Enzymatic synthesis of ω-carboxy-β-hydroxy-(l)-α-amino acids

Commercially available ω-carboxy-aldehydes and glycine have been subjected to the catalytic action of an l-threonine aldolase from Escherichia coli to give the corresponding β-hydroxy-α-(l)-amino acids as a mixture of erythro/threo epimers.Specifically, the reaction with glyoxylic acid (2) gave the epimeric β-hydroxy-(l)-aspartates (t,e)-9 that could be isolated by ion-exchange chromatography in 67% yield. Following esterification and N-Boc protection, the two epimers could be isolated as pure compounds.Similarly, the aldolase-catalyzed addition of glycine to succinic semialdehyde (4) gave the expected mixture of β-hydroxy-l-α-aminoadipic acids (t)-12 and (e)-12 in 34% yield.     

Threonine aldolases—an emerging tool for organic synthesis

In a systematic study, 21 ring-substituted benzaldehydes were reacted with glycine under catalysis with a l-threonine aldolase (lTA) from Pseudomonas putida and a d-threonine aldolase (dTA) from Alcaligenes xylosoxidans to form the corresponding β-hydroxy-α-amino acids 1-18. dTA proved to be highly selective with ee's >99% (d) and de's up to 99% (syn). Two thiamphenicol precursors were synthesized utilizing dTA on a preparative scale. lTA-catalyzed reactions led to ee's >99% (l) but low to moderate de's (20-50%, syn).     

Combining spiro-fused cyclohexadienone – tetrahydrofuran ring system with glycine: Asymmetric synthesis of a new class of α-amino acid derivatives

Herein, we present the asymmetric synthesis of spiro-fused cyclohexadienone – tetrahydrofuran-embedded glycine derivatives as a new class of nonproteinogenic α-amino acid derivatives. Starting from commercially available 2-allylphenols, key β-hydroxy-α-amino esters were synthesized via high-yielding multi-step reaction sequences involving Sharpless asymmetric dihydroxylation as the chirality induction step. PhI(OAc)₂-mediated oxidative dearomatization – spirocyclization of phenol-tethered β-hydroxy-α-amino esters efficiently produced the corresponding spiro-fused cyclohexadienone – tetrahydrofuran-embedded glycine derivatives, providing a general route to this hitherto-unreported class of compounds that are equipped with three privileged scaffolds (cyclohexadienone – tetrahydrofuran – α-amino ester).     

Trading N and O. Part 4: Asymmetric synthesis of syn-β-substituted-α-amino acids

A total of nine enantiopure syn-β-substituted-α-amino acids have been synthesised, comprising both syn-β-hydroxy-α-amino acids and syn-β-fluoro-α-amino acids. The key step in the synthetic strategy towards these syn-β-substituted-α-amino acids involves a stereospecific rearrangement, which proceeds via the intermediacy of the corresponding aziridinium ions. The requisite enantiopure syn-α-hydroxy-β-amino esters were prepared via asymmetric aminohydroxylation of the corresponding α,β-unsaturated esters followed by epimerisation of the resultant anti-α-hydroxy-β-amino esters at the C(2)-position. Subsequent activation of the α-hydroxy moiety as a leaving group followed by displacement by the β-amino substituent gave the corresponding aziridinium species. Regioselective in situ ring-opening of the aziridinium intermediates with either water or fluoride gave the corresponding syn-β-hydroxy-α-amino ester or syn-β-fluoro-α-amino ester, respectively, and N-deprotection and ester hydrolysis afforded the target syn-β-substituted-α-amino acids as single diastereoisomers in good overall yield.     

Trading N and O. Part 2: Exploiting aziridinium intermediates for the synthesis of β-hydroxy-α-amino acids

The β-hydroxy-α-amino acids (S,S)-allo-threonine, (S,S)-β-hydroxyleucine and a range of aryl substituted (S,S)-β-hydroxyphenylalanines were prepared from the corresponding enantiopure anti-α-hydroxy-β-amino esters via a rearrangement protocol, which proceeds via the intermediacy of the corresponding aziridinium ions. The starting anti-α-hydroxy-β-amino esters were prepared in >99:1 dr using our diastereoselective aminohydroxylation procedure, whereby conjugate addition of lithium (R)-N-benzyl-N-(α-methylbenzyl)amide to an α,β-unsaturated ester is followed by oxidation of the resultant enolate with (−)-camphorsulfonyloxaziridine. Subsequent activation of the hydroxyl group within the anti-α-hydroxy-β-amino esters promoted aziridinium ion formation [which proceeds with inversion of configuration at C(2)], and regioselective ring-opening of the intermediate aziridinium ions with H₂O [which proceeds with inversion of configuration at C(3)] gave the corresponding anti-β-hydroxy-α-amino esters as single diastereoisomers (>99:1 dr). Deprotection of these substrates via sequential hydrogenolysis and ester hydrolysis gave the corresponding β-hydroxy-α-amino acids in good yield and high diastereoisomeric and enantiomeric purity.     

