaction, with a two-step yield of 2a of 9% (entry 1). By contrast, the use of 4b allowed the reaction to proceed more efficiently in 20 hours. The solution containing 3a was subjected to concentration, and the solvent was switched to methanol to realize cyclization. Initially, a 72-hour reaction[5] was attempted, but due to the formation of undesired byproducts, the yield was only 7% (entry 2). After attempting a longer reaction time with no improvement, we monitored the reaction progress again by TLC and MS analyses. It was found that the target compound 2a was formed after 24 hours, and the yield improved to 45% (entry 3). Finally, the protecting groups of 2a were removed using TFA; thereby, the total synthesis of deoxypyridinoline (1) was achieved (Table [1]).[12]

Table 1 Synthesis of Deoxypyridinoline (1)a

Entry

X

Time (h)

Yield (%)b 2a

1

I

72

9

2

Br

96

7

3

Br

24

45

a Reaction conditions: a) tert-butyl 2,2,2-trichloroacetimidate, CH2Cl2, r.t., 20 h, 84%; b) 10% Pd/C (5 mol%), H2, MeOH, r.t., 1.5 h, quant.; c) K2CO3, MeCN, r.t., 20 h; d) TFA/H2O (95:5), r.t., 3 h, 57%.

b Two-step yield from 5a.

Next, for the synthesis of 1-13C2,15N1 , a strategy was devised based on the synthesis of 1 (Scheme [1]). The target compound was expected to be obtained through the closure of tertiary amine 3b and subsequent deprotection. 3b was planned to be synthesized by N-alkylation of primary amine 5b with α-bromo ketone 4c. Both 4c and 5b were to be synthesized from a common intermediate 7.

The synthesis of α-bromo ketone 4c commenced with DIBAL-H reduction of the common intermediate 7 (Scheme [3]). After obtaining aldehyde 8 in good yield, Wittig reaction using a phosphonium salt containing isotopic element 13C was attempted. The phosphonium salt was synthesized using iodomethane-13 C and triphenylphosphine. Obtained olefin 13 was subjected to epoxidation, ring opening, and IBX oxidation in the same manner as the preparation of 4b, completing the synthesis of the isotopically labeled α-bromo ketone 4c.

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Scheme 3 Synthesis of 13C α-bromo ketone 4c. Reagents and conditions: a) DIBAL-H, Et2O, –90 °C, 15 min, 82%; b) 13CH3PPh3I, nBuLi, THF, –90 °C to 0 °C, 15 min, 39%; c) mCPBA, CH2Cl2, r.t., 18 h, 90%; d) LiBr, AcOH, THF, r.t., 22 h, 81%; e) IBX, (CH2Cl)2, 65 °C, 12 h, 85%.

Using the glutamic acid derivative 7 synthesized in Scheme [2], we attempted the synthesis of a primary amine containing isotopic elements (Scheme [4]). Initially, DIBAL-H reduction was performed to obtain primary alcohol 16 with a yield of 43%. In this method, the relatively low yield of 16 is presumably due to a side reaction, where reduction also occurred at the other ester site. Next, an Appel reaction[18] was carried out to synthesize the bromide 17, which was followed by an SN2 reaction using KC15N to furnish nitrile 18. Finally, hydrogenation was performed at 2 atm pressure to reduce the nitrile, resulting in the synthesis of primary amine 5b.[14]

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Scheme 4 Synthesis of 15N amine 5b. Reagents and conditions: a) DIBAL-H, THF, –90 °C to 0 °C, 15 min, 43%; b) PPh3, CBr4, CH2Cl2, 0 °C, 20 min, 90%; c) KC15N, DMF/H2O, r.t., 6 h, 94%; d) PtO2 (10 mol%), H2 (2 atm), 2-propanol, HCl, r.t., 2 d; then, 1 N aq NaOH, quant.

Using the prepared bromo ketone 4c, N-alkylation of amine 5b was carried out in MeCN, in the presence of potassium carbonate (Scheme [5]). After monitoring the progress of the reaction by TLC, the solvent was removed, and methanol along with additional potassium carbonate was added to induce cyclization, resulting in the synthesis of the protected compound 2b. Finally, deprotection was performed using acid, leading to the successful synthesis of deoxypyridinoline-13C2,15N1 (1-13C2,15N1 ).

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Scheme 5 Synthesis of deoxypyridinoline-13C2,15N1 (1-13C2,15N1 ). Reagents and conditions: a) K2CO3, MeCN, r.t., 20 h; b) K2CO3, O2, MeOH, r.t., 24 h, 35% (2 steps); c) TFA/H2O (95:5), r.t., 2 h, 91%.

In conclusion, in this study we achieved the synthesis of deoxypyridinoline (1) in five steps with an overall yield of 20% using commercially available glutamic acid and lysine derivatives. Additionally, using a similar glutamic acid derivative, the synthesis of deoxypyridinoline-13C2,15N1 (1-13C2,15N1 ) was completed in nine steps with an overall yield of 3.1%. Moving forward, we plan to conduct quantitative analysis of deoxypyridinoline using this isotopically labeled compound as an internal standard and explore its potential as a biomarker.

All nonaqueous reactions were conducted under an atmosphere of nitrogen with magnetic stirring unless otherwise indicated. For room temperature reactions, the temperature range was between 24 and 28 °C. Organic solvents were purchased from commercial suppliers. All reagents were used without further purification unless otherwise stated. Analytical TLC was performed on silica gel 60 F254 plates produced by Merck (Rahway, NJ). Column chromatography was performed with acidic silica gel 60 (spherical, 40–50 μm) or neutral silica gel 60N (spherical, 40–50 μm) produced by Kanto Chemicals (Tokyo, Japan) or Cosmosil 140C18-OPN produced by Nacalai Tesque (Kyoto, Japan). Removal of small amounts of solvent was performed with a Smart Evaporator CEV1B-SQ/SU/GR/SK-V1A-GR-P2 (Kanagawa, Japan). Optical rotations were measured on a JASCO (Tokyo, Japan) P-2200 digital polarimeter using the sodium lamp D line (λ = 589 nm) and are reported as follows: [α]D T (c g/100 mL, solvent). 1H and 13C NMR spectra were recorded on a JEOL (Tokyo, Japan) JNM-ECA 500 spectrometer (500 MHz). 1H NMR data are reported as follows: chemical shift (δ, ppm), multiplicity (s, singlet; d, doublet; t, triplet; q, quartet; m, multiplet), coupling constant(s) (J) in Hz, integration, assignment. 13C NMR data are reported in terms of chemical shift (δ, ppm). Electrospray ionization mass spectrometry (ESI-MS) data with time-of-flight (TOF) detection for high-resolution measurements were recorded on a JEOL JMS-T100LC instrument and are reported as mass-to-charge ratio (m/z). The NMR carbon numberings of deoxypyridinoline are shown in Figure [2].

