Biosynthesis of l‐4‐Chlorokynurenine,an Antidepressant Prodrug and a Non‐Proteinogenic Amino Acid Found in Lipopeptide Antibiotics

l ‐4‐Chlorokynurenine (l ‐4‐Cl‐Kyn) is a neuropharmaceutical drug candidate that is in development for the treatment of major depressive disorder. Recently, this amino acid was naturally found as a residue in the lipopeptide antibiotic taromycin. Herein, we report the unprecedented conversion of l ‐tryptophan into l ‐4‐Cl‐Kyn catalyzed by four enzymes in the taromycin biosynthetic pathway from the marine bacterium Saccharomonospora sp. CNQ‐490. We used genetic, biochemical, structural, and analytical techniques to establish l ‐4‐Cl‐Kyn biosynthesis, which is initiated by the flavin‐dependent tryptophan chlorinase Tar14 and its flavin reductase partner Tar15. This work revealed the first tryptophan 2,3‐dioxygenase (Tar13) and kynurenine formamidase (Tar16) enzymes that are selective for chlorinated substrates. The substrate scope of Tar13, Tar14, and Tar16 was examined and revealed intriguing promiscuity, thereby opening doors for the targeted engineering of these enzymes as useful biocatalysts.     

Biosynthesis of l‐4‐Chlorokynurenine,an Antidepressant Prodrug and a Non‐Proteinogenic Amino Acid Found in Lipopeptide Antibiotics

l ‐4‐Chlorokynurenine (l ‐4‐Cl‐Kyn) is a neuropharmaceutical drug candidate that is in development for the treatment of major depressive disorder. Recently, this amino acid was naturally found as a residue in the lipopeptide antibiotic taromycin. Herein, we report the unprecedented conversion of l ‐tryptophan into l ‐4‐Cl‐Kyn catalyzed by four enzymes in the taromycin biosynthetic pathway from the marine bacterium Saccharomonospora sp. CNQ‐490. We used genetic, biochemical, structural, and analytical techniques to establish l ‐4‐Cl‐Kyn biosynthesis, which is initiated by the flavin‐dependent tryptophan chlorinase Tar14 and its flavin reductase partner Tar15. This work revealed the first tryptophan 2,3‐dioxygenase (Tar13) and kynurenine formamidase (Tar16) enzymes that are selective for chlorinated substrates. The substrate scope of Tar13, Tar14, and Tar16 was examined and revealed intriguing promiscuity, thereby opening doors for the targeted engineering of these enzymes as useful biocatalysts.     

Scalable Hybrid Synthetic/Biocatalytic Route to Psilocybin

Psilocybin, the principal indole alkaloid of Psilocybe mushrooms, is currently undergoing clinical trials as a medication against treatment-resistant depression and major depressive disorder. The psilocybin supply for pharmaceutical purposes is met by synthetic chemistry. We replaced the problematic phosphorylation step during synthesis with the mushroom kinase PsiK. This enzyme was biochemically characterized and used to produce one gram of psilocybin from psilocin within 20 minutes. We also describe a pilot-scale protocol for recombinant PsiK that yielded 150 mg enzyme in active and soluble form. Our work consolidates the simplicity of tryptamine chemistry with the specificity and selectivity of enzymatic catalysis and helps provide access to an important drug at potentially reasonable cost.     

Mechanistic Insights into the Radical S‐adenosyl‐l‐methionine Enzyme NosL From a Substrate Analogue and the Shunt Products

The radical S‐adenosyl‐l ‐methionine (SAM) enzyme NosL catalyzes the transformation of l ‐tryptophan into 3‐methyl‐2‐indolic acid (MIA), which is a key intermediate in the biosynthesis of a clinically interesting antibiotic nosiheptide. NosL catalysis was investigated by using the substrate analogue 2‐methyl‐3‐(indol‐3‐yl)propanoic acid (MIPA), which can be converted into MIA by NosL. Biochemical assays with different MIPA isotopomers in D₂O and H₂O unambiguously indicated that the 5′‐deoxyadenosyl (dAdo)‐radical‐mediated hydrogen abstraction is from the amino group of l ‐tryptophan and not a protein residue. Surprisingly, the dAdo‐radical‐mediated hydrogen abstraction occurs at two different sites of MIPA, thereby partitioning the substrate into different reaction pathways. Together with identification of an α,β‐unsaturated ketone shunt product, our study provides valuable mechanistic insight into NosL catalysis and highlights the remarkable catalytic flexibility of radical SAM enzymes.     

