The economical synthesis of [2'-(13)C, 1,3-(15)N2]uridine; preliminary conformational studies by solid state NMR

The synthesis of [2'-(13)C, 1,3-(15)N2]uridine 11 was achieved as follows. An epimeric mixture of D-[1-(13)C]ribose 3 and D-[1-(13)C]arabinose 4 was obtained in excellent yield by condensation of K13CN with D-erythrose 2 using a modification of the Kiliani-Fischer synthesis. Efficient separation of the two aldose epimers was pivotally achieved by a novel ion-exchange (Sm3+) chromatography method. D-[2-(13)C]Ribose 5 was obtained from D-[1-(13)C]arabinose 4 using a Ni(II) diamine complex (nickel chloride plus TEMED). Combination of these procedures in a general cycling manner can lead to the very efficient preparation of specifically labelled 13C-monosaccharides of particular chirality. 15N-labelling was introduced in the preparation of [2'-(13)C, 1,3-(15)N2]uridine 11 via [15N2]urea. Cross polarisation magic angle spinning (CP-MAS) solid-state NMR experiments using rotational echo double resonance (REDOR) were carried out on crystals of the labelled uridine to show that the inter-atomic distance between C-2' and N-1 is closely similar to that calculated from X-ray crystallographic data. The REDOR method will be used now to determine the conformation of bound substrates in the bacterial nucleoside transporters NupC and NupG.     

Solid-state NMR spectroscopy detects interactions between tryptophan residues of the E. coli sugar transporter GalP and the alpha-anomer of the D-glucose substrate

An experimental approach is described in which high resolution 13C solid-state NMR (SSNMR) spectroscopy has been used to detect interactions between specific residues of membrane-embedded transport proteins and weakly binding noncovalent ligands. This procedure has provided insight into the binding site for the substrate D-glucose in the Escherichia coli sugar transport protein GalP. Cross-polarization magic-angle spinning (CP-MAS) SSNMR spectra of GalP in its natural membrane at 4 degrees C indicated that the alpha- and beta-anomers of D-[1-(13)C]glucose were bound by GalP with equal affinity and underwent fast exchange between the free and bound environments. Further experiments confirmed that by lowering the measurement temperature to -10 degrees C, peaks could be detected selectively from the substrate when restrained within the binding site. Dipolar-assisted rotational resonance (DARR) SSNMR experiments at -10 degrees C showed a selective interaction between the alpha-anomer of D-[1-(13)C]glucose and 13C-labels within [13C]tryptophan-labeled GalP, which places the carbon atom at C-1 in the alpha-anomer of D-glucose to within 6 A of the carbonyl carbon of one or more tryptophan residues in the protein. No interaction was detected for the beta-isomer. The role of tryptophan residues in substrate binding was investigated further in CP-MAS experiments to detect D-[1-(13)C]glucose binding to the GalP mutants W371F and W395F before and after the addition of the inhibitor forskolin. The results suggest that both mutants bind D-glucose with similar affinities, but have different affinities for forskolin. This work highlights a useful general experimental strategy for probing the binding sites of membrane proteins, using methodology which overcomes the problems associated with the unfavorable dynamics of weak ligands.     

Participation of the surface structure of Pharaonis phoborhodopsin, ppR and its A149S and A149V mutants, consisting of the C-terminal alpha-helix and E-F loop, in the complex-formation with the cognate transducer pHtrII, as revealed by site-directed 13C solid-state NMR

We have recorded 13C solid state NMR spectra of [3-13C]Ala-labeled pharaonis phoborhodopsin (ppR) and its mutants, A149S and A149V, complexed with the cognate transducer pharaonis halobacterial transducer II protein (pHtrII) (1-159), to gain insight into a possible role of their cytoplasmic surface structure including the C-terminal alpha-helix and E-F loop for stabilization of the 2:2 complex, by both cross-polarization magic angle spinning (CP-MAS) and dipolar decoupled (DD)-MAS NMR techniques. We found that 13C CP-MAS NMR spectra of [3-13C]Ala-ppR, A149S and A149V complexed with the transducer pHtrII are very similar, reflecting their conformation and dynamics changes caused by mutual interactions through the transmembrane alpha-helical surfaces. In contrast, their DD-MAS NMR spectral features are quite different between [3-13C]Ala-A149S and A149V in the complexes with pHtrII: 13C DD-MAS NMR spectrum of [3-13C]Ala-A149S complex is rather similar to that of the uncomplexed form, while the corresponding spectral feature of A149V complex is similar to that of ppR complex in the C-terminal tip region. This is because more flexible surface structure detected by the DD-MAS NMR spectra are more directly influenced by the dynamics changes than the CP-MAS NMR. It turned out, therefore, that an altered surface structure of A149S resulted in destabilized complex as viewed from the 13C NMR spectrum of the surface areas, probably because of modified conformation at the corner of the helix E in addition to the change of hydropathy. It is, therefore, concluded that the surface structure of ppR including the C-terminal alpha-helix and the E-F loops is directly involved in the stabilization of the complex through conformational stability of the helix E.     

