Biliary bile acids in birds of the Cotingidae family: taurine-conjugated (24R,25R)-3α,7α,24-trihydroxy-5β-cholestan-27-oic acid and two epimers (25R and 25S) of 3α,7α-dihydroxy-5β-cholestan-27-oic acid

Three C(27) bile acids were found to be major biliary bile acids in the capuchinbird (Perissocephalus tricolor) and bare-throated bellbird (Procnias nudicollis), both members of the Cotingidae family of the order Passeriformes. The individual bile acids were isolated by preparative RP-HPLC, and their structures were established by RP-HPLC, LC/ESI-MS/MS and NMR as well as by a comparison of their chromatographic properties with those of authentic reference standards of their 12α-hydroxy derivatives. The most abundant bile acid present in the capuchinbird bile was the taurine conjugate of C(27) (24R,25R)-3α,7α,24-trihydroxy-5β-cholestan-27-oic acid, a diastereomer not previously identified as a natural bile acid. The four diastereomers of taurine-conjugated (24ξ,25ξ)-3α,7α,24-trihydroxy-5β-cholestan-27-oic acid could be distinguished by NMR and were resolved by RP-HPLC. The RRT of the diastereomers (with taurocholic acid as 1.0) were found to be increased in the following order: (24R,25R)<(24S,25R)<(24S,25S)<(24R,25S). Two epimers (25R and 25S) of C(27) 3α,7α-dihydroxy-5β-cholestan-27-oic acid were also present (as the taurine conjugates) in both bird species. Epimers of the two compounds could be distinguished by their NMR spectra and resolved by RP-HPLC with the (25S)-epimer eluting before the (25R)-epimer. Characterization of the taurine-conjugated (24R,25R)-3α,7α,24-trihydroxy-5β-cholestan-27-oic acid and two epimers (25R and 25S) of 3α,7α-dihydroxy-5β-cholestan-27-oic acid should facilitate their detection in peroxisomal disease and inborn errors of bile acid biosynthesis.     

Efficient synthesis and biological evaluation of all A-ring diastereomers of 1alpha,25-dihydroxyvitamin D3 and its 20-epimer

An improved synthesis of the diastereomers of 1alpha,25-dihydroxyvitamin D3 (1) was accomplished utilizing our practical route to the A-ring synthon. We applied this procedure to synthesize for the first time all possible A-ring diastereomers of 20-epi-1alpha,25-dihydroxyvitamin D3 (2). Ten-step conversion of 1-(4-methoxyphenoxy)but-3-ene (6), including enantiomeric introduction of the C-3 hydroxyl group to the olefin by the Sharpless asymmetric dihydroxylation, provided all four possible stereoisomers of A-ring enynes (3). i.e., (3R,5R)-, (3R,5S)-, (3S,5R)- and (3S,5S)-bis[(tert-butyldimethylsilyl)oxy]oct-1-en-7-yne, in good overall yield. Palladium-catalyzed cross-coupling of the A-ring synthon with the 20-epi CD-ring portion (5), (E)-(20S)-de-A,B-8-(bromomethylene)cholestan-25-ol, followed by deprotection, afforded the requisite diastereomers of 20-epi-1alpha,25-dihydroxyvitamin D3 (2). The biological profiles of the synthesized stereoisomers were assessed in terms of affinities for vitamin D receptor (VDR) and vitamin D binding protein (DBP). HL-60 cell differentiation-inducing activity and in vivo calcium-regulating potency in comparison with the natural hormone.     

Reverse cross-coupling in the synthesis 3 alpha, 7 alpha-dihydroxy-5 beta-cholestanoic acid.

The present report describes the characterization of (24R and 24S)-27-nor-24-methyl-3 alpha, 7 alpha-dihydroxy-5 beta-cholestan-26-oic acids obtained in considerable amounts during the synthesis of (25RS)-3 alpha, 7 alpha-dihydroxy-5 beta-cholestan-26-oic acid by the electrolytic coupling of chenodeoxycholic acid and the half ester of methylsuccinic acid. The mixture of 24R and 24S diastereomers was resolved by analytical and preparative thin-layer chromatography and characterized by gas-liquid chromatography, proton magnetic resonance, and molecular rotation differences. For reference, the model compound, 27-nor-3 alpha, 7 alpha-dihydroxy-5 beta-cholestan-26-oic acid, was synthesized by electrolytic coupling of chenodeoxycholic acid and the half ester of succinic acid.     