Preparation of chiral amino acid materials and the study of their interactions with 1,1‐Bi‐2‐naphthol

The amino acid tryptophan has been converted into acrylamide monomers using L /D ‐tryptophan methyl ester forming the enantiopure chiral monomers. Attempts were made to polymerize these monomers via reversible addition fragmentation chain transfer (RAFT) polymerization to form poly(tryptophan). Unfortunately, this proved difficult, and instead, a postpolymerization modification route was used by first synthesizing poly(pentafluorophenyl acrylate) via RAFT, which was then substituted with L ‐tryptophan methyl ester to give poly(L ‐tryptophan). The interactions of the newly synthesized tryptophan monomers, as well as previously reported phenylalanine monomers, were studied in the presence of rac‐BINOL. It has been shown that the enantiomers of tryptophan have a stronger interaction with BINOL than phenylalanine and this has been attributed to the larger π system on the side chain. By monitoring the shifts and splitting of the phenolic protons of BINOL, it has been observed that S‐BINOL interacts more favorably with L ‐monomer enantiomers and R‐BINOL with D ‐monomer enantiomers. Similar interactions have also been seen with poly(phenylalanine) and the newly synthesized poly(tryptophan) materials. © 2012 Wiley Periodicals, Inc. J Polym Sci Part A: Polym Chem, 2012     

Determination of l‐tryptophan and l‐kynurenine derivatized with (R)‐4‐(3‐isothiocyanatopyrrolidin‐1‐yl)‐7‐(N,N‐dimethylaminosulfonyl)‐2,1,3‐benzoxadiazole by LC‐MS/MS on a triazole‐bonded column and their quantification in human serum

The concentrations of l ‐tryptophan (Trp) and the metabolite l ‐kynurenine (KYN) can be used to evaluate the in‐vivo activity of indoleamine 2,3‐dioxygenase (IDO) and tryptophan 2,3‐dioxygenase (TDO). As such, a novel method involving derivatization of l ‐Trp and l ‐KYN with (R)‐4‐(3‐isothiocyanatopyrrolidin‐1‐yl)‐7‐(N,N‐dimethylaminosulfonyl)‐2,1,3‐benzoxadiazole (DBD‐PyNCS) and separation by high‐performance liquid chromatography (HPLC) with tandem mass spectrometric (MS/MS) detection on a triazole‐bonded column (Cosmosil HILIC®) was developed to determine their concentrations. The optimized mobile phase, CH₃CN/10 mm ammonium formate in H₂O (pH 5.0) (90:10, v/v) eluted isocratically, resulted in satisfactory separation and MS/MS detection of the analytes. The detection limits of l ‐Trp and l ‐KYN were approximately 50 and 4.0 pm , respectively. The column temperature affected the retention behaviour of the Trp and KYN derivatives, with increased column temperatures leading to increased capacity factors; positive enthalpy changes were revealed by van't Hoff plot analyses. Using the proposed LC‐MS/MS method, l ‐Trp and l ‐KYN were successfully determined in 10 μL human serum using 1‐methyl‐l ‐Trp as an internal standard. The precision and recovery of l ‐Trp were in the ranges 2.85–9.29 and 95.8–113%, respectively, while those of l ‐KYN were 2.51–16.0 and 80.8–98.2%, respectively. The proposed LC‐MS/MS method will be useful for evaluating the in vivo activity of IDO or TDO. Copyright © 2016 John Wiley & Sons, Ltd.     