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Figure 2

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5-Benzyl-1-(tert-butyl) N-(tert-Butoxycarbonyl)-l-glutamate (6)

To a stirred solution of Boc-Glu(OBzl)-OH (1.02 g, 3.03 mmol, 1.0 equiv) in CH2Cl2 (5.3 mL), tert-butyl 2,2,2-trichloroacetimidate (1.63 mL, 9.09 mmol, 3.0 equiv) was added at 0 °C. The resulting solution was stirred at room temperature for 20 h before being filtered through filter paper with hexane/EtOAc (97:3). Purification by silica gel column chromatography (hexane/EtOAc, 8:1) yielded 6 as a colorless oil (1.08 g, 2.76 mmol, 91%); Rf = 0.43 (hexane/EtOAc, 4:1).

1H NMR (500 MHz, CDCl3): δ = 7.38–7.31 (m, 5 H, Bn), 5.15–5.10 (m, 2 H, Bn), 5.06–5.05 (m, 1 H, NH), 4.22–4.21 (m, 1 H, CH), 2.51–2.37 (m, 2 H, CHCH2CH2 ), 2.37–2.14 (m, 1 H, CHCH 2CH2), 1.96–1.89 (m, 1 H, CHCH 2CH2), 1.46 (s, 9 H, Boc), 1.43 (s, 9 H, tBu).

13C NMR (125 MHz, CDCl3): δ = 172.8, 171.5, 155.5, 140.4, 136.0, 128.7, 128.4, 82.4, 80.0, 66.6, 53.5, 30.5, 29.1, 28.8, 28.6, 28.4, 28.2, 27.8.

ESI-HRMS: m/z calcd for C21H31NNaO6 [M + Na]+: 416.2049; found: 416.2061.


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5-Benzyl-1-(tert-butyl) N,N-Bis(tert-butoxycarbonyl)-l-glutamate (7)

To a stirred solution of 6 (1.19 g, 3.02 mmol, 1.0 equiv) in MeCN (9.4 mL), DMAP (73 mg, 0.60 mmol, 0.2 equiv) and di-tert-butyl dicarbonate (2.8 mL, 12.08 mmol, 4.0 equiv) were added, and the solution was stirred at room temperature for 16 h. The solvent was then removed on a rotary evaporator. Purification by silica gel column chromatography (hexane/EtOAc, 8:1) yielded 7 as a colorless oil (1.43 g, 2.90 mmol, quant); Rf = 0.6 (hexane/EtOAc, 4:1).

1H NMR (500 MHz, CDCl3): δ = 7.36–7.29 (m, 5 H, Bn), 5.11–5.07 (m, 2 H, Bn), 4.79–4.77 (m, 1 H, CH), 2.48–2.37 (m, 3 H, CHCH 2CH2 ), 2.20–2.11 (m, 1 H, CHCH 2CH2), 1.48 (s, 18 H, Boc), 1.44 (s, 9 H, tBu).

13C NMR (125 MHz, CDCl3): δ = 172.8, 169.4, 152.5, 136.1, 128.7, 128.5, 128.3, 83.1, 81.5, 66.4, 58.3, 31.2, 28.4, 28.1, 28.0, 24.7.

ESI-HRMS: m/z calcd for C26H39NNaO8 [M + Na]+: 516.2573; found: 516.2575.


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tert-Butyl (S)-2-(Bis(tert-butoxycarbonyl)amino)-5-oxopentanoate (8)[16]

DIBAL-H (1 M solution in hexane; 6.2 mL, 6.2 mmol, 1.4 equiv) was added dropwise to a –90 °C cooled solution of 7 (2.2 g, 4.5 mmol, 1.0 equiv) in Et2O (60 mL). The reaction mixture was stirred for 15 min, then quenched and warmed to room temperature. The white solid was then filtered through Celite and washed with Et2O. The filtrate was concentrated and purified by silica gel column chromatography (hexane/EtOAc, 8:1) to yield 8 as a colorless oil (1.4 g, 3.7 mmol, 82%); Rf = 0.43 (hexane/EtOAc, 5:1).

1H NMR (500 MHz, CDCl3): δ = 9.76 (s, 1 H, CHO), 4.75–4.72 (m, 1 H, CH), 2.56–2.41 (m, 3 H, CHCH 2CH2 ), 2.14–2.13 (m, 1 H, CHCH 2CH2), 1.50 (s, 18 H, Boc), 1.45 (s, 9 H, tBu).

ESI-MS: m/z calcd for C19H33NNaO7 [M + Na]+: 410.21; found: 410.17.


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tert-Butyl (S)-2-(Bis(tert-butoxycarbonyl)amino)hex-5-enoate (9)[16]

nBuLi (2.6 M solution in hexane; 0.46 mL, 1.20 mmol, 2.0 equiv) was dropwise added to a suspension of methyltriphenylphosphonium bromide (485 mg, 1.36 mmol, 2.2 equiv) in THF (5 mL) at –78 °C. After the mixture was stirred for 25 min at 0 °C, 8 (232 mg, 0.60 mmol, 1.0 equiv) in THF (2.5 mL) was added at –78 °C to the resulting orange ylide solution. The reaction mixture was stirred at 0 °C for 1.5 h, then quenched with saturated NH4Cl solution. The aqueous layer was then extracted with EtOAc. The combined organic layers were dried over Na2SO4 and concentrated in vacuo. Purification by silica gel column chromatography (hexane/EtOAc, 20:1) afforded 9 as a colorless oil (140 mg, 0.36 mmol, 60%); Rf = 0.63 (hexane/EtOAc, 5:1).

1H NMR (500 MHz, CDCl3): δ = 5.83–5.78 (m, 1 H, CH2=CH), 5.06–4.97 (dd, J = 15.0, 10.0 Hz, 2 H, CH2 =CH), 4.74–4.71 (dd, J = 5.0, 5.0 Hz, 1 H, CH), 2.18–2.08 (m, 3 H, CHCH 2CH2 ), 1.98–1.94 (m, 1 H, CHCH 2CH2), 1.51 (s, 18 H, Boc), 1.45 (s, 9 H, tBu).

ESI-MS: m/z calcd for C20H35NNaO6 [M + Na]+: 408.24; found: 408.20.


