- Recharacterization of the mammalian cytosolic type 2 (R)-β-hydroxybutyrate dehydrogenase as 4-oxo-L-proline reductase (EC 1.1.1.104)
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Early studies revealed that chicken embryos incubated with a rare analog of L-proline, 4-oxo-L-proline, showed increased levels of the metabolite 4-hydroxy-L-proline. In 1962, 4-oxo-L-proline reductase, an enzyme responsible for the reduction of 4-oxo-L-proline, was partially purified from rabbit kidneys and characterized biochemically. However, only recently was the molecular identity of this enzyme solved. Here, we report the purification from rat kidneys, identification, and biochemical characterization of 4-oxo-L-proline reductase. Following mass spectrometry analysis of the purified protein preparation, the previously annotated mammalian cytosolic type 2 (R)-βhydroxybutyrate dehydrogenase (BDH2) emerged as the only candidate for the reductase. We subsequently expressed rat and human BDH2 in Escherichia coli, then purified it, and showed that it catalyzed the reversible reduction of 4-oxo-L-proline to cis-4-hydroxy-L-proline via chromatographic and tandem mass spectrometry analysis. Specificity studies with an array of compounds carried out on both enzymes showed that 4-oxo-L-proline was the best substrate, and the human enzyme acted with 12,500-fold higher catalytic efficiency on 4-oxo-L-proline than on (R)-β-hydroxybutyrate. In addition, human embryonic kidney 293T (HEK293T) cells efficiently metabolized 4-oxo-L-proline to cis-4-hydroxy-L-proline, whereas HEK293T BDH2 KO cells were incapable of producing cis-4-hydroxy-L-proline. Both WT and KO HEK293T cells also produced trans-4-hydroxy-L-proline in the presence of 4-oxo-L-proline, suggesting that the latter compound might interfere with the trans-4-hydroxy-L-proline breakdown in human cells. We conclude that BDH2 is a mammalian 4-oxo-L-proline reductase that converts 4-oxo-L-proline to cis-4-hydroxy-L-proline and not to trans-4-hydroxy-L-proline, as originally thought. We also hypothesize that this enzyme may be a potential source of cis-4-hydroxy-L-proline in mammalian tissues.
- Bozko, Maria,Drozak, Jakub,Jagielski, Adam K.,Kocdemir, Kubra,Kwiatkowski, Sebastian,Witecka, Apolonia,Zarod, Michal
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- Discovery of New Fe(II)/α-Ketoglutarate-Dependent Dioxygenases for Oxidation of l-Proline
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Genome mining for novel Fe(II)/α-ketoglutarate-dependent dioxygenases (αKGDs) to expand the enzymatic repertoire in the oxidation of l-proline is reported. Through clustering of proteins, we predicted regio- and stereoselectivity in the hydroxylation reaction and validated this hypothesis experimentally. Two novel byproducts in the reactions with enzymes from Bacillus cereus and Streptomyces sp. were isolated, and the structures were determined to be a 3,4-epoxide and a 3,4-diol, respectively. The mechanism for the formation of the epoxide was investigated by performing an 18O-labeling experiment. We propose that the mechanism proceeds via initial cis-3-hydroxylation followed by ring closure. A biocatalytic step was run on subgram quantities of starting material without any significant optimization of the conditions. However, the substrate concentration was 40-fold higher than the usual reported titers for recombinant P450-mediated hydroxylations, showing the synthetic potential of αKGDs on a preparative scale.
- Dussauge, Solene,Moore, Charles,Snajdrova, Radka,Tassano, Erika,Vargas, Alexandra
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supporting information
(2022/02/09)
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- Modular Chemoenzymatic Synthesis of GE81112 B1 and Related Analogues Enables Elucidation of Its Key Pharmacophores
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The GE81112 complex has garnered much interest due to its broad antimicrobial properties and unique ability to inhibit bacterial translation initiation. Herein we report the use of a chemoenzymatic strategy to complete the first total synthesis of GE81112 B1. By pairing iron and α-ketoglutarate dependent hydroxylases found in GE81112 biosynthesis with traditional synthetic methodology, we were able to access the natural product in 11 steps (longest linear sequence). Following this strategy, 10 GE81112 B1 analogues were synthesized, allowing for identification of its key pharmacophores. A key feature of our medicinal chemistry effort is the incorporation of additional biocatalytic hydroxylations in modular analogue synthesis to rapidly enable exploration of relevant chemical space.