Uridine binding by Zn(II) macrocyclic complexes: diversion of RNA cleavage catalysts

Zn(II) complexes of 1-oxa-4,7,10-triazacyclododecane (12[ane]N3O), 1,5,9-triazacyclododecane (12[ane]N3), and 1-hydroxyethyl-1,4,7-triazacyclononane (9[ane]N3OH) promote cleavage of the RNA analogue, 2-hydroxypropyl-4-nitrophenyl phosphate (HpPNP) at pH 8.0, I=0.10 M (NaCl), 25 degrees C with second-order rate constants of 8.9x10(-3), 9.0x10(-3), and 3.3x10(-3) M-1 s-1, respectively. Cleavage of HpPNP by these catalysts is inhibited by uridine with inhibition constants (Ki) of 1.2, 0.46, and 45 mM, respectively, under these conditions. Binding constants derived from these inhibition constants are 2-200-fold larger than those for binding of related Zn(II) complexes to phosphate diesters under similar conditions, suggesting that uridine sequences in RNA will inhibit Zn(II)-catalyzed cleavage by competing with phosphate diester binding sites. Further studies are carried out that utilize pH-potentiometric titrations to monitor uridine binding to five Zn(II) macrocyclic complexes in aqueous solution at 25 degrees C, I=0.10 M (NaCl). The data are consistent with binding of the Zn(II) complexes to the N3-deprotonated form of uridine to give log KU.-values of 5.29, 4.57, 4.56, 3.47, and 2.65 for the Zn(II) complexes of 12[ane]N3, 12[ane]N4, 12[ane]N3O, 15[ane]N3O2, and 9[ane]N3OH, respectively (12[ane]N4=1,4,7,10-tetraazacyclododecane, 15[ane]N3O2=1,4-dioxa-7,10,13-triazacyclopentadecane). For the five Zn(II) complexes studied, there is a linear relationship between uridine anion binding constants and hydroxide binding constants.     

Synthesis and characterization of chemically bonded stationary phases on hydride surfaces by hydrosilation of alkynes and dienes

A hydrosilation reaction was used to bind four compounds with one or more alkyne groups or two alkene functionalities. The diffuse reflectance infrared Fourier transform, the 29Si cross-polarization magic-angle spinning (CP-MAS) NMR, and the 13C CP-MAS NMR spectra were used to characterize the various bonded materials. The bonded phase density was determined from carbon elemental analysis. The two ten-carbon hydrophobic stationary phases were characterized chromatographically and static stability tests were run in acidic and basic solutions.     

Non-radioactive detection of MHC class II-peptide antigen complexes in the sub-picomole range by high-performance size-exclusion chromatography with fluorescence detection

In order to avoid chemical or structural modification of T-cell epitopes by labelling, a high-performance size-exclusion chromatographic fluorescence binding assay was developed, based on the intrinsic Trp fluorescence of major histocompatibility complex (MHC) proteins. The increase in Trp fluorescence intensity of the isolated human MHC product HLA-DR 1 on complex formation with unlabelled influenza matrix peptide[18-29] (IM[18-29]) was examined. Binding of IM[18-29] to the heterodimeric form of HLA-DR 1 (Kd = 4.8 mM) and to the disassembled alpha-and beta-subunits (Kd = 9.2 mM) could be demonstrated. In addition, the assay showed the peptide-induced formation of a dimeric conformer of HLA-DR 1, the nature of which is still undefined. Detection of HLA-DR 1 subunit-peptide complexes was possible in amounts of 25 ng in 10 microliter (80 fmol/microliter). The technique proved to be reproducible and less time consuming than common methods that need fluorescence or radioactive labelling.     