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tert-Butyl (2S)-2-(Bis(tert-butoxycarbonyl)amino)-4-(oxiran-2-yl)butanoate (10)

mCPBA (containing ca. 30% H2O; 98 mg, 0.407 mmol, 1.6 equiv) was added to 9 (96 mg, 0.25 mmol, 1.0 equiv) in CH2Cl2 (2.0 mL) at 0 °C. Then, the mixture was warmed to room temperature and stirred for 18 h. The reaction mixture was quenched with saturated NaHSO3 solution. The aqueous layer was then extracted with EtOAc. The combined organic layers were dried over Na2SO4 and concentrated in vacuo. Purification by silica gel column chromatography (hexane/EtOAc, 5:1) afforded 10 as a colorless solid (91 mg, 0.23 mmol, 91%); Rf = 0.37 (hexane/EtOAc, 5:1).

1H NMR (500 MHz, CDCl3): δ = 4.78–4.73 (m, 1 H, CH), 2.93 (m, 1 H, CH2), 2.74 (m, 1 H, CH2), 2.50–2.45 (m, 1 H, CHOCH2), 2.24–2.21 (m, 1 H, CH 2CH2CH), 2.03–1.99 (m, 1 H, CH 2CH2CH), 1.61–1.58 (m, 2 H, CH2CH2 CH), 1.51 (s, 18 H, Boc), 1.45 (s, 9 H, tBu).

13C NMR (125 MHz, CDCl3): δ = 169.8, 169.7, 152.6, 83.1, 83.0, 81.5, 66.6, 58.7, 58.5, 52.0, 51.8, 47.3, 47.2, 29.6, 29.5, 28.2, 28.1, 25.8, 25.7.

ESI-HRMS: m/z calcd for C20H36NO7 [M + H]+: 402.2492; found: 402.2476.


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tert-Butyl (2S)-2-(Bis(tert-butoxycarbonyl)amino)-5-hydroxy-6-iodohexanoate (11)

AcOH (16 μL) was added to 10 (30 mg, 0.075 mmol, 1.0 equiv) in THF (0.8 mL). Then, NaI (18 mg, 0.12 mmol, 1.6 equiv) was added. The reaction mixture was stirred for 1.5 h, then diluted with H2O. The aqueous layer was then extracted with Et2O. The combined organic layers were dried over Na2SO4 and concentrated in vacuo. Purification by silica gel column chromatography (hexane/EtOAc, 5:1) afforded 11 as a colorless oil (39 mg, 0.074 mmol, quant); Rf = 0.23 (hexane/EtOAc, 5:1).

1H NMR (500 MHz, CDCl3): δ = 4.75–4.74 (m, 1 H, CH), 3.58 (m, 1 H, CHOH), 3.39–3.35 (m, 1 H, CH2I), 3.24–3.21 (m, 1 H, CH2I), 2.25–2.16 (m, 1 H, CH 2CH2CH), 1.98–1.89 (m, 1 H, CH 2CH2CH), 1.67–1.55 (m, 2 H, CH2CH2 CH), 1.51 (s, 18 H, Boc), 1.45 (s, 9 H, tBu).

13C NMR (125 MHz, CDCl3): δ = 169.8, 169.6, 152.5, 152.4, 83.0, 82.9, 81.4, 70.8, 70.3, 58.7, 58.4, 33.3, 30.0, 28.0, 27.9, 25.6, 25.5, 16.1, 15.9.

ESI-HRMS: m/z calcd for C20H36INNaO7 [M + Na]+: 552.1428; found: 552.1428.


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tert-Butyl (2S)-2-(Bis(tert-butoxycarbonyl)amino)-6-bromo-5-hydroxyhexanoate (12)

AcOH (350 μL) was added to 10 (820 mg, 2.04 mmol, 1.0 equiv) in THF (21 mL). Then, LiBr (566 mg, 6.52 mmol, 3.2 equiv) was added. The reaction mixture was stirred for 22 h, then diluted with H2O. The aqueous layer was then extracted with Et2O. The combined organic layers were dried over Na2SO4 and concentrated in vacuo. Purification by silica gel column chromatography (hexane/EtOAc, 5:1) afforded 12 as a colorless oil (853 mg, 1.77 mmol, 87%); Rf = 0.27 (hexane/EtOAc, 5:1).

1H NMR (500 MHz, CDCl3): δ = 4.75–4.71 (m, 1 H, CH), 3.81 (m, 1 H, CHOH), 3.54–3.51 (m, 1 H, CH2Br), 3.41–3.36 (m, 1 H, CH2Br), 2.27–2.15 (m, 1 H, CH 2CH2CH), 2.02–1.88 (m, 1 H, CH 2CH2CH), 1.66–1.57 (m, 2 H, CH2CH2 CH), 1.51 (s, 18 H, Boc), 1.41 (s, 9 H, tBu).

13C NMR (125 MHz, CDCl3): δ = 169.9, 169.7, 152.7, 152.6, 83.2, 83.1, 81.6, 81.5, 71.0, 70.5, 58.9, 58.6, 58.5, 40.3, 32.1, 31.9, 29.8, 29.5, 28.3, 28.2, 28.1, 27.9, 25.7, 25.6, 22.8, 18.5, 14.3.

ESI-HRMS: m/z calcd for C20H36BrNNaO7 [M + Na]+: 504.1567; found: 504.1567 [Isotope ratio: calcd for 504.16 (41%), 505.16 (9%), 506.16 (41%), 507.16 (9%); found: 504.16 (32%), 505.16 (12%), 506.16 (36%), 507.16 (20%)].


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tert-Butyl (S)-2-(Bis(tert-butoxycarbonyl)amino)-6-iodo-5-oxohexanoate (4a)

IBX (185 mg, 0.66 mmol, 3.5 mmol) was added to 11 (100 mg, 0.19 mmol, 1.0 equiv) in CH2Cl2 (1 mL). The reaction mixture was stirred at 50 °C for 6 h, then diluted with CH2Cl2 and filtered through filter paper with CH2Cl2. Then, the aqueous layer was extracted with Et2O. The combined organic layers were dried over Na2SO4 and concentrated in vacuo. Purification by silica gel column chromatography (hexane/EtOAc, 5:1) afforded 4a as a colorless oil (83 mg, 0.16 mmol, 83%); Rf = 0.43 (hexane/EtOAc, 5:1).

