Application of biosynthetic 13C-enrichment using [1-13C]-, [2-13C]- and [1,2-13C2]-acetate as precursor for 13C NMR spectral assignment of higher plant metabolites. The assignments of some bryonolic acid derivatives

The ¹³C NMR spectra of some derivatives of bryonolic acid (1) (D:C-friedoolean-8-en-3β-ol-29-oic acid) were assigned by means of ¹³C-enrichment, lanthanide-induced shifts (LIS) and comparison of chemical shift data between derivatives. The ¹³C-enriched species of 1, i.e., 1a, 1b and 1c were biosynthesized by Luffa cylindrica (Cucurbitaceae) callus fed with [1-¹³C]-, [2-¹³C]- or [1,2-¹³C₂]-acetate, respectively. Methyl acetylbryonolates 2, 2a, 2b and 2c, methyl bryonolates 3, 3a, 3b and 3c, methyl bryononates 4 and 4a, diacetyl-3β,29-diols (3,29-diacetyl-D:C-friedoolean-8-en-β,29-diol) 5, 5a, 5b and 5c, and 3-acetyl-3β,29-diols 6, 6a and 6b were prepared from 1, 1a, 1b and 1c, and their ¹³C NMR spectra were recorded. The ¹³C concentration of the ¹³C-enriched species was high enough to exhibit the satellite peaks clearly, and the analysed data were very useful for this study. Thus, total assignments for 2, 3, 4, 5 and 6 were established. It was found that conversion of the methoxycarbonyl group at C-29 into an acetoxymethyl group caused complex changes in the chemical shifts of the C, D- and E-ring carbons and those of the methyl carbons linked to these rings.     

Reverse phase high pressure liquid chromatography for the separation of peptide hormone diastereoisomers

Diastereoisomers of specifically labeled oxytocin derivatives were resolved using reverse phase high pressure liquid chromatography. The peptides [1-hemi-DL-[alpha-2H]cystine]oxytocin, [6-hemi-DL-[alpha-2H]cystine]oxytocin, [2-DL-[alpha-2H]tyrosine]oxytocin and[8-DL-[2-13C]leucine]oxytocin were readily separated using the conditions described. The diastereoisomers of the oxytocin analog [3-DL-[2-13C]leucine]oxytocin also demonstrated baseline resolution under the same conditions. The procedure offers the investigator a rapid method for screening synthetic oxytocin peptides for undesirable diastereoisomeric by-products.     

Biosynthesis of the antibiotic verrucarin E use of [1-13C]-, [2-13C]-, [1,2-13C]- and [2-13C, 2-2H3]-acetates. Verrucarins and roridins, 37th communication [1]

The origin of the carbon skeleton of verrucarin E (1) from acetate as precursor is confirmed. Incorporation studies with [1,2-¹³C]-acetate have demonstrated that two acetoacetate units couple together as shown in pattern A (Scheme 2) and not as in B . Analysis of the deuterium distribution in both verrucarin E (1) isolated after the incorporation of [2-¹³C,2-²H₃]-acetate and in sodium acetate obtained after Kuhn-Roth oxidation of the metabolite demonstrated that C(7) is derived from the starter unit of one of the acetoacetate moieties. The deuterium exchange in verrucarin E (1) occurring during fermentation was investigated.     

The mass spectra of [17-13C]phyllocladene and [3-13C]methylenecholestane—III

The mass spectra of [17-¹³C]phyllocladene and [3-¹³C]methylenecholestane have been examined. It is shown that there are some rearrangements at 70 e V as in the case of [17-¹³C]kaurene. However, no extensive randomization is evident at the molecular ion level. The results are interesting because very little is known about ¹³C randomization in polycyclic aliphatic hydrocarbons. The percentage retention of label was calculated for each ion.     