Preparation and configuration of racemic and optically active analgesic dialkylaminoalkylnaphthalenes

The alkylaminoalkylnaphthalene 3 shows interesting opioid-like analgesic properties, μ-selective ligand competition, and enkephalin hydrolyzing enzyme inhibition. 3 possesses two chiral centers and can exist as two racemic pairs and four diastereomers. Since the binding of opioids with the receptor is stereoselective, it was important to have the two racemic pairs as well as the four diastereomers. In this paper the synthesis of the (1R,2R/1S,2S)- and (1R,2S/1S,2R)-racemates and the (1R,2R)- and (1S,2S)-enantiomers of the 1-ethyl-1-hydroxy-1-[2-(6-hydroxynaphthyl)]-2-methyl-3-dimethylaminopropane 3 is considered and the determination of absolute configuration is described. The (1R,2R/1S,2S)- 3 and (1R,2S/1S,2R)- 3 racemates and the (1R,2R)- 3 and (1S,2S)- 3 enantiomers were prepared by reaction of the racemic and optically active 1-dimethylamino-2-methylpentan-3-one 2 , respectively, with the lithiation product obtained from 2-bromo-6-tetrahydropyranyloxynaphthalene and acidic hydrolysis. The optical resolution of aminoketone 2 was carried out via fractional crystallization of salts (+)- and (?)-dibenzoyltartrates. The configuration of the optically active compounds was determined by X-ray analysis of a crystal of (+)-(1R,2R)- 3 · HCl · H₂O. Preliminary pharmachological tests showed that (+)-(1R,2R)- 3 enantiomer is able to induce opioid-like analgesia with a relative potency 2.5 times that of (1R,2R/1S,2S)- 3 and about 4 times that of morphine. © 1994 Wiley-Liss, Inc.     

Preparation and configuration of racemic and optically active analgesic cycloaminoalkylnaphthalenes

Cycloaminoalkylnaphthalene 3 shows interesting opioid‐like analgesic properties. It possesses two chiral centers and can exist as two racemic pairs and four diastereomers. Since the binding of opioids with receptors is stereoselective, it was important to have the two racemic pairs as well as the four diastereomers. In this paper the synthesis of the (2R,3S/2S,3R) racemate and the (2R,3S) and (2S,3R) enantiomers of the 1,2‐dimethyl‐3‐[2‐(6‐hydroxynaphthyl)]‐3‐hydroxypyrrolidine 3 is considered and the determination of absolute configuration is described. The (2R,3S/2S,3R)‐ 3 racemate and the (2R,3S)‐ 3 and (2S,3R)‐ 3 enantiomers were prepared by reaction of the racemic and optically active 1,2‐dimethyl‐3‐pyrrolidone 2, respectively, with the lithiation product obtained from 2‐bromo‐6‐tetrahydropyranyloxy‐naphthalene 1 and acidic hydrolysis. The above‐mentioned enantiomers of 3 were also obtained by optical resolution via fractional crystallization of the salts with d ‐ and l ‐tartaric acids. The configuration of the optically active compounds was determined by X‐ray analysis of a crystal of (−)‐(2S,3R)‐ 3 · HCl · H₂O. The pharmacological test HPT showed that (−)‐(2S,3R)‐ 3 · HCl · H₂O enantiomer is able to induce opioid‐like analgesia with a relative potency 1.5 times that of (2R,3S/2S,3R)‐ 3 and ∼1.5 times that of morphine. Chirality 11:21–28, 1999. © 1999 Wiley‐Liss, Inc.     

Stereochemistry of the kynureninase reaction

The steric course of the replacement of the anthranilyl group of kynurenine by hydrogen was determined by conversion of (2S,3R)- and (2S,3S)-[3-3H]kynurenine into alanine with kynureninase in D2O, followed by chirality analysis of the alanine methyl group. To minimize enolization, the labeled substrates were generated in situ from the corresponding stereospecifically tritiated tryptophan species. The result, replacement in a retention mode, together with the finding that tritium from the alpha-position of the substrate is recycled and appears both at C alpha and C beta of the product suggests a single base mechanism and an active site geometry of the pyridoxal phosphate-substrate complex in which H alpha and the beta-substituent are syn oriented.     

