Butelase‐Mediated Macrocyclization of d‐Amino‐Acid‐Containing Peptides

Macrocyclic compounds have received increasing attention in recent years. With their large surface area, they hold promise for inhibiting protein–protein interactions, a chemical space that was thought to be undruggable. Although many chemical methods have been developed for peptide macrocyclization, enzymatic methods have emerged as a promising new economical approach. Thus far, most enzymes have been shown to act on l ‐peptides; their ability to cyclize d ‐amino‐acid‐containing peptides has rarely been documented. Herein we show that macrocycles consisting of d ‐amino acids, except for the Asn residue at the ligating site, were efficiently synthesized by butelase 1, an Asn/Asp‐specific ligase. Furthermore, by using a peptide‐library approach, we show that butelase 1 tolerates most of the d ‐amino acid residues at the P1′′ and P2′′ positions.     

Introduction of d‐Amino Acids in Minimalistic Peptide Substrates by an S‐Adenosyl‐l‐Methionine Radical Epimerase

Post‐translational modifying enzymes from the S‐adenosyl‐l ‐methionine (AdoMet) radical superfamily garner attention due to their ability to accomplish challenging biochemical reactions. Among them, a family of AdoMet radical epimerases catalyze irreversible l ‐ to d ‐amino acid transformations of diverse residues, including 18 sites in the complex sponge‐derived polytheonamide toxins. Herein, the in vitro activity of the model epimerase OspD is reported and its catalytic mechanism and substrate flexibility is investigated. The wild‐type enzyme was capable of leader‐independent epimerization of not only the stand‐alone core peptide, but also truncated and cyclic core variants. Introduction of d ‐amino acids can drastically alter the stability, structure, and activity of peptides; thus, epimerases offer opportunities in peptide bioengineering.     

Novel N‐(2,2‐Dimethyl‐2H‐azirin‐3‐yl)‐L‐prolinates as Aib‐Pro Synthons

The syntheses of phenacyl N‐(2,2‐dimethyl‐2H‐azirin‐3‐yl)‐L ‐prolinate and allyl N‐(2,2‐dimethyl‐2H‐azirin‐3‐yl)‐L ‐prolinate are reported. Reactions of these 2H‐azirin‐3‐amine derivatives with Z‐protected amino acids have shown them to be suitable synthons for the Aib‐Pro unit in peptide synthesis. After incorporation into the peptide by means of the ‘azirine/oxazolone method’, the C‐termini of the resulting peptides were deprotected selectively with Zn in AcOH or by a mild Pd⁰‐promoted procedure, respectively.     

Synthesis of β‐Peptides with β‐Helices from New C‐Linked Carbo‐β‐Amino Acids: Study on the Impact of Carbohydrate Side Chains

The design and synthesis of β‐peptides from new C‐linked carbo‐β‐amino acids (β‐Caa) presented here, provides an opportunity to understand the impact of carbohydrate side chains on the formation and stability of helical structures. The β‐amino acids, Boc‐(S)‐β‐Caa_(g)‐OMe 1 and Boc‐(R)‐β‐Caa_(g)‐OMe 2 , having a D ‐galactopyranoside side chain were prepared from D ‐galactose. Similarly, the homo C‐linked carbo‐β‐amino acids (β‐hCaa); Boc‐(S)‐β‐hCaa_(x)‐OMe 3 and Boc‐(R)‐β‐hCaa_(x)‐OMe 4 , were prepared from D ‐glucose. The peptides derived from the above monomers were investigated by NMR, CD, and MD studies. The β‐peptides, especially the shorter ones obtained from the epimeric (at the amine stereocenter C_β) 1 and 2 by the concept of alternating chirality, showed a much smaller propensity to form 10/12‐helices. This substantial destabilization of the helix could be attributed to the bulkier D ‐galactopyranoside side chain. Our efforts to prepare peptides with alternating 3 and 4 were unsuccessful. However, the β‐peptides derived from alternating geometrically heterochiral (at C_β) 4 and Boc‐(R)‐β‐Caa_(x)‐OMe 5 (D ‐xylose side chain) display robust right‐handed 10/12‐helices, while the mixed peptides with alternating 4 and Boc‐β‐hGly‐OMe 6 (β‐homoglycine), resulted in left‐handed β‐helices. These observations show a distinct influence of the side chains on helix formation as well as their stability.     

