Enantiomeric separation of d‐ and l‐serine using high‐performance liquid chromatography with electrochemical detection following pre‐column diastereomer derivatization

Enantiomeric separation of d ‐ and l ‐serine on an octadecylsilica column was investigated using (2R)‐2,5‐dioxopyrrolidin‐1‐yl‐2,5,7,8‐tetramethyl‐6‐(tetrahydro‐2H‐pyran‐2‐yloxy)chroman‐2‐carboxylate (R‐NPCA), which was developed for a pre‐column derivatization reagent for electrochemical detection. In addition, (2S)‐2,5‐dioxopyrrolidin‐1‐yl‐2,5,7,8‐tetramethyl‐6‐(tetrahydro‐2H‐pyran‐2‐yloxy)chroman‐2‐carboxylate (S‐NPCA) was newly synthesized from (S)‐(?)‐6‐hydroxy‐2,5,7,8‐tetramethylchroman‐2‐carboxylic acid (S‐α‐CA), and the enantiomeric separation of d ‐ and l ‐serine using S‐NPCA was also examined. The enantiomeric separation of d ,l ‐serine was achieved using the R‐ or S‐NPCA as a chiral derivatization reagent, and the elution orders of the enantiomers were reversed between R‐ and S‐NPCA. The elution orders of d ‐ and l ‐serine unexpectedly reversed between the phosphate buffer at pH 4.0 and pH 2.2, both of which were used in the mobile phase. Separation factors obtained using R‐ and S‐NPCA were similar—1.09 and 1.07, respectively. The detection limit was approximately 940 fmol on the column (signal‐to‐noise ratio 3) when the applied voltage was +650 mV. Copyright © 2009 John Wiley & Sons, Ltd.     

l‐Isoleucyl‐l‐serine 0.33‐hydrate,l‐phenyl­alanyl‐l‐serine and l‐methionyl‐l‐serine 0.34‐hydrate

The structures of the title dipeptides, C₉H₁₈N₂O₄·0.33H₂O, C₁₂H₁₆N₂O₄ and C₈H₁₆N₂O₄S·0.34H₂O, complete a series of investigations focused on l ‐Xaa‐l ‐serine peptides, where Xaa is a hydro­phobic residue. All three structures are divided into hydro­philic and hydro­phobic layers. The hydro­philic layers are thin for l ‐phenyl­alanyl‐l ‐serine, rendered possible by an unusual peptide conformation, and thick for l ‐isoleucyl‐l ‐serine and l ‐methionyl‐l ‐serine, which include cocrystallized water mol­ecules on the twofold axes.     

Substrate‐induced formation of a recognition structure in a four‐polypeptide–lipid monolayer system

Poly(γ‐methyl L ‐glutamate)s with Ser, His, Asp, and Glu residues at the amino terminal as the serine protease catalytic site were prepared. The number‐average degree of polymerization of the polypeptides was 51. A dipalmitoylphosphatidylcholine monolayer containing the polypeptides was formed at the air–water interface and was transferred onto gold‐deposited glass plates. The binding of N‐acetyltyrosine ethyl ester, a typical substrate of the serine protease, to the monolayer was characterized by surface plasmon resonance measurements. The four‐polypeptide–lipid monolayer system conditioned on an aqueous solution containing the substrate N‐acetyltyrosine ethyl ester exhibited Langmuir‐type binding of the substrate. Its binding constant of 6.1 × 10⁴ M⁻¹ was about 20 times larger than that observed for a monolayer prepared on pure water. The behavior may have arisen from a substrate‐induced rearrangement of the four kinds of polypeptides in the monolayer, forming a substrate‐binding structure similar to that found in serine protease. © 2000 John Wiley & Sons, Inc. J Polym Sci A: Polym Chem 38: 2186–2191, 2000     

Determination of D,L‐serine in midbrain of Parkinson's disease mouse by capillary electrophoresis with in‐column light‐emitting diode induced fluorescence detection

A capillary electrophoresis method with in‐column light‐emitting diode induced fluorescence detection is described for simultaneous determination of D ,L ‐serine in the midbrain of a Parkinson's disease mouse. D ,L ‐Serine was derivatized with fluorescein isothiocyanate, and chiral separation and determination of D ,L ‐serine derivatives were performed on a laboratory‐built capillary electrophoresis system with in‐column light‐emitting diode induced fluorescence detector using γ‐cyclodextrin as chiral selector. Using this method, the levels of D ‐ and L ‐serine in the midbrains of Parkinson's disease mice were determined. When compared to controls, the levels of D ‐ and L ‐serine showed significant differences. The result suggested that the biosynthesis and the transportation of endogenous D ,L ‐serine may participate in Parkinson's disease pathogenesis.     