1H NMR (500 MHz, CDCl3): δ = 4.71–4.68 (m, 1 H, CH), 3.83–3.79 (q, J = 8.3 Hz, 2 H, CH2I), 2.82–2.78 (m, 2 H, CH2CH2 CH), 2.45–2.41 (m, 1 H, CH 2CH2CH), 2.13–2.11 (m, 1 H, CH 2CH2CH), 1.51 (s, 18 H, Boc), 1.45 (s, 9 H, tBu).

13C NMR (125 MHz, CDCl3): δ = 202.0, 201.6, 169.2, 169.1, 152.4, 83.1, 81.5, 58.0, 48.2, 36.3, 35.8, 28.0, 23.9, 23.2.

ESI-HRMS: m/z calcd for C20H34INNaO7 [M + Na]+: 550.1272; found: 550.1276.


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tert-Butyl (S)-2-(Bis(tert-butoxycarbonyl)amino)-6-bromo-5-oxohexanoate (4b)

IBX (436 mg, 1.56 mmol, 3.0 equiv) was added to 12 (250 mg, 0.52 mmol, 1.0 equiv) in (CH2Cl)2 (1 mL). The reaction mixture was stirred at 65 °C for 12 h, then diluted with CH2Cl2 and filtered through filter paper with CH2Cl2. The aqueous layer was then extracted with Et2O. The combined organic layers were dried over Na2SO4 and concentrated in vacuo. Purification by silica gel column chromatography (hexane/EtOAc, 5:1) afforded 4b as a colorless oil (235 mg, 0.49 mmol, 94%); Rf = 0.43 (hexane/EtOAc, 5:1).

1H NMR (500 MHz, CDCl3): δ = 4.71–4.68 (q, J = 4.8 Hz, 1 H, CH), 3.91–3.86 (q, J = 9.3 Hz, 2 H, CH2Br), 2.81–2.66 (m, 2 H, CH2CH2 CH), 2.47–2.40 (m, 1 H, CH 2CH2CH), 2.15–2.09 (m, 1 H, CH 2CH2CH), 1.50 (s, 18 H, Boc), 1.44 (s, 9 H, tBu).

13C NMR (125 MHz, CDCl3): δ = 201.2, 169.3, 152.6, 83.3, 81.6, 58.1, 36.6, 34.5, 29.8, 28.2, 28.1, 23.6.

ESI-HRMS: m/z calcd for C20H34BrNNaO7 [M + Na]+: 502.1410; found: 502.1406 [Isotope ratio: calcd for 502.14 (41%), 503.14 (9%), 504.14 (41%), 505.14 (9%); found: 502.14 (41%), 503.14 (9%), 504.14 (41%), 505.14 (9%)].


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tert-Butyl N 6-((Benzyloxy)carbonyl)-N 2-(tert-butoxycarbonyl)-l-lysinate (19)[16]

Boc-Lys(Z)-OH (350 mg, 0.92 mmol, 1.0 equiv) was added to CH2Cl2 (9.0 mL) and cooled to 0 °C. tert-Butyl 2,2,2-trichloroacetimidate (0.5 mL, 2.76 mmol, 3.0 equiv) was added. After being stirred for 20 h at room temperature, the solution was isolated by filtration and the remaining solid was washed with hexane/EtOAc (97:3). Purification by silica gel column chromatography (hexane/EtOAc, 8:1) yielded 19 as a colorless oil (338.8 mg, 0.777 mmol, 84%); Rf = 0.17 (hexane/EtOAc, 5:1).

1H NMR (500 MHz, CDCl3): δ = 7.36–7.31 (m, 5 H, Cbz), 5.09 (s, 2 H, Cbz), 5.02 (m, 1 H, NH), 4.80 (m, 1 H, NH), 4.15 (m, 1 H, CH), 3.19 (m, 2 H, NHCH2 ), 1.89–1.52 (m, 6 H, CHCH2 CH2 CH2 ), 1.45 (s, 9 H, Boc), 1.43 (s, 9 H, tBu).

13C NMR (125 MHz, CDCl3): δ = 172.1, 156.6, 155.6, 136.7, 128.6, 128.5, 128.3, 128.2, 128.0, 82.0, 79.8, 66.7, 53.8, 40.9, 32.8, 29.8, 29.5, 28.5, 28.3, 28.1, 22.4.

ESI-MS: m/z calcd for C23H36N2NaO6 [M + Na]+: 459.25; found: 459.26.


#

tert-Butyl N 2-(tert-Butoxycarbonyl)-l-lysinate (5a)[16]

19 (0.124 g, 0.284 mmol, 1.0 equiv) in MeOH (1.53 mL) was treated with 10% Pd/C (14.9 mg, 0.014 mmol, 5.0 mol%) and hydrogenated at balloon pressure at room temperature. After stirring for 1.5 h at room temperature, the insoluble material was separated by filtration through Celite eluting with MeOH. The filtrate was then concentrated in vacuo. 5a was obtained as a colorless solid (0.102 g, 0.340 mmol, quant); Rf = 0.18 (hexane/EtOAc, 1:1).

1H NMR (500 MHz, CDCl3): δ = 5.05 (d, J = 8.0 Hz, 1 H, CH), 4.16 (m, 1 H, NH), 2.74 (s, 2 H, NH2CH2 ), 2.13–1.51 (m, 6 H, CH2 CH2 CH2 CH), 1.47 (s, 9 H, Boc), 1.44 (s, 9 H, tBu).

13C NMR (125 MHz, CDCl3): δ = 172.1, 155.6, 81.9, 79.7, 53.9, 41.4, 32.8, 31.8, 29.8, 28.5, 28.3, 28.1, 22.6.

ESI-MS: m/z calcd for C15H30N2NaO4 [M + Na]+: 325.21; found: 325.23.


#

Di-tert-butyl 6,6′-(((S)-6-(tert-Butoxy)-5-((tert-butoxycarbonyl)amino)-6-oxohexyl)azanediyl)(2S,2′S)-bis(2-(bis(tert-butoxycarbonyl)amino)-5-oxohexanoate) (3a)

4b (235 mg, 0.489 mmol, 2.0 equiv) was added to MeCN (8.0 mL). 5a (73 mg, 0.241 mmol, 1.0 equiv) was added to this solution, and then K2CO3 (117 mg, 0.846 mmol, 3.5 equiv) was added. After stirring for 20 h at room temperature, this solution was concentrated in vacuo to obtain the crude residue; Rf = 0.50 (hexane/EtOAc, 2:1).

ESI-HRMS: m/z calcd for C55H96N4NaO18 [M + Na]+: 1123.6612; found: 1123.6633.