Structure‐guided RP‐HPLC chromatography of diastereomeric α‐helical peptide analogs substituted with single amino acid stereoisomers

An α‐helical model peptide (Ac‐EAEKAAKE‐X‐EKAAKEAEK‐amide) was used as a template to examine the efficacy of conventional reversed‐phase high‐performance liquid chromatography (RP‐HPLC) in separating peptide analogs with single substitutions (at position X) of diasteromeric amino acids Ile, allo‐Ile, d ‐Ile and d ‐allo‐Ile. We compared differences in peptide retention behavior on a C₈ column and a C₁₈ column at different temperatures. We demonstrated how subtle differences in peptide secondary structure affected by the different substitutions of amino acids with identical overall hydrophobicity enabled effective resolution of these peptide analogs. We also demonstrated the ability of RP‐HPLC to separate Ile‐ and allo‐Ile‐substituted analogs of a 26‐residue α‐helical antimicrobial peptide (AMP), with the substitution site towards the C‐terminus of the α‐helix. These peptides show different values of antibacterial activity and hemolytic activity, and different selectivity against bacteria and human cells. Our results underline the ability of RP‐HPLC to resolve even difficult diasteromeric peptide mixtures as well as its value in monitoring very subtle hydrophobicity changes in de novo‐designed AMP. Copyright © 2013 John Wiley & Sons, Ltd.     

The self‐assembled of cyclic D,L‐α‐peptide systems: Insights into the structure and energetics

Cyclic D,L ‐α‐peptides are able to self‐assemble to nanotubes, although the inherent reason of the stability of this kind of nanotube as well as the intrinsic driving force of self‐assembly of the cyclic D ,L ‐α‐peptides still remain elusive. In this work, using several computational approaches, we investigated the structural and energy characteristics of a series of cyclo[(‐L ‐Phe‐D ‐Ala‐)₄] and cyclo[(‐L ‐Ala‐D ‐Ala‐)₄] oligomers. The results reveal that the thermodynamic stability, cooperativity, and self‐assembly patterns of cyclic D ,L ‐α‐peptide nanotubes are mainly determined by the interactions between cross‐strand side chains instead of those between backbones. For cyclo[(‐L ‐Phe‐D ‐Ala‐)₄] oligomers, the steric interaction between cross‐strand side chains, especially the electrostatic repulsion between the phenyls in Phe residues, brings anticooperative effect into parallel stacking mode, which is responsible for the preference of self‐assembling nanotube in antiparallel vs. parallel stacking orientation. Based on our results, a novel self‐assembling mechanism is put forward—it is the L ‐L antiparallel dimer of cyclo[(‐L ‐Phe‐D ‐Ala‐)₄], instead of the commonly presumed monomer, that acts as the basic building block in self assembly. It explains why these cyclic peptides uniquely self‐assemble to form antiparallel nanotubes. © 2009 Wiley Periodicals, Inc. Int J Quantum Chem, 2010     

β‐Hairpins Generated from Hybrid Peptide Sequences Containing both α‐ and β‐Amino Acids