Non‐Hydrolytic β‐Lactam Antibiotic Fragmentation by l,d‐Transpeptidases and Serine β‐Lactamase Cysteine Variants

Enzymes often use nucleophilic serine, threonine, and cysteine residues to achieve the same type of reaction; the underlying reasons for this are not understood. While bacterial d,d ‐transpeptidases (penicillin‐binding proteins) employ a nucleophilic serine, l,d ‐transpeptidases use a nucleophilic cysteine. The covalent complexes formed by l,d ‐transpeptidases with some β‐lactam antibiotics undergo non‐hydrolytic fragmentation. This is not usually observed for penicillin‐binding proteins, or for the related serine β‐lactamases. Replacement of the nucleophilic serine of serine β‐lactamases with cysteine yields enzymes which fragment β‐lactams via a similar mechanism as the l,d ‐transpeptidases, implying the different reaction outcomes are principally due to the formation of thioester versus ester intermediates. The results highlight fundamental differences in the reactivity of nucleophilic serine and cysteine enzymes, and imply new possibilities for the inhibition of nucleophilic enzymes.     

Enantiospecific Synthesis of (R)‐3‐Amino‐4‐(2,4,5‐trifluorophenyl)butanoic Acid Using (S)‐Serine as a Chiral Pool

Starting from (S)‐serine, a new method was developed for the synthesis of the β‐amino acid part of sitagliptin in ten steps and with an overall yield of 30%. The crucial step of the synthesis was the ring opening of N‐ and O‐protected (R)‐aziridin‐2‐methanol with (2,4,5‐trifluorophenyl)magnesium bromide to give N‐ and O‐protected (R)‐2‐amino‐3‐(2,4,5‐trifluorophenyl)propan‐1‐ol.     

Synthesis and quantitative structure–activity relationship of a new series of chiral 4‐alkoxycarbonyl‐2‐(alkylamino)‐1,3,2‐oxa or thiazaphospholidine‐2‐ones

Twenty‐one of the chiral 4‐alkoxycarbonyl‐2‐(α‐alkyl‐α‐ethoxycarbonyl methylamino)‐1,3‐2‐thia or oxazaphospholidine‐2‐ones have been synthesized by cyclization of L‐serine or L‐cysteine ethyl or n‐octyl ester with phosphoryl chloride followed by reaction with a suitable L‐amino acid ethyl ester. Proton NMR, IR, and mass spectra of these compounds have been discussed in detail. These compounds inhibited up to 68.52% of acetylcholinesterase (AChE) at the 1 ppm concentration level. Regression analysis showed that AChE inhibition was determined by both the steric and electronic effects of the alkyl groups of the amino acid. The enzyme inhibition correlated directly with the steric bulk of the alkyl groups, indicating a steric requirement for maximizing inhibitor–enzyme interaction and an inverse relationship with the electron‐donating ability of the alkyl groups. This supports the concept of a nucleophilic attack mechanism of a hydroxyl group of a serine amino acid in the enzyme active center on the partially positive phosphorus atom of the oxazaphospholidines and thiazaphospholidines, with correlation coefficients of 0.999 and 0.838, respectively. Results also indicated that the steric requirement was more important than the electronic factor in affecting the inhibition process, which explained the high activity of compounds containing the isoleucine moiety. The high AChE inhibition activity of these compounds and the expected nontoxic products of their in vivo hydrolysis make them eligible for pesticidal application. © 1999 John Wiley & Sons, Inc. Heteroatom Chem 10: 475–480, 1999     

Leader Peptide‐Free In Vitro Reconstitution of Microviridin Biosynthesis Enables Design of Synthetic Protease‐Targeted Libraries

Microviridins are a family of ribosomally synthesized and post‐translationally modified peptides with a highly unusual architecture featuring non‐canonical lactone as well as lactam rings. Individual variants specifically inhibit different types of serine proteases. Here we have established an efficient in vitro reconstitution approach based on two ATP‐grasp ligases that were constitutively activated using covalently attached leader peptides and a GNAT‐type N‐acetyltransferase. The method facilitates the efficient in vitro one‐pot transformation of microviridin core peptides to mature microviridins. The engineering potential of the chemo‐enzymatic technology was demonstrated for two synthetic peptide libraries that were used to screen and optimize microviridin variants targeting the serine proteases trypsin and subtilisin. Successive analysis of intermediates revealed distinct structure–activity relationships for respective target proteases.     