#

4-((S)-2-(Bis(tert-butoxycarbonyl)amino)-3-(tert-butoxy)-3-oxopropyl)-3-((S)-3-(bis(tert-butoxycarbonyl)amino)-4-(tert-butoxy)-4-oxobutyl)-1-((S)-6-(tert-butoxy)-5-((tert-butoxycarbonyl)amino)-6-oxohexyl)-5-hydroxypyridin-1-ium (2a)

The crude residue was added to MeOH (8.0 mL) and additional K2CO3 (50 mg, 0.362 mmol, 1.5 equiv). Then, this solution was oxygenated at balloon pressure at room temperature. After stirring for 24 h at room temperature, the mixture was purified by silica gel column chromatography (CH2Cl2/MeOH, 100:7). 2a was obtained as an orange oil (0.126 g, 0.108 mmol, 45%); Rf = 0.47 (CH2Cl2/MeOH, 10:1); aromatic protons confirmed (δ 7.19, 6.89).

ESI-HRMS: m/z calcd for C55H93N4O17 + [M]+: 1081.6530; found: 1081.6535.


#

Deoxypyridinoline (1)

TFA (1.0 mL) and distilled H2O (53 μL) were added to 2a (100 mg, 0.086 mmol, 1.0 equiv). After stirring for 3 h at room temperature, this solution was concentrated in vacuo to obtain the crude residue. Then, purification by C18 silica gel column chromatography (H2O/MeOH, 7:3, 0.1% TFA in solvents) afforded 1 as the TFA salt (25 mg, 0.047 mmol, 57%); [α]D 20 +29.1 (c 0.25, H2O).

1H NMR (500 MHz, D2O): δ = 8.24 (s, 1 H, H2), 8.17 (s, 1 H, H2), 4.45 (t, J = 7.0 Hz, 2 H, H7), 4.09 (t, J = 7.3 Hz, 1 H, H18), 3.87 (t, J = 5.3 Hz, 1 H, H15), 3.76 (t, J = 5.5 Hz, 1 H, H11), 3.32 (m, 2 H, H17), 2.95 (m, 1 H, H13), 2.83 (m, 1 H, H13), 2.14 (m, 2 H, H14), 2.00 (m, 2 H, H8), 1.87 (m, 2 H, H10), 1.45 (m, 1 H, H9), 1.36 (m, 1 H, H9).

13C NMR (125 MHz, D2O): δ = 174.5 (C12), 173.1 (C19), 171.0 (C16), 164.0 (TFA), 163.7 (TFA), 163.4 (TFA), 163.2 (TFA), 156.0 (C3), 141.8 (C4), 141.7 (C5), 136.2 (C6), 129.3 (C2), 120.4 (TFA), 118.1 (TFA), 115.7 (TFA), 61.59 (C7), 54.54 (C11), 54.11 (C15), 53.06 (C18), 31.01 (C14), 30.44 (C8), 30.11 (C10), 28.26 (C19), 26.04 (C13), 21.53 (C9).

ESI-HRMS: m/z calcd for C18H29N4O7 + [M]+: 413.2031; found: 413.2037.


#

(Methyl-13 C)triphenylphosphonium Iodide (20)

PPh3 (2.22 g, 8.46 mmol, 1.0 equiv) was added to toluene (11.1 mL) under N2 atmosphere. Then, the mixture was cooled at –5 °C. Iodomethane-13 C (0.63 mL, 10.1 mmol, 1.2 equiv) was added, and the mixture was stirred at room temperature for 24 h. The crude residue was filtered and washed with toluene/hexane (1:5). Finally, the solid was dried in vacuo to afford 20 as a white solid (3.43 g, 8.49 mmol, quant).

1H NMR (500 MHz, CDCl3): δ = 7.82–7.68 (m, 15 H, 3 × Ph), 3.39–3.36, 3.12–3.09 (dd, J = 15.0, 15.0 Hz, 3 H, 13CH3).


#

tert-Butyl (S)-2-(Bis(tert-butoxycarbonyl)amino)hex-5-enoate-6-13 C (13)

nBuLi (1.55 M solution in hexane; 2.11 mL, 3.27 mmol, 1.1 equiv) was dropwise added to a suspension of (methyl-13 C)triphenylphosphonium iodide (1.56 g, 3.86 mmol, 1.3 equiv; dried for 1 h at 60 °C in vacuo­) in THF (11 mL) at –78 °C. After the mixture was stirred for 10 min at 0 °C, 8 (1.15 g, 2.97 mmol, 1.0 equiv) in THF (9.0 mL) was added at –78 °C to the resulting orange ylide solution. The reaction mixture was stirred at 0 °C for 15 min, then quenched with saturated NH4Cl solution. The aqueous layer was then extracted with EtOAc. The combined organic layers were dried over Na2SO4 and concentrated in vacuo. Purification by silica gel column chromatography (hexane/EtOAc, 20:1) afforded 13 as a colorless oil (443 mg, 1.15 mmol, 39%); Rf = 0.63 (hexane/EtOAc, 5:1).

1H NMR (500 MHz, CDCl3): δ = 5.83–5.78 (m, 1 H, CH2=CH), 5.21–5.13 (dd, J = 17.5, 10.0 Hz, 1 H, 13CH 2=CH), 4.90–4.81 (dd, J = 17.0, 10.0 Hz, 1 H, 13CH 2=CH), 4.74–4.71 (dd, J = 5.0, 5.0 Hz, 1 H, CH), 2.18–2.09 (m, 3 H, CHCH 2CH2 ), 1.98–1.94 (m, 1 H, CHCH 2CH2), 1.51 (s, 18 H, Boc), 1.45 (s, 9 H, tBu).

13C NMR (125 MHz, CDCl3): δ = 170.0, 152.6, 138.0, 137.5, 115.7, 115.4, 115.1, 83.4, 82.8, 81.3, 58.5, 30.7, 28.8, 28.8, 28.2, 28.1.

ESI-HRMS: m/z calcd for 12C19 13CH35NNaO6 [M + Na]+: 409.2390; found: 409.2390.


#

tert-Butyl (2S)-2-(Bis(tert-butoxycarbonyl)amino)-4-(oxiran-2-yl-3-13 C)butanoate (14)

mCPBA (containing ca. 30% H2O; 162 mg, 0.66 mmol, 1.6 equiv) was added to 13 (159 mg, 0.41 mmol, 1.0 equiv) in CH2Cl2 (4 mL) at 0 °C. The reaction mixture was stirred for 18 h at room temperature, then quenched with saturated NaHSO3 solution. The aqueous layer was then extracted with EtOAc. The combined organic layers were dried over Na2SO4 and concentrated in vacuo. Purification by silica gel column chromatography (hexane/EtOAc, 5:1) afforded 14 as a colorless solid (150 mg, 0.37 mmol, 90%); Rf = 0.37 (hexane/EtOAc, 5:1).