Hyperolactone C: determination of its absolute configuration by comparison of experimental and calculated CD spectra

A detailed conformational analysis of hyperolactone C diastereomers and enantiomers ((5R,9R),(5S,9S) and (5S,9R),(5R,9S)) was done with molecular mechanics and density functional theory methods. Time-dependent density functional theory (B3PW91/TZVP) was used to calculate electronic transition energies (UV/vis spectra) and rotational strengths of the respective conformations. The effect of solvation (acetonitrile solution) on excitation energies and electronic circular dichroism was approximated by the polarizable continuum model. By comparison of the simulated CD spectrum with that measured for hyperolactone C isolated from Hypericum lloydii, its absolute configuration can be assigned as (5S,9S).     

Studies on the synthesis of safflomin-A, a yellow pigment in safflower petals: oxidation of 3-C-beta-d-glucopyranosyl-5-methylphloroacetophenone

The direct C-glycosylation of methylphloroacetophenone 8 with d-glucose gave C-beta-d-glucopyranosylmethylphloroacetophenone (7) in 65% yield, which, on oxidation in the presence of small amount of pyridine under an oxygen atmosphere afforded the quinone 9, oxidized at the methylated position of the benzene ring as a pair of diastereomers in 27% yield. A detailed NMR analysis and a comparison of the UV-vis and CD spectra of their acetates indicated that the structure and stereochemistry of 9 was (1R,1'S,2R,3S,3aS,5R and 1R,1'S,2R,3S,3aS,5S)-7-acetyl-2-(1',2'-dihydroxyethyl)-5-methyl-3,5,6-trihydroxy-8-oxofuro[3,2-d]benzo[b]furan.     

Synthesis of the diastereomers of thymidine glycol, determination of concentrations and rates of interconversion of their cis-trans epimers at equilibrium and demonstration of differential alkali lability within DNA.

5,6-dihydroxy-5,6-dihydrothymidine (thymidine glycol) is a major product of the reaction of thymidine with reactive oxygen species, including those generated by ionizing radiation. Thymidine glycol exists as 2 diastereomeric pairs by virtue of the chirality of the C(5) and C(6) atoms. A simple procedure is described for synthesizing and purifying each of the diastereomeric pairs separately. After brominating thymidine, the two trans 5-bromo-6-hydroxy-5,6-dihydrothymidine (thymidine bromohydrin) C(5) diastereomers were easily separated by High Performance Liquid Chromatography. Each thymidine bromohydrin was quantitatively converted to the corresponding diastereomeric thymidine glycol pair by reflux in aqueous solution. The concentrations at equilibrium of the cis (5S,6R),(5R,6S) and trans (5S,6S),(5R,6R) forms of the thymidine glycol diastereomers were determined and were 80% cis and 20% trans for the 5S pair and 87% cis and 13% trans for the 5R pair. At equilibrium, the rate of cis-trans epimerization of the two sets of diastereomers was essentially identical. The 5S diastereomeric pair was significantly more alkali labile than the 5R pair due to the higher concentration of the 5S trans epimer at equilibrium. This differential alkali lability was also manifest when the thymine glycol moiety was present in chemically oxidized poly(dA-dT).poly(dA-dT) indicating that the chemical differences between the diastereomeric pairs are preserved in DNA. These chemical differences may affect the biological properties of this important oxidative derivative of thymine in DNA.     

Isolation and Identification of trans-2- and trans-3-Hydroxy-1,8-cineole Glucosides from Alpinia galanga

Three hydroxy-1,8-cineole glucopyranosides, (1R,2R,4S)- and (1S,2S,4R)-trans-2-hydroxy-1,8-cineole β-D-glucopyranosides, and (1R,3S,4S)-trans-3-hydroxy-1,8-cineole β-D-glucopyranoside, which are possible precursors of acetoxy-1,8-cineoles as unique aroma components, were isolated from the rhizomes of greater galangal (Alpinia galanga W.). Their structures were analyzed by FAB-MS and NMR spectrometry, and the absolute configulation of each aglycone was determined by using a GC-MS analysis with a capillary column coated with a chiral stationary phase. The composition of the diastereomers of (1R,2R,4S)- and (1S,2S,4R)- trans-2-hydroxy-1,8-cineole β-D-glucopyranosides in the rhizomes was determined as 3:7 by a GC-MS analysis after preparing the trifluoroacetate derivatives of the glucosides.     

Synthesis of nucleic acid methylphosphonothioates.

The reagent obtained in situ by treating methylphosphonothioic dichloride with 1-hydroxy-6-trifluoromethylbenzotriazole could be used for the introduction of methylphosphonothioate linkages. The individual diastereomers of the protected dimer d-Tp(S,Me)A were applied in the synthesis of the chiral pure (R or S) hexamers d-[CpCpTp(S,Me)ApGpG]. The reagent showed also to be very effective for the preparation of the 3',5'-cyclic methylphosphonothioate of uridine.     