Synthesis of 2-dichloromethyl-2-methyl [2-14C]-1,3-dioxolane

The labelled compound was prepared by chlorination of [2-¹⁴C]acetone obtained from the barium salt of [1-¹⁴C]acetic acid by pyrolysis. The reaction product 1,1-dichloro [2-¹⁴C]acetone was converted to 2-dichloromethyl-2-methyl [2-¹⁴C]-1,3-dioxolane by condensation with ethylene glycol in the presence of thionyl chloride. Radiochemical yield: 62% based on [1-¹⁴C]acetic acid.     

Chemoenzymatic synthesis of [3,9-C]-labeled NeuAc and KDN

The chemoenzymatic synthesis of ¹³C-labeled sialic acid (NeuAc) and 3-deoxy-d-glycero-d-galacto-2-nonulosonic acid (KDN) as useful molecular probes for studying the conformation of sialyl or KDN oligosaccharides attached to proteins was performed by using [6-¹³C]-ManNAc, [6-¹³C]-Man and [3-¹³C]-pyruvic acid sodium salt. In the synthesis of the compounds, 5,6-anhydro intermediates were found to easily provide not only 6-¹³C-labeled but also 5-, and 6-modified NeuAc and KDN analogs. Furthermore, it was demonstrated that identical results are obtained by NMR for both [3,9-¹³C]-NeuAc and 1:1 mixtures of [3-¹³C]- and [9-¹³C]-NeuAc.     

Practical synthesis of [1-C]- and [6-C]-d-galactose

The chemical synthesis of ¹³C-labeled d-galactose as useful molecular probes for studying the conformation of oligosaccharides attached to proteins was performed. The method for synthesizing the title labeled compounds was newly developed via the corresponding 1-ene and 5-ene compounds derived from 1,2:5,6-di-O-isoproppylidene-α-d-galactofuranose by considering the efficient introduction of the atom. All protons of galactose from H-1 to H-6 were observed by the HMQC-HOHAHA technique using 1:1 mixtures of methyl [1-¹³C]- and [6-¹³C]-β-d-galactopyranoside, which were prepared from the title compounds.     

Order-Unity 13C Nuclear Polarization of [1-13C]Pyruvate in Seconds and the Interplay of Water and SABRE Enhancement

Signal Amplification By Reversible Exchange in SHield Enabled Alignment Transfer (SABRE-SHEATH) is investigated to achieve rapid hyperpolarization of ¹³C₁ spins of [1-¹³C]pyruvate, using parahydrogen as the source of nuclear spin order. Pyruvate exchange with an iridium polarization transfer complex can be modulated via a sensitive interplay between temperature and co-ligation of DMSO and H₂O. Order-unity ¹³C (>50 %) polarization of catalyst-bound [1-¹³C]pyruvate is achieved in less than 30 s by restricting the chemical exchange of [1-¹³C]pyruvate at lower temperatures. On the catalyst bound pyruvate, 39 % polarization is measured using a 1.4 T NMR spectrometer, and extrapolated to >50 % at the end of build-up in situ. The highest measured polarization of a 30-mM pyruvate sample, including free and bound pyruvate is 13 % when using 20 mM DMSO and 0.5 M water in CD₃OD. Efficient ¹³C polarization is also enabled by favorable relaxation dynamics in sub-microtesla magnetic fields, as indicated by fast polarization buildup rates compared to the T₁ spin-relaxation rates (e. g., ∼0.2 s⁻¹ versus ∼0.1 s⁻¹, respectively, for a 6 mM catalyst-[1-¹³C]pyruvate sample). Finally, the catalyst-bound hyperpolarized [1-¹³C]pyruvate can be released rapidly by cycling the temperature and/or by optimizing the amount of water, paving the way to future biomedical applications of hyperpolarized [1-¹³C]pyruvate produced via comparatively fast and simple SABRE-SHEATH-based approaches.     