Enantioseparation of aromatic amino acids using CEC monolith with novel chiral selector,N‐methacryloyl‐l‐histidine methyl ester

A new type of polymethacrylate‐based monolithic column with chiral stationary phase was prepared for the enantioseparation of aromatic amino acids, namely d ,l ‐phenylalanine, d ,l ‐tyrosine, and d ,l ‐tryptophan by CEC. The monolithic column was prepared by in situ polymerization of butyl methacrylate (BMA), N‐methacryloyl‐l ‐histidine methyl ester (MAH), and ethylene dimethacrylate (EDMA) in the presence of porogens. The porogen mixture included DMF and phosphate buffer. MAH was used as a chiral selector. FTIR spectrum of the polymethacrylate‐based monolith showed that MAH was incorporated into the polymeric structure via in situ polymerization. Some experimental parameters including pH, concentration of the mobile phase, and MAH concentration with regard to the chiral CEC separation were investigated. Single enantiomers and enantiomer mixtures of the amino acids were separately injected into the monolithic column. It was observed that l ‐enantiomers of aromatic amino acids migrated before d ‐enantiomers. The reversal enantiomer migration order for tryptophan was observed upon changing of pH. Using the chiral monolithic column (100 μm id and 375 μm od), the best chiral separation was performed in 35:65% ACN/phosphate buffer (pH 8.0, 10 mM) with an applied voltage of 12 kV in CEC. SEM images showed that the chiral monolithic column has a continuous polymeric skeleton and large through‐pore structure.     

Simultaneous detection of stable isotope‐labeled and unlabeled l‐tryptophan and of its main metabolites,l‐kynurenine,serotonin and quinolinic acid,by gas chromatography/negative ion chemical ionization mass spectrometry

A method for the detection of unlabeled and ¹⁵N₂‐labeled l ‐tryptophan (l ‐Trp), l ‐kynurenine (l ‐Kyn), serotonin (5‐HT) and quinolinic acid (QA) in human and rat plasma by GC/MS is described. Labeled and unlabeled versions of these four products were analyzed as their acyl substitution derivatives using pentafluoropropionic anhydride and 2,2,3,3,3‐pentafluoro‐1‐propanol. Products were then separated by GC and analyzed by selected ion monitoring using negative ion chemical ionization mass spectrometry. l ‐[¹³C₁₁, ¹⁵N₂]‐Trp, methyl‐serotonin and 3,5‐pyridinedicarboxylic acid were used as internal standards for this method. The coefficients of variation for inter‐assay repeatability were found to be approximately 5.2% for l ‐Trp and ¹⁵N₂‐Trp, 17.1% for l ‐Kyn, 16.9% for 5‐HT and 5.8% for QA (n = 2). We used this method to determine isotope enrichments in plasma l ‐Trp over the course of a continuous, intravenous infusion of l ‐[¹⁵N₂]Trp in pregnant rat in the fasting state. Plasma ¹⁵N₂‐Trp enrichment reached a plateau at 120 min. The free Trp appearance rate (Ra) into plasma was 49.5 ± 3.35 µmol/kg/h. The GC/MS method was applied to determine the enrichment of ¹⁵N‐labeled l ‐Trp, l ‐Kyn, 5‐HT and QA concurrently with the concentration of non‐labeled l ‐Trp, l ‐Kyn, 5‐HT and QA in plasma. This method may help improve our understanding on l ‐Trp metabolism in vivo in animals and humans and potentially reveal the relative contribution of the four pathways of l ‐Trp metabolism. Copyright © 2014 John Wiley & Sons, Ltd.     

Development of a validated strategy for the determination of tryptamine in human cerebrospinal fluid in the presence of competitors using molecularly imprinted polymers

This study presents a validated strategy for the determination of tryptamine in the presence of its competitors, which involves the molecularly imprinted solid‐phase extraction combined with high‐performance liquid chromatography coupled with fluorimetric detection. Tryptamine‐imprinted microscale sorbent was produced from 4‐vinylbenzoic acid and ethylene glycol dimethacrylate in methanol by precipitation polymerization, and its imprinting factor was equal to 15.4 in static experiments or 18.6 in dynamic binding experiments. The method for tryptamine determination in the presence of serotonin and l ‐tryptophan was validated using a complex matrix of bovine serum albumin yielding the recoveries of tryptamine that ranged between 98.7 and 107.0%. Very low limits of detection and limits of quantification for tryptamine (19.9 and 60.3 nmol/L, respectively) allow the quantification of tryptamine in human cerebrospinal fluid in the presence of tryptophan and serotonin.     