Isolation and identification of trans-2- and trans-3-hydroxy-1,8-cineole glucosides from Alpinia galanga

Three hydroxy-1,8-cineole glucopyranosides, (1R, 2R, 4S)- and (1S, 2S, 4R)-trans-2-hydroxy-1,8-cineole beta-D-glucopyranosides, and (1R, 3S, 4S)-trans-3-hydroxy-1,8-cineole beta-D-glucopyranoside, which are possible precursors of acetoxy-1,8-cineoles as unique aroma components, were isolated from the rhizomes of greater galangal (Alpinia galanga W.). Their structures were analyzed by FAB-MS and NMR spectrometry, and the absolute configulation of each aglycone was determined by using a GC-MS analysis with a capillary column coated with a chiral stationary phase. The composition of the diastereomers of (1R, 2R, 4S)- and (1S, 2S, 4R)-trans-2-hydroxy-1,8-cineole beta-D-glucopyranosides in the rhizomes was determined as 3:7 by a GC-MS analysis after preparing the trifluoroacetate derivatives of the glucosides.     

Metal ion dependence of phosphorothioate ATP analogues in the Bacillus stearothermophilus tyrosyl-tRNA synthetase reaction

Pre-steady-state kinetic analyses on the formation of tyrosyl adenylate from tyrosine and each of the four diastereomers of alpha- and beta-phosphorothioate adenosine triphosphates [ATP alpha S and ATP beta S; Eckstein, F., & Goody, R. (1976) Biochemistry 15, 1685-1691; Yee, D., Armstrong, V. W., & Eckstein, F. (1979) Biochemistry 18, 4116-4123] were performed in the presence of Mg2+, Co2+, and Cd2+ as the divalent metal ion cofactor. A modest preference of 5.5-fold in kappa 3/KA' (where kappa 3 is the rate constant for tyrosyl adenylate formation and KA' is the dissociation constant for ATP, or phosphorothioate ATP, from the E.Tyr.metal.ATP complex) for the Sp ATP alpha S diastereomer and the absence of an inversion of preference when the metal ion is changed suggest that there is a stereospecific enzyme-alpha-phosphate interaction and that there is no direct metal ion interaction with the alpha-phosphate. The extent of reaction of the ATP alpha S diastereomers (30-50%) implies that these analogues are more susceptible to the hydrolytic site reaction previously reported for this enzyme [Wells, T. N. C., & Fersht, A. R. (1986) Biochemistry 25, 1881-1886]. The strong preference in kappa 3/KA' for the RP ATP beta S diastereomer (16-fold for Mg2+ and 50-fold for Co2+) is indicative of a stereospecific interaction with the pro SP beta oxygen of ATP.(ABSTRACT TRUNCATED AT 250 WORDS)     

Synthesis of (2S,3R)-beta-methyltyrosine catalyzed by tyrosine phenol-lyase.

A one-step enzymatic synthesis of the conformationally restrained tyrosine analog (2S,3R)-beta-methyltyrosine is reported. This synthesis extends the preparative chemistry associated with tyrosine phenol-lyase. This beta-methyltyrosine derivative was shown to be an efficient protein tyrosine kinase substrate, suggesting that conformational restraint may ultimately be used to enhance tyrosine kinase recognition of substrates.     

Structure and synthesis of histopine, a histidine derivative produced by crown gall tumors

Histopine, an unusual amino acid derivative of histidine isolated from crown gall tumors of sunflowers (Helianthus annus) inoculated with Agrobacterium tumefaciens strain B6, was previously assigned the gross structure N-(1-carboxyethyl) histidine (2). A diastereomeric mixture containing histopine (2a and 2b) was readily prepared by reductive alkylation of (S)-histidine (1) with pyruvic acid and sodium cyanoborohydride. The individual diastereomers were prepared by reaction of (S)-histidine with (R)- and (S)-2-bromopropionic acid. (R)-N-(1-Carboxyethyl)-(S)-histidine (2a) supports the growth of A. tumefaciens whereas (S)-N-(1-carboxyethyl)-(S)-histidine (2b) is inactive. Therefore, we assign structure 2a to histopine.     