Amino acids of ricin and its polypeptides

Ricin and its corresponding polypeptides (A & B chain) were purified from castor seed. The molecular weight of ricin subunits were 29,000 and 28,000 daltons. The amino acids in ricin determined were Asp45 The22 Ser40 Glu53 Cys4 Gly96 His5 Ile21 Leu33 Lys20 Met4 Phe13 Pro37 Tyr11 Ala45 Val23 Arg20 indicating that ricin contains approximately 516 amino acid residues. The amino acids of the two subunits of ricin A and B chains were Asp23 The12 Ser21 Glu29 Cys2 Gly48 His3 Ile12, Leu17 Lys10 Met2 Phe6 Pro17 Tyr7 Ala35 Val13 Arg13 while in B chain the amino acids were Asp22 The10 Ser19 Glu25 Cys2 Gly47 His1 Ile10, Leu15 Lys11 Met1 Phe7 Pro6 Tyr5 Ala32Val11 Arg10. The total helical content of ricin came around 53.6% which is a new observation.     

Stereoselective synthesis of β-hydroxy-α-amino acids β-substituted with non-aromatic heterocycles

We have stereoselectively synthesised β-hydroxy-α-amino acids β-substituted with non-aromatic heterocycles by means of a condensation reaction between enantiomerically pure heterocyclic aldehydes and the (R)-(+)-2,5-dihydro-3,6-dimethoxy-2-isopropylpyrazine (Schöllkopf’s reagent) as a chiral auxiliary. The stereocontrolled addition gave mixtures of diastereoisomers whose steric configurations were assigned on the basis of spectroscopic data and X-ray analysis. Upon controlled hydrolysis, the adducts were transformed into the corresponding methyl esters of β-hydroxy-β-heterocyclic substituted α-amino acids.     

Stereoselective synthesis of δ-heteroaryl substituted β-hydroxy-γ,δ-unsaturated α-amino acids

Enantiomerically pure δ-heteroaryl substituted β-hydroxy-γ,δ-unsaturated α-amino acids were stereoselectively synthesized starting from (2R)-(+)-2,5-dihydro-3,6-dimethoxy-2-isopropylpyrazine (Schöllkopf’s reagent) and suitable β-heteroaryl-α,β-unsaturated aldehydes. The stereocontrolled addition gave a mixture of two diastereoisomers whose configurations were assigned on the basis of spectroscopic data and the accepted model for aldol condensation of the Schöllkopf’s reagent. Upon controlled hydrolysis the adducts were transformed into the corresponding methyl esters of δ-heteroaryl substituted β-hydroxy-γ,δ-unsaturated α-amino acids.     

N-Hydroxymethyl derivatives of α-amino aldehydes used for the stereoselective syntheses of β-amino-α-hydroxy acids

The N-hydroxymethyl derivatives of α-amino aldehydes 1 were utilized for the effective synthesis of several β-amino-α-hydroxy acid derivatives in a one-pot process starting from the corresponding α-amino aldehydes. Properly protected methyl esters 3 were prepared in 65–79% yields from α-amino aldehyde derivatives 1 with more than 20:1 stereoselectivity. The application of suitably protected β-amino-α-hydroxy esters was shown by an efficient synthesis of the bioactive peptide, bestatin, and its more potent analogue, AHPBA-Val, in high yields from ent-3a.     

Dehydration of β-Hydroxy-α-amino Acid Derivatives With Phenyltriflimide

Conversion of N-protected β-hydroxy-α-amino esters to corresponding didehydroamino esters has been achieved using phenyltriflimide/triethylamine as a mild dehydrating agent. The selective formation of didehydroalanine derivatives from dipeptides containing, beside a serine residue, another β-hydroxy-α-amino acid, is also described.     

Cascade Dissociations of Peptide Cation-Radicals. Part 1. Scope and Effects of Amino Acid Residues in Penta-, Nona-, and Decapeptides