HPLC enantioseparation of racemic bupropion,baclofen and etodolac: modification of conventional ligand exchange approach by pre‐column formation of chiral ligand exchange complexes

Separation of racemic mixture of (RS)‐bupropion, (RS)‐baclofen and (RS)‐etodolac, commonly marketed racemic drugs, has been achieved by modifying the conventional ligand exchange approach. The Cu(II) complexes were first prepared with a few l ‐amino acids, namely, l ‐proline, l ‐histidine, l ‐phenylalanine and l ‐tryptophan, and to these was introduced a mixture of the enantiomer pair of (RS)‐bupropion, or (RS)‐baclofen or (RS)‐etodolac. As a result, formation of a pair of diastereomeric complexes occurred by ‘chiral ligand exchange’ via the competition between the chelating l ‐amino acid and each of the two enantiomers from a given pair. The diastereomeric mixture formed in the pre‐column process was loaded onto HPLC column. Thus, both the phases during chromatographic separation process were achiral (i.e. neither the stationary phase had any chiral structural feature of its own nor did the mobile phase have any chiral additive). Separation of diastereomers was successful using a C₁₈ column and a binary mixture of MeCN and TEAP buffer of pH 4.0 (60:40, v/v) as mobile phase at a flow rate of 1 mL/min and UV detection at 230 nm for (RS)‐Bup, 220 nm for (RS)‐Bac and 223 nm for (RS)‐Etd. Baseline separation of the two enantiomers was obtained with a resolution of 6.63 in <15 min. Copyright © 2016 John Wiley & Sons, Ltd.     

Injury‐Triggered Blueing Reactions of Psilocybe “Magic” Mushrooms

Upon injury, psychotropic psilocybin‐producing mushrooms instantly develop an intense blue color, the chemical basis and mode of formation of which has remained elusive. We report two enzymes from Psilocybe cubensis that carry out a two‐step cascade to prepare psilocybin for oxidative oligomerization that leads to blue products. The phosphatase PsiP removes the 4‐O‐phosphate group to yield psilocin, while PsiL oxidizes its 4‐hydroxy group. The PsiL reaction was monitored by in situ ¹³C NMR spectroscopy, which indicated that oxidative coupling of psilocyl residues occurs primarily via C‐5. MS and IR spectroscopy indicated the formation of a heterogeneous mixture of preferentially psilocyl 3‐ to 13‐mers and suggest multiple oligomerization routes, depending on oxidative power and substrate concentration. The results also imply that phosphate ester of psilocybin serves a reversible protective function.     

Urinary l‐kynurenine quantification and selective extraction through a molecularly imprinted solid‐phase extraction device

l ‐Kynurenine is an endogenous metabolite generated by the catabolic pathway of l ‐tryptophan and it could be a potential biomarker to test the efficacy of several checkpoint inhibitors that have already reached the clinical trials in the antitumor therapy. Thus, a molecularly imprinted polymer specific for the recognition of this metabolite was synthesized and used as innovative system in solid‐phase extraction technique for the specific extraction and quantification of l ‐kynurenine in human urine. The off‐line system was firstly tested on l ‐kynurenine standard solutions, allowing recoveries up to 97.7% (relative standard deviation = 2.2%) and then applied to fortified and deproteinated human urine samples, where a recovery of 84.1% (relative standard deviation = 3.1%) was obtained. The method was validated and it revealed a good linearity in the range of 0.157–20 μg/mL (r² = 0.9992). The optimized procedure demonstrated a good feasibility on biological samples, allowing a ready quantification of l ‐kynurenine in the human urine, where the metabolite was found at a very low concentration (0.80 μg/mL). The extraction system developed could attract attention of pharmaceutical industries for l ‐kynurenine production as potential drug in the treatment of autoimmune disorders through its extraction and purification from biological matrixes.     