Enzymic conversion of 11,12-leukotriene A4 to 11,12-dihydroxy-5,14-cis-7,9-trans-eicosatetraenoic acid. Purification of an epoxide hydrolase from the guinea pig liver cytosol

(11S,12S)-Epoxy-5,14-cis-7,9-trans-eicosatetraenoic acid (11,12-leukotriene A4) was nonenzymically converted to seven compounds: two diastereomers of (12S)-hydroxyeicosatetraeno-delta-lactones (major products), two diastereomers of (5,12S)-dihydroxyeicosatetraenoic acid and three stereoisomers of (11,12S)-dihydroxyeicosatetraenoic acid. Among these compounds, (11R,12S)-dihydroxy-5,14-cis-7,9-trans-eicosatetraenoic acid proved to be the only enzymic product. This hydrolysis activity was present in the cytosol fractions of various tissues of guinea pig such as liver, adrenal gland, small intestine, and brain. We purified the epoxide hydrolase to an apparent homogeneity from the guinea pig liver. The enzyme had a molecular weight of 60,000 as determined by sodium dodecyl sulfate-polyacrylamide gel electrophoresis, and an isoelectric point of 7.3. The partial amino acid sequence was different from that of the microsomal enzyme. Km and Vmax values for 11,12-leukotriene A4 were 18 microM and 2.4 mumol/min/mg protein, respectively. These results indicate that 11,12-dihydroxyeicosatetraenoic acid is enzymically synthesized from 11,12-leukotriene A4 by the action of the cytosolic epoxide hydrolase in vitro.     

Preparation of (6R)-tetrahydrofolic acid and (6R)-5-formyltetrahydrofolic acid of high stereochemical purity

Commercially available 5-formyltetrahydrofolate (5-CHO-H4PteGlu) is chemically prepared in a reaction that introduces an asymmetric center at the 6 carbon, and hence is the mixture of diastereomers differing in chirality about this position. (6R)-5-CHO-H4PteGlu, the diastereomer that is not normally found in vivo, was prepared from folic acid. Folic acid was chemically reduced and (6R)-tetrahydrofolate (H4PteGlu) was obtained from the resultant (6R,S)-H4PteGlu by enzymatic consumption of the natural diastereomer of (6R,S)-5,10-CH2-H4PteGlu (reversibly formed from (6R,S)-H4PteGlu in the presence of formaldehyde) with Lactobacillus casei thymidylate synthase. The 5 position of purified (6R)-H4PteGlu was directly formylated in a carbodiimide-catalyzed reaction. The level of contamination of these preparations with the corresponding 6S diastereomers was estimated using the binding of fluorodeoxyuridylate to thymidylate synthase promoted by folate cofactor (for H4PteGlu) and by the growth of folate requiring bacteria (for 5-CHO-H4PteGlu). Purified preparations of (6R)-H4PteGlu promoted the binding of fluorodeoxyuridylate to L. casei thymidylate synthase (in the presence of formaldehyde) only at concentrations greater than 1000-fold higher than equiactive levels of (6S)-H4PteGlu. Likewise, the (6R)-5-CHO-H4PteGlu made by this method was 600 times less active as a growth factor for Pediococcus cerevisiae than was authentic (6S)-5-CHO-H4PteGlu. Hence, the minimum stereochemical purity of these preparations was 99.9% for (6R)-H4PteGlu and 99.8% for (6R)-5-CHO-H4PteGlu.     

Preparation of optically pure 4-(hydroxymethyl)-2-pentadecyl-1,3-dioxolanes and their corresponding phosphodiester ether lipid derivatives.

Semi-preparative HPLC on a chiral stationary phase (Chiracel OD) was utilized in the course of this synthesis to separate the four possible diastereomers [cis-(2R,4S)-2a, trans-(2S,4S)-2b, cis-(2S,4R)-2a', and trans-(2R,4R)-2b'] of a 2,4-disubstituted-1,3-dioxolane into optically pure forms (100% de, 100% ee). The syntheses of phosphodiester head group derivatives from each of these four conformationally constrained diastereomeric dioxolanes gave phospholipids which are monocyclic ether lipid analogs. First, the series of four [[(2-pentadecyl-1,3-dioxolan-4-yl)methyl]oxy]phosphocholines 5 were synthesized to give optically pure conformationally constrained analogues of ET-16-OCH(3). A head group variation was also demonstrated by the syntheses of the four diastereomeric [[(2-pentadecyl-1,3-dioxolan-4-yl)-methyl]oxy]phospho-beta-(N-methylmorpholino)ethanols 6.     