1H NMR (500 MHz, CDCl3): δ = 4.78–4.73 (m, 1 H, CH), 2.93 (m, 1.5 H, CH2), 2.68–2.63 (m, 0.5 H, CHO13CH2), 2.58 (m, 0.5 H, CH2), 2.33–2.28 (m, 0.5 H, CHO13CH2), 2.27–2.18 (m, 1 H, CH 2CH2CH), 2.05–1.94 (m, 1 H, CH 2CH2CH), 1.61–1.58 (m, 2 H, CH2CH2 CH), 1.51 (s, 18 H, Boc), 1.45 (s, 9 H, tBu).

13C NMR (125 MHz, CDCl3): δ = 169.8, 169.7, 152.6, 115.4, 83.0, 81.5, 65.1, 65.0, 58.8, 58.5, 52.1, 51.9, 51.7, 47.3, 47.2, 40.3, 29.6, 29.6, 28.2, 28.1, 25.8.

ESI-HRMS: m/z calcd for 12C19 13CH35NNaO7 [M + Na]+: 425.2339; found: 425.2339.


#

tert-Butyl (2S)-2-(Bis(tert-butoxycarbonyl)amino)-6-bromo-5-hydroxyhexanoate-6-13 C (15)

AcOH (120 μL) was added to 14 (150 mg, 0.37 mmol, 1.0 equiv) in THF (4.2 mL). Then, LiBr (104 mg, 1.19 mmol, 3.2 equiv) was added. The reaction mixture was stirred for 22 h, then diluted with H2O. The aqueous layer was then extracted with Et2O. The combined organic layers were dried over Na2SO4 and concentrated in vacuo. Purification by silica gel column chromatography (hexane/EtOAc, 5:1) afforded 15 as a colorless oil (145 mg, 0.30 mmol, 81%); Rf = 0.27 (hexane/EtOAc, 5:1).

1H NMR (500 MHz, CDCl3): δ = 4.75–4.71 (m, 1 H, CH), 3.82 (m, 1 H, CHOH), 3.69–3.65 (m, 0.5 H, 13CH2Br), 3.56–3.51 (m, 0.5 H, 13CH2Br), 3.39–3.35 (m, 0.5 H, 13CH2Br), 3.26–3.21 (m, 1 H, 13CH2Br), 2.26–2.16 (m, 1 H, CH 2CH2CH), 2.03–1.88 (m, 1 H, CH 2CH2CH), 1.63–1.57 (m, 2 H, CH2CH2 CH), 1.51 (s, 18 H, Boc), 1.44 (s, 9 H, tBu).

13C NMR (125 MHz, CDCl3): δ = 169.9, 169.7, 152.7, 152.6, 83.2, 83.1, 81.6, 81.5, 71.2, 70.9, 70.7, 70.4, 67.4, 66.8, 58.9, 58.6, 46.0, 40.5, 40.3, 40.2, 32.1, 31.9, 28.2, 28.1, 25.7, 25.7, 25.6, 25.6.

ESI-HRMS: m/z calcd for 12C19 13CH36BrNNaO7 [M + Na]+: 505.1601; found: 505.1588 [Isotope ratio: calcd for 505.16 (41%), 506.16 (9%), 507.16 (41%), 508.16 (9%); found: 505.16 (41%), 503.14 (9%), 504.14 (41%), 505.14 (9%)].


#

tert-Butyl (S)-2-(Bis(tert-butoxycarbonyl)amino)-6-bromo-5-oxohexanoate-6-13 C (4c)

IBX (253 mg, 0.90 mmol, 3.0 equiv) was added to 15 (145 mg, 0.30 mmol, 1.0 equiv) in (CH2Cl)2 (0.5 mL). The reaction mixture was stirred at 65 °C for 12 h, then diluted with CH2Cl2 and filtered through filter paper with CH2Cl2. The aqueous layer was then extracted with Et2O. The combined organic layers were dried over Na2SO4 and concentrated in vacuo. Purification by silica gel column chromatography (hexane/EtOAc, 5:1) afforded 4c as a colorless oil (123 mg, 0.26 mmol, 85%); Rf = 0.43 (hexane/EtOAc, 5:1).

1H NMR (500 MHz, CDCl3): δ = 4.71–4.68 (dd, J = 6.0, 5.0 Hz, 1 H, CH), 4.07–4.01 (m, 1 H, 13CH2Br), 3.77–3.71 (m, 1 H, 13CH2Br), 2.82–2.67 (m, 2 H, CH2CH2 CH), 2.47–2.40 (m, 1 H, CH 2CH2CH), 2.16–2.08 (m, 1 H, CH 2CH2CH), 1.51 (s, 18 H, Boc), 1.45 (s, 9 H, tBu).

13C NMR (125 MHz, CDCl3): δ = 201.4, 201.0, 192.4, 192.4, 169.3, 152.6, 83.3, 81.6, 68.7, 58.1, 36.6, 36.5, 34.5, 34.3, 28.2, 28.1, 23.7.

ESI-HRMS: m/z calcd for 12C19 13CH34BrNNaO7 [M + Na]+: 503.1444; found: 503.1436 [Isotope ratio: calcd for 503.14 (42%), 504.14 (9%), 505.14 (41%), 506.14 (8%); found: 503.14 (41%), 504.14 (9%), 505.14 (41%), 506.14 (9%)].


#

tert-Butyl (S)-2-(Bis(tert-butoxycarbonyl)amino)-5-hydroxypentanoate (16)

DIBAL-H (1 M solution in hexane; 7.61 mL, 7.61 mmol, 2.5 equiv) was added dropwise to a –90 °C cooled solution of 7 (1.50 g, 3.04 mmol, 1.0 equiv) in THF (30 mL). The reaction mixture was stirred at 0 °C for 15 min, then quenched with H2O and warmed to room temperature. The white solid was then filtered through Celite and washed with Et2O. The filtrate was concentrated and purified by silica gel column chromatography (hexane/EtOAc, 8:1) to yield 16 as a colorless oil (512.2 mg, 1.32 mmol, 43%); Rf = 0.20 (hexane/EtOAc, 4:1).