One‐Pot Bioconversion of l‐Arabinose to l‐Ribulose in an Enzymatic Cascade

This work reports the one‐pot enzymatic cascade that completely converts l ‐arabinose to l ‐ribulose using four reactions catalyzed by pyranose 2‐oxidase (P2O), xylose reductase, formate dehydrogenase, and catalase. As wild‐type P2O is specific for the oxidation of six‐carbon sugars, a pool of P2O variants was generated based on rational design to change the specificity of the enzyme towards the oxidation of l ‐arabinose at the C2‐position. The variant T169G was identified as the best candidate, and this had an approximately 40‐fold higher rate constant for the flavin reduction (sugar oxidation) step, as compared to the wild‐type enzyme. Computational calculations using quantum mechanics/molecular mechanics (QM/MM) molecular dynamics (MD) showed that this improvement is due to a decrease in the steric effects at the axial C4‐OH of l ‐arabinose, which allows a reduction in the distance between the C2‐H and flavin N5, facilitating hydride transfer and enabling flavin reduction.     

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 Combined Effect of Thermal and Enzymatic Racemization for d‐Aspartic Acid to l‐Aspartic Acid by High‐Performance Liquid Chromatography Assay

The racemization of d ‐aspartic acid to l ‐aspartic acid has been successfully performed with a coupled enzyme system at 90 °C and a pH of about 4.0 by the assay of high‐performance liquid chromatography. This coupled enzymatic racemization is a successive two‐step reaction first induced by d ‐amino acid oxidase and a subsequent coupled reaction by an aminotransferase clonezyme with the help of coenzyme pyridoxal 5′‐phosphate and cosubstrate l ‐glutamate. Due to the very high temperature, part of the l ‐aspartic acid is produced by the thermal effect. In fact the thermal racemization for aspartic acid can proceed from either d ‐ or l ‐aspartic acid via an intermediate fumaric acid and leads to the formation of d ,l ‐malic acid. The formation of α‐oxalacetic acid formed irreversibly from d ‐aspartic acid with d ‐amino acid oxidase can induce a side reaction to l ‐alanine. The thermal effect may also be responsible for the production of d ‐, and l ‐alanine.     

Simultaneous determination of l‐tetrahydropalmatine and its active metabolites in rat plasma by a sensitive ultra‐high‐performance liquid chromatography with tandem mass spectrometry method and its application in a pharmacokinetic study

A sensitive and reliable ultra‐high‐performance liquid chromatography with tandem mass spectrometry (UHPLC–MS/MS) method was developed and validated for simultaneous determination of l ‐tetrahydropalmatine (l ‐THP) and its active metabolites l ‐isocorypalmine (l ‐ICP) and L ‐corydalmine (l ‐CD) in rat plasma. The analytes were extracted by liquid–liquid extraction and separated on a Bonshell ASB C₁₈ column (2.1 × 100 mm; 2.7 μm; Agela) using acetonitrile–formic acid aqueous as mobile phase at a flow rate of 0.2 mL/min in gradient mode. The method was validated over the concentration range of 4.00–2500 ng/mL for l ‐THP, 0.400–250 ng/mL for l ‐ICP and 1.00–625 ng/mL for l ‐CD. Intra‐ and inter‐day accuracy and precision were within the acceptable limits of <15% at all concentrations. Correlation coefficients (r ) for the calibration curves were >0.99 for all analytes. The quantitative method was successfully applied for simultaneous determination of l ‐THP and its active metabolites in a pharmacokinetic study after oral administration with l ‐THP at a dose of 15 mg/kg to rats.     

Metabonomic analysis of the anti‐inflammatory effects of volatile oils of Angelica sinensis on rat model of acute inflammation

Metabonomics based on GC‐MS was used to study the possible anti‐inflammatory mechanisms of volatile oils of Angelica sinensis (VOAS) in rats with acute inflammation. Acute inflammation was induced by subcutaneous injection of carrageenan in rats. The levels of prostaglandin E₂ (PGE₂), histamine (HIS) and 5‐hydroxytryptamine (5‐HT) in the inflammatory fluid were detected. Principal component analysis and orthogonal partial least squares‐discriminant analysis models were performed for pattern recognition analysis. After the administration of VOAS, the levels of PGE₂, HIS, and 5‐HT returned to levels observed in normal group. According to GC‐MS analysis, the intervention of VOAS in rats with acute inflammation induced substantial and characteristic changes in their metabolic profiles. Fourteen metabolite biomarkers, namely, lactic acid, malic acid, citric acid, trans‐dehydroandrosterone, aldosterone, linoleic acid, hexadecanoic acid, pregnenolone, octadecenoic acid, myristic acid, l ‐histidine, octadecanoic acid, arachidonic acid (AA) and l ‐tryptophan, were detected in the inflammatory fluid. The levels of all biomarkers either increased or decreased significantly in model groups. VOAS possibly intervened in the metabolic process of inflammation by altering histidine metabolism, tryptophan metabolism, AA metabolism, steroid hormone biosynthesis, fatty acid metabolism and energy metabolism. Metabonomics was used to reflect an organism's physiological and metabolic state comprehensively, and it is a potentially powerful tool that reveals the anti‐acute‐inflammatory mechanism of VOAS. Copyright © 2014 John Wiley & Sons, Ltd.     