Density Functional Theory Investigations on Vibrational Spectra,Molecular Structure,and Properties of the L‐Serine,L‐Cysteine,and L‐Aspartic Acid Molecules

In this paper, we present a thorough investigation of the conformational space to characterize all possible gas‐phase structures of the neutral L‐serine, L‐cysteine, and L‐aspartic acid molecules. A total of 120 trial structures were generated for L‐aspartic acid and 96 trial structures for L‐serine and L‐cysteine by combining all internal single‐bond rotamers. Various combinations of the Hartree–Fock and density functional theory/B3LYP methods with different bases were used to optimize all possible trial structures. The theoretical studies on the structure, harmonic vibrational spectra, and molecular properties of these amino acids are presented. The assignments of the calculated wave numbers resulting from potential energy distributions were performed using the VEDA 4 program to allow a good interpretation of the theoretical vibrational spectra of the title compounds. The fundamental harmonic frequencies were found to be in good agreement with data in the literature. A natural bond orbital analysis was performed to investigate the charge delocalization throughout the molecules for the three test compounds. Moreover, an extensive discussion of the highest occupied molecular orbital–lowest unoccupied molecular orbital energy gap as well as other related molecular properties are reported.     

The monohydrates of the four polar dipeptides l‐seryl‐l‐asparagine,l‐seryl‐l‐tyrosine,l‐tryptophanyl‐l‐serine and l‐tyrosyl‐l‐tryptophan

The crystal structures of the four dipeptides l ‐seryl‐l ‐asparagine monohydrate, C₇H₁₃N₃O₅·H₂O, l ‐seryl‐l ‐tyrosine monohydrate, C₁₂H₁₆N₂O₅·H₂O, l ‐tryptophanyl‐l ‐serine monohydrate, C₁₄H₁₇N₃O₄·H₂O, and l ‐tyrosyl‐l ‐tryptophan monohydrate, C₂₀H₂₁N₃O₄·H₂O, are dominated by extensive hydrogen‐bonding networks that include cocrystallized solvent water molecules. Side‐chain conformations are discussed on the basis of previous observations in dipeptides. These four dipeptide structures greatly expand our knowledge on dipeptides incorporating polar residues such as serine, asparagine, threonine, tyrosine and tryptophan.     

A New Mechanism for β‐Lactamases: Class D Enzymes Degrade 1β‐Methyl Carbapenems through Lactone Formation

β‐Lactamases threaten the clinical use of carbapenems, which are considered antibiotics of last resort. The classical mechanism of serine carbapenemase catalysis proceeds through hydrolysis of an acyl‐enzyme intermediate. We show that class D β‐lactamases also degrade clinically used 1β‐methyl‐substituted carbapenems through the unprecedented formation of a carbapenem‐derived β‐lactone. β‐Lactone formation results from nucleophilic attack of the carbapenem hydroxyethyl side chain on the ester carbonyl of the acyl‐enzyme intermediate. The carbapenem‐derived lactone products inhibit both serine β‐lactamases (particularly class D) and metallo‐β‐lactamases. These results define a new mechanism for the class D carbapenemases, in which a hydrolytic water molecule is not required.     

Expedient Synthesis of Chiral α‐Amino Acids through Nickel‐Catalyzed Reductive Cross‐Coupling

A novel method for the synthesis of non‐natural L ‐ and D ‐amino acids by a Ni‐catalyzed reductive cross‐coupling reaction is described. This strategy enables the racemization‐free cross‐coupling of serine/homoserine‐ derived iodides with aryl/acyl/alkyl halides. It provides convenient access to varieties of enantiopure and functionalized amino acids, which are important building blocks in bioactive compounds and pharmaceuticals.     