Solid-state nuclear magnetic resonance studies delineate the role of the protein in activation of both aromatic rings of thiamin

Knowledge of the state of ionization and tautomerization of heteroaromatic cofactors when enzyme-bound is essential for formulating a detailed stepwise mechanism via proton transfers, the most commonly observed contribution to enzyme catalysis. In the bifunctional coenzyme, thiamin diphosphate (ThDP), both aromatic rings participate in catalysis, the thiazolium ring as an electrophilic covalent catalyst and the 4'-aminopyrimidine as acid-base catalyst involving its 1',4'-iminopyrimidine tautomeric form. Two of four ionization and tautomeric states of ThDP are well characterized via circular dichroism spectral signatures on several ThDP superfamily members. Yet, the method is incapable of providing information about specific proton locations, which in principle may be accessible via NMR studies. To determine the precise ionization/tautomerization states of ThDP during various stages of the catalytic cycle, we report the first application of solid-state NMR spectroscopy to ThDP enzymes, whose large mass (160,000-250,000 Da) precludes solution NMR approaches. Three de novo synthesized analogues, [C2,C6'-(13)C(2)]ThDP, [C2-(13)C]ThDP, and [N4'-(15)N]ThDP used with three enzymes revealed that (a) binding to the enzymes activates both the 4'-aminopyrimidine (via pK(a) elevation) and the thiazolium rings (pK(a) suppression); (b) detection of a pre-decarboxylation intermediate analogue using [C2,C6'-(13)C(2)]ThDP, enables both confirmation of covalent bond formation and response in 4'-aminopyrimidine ring's tautomeric state to intermediate formation, supporting the mechanism we postulate; and (c) the chemical shift of bound [N4'-(15)N]ThDP provides plausible models for the participation of the 1',4'-iminopyrimidine tautomer in the mechanism. Unprecedented detail is achieved about proton positions on this bifunctional coenzyme on large enzymes in their active states.     

Efficient synthesis of [2'-18O]uridine and its incorporation into oligonucleotides: a new tool for mechanistic study of nucleotidyl transfer reactions by isotope effect analysis

Lack of sufficient quantities of isotopically labeled materials has precluded the use of heavy atom isotope effects to investigate mechanisms of nucleotidyl transfer reactions in nucleic acids. Here we achieve regioselective opening of 2,2'-cyclouridine with [(18)O2]benzoic acid/potassium hydride, allowing an efficient "one-pot" synthesis of [2'-18O]uridine in 88% yield. Conversion to the corresponding phosphoramidite enables solid-phase synthesis of [2'-(18)O] RNA substrates for isotope effect studies with nucleotidyl transferases and hydrolases.     

Transition state structure of E. coli tRNA-specific adenosine deaminase

Bacterial tRNA-specific adenosine deaminase (TadA) catalyzes the essential deamination of adenosine to inosine at the wobble position of tRNAs and is necessary to permit a single tRNA species to recognize multiple codons. The transition state structure of Escherichia coli TadA was characterized by kinetic isotope effects (KIEs) and quantum chemical calculations. A stem loop of E. coli tRNA(Arg2) was used as a minimized TadA substrate, and its adenylate editing site was isotopically labeled as [1'-(3)H], [5'-(3)H2], [1'-(14)C], [6-(13)C], [6-(15)N], [6-(13)C, 6-(15)N] and [1-(15)N]. The intrinsic KIEs of 1.014, 1.022, 0.994, 1.014 and 0.963 were obtained for [6-(13)C]-, [6-(15)N]-, [1-(15)N]-, [1'-(3)H]-, [5'-(3)H2]-labeled substrates, respectively. The suite of KIEs are consistent with a late SNAr transition state with a complete, pro-S-face hydroxyl attack and nearly complete N1 protonation. A significant N6-C6 dissociation at the transition state of TadA is indicated by the large [6-(15)N] KIE of 1.022 and corresponds to an N6-C6 distance of 2.0 A in the transition state structure. Another remarkable feature of the E. coli TadA transition state structure is the Glu70-mediated, partial proton transfer from the hydroxyl nucleophile to the N6 leaving group. KIEs correspond to H-O and H-N distances of 2.02 and 1.60 A, respectively. The large inverse [5'-(3)H] KIE of -3.7% and modest normal [1'-(3)H] KIE of 1.4% indicate that significant ribosyl 5'-reconfiguration and purine rotation occur on the path to the transition state. The late SNAr transition-state established here for E. coli TadA is similar to the late transition state reported for cytidine deaminase. It differs from the early SNAr transition states described recently for the adenosine deaminases from human, bovine, and Plasmodium falciparum sources. The ecTadA transition state structure reveals the detailed architecture for enzymatic catalysis. This approach should be readily transferable for transition state characterization of other RNA editing enzymes.     