Biosynthesis of the cytochalasans. Part III. C-NMR. of cytochalasin B (phomin) and cytochalasin D. Incorporation of [1-13C]- and [2-13C]-sodium acetate

Sequential single frequency decoupling and partially relaxed Fourier transform (PRFT) are used to assign the natural abundance ¹³C-NMR. spectra of cytochalasin B (phomin) ( 1 ) and cytochalasin D ( 2 ). Cultures of Phoma spec. S 298 were fed [2-¹³C]-sodium acetate, and the distribution of this precursor in cytochalasin B (phomin) ( 1 ) was determined by ¹³C-NMR. spectroscopy. Likewise, the labelling patterns in cytochalasin D (zygosporin A) ( 2 ) from Zygos-posium masonii could be identified after incorporation of [2-¹³C]-sodium acetate and [l-¹³C]-sodium acetate. The results confirm previous proposals for the biogenesis of the cytochalasans from phenylalanine, methionine, and a C₁₈, or C₁₆, polyketide part.     

An Efficient Synthesis of Optically Active [4-13C] Labelled Quorum Sensing Signal Autoinducer-2

A new synthetic route for the quorum sensing signal Autoinducer-2 (AI-2) is described and used for the preparation of [4-¹³C]-AI-2 starting from [1-¹³C]-bromoacetic acid. The key step in this process was the enantioselective reduction of an intermediate ketone. This synthesis provides, selectively, both enantiomers of the labelled or unlabelled parent compound, (R) or (S)-4,5-dihydroxypentane-2,3-dione (DPD) and was used for an improved synthesis of [1-¹³C]-AI-2.     

Furopyridines. XI. 13C NMR Spectra of 2- or 3-Substituted Furo[2,3-b]-, Furo[3,2-b]-, Furo[2,3-c]- and Furo[3,2-c]pyridine

The ¹³C nmr spectra of 2- or 3-monosubstituted furo[2,3-b]- 1a-1j , furo[3,2-b]- 2a-2j , furo[2,3-c]- 3a-3j and furo[3,2-c]pyridine derivatives 4a-4j are reported. Effects by change in annelation and substituent effects on ¹³C chemical shifts and carbon-proton coupling constants are discussed. The spectra of benzo[b]furan derivatives 5a-5j having the corresponding substituent are also reported for comparison.     

Biosynthese de la botryodiplodine,mycotoxine de penicillium roqueforti: Incorporations d'acetate[1-13C], [2-13C], [1-2 13C]et d'acide orsellinique 2-13C-carboxyle 13C], [3-4 13C]

Incorporations of [1-¹³C], [2-¹³C], [1-2¹³C]acetate and [2-¹³C, carboxyl-¹³C], [3-4¹³C]orsellinic acid into botryodiplodin indicate that this mycotoxin is biosynthesized by the polyketide pathway. Orsellinic acid is a precursor of botryodiplodin. A biosynthetic pathway, using orsellinic acid as precursor, is proposed.     

Synthesis of [1, 2-13C2]-S-Lysine Hydrochloride

Cobalt catalyzed hydroformylation-amidocarbonylation of 3-cyanopropene by carbon-13 monoxide and acetamide produced a [1,2-¹³C₂]-labelled mixture of 5-cyano-2-acetamidopentanoic acid and 4-cyano-3-methyl-2-acetamidobutanoic acid (n to b ratio of 7 to 3. 55% yield). Acylase I resolution of this mixture gave the free S-amino acids which could be separated by crystallization. Catalytic reduction of the cyano group of the straight chain acid furnished [1,2-¹³C₂] -S-lysine. HCl.     

An asymmetric synthesis of l-[3-C]phenylalanine and l-[3-C]tyrosine from [C]carbon monoxide

-[3-¹³C]Phenylalanine and -[3-¹³C]tyrosine were synthesized. [α-¹³C]Benzyl bromides were prepared from [¹³C]carbon monoxide via the palladium-catalyzed carboalkoxylation of aryl halides. The asymmetric carbon corresponding to the 2-position in phenylalanine was introduced by the diastereoselective alkylation of Dellaria's oxazinone with [α-¹³C]benzyl bromides. Finally, ethanolysis, deprotection, hydrogenolysis and acid hydrolysis of the resulting alkylated oxazinones gave -[3-¹³C]phenylalanine and -[3-¹³C]tyrosine in high optical purity.     