- Zwick, Christian R.,Sosa, Max B.,Renata, Hans
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supporting information
p. 1673 - 1679
(2021/01/25)
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- Studies on the selectivity of proline hydroxylases reveal new substrates including bicycles
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Studies on the substrate selectivity of recombinant ferrous-iron- and 2-oxoglutarate-dependent proline hydroxylases (PHs) reveal that they can catalyse the production of dihydroxylated 5-, 6-, and 7-membered ring products, and can accept bicyclic substrates. Ring-substituted substrate analogues (such hydroxylated and fluorinated prolines) are accepted in some cases. The results highlight the considerable, as yet largely untapped, potential for amino acid hydroxylases and other 2OG oxygenases in biocatalysis.
- Smart, Tristan J.,Hamed, Refaat B.,Claridge, Timothy D.W.,Schofield, Christopher J.
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supporting information
(2019/11/26)
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- Production of cis-4-hydroxyproline
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PROBLEM TO BE SOLVED: To produce cis-4-hydroxyproline useful as a raw material of medicines and agrochemicals by an industrially suitable method.SOLUTION: The method for producing cis-4-hydroxyproline includes: hydrolyzing a hydroxyproline derivative represented by formula (1), wherein Rdenotes a 1-6C alkylcarbonyl group, an arylcarbonyl group, an alkoxycarbonyl group, an aryloxycarbonyl group, or an aralkyloxycarbonyl group, in the presence of a hydrochloric acid catalyst; neutralizing the resultant with an organic base; and thereafter diluting the resultant with an alcohol.
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- A short diastereoselective synthesis of cis-(2S,4S) and cis-(2R,4R)-4-hydroxyprolines
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A concise synthesis of (2R,4R)-4-hydroxyproline (1) and (2S,4S)-4-hydroxyproline (2) has been developed in enantiomerically pure form from commercially available starting materials with excellent diastereoselectivity. The tightly bound chelation controlled transition state formed during the 5-exo-tet ring closure reaction is assumed to be the origin of high diastereoselectivity.
- Gajare, Vikas S.,Khobare, Sandip R.,Malavika,Rajana, Nagaraju,Venkateswara Rao,Syam Kumar
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supporting information
p. 3743 - 3746
(2015/06/08)
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- Regio- and stereoselective oxygenation of proline derivatives by using microbial 2-oxoglutarate-dependent dioxygenases
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We evaluated the substrate specificities of four proline cis-selective hydroxylases toward the efficient synthesis of proline derivatives. In an initial evaluation, 15 proline-related compounds were investigated as substrates. In addition to L-proline and L-pipecolinic acid, we found that 3,4-dehydro-L-proline, L-azetidine-2-carboxylic acid, cis-3-hydroxy-L-proline, and L-thioproline were also oxygenated. Subsequently, the product structures were determined, revealing cis-3,4-epoxy-L-proline, cis-3-hydroxy-L-azetidine-2-carboxylic acid, and 2,3-cis-3,4-cis-3,4-dihydroxy-L-proline.
- Hara, Ryotaro,Uchiumi, Naoko,Okamoto, Naoko,Kino, Kuniki
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p. 1384 - 1388
(2015/07/20)
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- Synthesis of (2S,4S)-4-hydroxyproline from D-glucose
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Diacetone-D-glucose 1 gives 3-O-methylxanthate 2 on reaction with NaH=Me I. Reductive deoxygenation of compound 2 by Bu3SnH gives the corresponding 3-deoxy glucose derivative 3 and on acid-catalyzed regioselective deprotection of C-5,6-acetonide gives the diol 4. The diol on oxidative cleavage with NaIO4 gives the aldehyde 5, which on further condensation with benzylamine followed by reduction with NaBH4 gives the amine 7. Z-Protection of the amine followed by methanolysis gives methyl furanoside 9. Reaction of 9 with methanesulfonyl chloride=Et3N gives the corresponding C-3-O-mesylate derivative 10. Catalytic hydrogenation of compound 10 (Pd=C=H2=MeOH 3 kg) gives bicyclic oxaazo compound 11, due to deprotection of the N-benzyl- and Z-protecting groups and intramolecular nucleophilic displacement of the C-2-O-mesylate by the C-5 amine in a one-pot reaction. Z-Protection of the amine 11 followed by acid-catalyzed hydrolysis gives acetal 13. Reduction of acetal by use of NaBH4 gives Z-prolinol 14. Selective oxidation of diol 14 by (2,2,6,6-tetramethylpiperidin-1-yl)-oxyl (TEMPO)=[(bis)(acetoxy)iodo]-benzene (BAIB) and NaClO2=NaH 2PO4, followed by Z-deprotection, gives the title compound I in 3.5% overall yield from D-glucose. Copyright Taylor & Francis Group, LLC.