ATP-GTP-BINDING PROTEINS AND ENDOGENOUS ADP-RIBOSYL TRANSFERASE IN Lemna paucicostata 441

Abstract— A crude extract containing membrane components of Lemna paucicostata was treated with 1% Lubrol PX and fractionated by gel nitration. Binding activities to non-hydrolyzable analogues of ATP, [³⁵S]ATPγS (adenosine 5'[;γ-thio]triphosphate) and that of GTP, [³⁵S]GTPγS (guanosine 5'[γ-thiojtriphosphate) were detected in some fractions, and these activities were prevented in the presence of 0.1 mM ATP or GTP. ATP and GTP were 2 to 3 orders of magnitude more effective than CTP or UTP in preventing this binding activity. These fractions showed ATPase and GTPase activities with 1 nM [γ-³²P]ATP or [γ–³²P]GTP substrate. Analyses by sodium dodecyl sulfate polyacrylamide gel electrophoresis of these fractions after binding with [³⁵S]ATPγS or [³⁵S]GTP-γ S revealed that these fractions contained [³⁵S]ATPγS and [³⁵S]GTPγS binding proteins with molecular weights of 53 000 and 60 000, respectively. Both of these proteins were [³²P]ADP-ribosylated by endogenous ADP-ribosyl transferase. Three proteins with molecular weights of 11 000, 12 000 and 13 000 which could bind [³⁵S]ATP7S or [-³⁵S]GTP-γ S were ADP-ribosylated by endogenous ADP-ribosyl transferase. Pertussis toxin stimulated ADP-ribosylation of these proteins. Four proteins with molecular weight of 37 000, 50 000, 80 000 and 115 000 with P^SS]ATP7S and [^,3S]GTP7S binding activities were also detected. The signal transduction of light to underlying clock mechanism in Lemna may be controlled by ATP-GTP-binding proteins and by the ADP-ribosylation of these proteins.     

Zirconium(IV) complexes of oxydiacetic acid in aqueous solution and in the solid state as studied by multinuclear NMR and X-ray crystallography

The structures of complexes of Zr(IV) and oxydiacetate (ODA2-) in aqueous solutions of pH 0-7 were investigated with the use of 1H, 13C, and 17O NMR spectroscopy. Equilibria of mononuclear [Zr(oda)]2+, [Zr(oda)2], and [Zr(oda)3]2- complexes have been observed. In all complexes ODA2- is bound in a tridentate fashion through the two carboxylate groups and the ether oxygen. No di- or oligonuclear species containing ODA2- were observed. An excess of free Zr(IV) remains in solution, probably as a result of weak electrostatic interactions between negatively charged Zr-ODA complexes or free ODA2- and a positively charged cyclic tetranuclear hydroxy zirconium complex. CP-MAS 13C NMR spectra of solid compounds isolated from the samples indicated that the structures of the [Zr(oda)2] and [Zr(oda)3]2- complexes in solution are similar to those in the solid state. This is corroborated by the single-crystal X-ray structure of Na2[Zr(oda)3] x 5.5 H2O, which was obtained from a solution containing exclusively the [Zr(oda)3]2- complex. In this structure Zr(IV) is nine-coordinate with the three ODA2- ligands bound in a tricapped trigonal prismatic geometry. The negative charge of this [Zr(oda)3]2complex is balanced by two Na+ ions, one of which is on a center of symmetry between delta and lambda enantiomers of [Zr(oda)3]2-. This Na+ is octahedrally coordinated to six (non Zr(IV)-bound) carboxylate oxygen atoms of six different [Zr(oda)3]2- units.     

