Synthesis of 7-(15)N-Oroidin and evaluation of utility for biosynthetic studies of pyrrole-imidazole alkaloids by microscale (1)H-(15)N HSQC and FTMS

Abstract

Numerous marine-derived pyrrole-imidazole alkaloids (PIAs), ostensibly derived from the simple precursor oroidin, 1a, have been reported and have garnered intense synthetic interest due to their complex structures and in some cases biological activity; however very little is known regarding their biosynthesis. We describe a concise synthesis of 7-(15)N-oroidin (1d) from urocanic acid and a direct method for measurement of (15)N incorporation by pulse labeling and analysis by 1D (1)H-(15)N HSQC NMR and FTMS. Using a mock pulse labeling experiment, we estimate the limit of detection (LOD) for incorporation of newly biosynthesized PIA by 1D (1)H-(15)N HSQC to be 0.96 microg equivalent of (15)N-oroidin (2.4 nmole) in a background of 1500 microg of unlabeled oroidin (about 1 part per 1600). 7-(15)N-Oroidin will find utility in biosynthetic feeding experiments with live sponges to provide direct information to clarify the pathways leading to more complex pyrrole-imidazole alkaloids.

Figure 1

Oroidin (1a) and other representatives of the pyrrole-imidazole alkaloid (PIA) family from marine sponges.

Figure 2

MALDI FTMS (7T) expansions of the pseudomolecular ion [M+H]⁺ of oroidin for (a) natural 1a (C₁₁H₁₂Br₂¹⁴N₅NO) and (b) 7-¹⁵N-oroidin (1d), C₁₁H₁₂Br₂Br₂¹⁴N₄¹⁵NO, >98% atom ¹⁵N. Mass accuracy = 1ppm.

Figure 3

MALDI FTMS (7T) expansions of m/z and peak height, h, for the ‘M+1’ peak of the pseudomolecular ions of oroidin (C₁₁H₁₁Br₂N₅O). Mass accuracy = 1 ppm (R = 240,000). (a) natural 1a. (b) 1a + 1d (0.04 mol equiv). (c) 1a + 1d (0.10 mol equiv). Peak assignments of [M+H]⁺ in (c): m/z 388.94372, ¹²C₁₀¹³CH₁₂Br₂N₅O⁺; m/z 388.93739; ¹²C₁₁H₁₂Br₂N₄¹⁵NO⁺. The unrelated peak at m/z 388.9305 (indicated with an ‘*’) is tentatively assigned to some loss of H from the dominant ⁷⁹Br/⁸¹Br isotopomer, C₁₁H₁₀⁷⁹Br⁸¹BrN₅O⁺ ([M]⁺, Δmmu = –0.5).

Figure 4

2D ¹H-¹⁵N HSQC (600 MHz, 1:1 DMSO-d₆/benzene-d₆) of natural oroidin (1a, depicted tautomer is arbitrary). N locants are labeled following the numbering scheme of Assman and Köck (Ref.xxxii). ns = 16, T2, T1 = 2K × 256; F2, F1 = 2K × 1K; d1 =1.5s

Figure 5

1D ¹H-¹⁵N HSQC experiments (¹H δ, 1.7 mm microcryoprobe, 600 MHz) of 1a with N-H assignments. Labels indicate N atom assignments made from a separate ¹H-¹⁵N HMBC experiment (not shown). (a) 1500 μg of 1a, no added [¹⁵N]-1d. (b) 1500 μg 1a + 10 μg 1d. (c) 1500 μg 1a + 5.0 μg 1d. (d) 1500 μg 1a + 2.5 μg. Relaxation delay, d1 = 1.50 s, optimized for ¹J_N-H = 90 Hz; ¹H π/2 pulse = 12 μS, ¹⁵N π/2 pulse = 34 μS; NA = 6K; dummy scans = 16; NI = 1; T2 = F2= 8K points (no zero fill). See Supporting Information for calculations of limit of detection (LOD), mean and SD.

Figure 6

Linear regression of ¹H-¹⁵N HSQC cross-peak integral ratio, (I–I₀)/I₀ for the NH(C=O) signal in ‘mock’ pulse labeling experiments with natural abundance 1a (1500 μg, I₀) spiked with measured aliquots of 1d (μg, ‘x-scale'). Observed values, I, for the NH-C=O ¹H-¹⁵N couplet are normalized to the intensity of I_py the pyrrole N-H cross peak. For the NH(C=O) peak in the natural abundance sample (no added 1d), I = I₀ and I₀/I_py = 1.00. Error bars are ± SD.

Figure 7

Possible biosynthesis of oroidin (1a) and conversion to stevensine (17)

Scheme 1

Retrosynthetic analysis of 7-¹⁵N-oroidin (1d) from urocanic acid.

Scheme 2

Synthesis of ¹⁵N-labeled oroidin.^{a a}Reagents and conditions: (a) AcCl, MeOH, reflux, quant.; (b) TrCl, Et₃N, DMF; (c) DIBAL-H, THF; (d) TBSCl, imidazole, 74% for 3 steps; (e) n-BuLi, TsN₃, THF, 94%; (f) TBAF, THF, 82%; (g) MsCl, Et₃N, THF; (h) potassium phthalimide-¹⁵N, DMF, 42% for 2 steps; (i) NH₂NH₂, EtOH, 50 °C; (j) 4,5-dibromo-2-trichloroacetylpyrrole, Na₂CO₃, DMF, 76% for 2 steps; (k) AcCl, MeOH/EtOAc, 99%; (l) H₂/Pd-Lindlar, MeOH/THF, 84%.