Amino acid residue-specific backbone and side-chain dissociations of peptide z ions in MS(3) spectra were elucidated for over 40 pentapeptides with arginine C-terminated sequences of the AAXAR and AAHXR type, nonapeptides of the AAHAAXX"AR and AAHAXAX"AR type, and AAHAAXX"AAR decapeptides. Peptide z(n) ions containing amino acid residues with readily transferrable benzylic or tertiary β-hydrogen atoms (Phe, Tyr, His, Trp, Val) underwent facile backbone cleavages to form dominant z(n-2) or z(n-3) ions. These backbone cleavages are thought to be triggered by a side-chain β-hydrogen atom transfer to the z ion C(α) radical site followed by homolytic dissociation of the adjacent C(α)-CO bond, forming x(n-2) cation-radicals that spontaneously dissociate by loss of HNCO. Amino acid residues that do not have readily transferrable β-hydrogen atoms (Gly, Ala) do not undergo the z(n) → z(n-2) dissociations. The backbone cleavages compete with side-chain dissociations in z ions containing Asp and Asn residues. Side-chain dissociations are thought to be triggered by α-hydrogen atom transfers that activate the C(β)-C(γ) or C(β)-heteroatom bonds for dissociations that dominate the MS(3) spectra of z ions from peptides containing Leu, Cys, Lys, Met, Ser, Arg, Glu, and Gln residues. The Lys, Arg, Gln, and Glu residues also participate in γ-hydrogen atom transfers that trigger other side-chain dissociations.     

Sequential aldol condensation catalyzed by DERA mutant Ser238Asp and a formal total synthesis of atorvastatin

A mutant d-2-deoxyribose-5-phosphate aldolase (DERA), Ser238Asp, was used to prepare β-hydroxy-δ-lactol synthons and a key intermediate for atorvastatin synthesis.     

Asymmetric syntheses of the N-terminal α-hydroxy-β-amino acid components of microginins 612, 646 and 680

The asymmetric syntheses of the N-terminal α-hydroxy-β-amino acid components of microginins 612, 646 and 680 are reported. Conjugate addition of lithium (R)-N-benzyl-N-(α-methylbenzyl)amide to the requisite (E)-α,β-unsaturated ester followed by in situ enolate oxidation with (?)-(camphorsulfonyl)oxaziridne (CSO) gave the corresponding anti-α-hydroxy-β-amino esters. Sequential Swern oxidation followed by diastereoselective reduction gave the corresponding syn-α-hydroxy-β-amino esters. Subsequent N-debenzylation (i.e., hydrogenolysis for microginin 612, and NaBrO₃-mediated oxidative N-debenzylation for microginins 646 and 680) followed by acid catalysed ester hydrolysis gave the corresponding syn-α-hydroxy-β-amino acids, the N-terminal components of microginins 612, 646 and 680, in good yield. An analogous strategy for elaboration of the enantiopure anti-α-hydroxy-β-amino esters facilitated the asymmetric synthesis of the corresponding C(2)-epimeric α-hydroxy-β-amino acids.     

Trading N and O: asymmetric syntheses of β-hydroxy-α-amino acids via α-hydroxy-β-amino esters

Both diastereoisomers of 2-amino-3-hydroxybutanoic acid and 2-amino-3-hydroxy-3-phenylpropanoic acid have been prepared from enantiopure α-hydroxy-β-amino esters via the intermediacy of the corresponding cis- and trans-aziridines. Aminohydroxylation of two α,β-unsaturated esters produced enantiopure 2,3-anti-α-hydroxy-β-amino esters in >99:1 dr. Subsequent epimerisation at the C(2)-position via a sequential oxidation/diastereoselective reduction protocol gave the corresponding enantiopure 2,3-syn-α-hydroxy-β-amino esters in >99:1 dr. These syn- and anti-substrates were then converted into the corresponding N-Boc protected cis- and trans-aziridines, respectively, via a three step reaction sequence: (i) hydrogenolysis and in situ N-Boc protection; (ii) OH-activation; and (iii) aziridine formation. Subsequent regioselective ring-opening of the C(3)-methyl-aziridines with Cl₃CCO₂H proceeded with inversion of configuration to give the corresponding 2-amino-3-trichloroacetate esters, whereas the analogous reaction with the C(3)-phenyl-aziridines resulted in rearrangement to the corresponding oxazolidin-2-ones with retention of configuration. In each case, hydrolysis of the products from these ring-opening reactions produced the corresponding enantiopure β-hydroxy-α-amino acids as single diastereoisomers.     

Efficient synthesis of β-halogeno protected l-alanines and their β-phosphonium derivatives

Ring opening of oxazolines, prepared from l-serinates, with trimethylsilyl halides (TMSX) led to β-halogeno-N-benzoyl-α-amino esters in good to excellent yields. Quaternization of triphenylphosphine by the β-bromo or -iodo amino esters gave the corresponding β-phosphonium salts in overall yields of up to 93% and with e.e. >96%. Hydrolysis of the ester function afforded the phosphonium salt bearing an N-benzoyl-α-amino acid substituent, with partial racemization. However, the reaction of the TMSX with the carboxylic salt, prepared by saponification of the starting oxazoline ester, furnished the corresponding β-halogeno-N-benzoyl-α-amino acids in 70–95% yields. Quaternization of triphenylphosphine by the bromo or iodo derivatives led to the phosphonium salts bearing a free acid function in 95% yield, without racemization. The efficiency of this synthesis was demonstrated by the preparation of these phosphonium salts in excellent overall yields, by a one-pot procedure starting from the oxazoline.     