Mononuclear-dinuclear equilibrium of grafted copper complexes confined in the nanochannels of MCM-41 silica

Following the structural concept of copper-containing proteins in which dinuclear copper centers are connected by hydroxide bridging ligands, a bidentate copper(II) complex has been incorporated into nano-confined MCM-41 silica by a multistep sequential grafting technique. Characterization by a combination of EPR spectroscopy, X-ray photoelectron spectroscopy (XPS), UV/Vis spectroscopy, IR spectroscopy , and solid-state (13)C and (29)Si cross-polarization magic-angle spinning (CP-MAS) NMR suggests that dinuclear Cu complexes are bridged by hydroxide and other counterions (chloride or perchlorate ions), similar to the situation for EPR-undetectable [Cu(II)···Cu(II)] dimer analogues in biological systems. More importantly, a dynamic mononuclear-dinuclear equilibrium between different coordination modes of copper is observed, which strongly depends on the nature of the counterions (Cl(-) or ClO(4)(-)) in the copper precursor and the pore size of the silica matrix (the so-called confinement effect). A proton-transfer mechanism within the hydrogen-bonding network is suggested to explain the dynamic nature of the dinuclear copper complex supported on the MCM-41 silica.     

Sensitivity to antitumor drugs and vinblastine binding to membrane in rat ascites hepatoma AH66 cells.

Rat ascites hepatoma AH66 cells have lower sensitivity to Vinca alkaloids and anthracycline antibiotics than AH66F cells, a subline of AH66 cells. AH66 cells expressed P-glycoprotein, while the protein was not detectable in AH66F cells. There are two affinity sites for [3H]vinblastine binding in the AH66 cell membrane, while AH66F cells have only one affinity site. The high affinity [3H]vinblastine binding in AH66 cells was inhibited by Adriamycin, verapamil, nicardipine, and reserpine. The high affinity site of the binding may be the multidrug transporter, P-glycoprotein. [3H]Vinblastine binding was not influenced by adenosine 3'-5'-monophosphate (AMP), adenosine triphosphate (ATP), or guanosine triphosphate (GTP). The multidrug resistance in AH66 cells may depend on P-glycoprotein which is not modulated by nucleotide.     

Simultaneous determination of stable isotopically labelled L-histidine and urocanic acid in human plasma by stable isotope dilution mass spectrometry.

A capillary gas chromatographic-mass spectrometric method for the simultaneous determination of stable isotopically labelled L-histidine (L-[3,3-2H2,1',3'-15N2]histidine, L-His-[M + 4]) and urocanic acid ([3-2H,1',3'-15N2]urocanic acid, UA-[M + 3]) in human plasma was developed using DL-[2,3,3,5'-2H4,2'-13C,1',3'-15N2]histidine (DL-His-[M + 7]) and [2,3,5'-2H3,2'-13C,1',3'-15N2]urocanic acid (UA-[M + 6]) as internal standards. L-Histidine and urocanic acid were derivatized to alpha N-(trifluoroacetyl)-imN-(ethoxycarbonyl)-L-histidine n-butyl ester and imN-(ethoxycarbonyl)urocanic acid n-butyl ester. Quantification was carried out by selected ion monitoring of the molecular ions of the respective derivatives of L-His-[M + 4], DL-His-[M + 7], UA-[M + 3] and UA-[M + 6]. The sensitivity, specificity, precision and accuracy of the method were demonstrated to be satisfactory for measuring plasma concentrations of L-His-[M + 4] and UA-[M + 3] following administration of trace amounts of L-His-[M + 4] to humans.     

Synthesis of [5'-13C]ribonucleosides and 2'-deoxy[5'-13C]ribonucleosides

The present efficient synthesis of [5'-13C]ribonucleosides and 2'-deoxy[5'-13C]ribonucleosides is characterized by the synthesis of the D-[5-13C]ribose derivative as an intermediate via the Wittig reaction of 4-aldehydo-D-erythrose dialkyl acetals with Ph3P13CH3I-BuLi to introduce the 13C label at the 5-position of a pentose. This was followed by the highly diastereoselective osmium dihydroxylation for the preparation of 2,3-di-O-benzyl-D-[5-13C]ribose dialkyl acetal and the cyclization from D-[5-13C]ribose dialkyl acetal derivatives to the alkyl D-[5-13C]ribofuranoside derivative by the use of LiBF(4). The obtained D-[5-13C]ribose derivative was converted into [5'-13C]ribonucleosides and subsequently into the corresponding 2'-deoxynucleosides.     