1H NMR (500 MHz, CDCl3): δ = 4.75–4.72 (m, 1 H, CHCH2CH2), 3.68–3.64 (m, 2 H, CH2 OH), 2.21–2.14 (m, 1 H, CHCH 2CH2), 1.94–1.86 (m, 1 H, CHCH 2CH2), 1.66–1.60 (m, 2 H, CHCH2CH2 ), 1.51 (s, 18 H, Boc), 1.45 (s, 9 H, tBu).

13C NMR (125 MHz, CDCl3): δ = 170.0, 152.7, 83.0, 81.4, 62.6, 58.8, 31.1, 29.8, 28.2, 28.1, 25.9, 18.6.

ESI-HRMS: m/z calcd for C19H35NNaO7 [M + Na]+: 412.2306; found: 412.2305.


#

tert-Butyl (S)-2-(Bis(tert-butoxycarbonyl)amino)-5-bromopentanoate (17)

A solution of 16 (512 mg, 1.32 mmol, 1.0 equiv) in CH2Cl2 (13 mL) was cooled to 0 °C and CBr4 (0.66 g, 1.98 mmol, 1.5 equiv) and PPh3 (0.69 g, 2.64 mmol, 2.0 equiv) were added. The reaction mixture was stirred at 0 °C for 20 min, then quenched with hexane/EtOAc (1:1). After passage through a short column, purification by neutral silica gel column chromatography (hexane/EtOAc, 4:1) yielded 17 as a colorless oil (0.53 g, 1.18 mmol, 90%); Rf = 0.73 (hexane/EtOAc, 4:1).

1H NMR (500 MHz, CDCl3): δ = 4.74–4.71 (dd, J = 5.0, 4.5 Hz, 1 H, CH), 3.47–3.40 (m, 2 H, CH2Br), 2.23–2.18 (m, 1 H, CH2CH 2CH), 2.06–1.98 (m, 1 H, CH2CH 2CH), 1.97–1.87 (m, 2 H, CH2 CH2CH), 1.51 (s, 18 H, Boc), 1.45 (s, 9 H, tBu).

13C NMR (125 MHz, CDCl3): δ = 169.6, 152.6, 83.1, 81.2, 58.2, 33.2, 29.8, 28.2, 28.1, 18.6.

ESI-HRMS: m/z calcd for C19H34BrNNaO6 [M + Na]+: 474.1462; found: 474.1454.


#

tert-Butyl (S)-2-(Bis(tert-butoxycarbonyl)amino)-5-(cyano-15 N)pentanoate (18)

Potassium cyanide-15 N (0.11 g, 1.65 mmol, 1.4 equiv) dissolved in DMF/H2O (8.63 mL/0.96 mL) was added to a solution of 17 (0.53 g, 1.18 mmol, 1.0 equiv) in DMF (16 mL). The reaction mixture was stirred for 6 h. The solution was filtered and the aqueous layer was then extracted with CH2Cl2. The combined organic layers were washed with brine, dried over Na2SO4, and concentrated in vacuo. Purification by silica gel column chromatography (hexane/EtOAc, 8:1) yielded 18 as a colorless solid (441 mg, 1.10 mmol, 94%); Rf = 0.53 (hexane/EtOAc, 4:1).

1H NMR (500 MHz, CDCl3): δ = 4.72–4.69 (dd, J = 6.0, 5.0 Hz, 1 H, CH), 2.44–2.34 (m, 2 H, CH2 CH2CH2CH), 2.22–2.15 (m, 1 H, CH2CH2CH 2CH), 2.05–1.98 (m, 1 H, CH2CH2CH 2CH), 1.76–1.69 (m, 2 H, CH2CH2 CH2CH), 1.51 (s, 18 H, Boc), 1.45 (s, 9 H, tBu).

13C NMR (125 MHz, CDCl3): δ = 169.3, 152.6, 119.5, 119.4, 83.3, 81.8, 58.0, 28.6, 28.2, 28.1, 22.8, 17.0, 17.0.

ESI-HRMS: m/z calcd for C20H34 14N15NNaO6 [M + Na]+: 422.2279; found: 422.2277.


#

tert-Butyl N 2,N 2-Bis(tert-butoxycarbonyl)-l-lysinate-6-15 N (5b)

18 (180 mg, 0.45 mmol, 1.0 equiv) was dissolved in 2-propanol (11.52 mL), together with 1 M HCl (0.50 mL, 0.50 mmol, 1.1 equiv) and platinum oxide (63.3 mg, 0.045 mmol, 0.1 equiv). The reaction mixture was stirred under 2 atm H2 for 48 h. Thereafter, the mixture was filtered through Celite and concentrated in vacuo. To the mixture was added 1 M aqueous NaOH solution (0.45 mL, 0.45 mmol, 1.0 equiv). After the mixture was stirred for 20 min, H2O was added, and the aqueous layer was then extracted with CH2Cl. The combined organic layers were washed with brine, dried over Na2SO4, and concentrated in vacuo to yield 5b as a yellow oil (177 mg, 0.44 mmol, quant); Rf = 0.18 (hexane/EtOAc, 1:1).

1H NMR (500 MHz, CDCl3): δ = 4.72–4.69 (dd, J = 5.5, 5.0 Hz, 1 H, CH), 2.44–2.34 (t, J = 6.5 Hz, 2 H, CH2 CH2CH2CH2CH), 2.09–2.02 (m, 1 H, CH2CH2CH2CH 2CH), 1.89–1.82 (m, 1 H, CH2CH2CH2CH 2CH), 1.51 (s, 18 H, Boc), 1.45 (s, 9 H, tBu), 1.41–1.32 (m, 4 H, CH2CH2 CH2 CH2CH).

13C NMR (125 MHz, CDCl3): δ = 207.1, 170.1, 152.7, 82.8, 81.3, 59.0, 31.1, 29.2, 28.5, 28.2, 28.1, 23.9.

ESI-HRMS: m/z calcd for C20H38 14N15NNaO6 [M + Na]+: 426.2592; found: 426.2592.


#

Di-tert-Butyl 6,6′-(((S)-5-(bis(tert-butoxycarbonyl)amino)-6-(tert-butoxy)-6-oxohexyl)azanediyl-15 N)(2S,2′S)-bis(2-(bis(tert-butoxycarbonyl)amino)-5-oxohexanoate-6-13 C) (3b)

4c (351 mg, 0.73 mmol, 2.0 equiv) was added to MeCN (11 mL). 5b (148 mg, 0.37 mmol, 1.0 equiv) was added to this solution, and then K2CO3 (152 mg, 1.1 mmol, 3.0 equiv) was added. After stirring for 20 h at room temperature, this solution was concentrated in vacuo to obtain the crude residue; Rf = 0.50 (hexane/EtOAc, 2:1).