An unexpected tetragonal unit cell for N‐(L‐2‐aminobutyryl)‐L‐serine

The title dipeptide {systematic name: (S)‐2‐[(S)‐2‐azaniumylbutanamido]‐3‐hydroxypropanoate}, C₇H₁₄N₂O₄, was synthesized in the anticipation that it would form nanoporous crystals with hexagonal symmetry. Single‐crystal X‐ray diffraction analysis showed that it had instead adopted a unit cell in the space group I4, similar to L‐alanyl‐L‐alanine [Fletterick, Tsai & Hughes (1970). J. Phys. Chem. 75 , 918–922]. The resulting packing arrangement has a high density for a peptide (1.462 Mg m⁻³), which is rendered possible by extensive disorder over two positions for the ethyl side chain of the 2‐aminobutyric acid fragment and over three positions for the serine side chain.<!?tpb=17.5pt>     

Controllable ring‐opening copolymerization of L‐lactide and (3S)‐benzyloxymethyl‐(6S)‐methyl‐morpholine‐2,5‐dione initiated by a biogenic compound creatinine acetate

Ring‐opening copolymerization (ROCP) of L ‐lactide (L ‐LA) and (3S)‐benzyloxymethyl‐(6S)‐methyl‐morpholine‐2,5‐dione [(3S, 6S)‐BMMD] initiated by creatinine acetate, a biogenic organic compound, was performed in the bulk at 130 °C. The copolymerization was well controlled as evidenced by that both the measured values of number‐average molecular weight (M_n,NMR(OH) and M_n,NMR(COOH)) and serine molar fraction (F_Bz.ser) of synthesized copolymers were close to the corresponding theoretical values; and that the higher isotacticity of synthesized copolymers (85–86%) and lower racemization degree of the ROCP. After removing O‐benzyls of the copolymers with Et₃SiH/Et₃N/CH₂Cl₂ under catalysis of PdCl₂, functional biodegradable copolymers of L ‐lactic acid (L ‐Lac) and L ‐Ser with designed molar fraction of serine (F_ser 1.35%, 3.57%, 5.41%), narrow molecular weight distribution (polydispersity index 1.10–1.36), and improved hydrophilicity (θ_stat 82.3–89.6°) were finally obtained. © 2012 Wiley Periodicals, Inc. J Polym Sci Part A: Polym Chem, 2012     

Molecular Mechanism of a Hotdog‐Fold Acyl‐CoA Thioesterase

Thioesterases are enzymes that hydrolyze thioester bonds between a carbonyl group and a sulfur atom. They catalyze key steps in fatty acid biosynthesis and metabolism, as well as polyketide biosynthesis. The reaction molecular mechanism of most hotdog‐fold acyl‐CoA thioesterases remains unknown, but several hypotheses have been put forward in structural and biochemical investigations. The reaction of a human thioesterase (hTHEM2), representing a thioesterase family with a hotdog fold where a coenzyme A moiety is cleaved, was simulated by quantum mechanics/molecular mechanics metadynamics techniques to elucidate atomic and electronic details of its mechanism, its transition‐state conformation, and the free energy landscape of the process. A single‐displacement acid‐base‐like mechanism, in which a nucleophilic water molecule is activated by an aspartate residue acting as a base, was found, confirming previous experimental proposals. The results provide unambiguous evidence of the formation of a tetrahedral‐like transition state. They also explain the roles of other conserved active‐site residues during the reaction, especially that of a nearby histidine/serine pair that protonates the thioester sulfur atom, the participation of which could not be elucidated from mutation analyses alone.     

Atomistic detailed free‐energy landscape of intrinsically disordered protein studied by multi‐scale divide‐and‐conquer molecular dynamics simulation

Calcineurin (CaN) is a eukaryotic serine/threonine protein phosphatase activated by both Ca²⁺ and calmodulin (CaM), including intrinsically disordered region (IDR). The region undergoes folding into an α‐helix form in the presence Ca²⁺‐loaded CaM. To sample the ordered structure of the IDR by conventional all atom model (AAM) molecular dynamics (MD) simulation, the IDR and Ca²⁺‐loaded CaM must be simultaneously treated. However, it is time‐consuming task because the coupled folding and binding should include repeated binding and dissociation. Then, in this study, we propose novel multi‐scale divide‐and‐conquer MD (MSDC‐MD), which combines AAM‐MD and coarse‐grained model MD (CGM‐MD). To speed up the conformation sampling, MSDC‐MD simulation first treats the IDR by CGM to sample conformations from wide conformation space; then, multiple AAM‐MD in a limited area is initiated using the resultant CGM conformation, which is reconstructed by homology modeling method. To investigate performance, we sampled the ordered conformation of the IDR using MSDC‐MD; the root‐mean‐square distance (RMSD) with respect to the experimental structure was 2.23 Å.     