Phosphorothioate analogues of 5-phosphoribosyl 1-diphosphate: synthesis, purification, and partial characterization

The 1-phosphorothioate analogues of 5-phosphoribosyl 1-diphosphate (P-Rib-PP) have been prepared enzymatically, in reactions catalyzed by P-Rib-PP synthetase from Salmonella typhimurium. 5-Phosphoribosyl 1-O-(2-thiodiphosphate) (P-Rib-PP beta S) was synthesized from ribose 5-phosphate (Rib-5-P) and the Mg2+ complex of adenosine 5'-O-(3-thiotriphosphate). The SP and RP diastereomers of 5-phosphoribosyl 1-O-(1-thiodiphosphate) (P-Rib-PP alpha S) were synthesized from Rib-5-P and the Mg2+ complex of adenosine 5'-O-(2-thiotriphosphate) (ATP beta S) (SP diastereomer, delta-configuration) and the Cd2+ complex of ATP beta S (RP diastereomer, delta-configuration), respectively. The strategy for the synthesis and stereochemical assignment of the P-Rib-PP alpha S diastereomers was based on the specificity of P-Rib-PP synthetase for the (delta)-beta, gamma-bidentate metal-nucleotide substrate and the stereochemical course of the synthetase reaction, leading to inversion of configuration at the P beta atom of the nucleotide [Li, T. M., Mildvan, A. S., & Switzer, R. L. (1978) J. Biol. Chem. 253, 3918-3923], and the known configurations of the Mg2+ and Cd2+ beta, gamma-bidentate complexes of the ATP beta S diastereomers [Jaffe, E. K., & Cohn, M. (1979) J. Biol. Chem. 254, 10839-10845]. The P-Rib-PP analogues were purified by gradient elution from DEAE-Sephadex and characterized by chemical analysis and 31P nuclear magnetic resonance [Smithers, G. W., & O'Sullivan, W. J. (1984) Biochemistry (following paper in this issue)]. A preliminary account of their interaction with human brain hypoxanthine phosphoribosyltransferase and yeast orotate phosphoribosyltransferase (OPRTase) is described.(ABSTRACT TRUNCATED AT 250 WORDS)     

Binding of (6R,S)-methyltetrahydrofolate to methyltransferase from Clostridium thermoaceticum: role of protonation of methyltetrahydrofolate in the mechanism of methyl transfer

The methyltetrahydrofolate:corrinoid/iron-sulfur protein methyltransferase (MeTr) from Clostridium thermoacetium catalyzes transfer of the N5-methyl group of (6S)-methyltetrahydrofolate (CH3-H4folate) to the cob(I)amide center of a corrinoid/iron-sulfur protein (CFeSP), forming H4folate and methylcob(III)amide. We have investigated binding of 13C-enriched (6R,S)-CH3-H4folate and (6R)-CH3-H4folate to MeTr by 13C NMR, equilibrium dialysis, fluorescence quenching, and proton uptake experiments. The results described here and in the accompanying paper [Seravalli, J., Shoemaker, R. K., Sudbeck, M. J., and Ragsdale, S. W. (1999) Biochemistry 38, 5728-5735] constitute the first evidence for protonation of the pterin ring of CH3-H4folate. The pH dependence of the chemical shift in the 13C NMR spectrum for the N5-methyl resonance indicates that MeTr decreases the acidity of the N5 tertiary amine of CH3-H4folate by 1 pK unit in both water and deuterium oxide. Binding of (6R,S)-CH3H4folate is accompanied by the uptake of one proton. These results are consistent with a mechanism of activation of CH3-H4folate by protonation to make the methyl group more electrophilic and the product H4folate a better leaving group toward nucleophilic attack by cob(I)amide. When MeTr is present in excess over (6R,S)-13CH3-H4folate, the 13C NMR signal is split into two broad signals that reflect the bound states of the two diastereomers. This unexpected ability of MeTr to bind both isomers was confirmed by the observation of MeTr-bound (6R)-13CH3-H4folate by NMR and by the measurement of similar dissociation constants for (6R)- and (6S)-CH3-H4folate diastereomers by fluorescence quenching experiments. The transversal relaxation time (T2) of 13CH3-H4folate bound to MeTr is pH independent between pH 5.50 and 7.0, indicating that neither changes in the protonation state of bound CH3-H4folate nor the previously observed pH-dependent MeTr conformational change contribute to broadening of the 13C resonance signal. The dissociation constant for (6R,S)-CH3-H4folate is also pH independent, indicating that the role of the pH-dependent conformational change is to stabilize the transition state for methyl transfer, and not to favor the binding of CH3-H4folate.