- Mereyala, Hari Babu,Pathuri, Gopal,Nagarapu, Lingaiah
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p. 1278 - 1287
(2012/04/17)
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- Tertiary alcohol preferred: Hydroxylation of trans-3-methyl-L-proline with proline hydroxylases
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The enzymatic synthesis of tertiary alcohols by the stereospecific oxidation of tertiary alkyl centers is a most-straightforward but challenging approach, since these positions are sterically hindered. In contrast to P450-monooxygenases, there is little known about the potential of non-heme iron(II) oxygenases to catalyze such reactions. We have studied the hydroxylation of trans-3-methyl-Lproline with the a-ketoglutarate (α-KG) dependent oxygenases, cis-3-proline hydroxylase type II and cis-4-proline hydroxylase (cis-P3H-II and cis-P4H). With cis-P3H-II, the tertiary alcohol product (3R)-3-hydroxy-3-methyl-L-proline was obtained exclusively but in reduced yield (～7%) compared to the native substrate L-proline. For cis-P4H, a complete shift in regioselectivity from C-4 to C-3 was observed so that the same product as with cis-P3H-II was obtained. Moreover, the yields were at least as good as in control reactions with L-proline (～110% relative yield). This result demonstrates a remarkable potential of non-heme iron(II) oxygenases to oxidize substrates selectively at sterically hindered positions. 10.3762/bjoc.7.193.
- Klein, Christian,Huettel, Wolfgang
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scheme or table
p. 1643 - 1647
(2012/01/19)
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- A simple procedure for selective hydroxylation of L -proline and l -pipecolic acid with recombinantly expressed proline hydroxylases
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Due to their diverse regio- and stereoselectivities, proline hydroxylases provide a straightforward access to hydroxprolines and other hydroxylated cylic amino acids, valuable chiral building blocks for chemical synthesis, which are often not available at reasonable expense by classical chemical synthesis. As yet, the application of proline hydroxylases is limited to a sophisticated industrial process for the production of two hydroxyproline isomers. This is mainly due to difficulties in their heterologues expression, their limited in vitro stability and complex product purification procedures. Here we describe a facile method for the production of cis-3-, cis-4- and trans-4-proline hydroxylase, and their application for the regio- and stereoselective hydroxylation of L-proline and its six-membered ring homologue l-pipecolic acid. Since in vitro catalysis with these enzymes is not very efficient and conversions are restricted to the milligram scale, an in vivo procedure was established, which allowed a quantitative conversion of 6 mM l-proline in shake flask cultures. After facile product purification via ion exchange chromatography, hydroxyprolines were isolated in yields of 35-61% (175-305 mg per flask). L-Pipecolic acid was converted with the isolated enzymes to prove the selectivities of the reactions. In transformations with optimized iron(II) concentration, conversions of 17-68% to hydroxylated products were achieved. The regio- and stereochemistry of the products was determined by NMR techniques. To demonstrate the applicability of the preparative in vivo approach for non-physiological substrates, L-pipecolic acid was converted with an E. coli strain producing trans-4-proline hydroxylase to trans-5-hydroxy-L-pipecolic acid in 61% yield. Thus, a synthetically valuable group of biocatalysts was made readily accessible for application in the laboratory without a need for special equipment or considerable development effort.
- Klein, Christian,Huettel, Wolfgang
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experimental part
p. 1375 - 1383
(2011/06/26)
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- Compounds and compositions for use in the prevention and treatment of obesity and related syndromes
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The invention relates to 4-hydroxyisoleucine, isomers, analogs, lactones, salts, and prodrugs thereof, to processes for their preparation, and to pharmaceutical compositions comprising the same. More particularly, the invention relates to the use of those compounds in the prevention and treatment of obesity and related syndromes.
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Page/Page column 60
(2010/11/24)
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- A new synthetic method for the preparation of α,β-didehydroamino acid derivatives by means of a wittig-type reaction. Syntheses of (2S, 4S)- and (2R, 4R)-4-hydroxyprolines
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Ethyl N-Boc- and N-Z-α-tosylglycinates were reacted with a variety of aldehydes in the presence of tributylphosphine and a base to afford the corresponding α,β-didehydroamino acid derivatives with high (Z)-selectivity in good yields. Moreover, ethyl (4S)- and (4R)-2-(N-Boc-amino)-4,5-isopropylidenedioxy-2-pentenoates prepared by the present method were converted to (2S, 4S)- and (2R, 4R)-4-hydroxyprolines, respectively.