Silk‐Inspired β‐Peptide Materials Resist Fouling and the Foreign‐Body Response

The functions of implants like medical devices are often compromised by the host's foreign‐body response (FBR). Herein, we report the development of low‐FBR materials inspired by serine‐rich sericin from silk. Poly‐β‐homoserine (β‐HS) materials consist of the hydrophilic unnatural amino acid β‐homoserine. Self‐assembled monolayers (SAMs) of β‐HS resist adsorption by diverse proteins, as well as adhesion by cells, platelets, and diverse microbes. Experiments lasting up to 3 months revealed that, while implantation with control PEG hydrogels induced obvious inflammatory responses, collagen encapsulation, and macrophage accumulation, these responses were minimal with β‐HS hydrogels. Strikingly, the β‐HS hydrogels induce angiogenesis in implant‐adjacent tissues. Molecular dynamics simulations indicated that the low FBR performance of β‐HS results from what we term “dual hydrogen bonding hydration”, wherein both the backbone amide groups and the sidechain hydroxyl groups of β‐HS undergo hydration.     

Engineered L‐Serine Hydroxymethyltransferase from Streptococcus thermophilus for the Synthesis of α,α‐Dialkyl‐α‐Amino Acids

α,α‐Disubstituted α‐amino acids are central to biotechnological and biomedical chemical processes for their own sake and as substructures of biologically active molecules for diverse biomedical applications. Structurally, these compounds contain a quaternary stereocenter, which is particularly challenging for stereoselective synthesis. The pyridoxal‐5′‐phosphate (PLP)‐dependent L ‐serine hydroxymethyltransferase from Streptococcus thermophilus (SHMT_Sth; EC 2.1.2.1) was engineered to achieve the stereoselective synthesis of a broad structural variety of α,α‐dialkyl‐α‐amino acids. This was accomplished by the formation of quaternary stereocenters through aldol addition of the amino acids D ‐Ala and D ‐Ser to a wide acceptor scope catalyzed by the minimalist SHMT_Sth Y55T variant overcoming the limitation of the native enzyme for Gly. The SHMT_Sth Y55T variant tolerates aromatic and aliphatic aldehydes as well as hydroxy‐ and nitrogen‐containing aldehydes as acceptors.     

Gas chromatography/time‐of‐flight mass spectrometry‐based metabonomics of hepatocarcinoma in rats with lung metastasis: elucidation of the metabolic characteristics of hepatocarcinoma at formation and metastasis

Hepatocarcinoma (HCC) has a very high mortality rate and the high recurrence and metastasis rates contribute to the poor prognosis of HCC patients. To understand HCC formation and metastasis, we assessed the metabonomics of rat HCC and HCC with lung metastasis (HLM). The HLM rat model was established by exposure to diethylnitrosamine (DEN). Levels of serum and urine metabolites were quantified with gas chromatography/time‐of‐flight mass spectrometry (GC/TOFMS), and data were analyzed with partial least‐squares discrimination analysis (PLS‐DA). Serum and urine levels of some metabolites differed significantly between the control, HCC, and HLM groups. The products and intermediates from glycolysis and glutamate metabolism were elevated, while the tricarboxylic acid (TCA) cycle was inhibited, in both HCC and HLM. HLM samples revealed enhanced metabolism of nucleic acids, amino acids and glucuronic acid. PLS‐DA indicated that principal component weighting was greatest for serum serine, phenylalanine, lactic acid, tyrosine and glucuronic acid, and urine glycine, serine, 5‐oxyproline, malate, hippuric acid and uric acid. These data provide novel information that will improve understanding of the pathophysiological processes involved in HCC and HLM, and revealed potential metabolic markers for HCC invasion and metastasis. Copyright © 2010 John Wiley & Sons, Ltd.     

Reversal of elution order of N‐(2,4‐dinitrophenyl)‐proline and N‐(2,4‐dinitrophenyl)‐serine in HPLC by BSA chiral stationary phase

N‐(2,4‐dinitrophenyl)‐proline and N‐(2,4‐dinitrophenyl)‐serine were enantiomerically resolved on the BSA chiral stationary phase by HPLC in reversed‐phase mode. Effects of chromatographic conditions on enantioseparation and elution order have been investigated in detail. For these two samples, reversal of enantiomer elution order was observed by changing buffer pH, the content of acetonitrile, or alcohol modifiers in mobile phase, which is firstly reported in the BSA chiral stationary phase studies. More interestingly, combined effect between buffer pH and the content of acetonitrile was also observed. In addition, coelution range of enantiomers varied along with the content of acetonitrile in mobile phase.