Transition-state analysis of Trypanosoma cruzi uridine phosphorylase-catalyzed arsenolysis of uridine

Uridine phosphorylase catalyzes the reversible phosphorolysis of uridine and 2'-deoxyuridine to generate uracil and (2-deoxy)ribose 1-phosphate, an important step in the pyrimidine salvage pathway. The coding sequence annotated as a putative nucleoside phosphorylase in the Trypanosoma cruzi genome was overexpressed in Escherichia coli , purified to homogeneity, and shown to be a homodimeric uridine phosphorylase, with similar specificity for uridine and 2'-deoxyuridine and undetectable activity toward thymidine and purine nucleosides. Competitive kinetic isotope effects (KIEs) were measured and corrected for a forward commitment factor using arsenate as the nucleophile. The intrinsic KIEs are: 1'-(14)C = 1.103, 1,3-(15)N(2) = 1.034, 3-(15)N = 1.004, 1-(15)N = 1.030, 1'-(3)H = 1.132, 2'-(2)H = 1.086, and 5'-(3)H(2) = 1.041 for this reaction. Density functional theory was employed to quantitatively interpret the KIEs in terms of transition-state structure and geometry. Matching of experimental KIEs to proposed transition-state structures suggests an almost synchronous, S(N)2-like transition-state model, in which the ribosyl moiety possesses significant bond order to both nucleophile and leaving groups. Natural bond orbital analysis allowed a comparison of the charge distribution pattern between the ground-state and the transition-state models.     

Supramolecular complex induced by the binding of sodium dodecyl sulfate to PAMAM dendrimers

Isothermal titration calorimetry (ITC) and dynamic light scattering (DLS) were employed to study the spontaneous supramolecular complexation of amine terminated PAMAM dendrimer (G3[EDA] PAMAM-NH2) induced by the binding of an anionic surfactant, sodium dodecyl sulfate (SDS). At pHor=10, the electrostatic binding ceased because the deprotonated PAMAM dendrimer was uncharged, and hence the surfactant-induced supramolecular assembly could not be formed.     

Pressure-induced isomerization of retinal on bacteriorhodopsin as disclosed by fast magic angle spinning NMR

Bacteriorhodopsin (bR) is a retinal protein in purple membrane of Halobacterium salinarum, which functions as a light-driven proton pump. We have detected pressure-induced isomerization of retinal in bR by analyzing 15N cross polarization-magic angle spinning (CP-MAS) NMR spectra of [zeta-15N]Lys-labeled bR. In the 15N-NMR spectra, both all-trans and 13-cis retinal configurations have been observed in the Lys N(zeta) in protonated Schiff base at 148.0 and 155.0 ppm, respectively, at the MAS frequency of 4 kHz in the dark. When the MAS frequency was increased up to 12 kHz corresponding to the sample pressure of 63 bar, the 15N-NMR signals of [zeta-15N]Lys in Schiff base of retinal were broadened. On the other hand, other [zeta-15N]Lys did not show broadening. Subsequently, the increased signal intensity of [zeta-15N]Lys in Schiff base of 13-cis retinal at 155.0 ppm was observed when the MAS frequency was decreased from 12 to 4 kHz. These results showed that the equilibrium constant of [all-trans-bR]/[13-cis-bR] in retinal decreased by the pressure of 63 bar. It was also revealed that the structural changes induced by the pressure occurred in the vicinity of retinal. Therefore, microscopically, hydrogen-bond network around retinal would be disrupted or distorted by a constantly applied pressure. It is, therefore, clearly demonstrated that increased pressure induced by fast MAS frequencies generated isomerization of retinal from all-trans to 13-cis state in the membrane protein bR.     

NMR confirmation that tryptophan dehydrogenation occurs with syn stereochemistry during the biosynthesis of CDA in Streptomyces coelicolor

Doubly labeled (2'S,3'R)-[3'-2H1,13C1]-tryptophan was fed to the Trp-His auxotrophic Streptomyces coelicolor strain WH101. Mass spectrometry showed single and double incorporation of the labeled Trp into the calcium-dependent lipopeptide antibiotic (CDA4a). From 13C NMR spectroscopy, it was apparent that the C3'-signal of the (Z)-2',3'-dehydrotryptophan (position 11 in CDA4) was a 1:1:1 triplet indicating that the deuterium atom in the pro-R position of the methylene group is retained during Trp-oxidation. This provides definitive proof that Trp dehydrogenation occurs through the loss of the 2' and pro-3'S hydrogen atoms with overall syn stereochemistry.