13C?13C spin coupling constants in tetrahydronaphthylene,hexahydrobenzanthracene and benzo[a]pyrene derivatives

The ¹³C? ¹³C spin coupling constants have been determined in substituted [1-¹³C]tetrahydronaphthylenes, [5-¹³C]hexahydrobenzanthracenes and [5-¹³C]benzanthracene. In addition, the ¹³C? ¹³C spin coupling constants for 7-hydroxy[7-¹³C]benzopyrene, trans-7,8-dihydro[7-¹³C]benzopyrene-7,8-diol and trans-7,8-dihydro[10-¹³C]benzopyrene-7,8-diol are reported, together with the one-bond carbon-carbon coupling constants between C-4 and C-5 in selected 4,5-disubstituted benzopyrenes. Values for the directly bonded coupling constants and long-range coupling constants are similar to those reported previously for other aromatic and aliphatic systems. Substituent effects on carbon-carbon coupling are compared for similarly substituted cyclic and acyclic systems.     

Biosynthesis of psilotin from [2′,3′-13C2,1′-14C,4-3H]phenylalanine studied with 13C-NMR

The 6-phenyl-dihydro-α-pyrone moiety of psilotin is formed from [2′,3′-¹³C₂,1′-¹⁴C,-4-³H]phenylalanine in the plant $\text{Psilotum}$$\text{nudum}$ with retention of all the isotopes.     

Enzymatic Synthesis of [3-14C]Cinnamic Acid

Convenient and efficient route of the synthesis of [3-¹⁴C] cinnamic acid is reported. [1-¹⁴C]Benzoic acid, prepared by carbonation of Grignard reagent with [¹⁴C]carbon dioxide, was reduced to [1-¹⁴C]benzyl alcohol. In the enzymatic step this alcohol was selectively oxidised to [1-¹⁴C]benzaldehyde using enzyme YADH (Ec. 1.1.1.1) and immediately condensed with malonic acid. This combined chemical and enzymatic approach allows to obtain [3-¹⁴C]cinnamic acid with radiochemical yield higher than 50% in respect to the starting alcohol.     

Biosynthesis of highly modified meroterpenoids in aspergillus variecolor. Incorporation of 13C-labelled acetates and methionine into anditomin and andilesin C

Incorporations of [1-¹³C]?, [2-¹³C]?, [1,2-¹³C₂]-acetates and [¹³C]-methionine into anditomin, a metabolite of $\text{Aspergillus variecolor}$, indicate its formation by a mixed polyketide-terpenoid biosynthetic pathway similar to that elucidated for andibenin; observations are made on the possible biosynthetic relationship of the $\text{A. variecolor}$ metabolites with austin and terretonin, mycotoxins recently isolated from $\text{A. ustus}$ and $\text{A. terreus}$ respectively.     

Total 1H and 13C chemical shift assignment of indolizino[3,4,5-a,b]-isoindole and 2-methylthiobenz[f]imidazo[5,1,2-c,d]indolizine

The ¹H and ¹³C nmr spectral assignment of indolizino[3,4,5-a,b]isoindole and 2-methylthiobenz[f]-imidazo[5,1,2-c,d]indolizine are described. A concerted interpretation of the HMQC, HMQC-TOCSY, HMBC and nOe-difference experiments were used to assign the ¹H and ¹³C resonances of indolizino-[3,4,5-a,b]isoindole, whereas for 2-methylthiobenz[f]imidazo[5,1,2-c,d]indolizine a concerted interpretation of the COSY, HMQC and HMBC experiments were used to generate spectral assignments.