- Kimura, Rumi,Nagano, Tanemasa,Kinoshita, Hideki
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p. 2517 - 2525
(2007/10/03)
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- An improved procedure for the enantioselective synthesis of (+)- and (-)-cis-4-hydroxyprolines and bulgecinines
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An efficient enantioselective synthesis of both the enantiomers of cis-4-hydroxy-proline and bulgecinine was accomplished starting from synthons 1 or 1'. The improved synthetic route to the aforesaid enantiomerically pure proline derivatives was established via a stereocontrolled double iodocyclization of 3(a,b) or 3'(a,b).
- Graziani, Lucia,Porzi, Gianni,Sandri, Sergio
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p. 1341 - 1346
(2007/10/03)
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- Stereoselective synthesis of unnatural aminoacids cis-4-hydroxyproline and bulgecinine
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A new stereoselective synthesis of both (2R,4R)- and (2S,4S)-4-hydroxy-proline and (+)- and (-)-bulgecinine was performed starting from synthons 1 or 1', respectively. The synthetic route has been established via a novel assisted cleavage of a disubstituted amide in mild conditions and successive stereocontrolled iodocyclization.
- Madau, Alessandra,Porzi, Gianni,Sandri, Sergio
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p. 825 - 830
(2007/10/03)
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- A Convenient Synthesis of (2S,4S)-4-Hydroxyproline
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A practical synthesis of (2S,4S)-4-hydroxyproline (1) based on DCC-induced inversion of the hydroxyl group of (2S,4R)-4-hydroxyproline (2) is described.
- Seki, Masahiko,Matsumoto, Kazuo
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p. 1161 - 1162
(2007/10/02)
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- Compounds of N-benzoylpyroline
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Compounds of the general formula (I): STR1 where A, R1, R2, R3, R4, R5 and R6 are defined in the description. Medicinal products.
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- Synthesis of De(hydroxymethyl)desulfo Analogues of Bulgecins A, B and C
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The syntheses of de(hydroxymethyl)desulfo analogues of Bulgecin A, B and C are described.Stereospecific β-glycosylation of aglycones was achieved using Schmidt's trichloroacetimidate metodology.
- Brown, Allan G.,Moss, Stephen F.,Southgate, Robert
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p. 451 - 454
(2007/10/02)
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- A Short Stereoselective Synthesis of cis- and trans-4-Hydroxy-L-proline
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A concise three-step synthesis of cis- and trans-4-hydroxy-L-proline on a preparative scale has been carried out using readily available starting materials.
- Mehlfuehrer, Michaela,Berner, Heinz,Thirring, Klaus
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p. 1291 - 1292
(2007/10/02)
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- Simple Synthesis of cis-4-Hydroxy-L-proline and Derivatives Suitable for Use as Intermediates in Peptide Synthesis
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An intramolecular Mitsunobu reaction in the presence of triphenylphosphine - diethyl azodicarboxylate (TPP-DEAD) resulted in the conversion of trans-4-hydroxy-N-trityl-L-proline to 5-triphenylmethyl-2-oxa-5-aza-bicycloheptan-3-one.This bicyclic lactone proved to be a key intermediate in the synthesis of cis-4-hydroxy-L-proline and derivatives thereof.TPP-DEAD-catalysed methanolysis of the key lactone gave the methyl ester of cis-4-hydroxy-N-trityl-L-proline, while ammonolysis in isopropyl alcohol provided the corresponding amide. p-Toluene-sulfonic acid treatmen t of the lactone, ester and amide led to detritylation and formation of the corresponding p-toluenesulfonates.Saponification of the key intermediate provided cis-4-hydroxy-N-trityl-L-proline which was first benzylated and then elaborated to the 1-hydroxybenzotriazolyl ester.This last ester and the three p-tolenesulfonates preparared above were utilised for the incorporation of cis-4-hydroxy-L-proline in the synthesis of model peptides.
- Papaioannou, Dionissios,Stavropoulos, George,Karagiannis, Kostas,Francis, George W.,Brekke, Trond,Aksnes, Dagfinn W.
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p. 243 - 251
(2007/10/02)
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