Label‐free aptamer‐based partial filling technique for enantioseparation and determination of dl‐tryptophan with micellar electrokinetic chromatography

In this study, a simple and reproducible method for enantioseparation and determination of dl ‐tryptophan (dl ‐T rp) was developed by using a partial filling technique in combination with MEKC . The corresponding l ‐T rp specific DNA aptamer was used as a chiral selector. Sodium cholate was used to form the chiral micelles and to enhance the enantioseparation of the enantiomers. Effects of aptamer concentration, filling time, buffer composition, and separation voltage on the enantioseparation were evaluated. The M g²⁺ and Na⁺ concentration in separation buffer was found to effectively affect the separation efficiency and reproducibility. Under the optimal conditions, d ‐ and l ‐T rp were completely enantioseparated in less than 9 min. This aptamer‐based partial‐filling approach has the potential to be extended to the separation of other enantiomers after the replacement of corresponding specific aptamers.     

Cyclic lipoundecapeptide amphisin from Pseudomonas sp. strain DSS73

The crystal structure of the lipoundecapeptide amphisin, presented here as the tetrahydrate, C₆₆H₁₁₄N₁₂O₂₀·4H₂O, originating from non‐ribosomal biosynthesis by Pseudomonas sp. strain DSS73, has been solved to a resolution of 0.65 Å. The primary structure of amphisin is β‐hydroxy­decanoyl‐d ‐Leu‐d ‐Asp‐d ‐allo‐Thr‐d ‐Leu‐d ‐Leu‐d ‐Ser‐l ‐Leu‐d ‐Gln‐l ‐Leu‐l ‐Ile‐l ‐Asp (Leu is leucine, Asp is aspartic acid, Thr is threonine, Ser is serine, Gln is glut­amine and Ile is isoleucine). The peptide is a lactone, linking Thr4 O_γ to the C‐terminal. The stereochemistry of the β‐hydroxy acid is R. The peptide is a close analogue of the cyclic lipopeptides tensin and pholipeptin produced by Pseudomonas fluorescens. The structure of amphisin is mainly helical (3₁₀‐helix), with the cyclic peptide wrapping around a hydrogen‐bonded water mol­ecule. This lipopeptide is amphiphilic and has biosurfactant and antifungal properties.     

Facile synthesis of poly(l‐tryptophan) through polycondensation of activated urethane derivatives

A facile and phosgene‐free synthetic route to poly(l ‐tryptophan) 2 by the polycondensation of N‐phenoxycarbonyl‐l ‐tryptophan 1 is described. The monomer 1 was synthesized via the carbamylation of tetrabutylammonium salt of L‐tryptophan with diphenyl carbonate. The polycondensation proceeded smoothly at 60 °C in N,N‐dimethylacetamide in the presence of amines (n‐butylamine, diethylamine, and triethylamine) along with the elimination of phenol and carbon dioxide. The structural analysis of the obtained 2 by Matrix‐assisted laser desorption/ionization time‐of‐flight mass spectrometry revealed that n‐butylamine or diethylamine was successfully incorporated into the chain end of the polypeptide. Furthermore, we have demonstrated the synthesis of a diblock copolymer by utilizing amine‐terminated poly(ethylene glycol) as a source of the polyether segment. The chain length of the polypeptide segment was controlled by varying feed ratio between 1 and the amino group of poly(ethylene glycol). © 2013 Wiley Periodicals, Inc. J. Polym. Sci., Part A: Polym. Chem. 2013 , 51, 4565–4571