A facile cleavage of oxirane with hydrazoic acid in dmf A new route to chiral β-hydroxy-α-amino acids

Chiral β-hydroxy-α-amino acids such as erythro-β-hydroxy-L-aspartic acid and erythro-β-hydroxymethyl-L-serine derivatives have been synthesized in optically pure form from L- and D-tartaric acids through a novel oxirane ring-opening reaction and a selective reduction of α-hydroxy ester as key steps.     

Asymmetric synthesis of chiral β-hydroxy-α-amino acid derivatives by organocatalytic aldol reactions of isocyanoesters with β,γ-unsaturated α-ketoesters

The first organocatalytic asymmetric aldol reaction of isocyanoesters with various β,γ-unsaturated α-ketoesters has been described. Using cinchona alkaloid-derived bifunctional thiourea as the catalyst, chiral β-hydroxy-α-amino acid derivatives can be obtained in excellent yields and enantioselectivities (up to 95% yield and 92% ee) after acidic hydrolysis. This protocol provides a straightforward method to access multiple substituted β-hydroxy-α-amino acid derivatives with high enantiomeric purity.     

A QM/MM study on the catalytic mechanism of pyruvate decarboxylase

Pyruvate decarboxylase (PDC) is a typical thiamin diphosphate (ThDP)-dependent enzyme with widespread applications in industry. Though studies regarding the reaction mechanism of PDC have been reported, they are mainly focused on the formation of ThDP ylide and some elementary steps in the catalytic cycle, studies about the whole catalytic cycle of PDC are still not completed. In these previous studies, a major controversy is whether the key active residues (Glu473, Glu50′, Asp27′, His113′, His114′) are protonated or ionized during the reaction. To explore the catalytic mechanism and the role of key residues in the active site, three whole-enzyme models were considered, and the combined QM/MM calculations on the nonoxidative decarboxylation of pyruvate to acetaldehyde catalyzed by PDC were performed. According to our computational results, the fundamental reaction pathways, the complete energy profiles of the whole catalytic cycle, and the specific role of key residues in the common steps were obtained. It is also found that the same residue with different protonation states will lead to different reaction pathways and energy profiles. The mechanism derived from the model in which the residues (Glu473, Glu50′, Asp27′, His113′, His114′) are in their protonated states is most consistent with experimental observations. Therefore, extreme care must be taken when assigning the protonation states in the mechanism study. Because the experimental determination of protonation state is currently difficult, the combined QM/MM method provides an indirect means for determining the active-site protonation state.     

Direct test of the Gaussian-chain model for treating residual charge-charge interactions in the unfolded state of proteins

The Gaussian-chain model for treating residual charge-charge interactions was critically tested by recent experimental pK(a) results for individual Asp, Glu, and His residues in the unfolded drkN SH3 domain. Predicted pK(a)'s were in good agreement with experiment. The clustering of Asp and Glu residues along the sequence was suggested to limit pK(a) shifts and contribute to the folding stability by destabilizing the unfolded state.     

Differential effects of the Zn-His-Bkb vs Zn-His-[Asp/Glu] triad on Zn-core stability and reactivity

The most common partner of the Zn-bound His is the Asp/Glu carboxylate side chain in catalytic Zn sites and the backbone (Bkb) carbonyl group in structural Zn sites. To elucidate the factors governing the selection of the second-shell partner of the Zn-bound His in structural/catalytic Zn sites, systematic studies using density functional theory and continuum dielectric calculations were performed to determine the relative contributions of the second-shell Bkb carbonyl and the Asp/Glu carboxylate to the Zn-core stability and reactivity. The results show that the contributions of the second-shell Bkb carbonyl and Asp/Glu carboxylate to the Zn-core stability depend mainly on the solvent accessibility of the Zn-site and the composition of the Zn-core. They reveal the advantage of a second-shell Bkb carbonyl in anionic Zn cavities: it stabilizes anionic, buried Zn-cores more than the corresponding negatively charged Asp/Glu carboxylate, thus explaining the absence of the Zn-His-Asp/Glu triad in structural [Zn(Cys)3(His)]- cores. They also reveal the advantage of a second-shell Asp/Glu carboxylate in catalytic Zn-cores: relative to a Bkb carbonyl group, it increases (i) the HOMO energy of the cationic/neutral zinc core, (ii) the reactivity of the attacking Zn-bound OH-, (iii) electron transfer to the substrate, and (iv) the stability of the metal complex upon electron transfer. Furthermore, a second-shell Asp/Glu carboxylate could facilitate product release in the common cationic catalytic cores, by acting as a proton acceptor of the Zn-bound His creating an Asp...His- dyad that stabilizes the zinc dication more than the respective Bkb...His0 dyad.