ESI-HRMS: m/z calcd for 12C58 13C2H104 14N3 15NKO20 [M + K]+: 1242.6913; found: 1242.6944.


#

4-((S)-2-(Bis(tert-butoxycarbonyl)amino)-3-(tert-butoxy)-3-oxopropyl)-3-((S)-3-(bis(tert-butoxycarbonyl)amino)-4-(tert-butoxy)-4-oxobutyl)-1-((S)-5-(bis(tert-butoxycarbonyl)amino)-6-(tert-butoxy)-6-oxohexyl)-5-hydroxypyridin-1-ium-2,6-13 C 2-1-15 N (2b)

The crude residue was added to MeOH (9.2 mL) and additional K2CO3 (76 mg, 0.55 mmol, 1.5 equiv). Then, this solution was oxygenated at balloon pressure at room temperature. After stirring for 24 h at room temperature, the mixture was purified by silica gel column chromatography (CH2Cl2/MeOH, 100:7). 2b was obtained as an orange oil (168 mg, 0.13 mmol, 35%); Rf = 0.47 (CH2Cl2/MeOH, 10:1); aromatic protons confirmed (δ 9.39, 9.01, 7.90, 7.53).

ESI-HRMS: m/z calcd for 12C58 13C2H101 14N3 15NO19 + [M]+: 1184.7092; found: 1184.7082.


#

Deoxypyridinoline-13C2,15N1 (1-13C2,15N1)

TFA (25.5 mL) and H2O were added to 2b (160 mg, 126 μmol, 1.0 equiv). After stirring for 2 h at room temperature, this solution was concentrated in vacuo to obtain the crude residue. Then, purification by C18 silica gel column chromatography (H2O/MeOH, 7:3, 0.1% TFA in solvents) afforded 1-13C2,15N1 as the TFA salt (100 mg, 115 μmol, 91%).

1H NMR (500 MHz, D2O): δ = 8.46, 8.08 (d, J = 190.0 Hz, 1 H, H2 or H6), 8.38, 8.01 (d, J = 185.0 Hz, 1 H, H2 or H6), 4.48 (t, J = 7.0 Hz, 2 H, H7), 4.14–4.10 (m, 1 H, H18), 3.92–3.88 (t, J = 6.0 Hz, 1 H, H15), 3.81–3.79 (t, J = 6.0 Hz, 1 H, H11), 3.39–3.31 (m, 2 H, H17), 3.00–2.95 (m, 1 H, H13), 2.90–2.84 (m, 1 H, H13), 2.22–2.13 (m, 2 H, H14), 2.07–2.00 (m, 2 H, H8), 1.93–1.88 (m, 2 H, H10), 1.50–1.45 (m, 1 H, H9), 1.40–1.30 (m, 1 H, H9).

13C NMR (125 MHz, D2O): δ = 174.4, 173.8, 173.0, 164.9, 164.8, 163.7, 163.5, 141.8, 136.3, 136.2, 136.0, 129.6, 129.4, 129.2, 129.1, 128.9, 127.6, 127.5, 118.1, 115.8, 103.4, 103.3, 61.6, 61.6, 54.5, 54.4, 54.2, 53.0, 31.0, 30.4, 30.3, 30.1, 28.3, 26.1, 21.5.

ESI-HRMS: m/z calcd for 12C16 13C2H29 14N3 15NO7 + [M]+: 416.2068; found: 416.2068.


#
#

Conflict of Interest

The authors declare no conflict of interest.

Supporting Information


Corresponding Author

Toyonobu Usuki
Department of Materials and Life Sciences, Faculty of Science and Technology, Sophia University
7-1 Kioicho, Chiyoda-ku, Tokyo 102-8554
Japan   

Publication History

Received: 07 April 2025

Accepted after revision: 22 April 2025

Article published online:
03 June 2025

© 2025. Thieme. All rights reserved

Georg Thieme Verlag KG
Oswald-Hesse-Straße 50, 70469 Stuttgart, Germany


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Figure 1 Structure of deoxypyridinoline (1) and deoxypyridinoline-13C2,15N1 (1-13C2,15N1 )
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Scheme 1 Retrosynthetic analysis of deoxypyridinoline (1) and deoxypyridinoline-13C2,15N1 (1-13C2,15N1 )
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Scheme 2 Synthesis of α-halo ketones 4a and 4b. Reagents and conditions: a) tert-butyl 2,2,2-trichloroacetimidate, CH2Cl2, r.t., 20 h, 91%; b) (Boc)2O, MeCN, r.t., 16 h, quant; c) DIBAL-H, Et2O, –90 °C, 15 min, 82%; d) MePPh3Br, nBuLi, THF, –90 °C to 0 °C, 1.5 h, 60%; e) mCPBA, CH2Cl2, r.t., 18 h, 91%; f) NaI, AcOH, THF, r.t., 1.5 h, quant; g) IBX, CH2Cl2, 50 °C, 6 h, 83%; h) LiBr, AcOH, THF, r.t., 22 h, 87%; i) IBX, (CH2Cl)2, 65 °C, 12 h, 94%.
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Scheme 3 Synthesis of 13C α-bromo ketone 4c. Reagents and conditions: a) DIBAL-H, Et2O, –90 °C, 15 min, 82%; b) 13CH3PPh3I, nBuLi, THF, –90 °C to 0 °C, 15 min, 39%; c) mCPBA, CH2Cl2, r.t., 18 h, 90%; d) LiBr, AcOH, THF, r.t., 22 h, 81%; e) IBX, (CH2Cl)2, 65 °C, 12 h, 85%.
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Scheme 4 Synthesis of 15N amine 5b. Reagents and conditions: a) DIBAL-H, THF, –90 °C to 0 °C, 15 min, 43%; b) PPh3, CBr4, CH2Cl2, 0 °C, 20 min, 90%; c) KC15N, DMF/H2O, r.t., 6 h, 94%; d) PtO2 (10 mol%), H2 (2 atm), 2-propanol, HCl, r.t., 2 d; then, 1 N aq NaOH, quant.
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Scheme 5 Synthesis of deoxypyridinoline-13C2,15N1 (1-13C2,15N1 ). Reagents and conditions: a) K2CO3, MeCN, r.t., 20 h; b) K2CO3, O2, MeOH, r.t., 24 h, 35% (2 steps); c) TFA/H2O (95:5), r.t., 2 h, 91%.
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Figure 2