The incorporation of the β‐amino acid residues into specific positions in the strands and β‐turn segments of peptide hairpins is being systematically explored. The presence of an additional torsion variable about the C(α) C(β) bond (θ) enhances the conformational repertoire in β‐residues. The conformational analysis of three designed peptide hairpins composed of α/β‐hybrid segments is described: Boc‐Leu‐Val‐Val‐^DPro‐β Phe ‐Leu‐Val‐Val‐OMe ( 1 ), Boc‐Leu‐Val‐β Val ‐^DPro‐Gly‐β Leu ‐Val‐Val‐OMe ( 2 ), and Boc‐Leu‐Val‐β Phe ‐Val‐^DPro‐Gly‐Leu‐β Phe ‐Val‐Val‐OMe ( 3 ). 500‐MHz ¹H‐NMR Analysis supports a preponderance of β‐hairpin conformation in solution for all three peptides, with critical cross‐strand NOEs providing evidence for the proposed structures. The crystal structure of peptide 2 reveals a β‐hairpin conformation with two β‐residues occupying facing, non‐H‐bonded positions in antiparallel β‐strands. Notably, βVal(3) adopts a gauche conformation about the C(α) C(β) bond (θ=+65°) without disturbing cross‐strand H‐bonding. The crystal structure of 2 , together with previously published crystal structures of peptides 3 and Boc‐β Phe ‐β Phe ‐^DPro‐Gly‐β Phe ‐β Phe ‐OMe, provide an opportunity to visualize the packing of peptide sheets with local ‘polar segments' formed as a consequence of reversal peptide‐bond orientation. The available structural evidence for hairpins suggests that β‐residues can be accommodated into nucleating turn segments and into both the H‐bonding and non‐H‐bonding positions on the strands.     

An Orthogonal D2O‐Based Induction System that Provides Insights into d‐Amino Acid Pattern Formation by Radical S‐Adenosylmethionine Peptide Epimerases

Radical S‐adenosyl methionine peptide epimerases (RSPEs) are an enzyme family that accomplishes regiospecific and irreversible introduction of multiple d ‐configured residues into ribosomally encoded peptides. Collectively, RSPEs can generate diverse epimerization patterns in a wide range of substrates. Previously, the lack of rapid methods to localize epimerized residues has impeded efforts to investigate the function and applicative potential of RSPEs. An efficient mass spectrometry‐based assay is introduced that permits characterization of products generated in E. coli . Applying this to a range of non‐natural peptide‐epimerase combinations, it is shown that the d ‐amino acid pattern is largely but not exclusively dictated by the core peptide sequence, while the epimerization order is dependent on the enzyme‐leader pair. RSPEs were found to be highly promiscuous, which allowed for modular introduction of peptide segments with defined patterns.     

Differently Glycosidated 2‐Amino‐2‐deoxy‐d‐glucopyranosiduronic Acids as Building Blocks in Peptide Synthesis

Seven differently glycosidated sugar amino acids (SSAs) derived from glucosamine have been prepared. Following standard solution‐phase peptide‐coupling procedures, the glycosidated 2‐amino‐2‐deoxy‐D ‐glucopyranosiduronic acids were condensed with natural amino acids to furnish useful heterodi‐ and ‐trimeric building blocks to be used in peptide synthesis. Combinations of these building blocks yielded hetero‐oligomeric peptides with two sugar amino acid units in different distances to each other. These were prepared to evaluate the influence of glycosidic side chains on the peptide backbone. Conformations of selected examples were examined by means of ROESY spectroscopy in combination with molecular dynamics (MD) simulations and circular‐dichroism (CD) studies.     

The Design of α/β‐Peptides: Study on Three‐Residue Turn Motifs and the Influence of Achiral Glycine on Helix and Turn

Novel three‐residue helix‐turn secondary structures, nucleated by a helix at the N terminus, were generated in peptides that have ‘β‐Caa‐L ‐Ala‐L ‐Ala,’ ‘β‐Caa‐L ‐Ala‐γ‐Caa,’ and ‘β‐Caa‐L ‐Ala‐δ‐Caa’ (in which β‐Caa is C‐linked carbo‐β‐amino acid, γ‐Caa is C‐linked carbo‐γ‐amino acid, and δ‐Caa is C‐linked carbo‐δ‐amino acid) at the C terminus. These turn structures are stabilized by 12‐, 14‐, and 15‐membered (mr) hydrogen bonding between NH(i)/CO(i+2) (i+2 is the last residue in the peptide) along with a 7‐mr hydrogen bond between CO(i)/NH(i+2). In addition, a series of α/β‐peptides were designed and synthesized with alternating glycine (Gly) and (S)‐β‐Caa to study the influence of an achiral α‐residue on the helix and helix‐turn structures. In contrast to previous results, the three ‘β–α–β’ residues at the C terminus (α‐residue being Gly) are stabilized by only a 13‐mr forward hydrogen bond, which resembles an α‐turn. Extensive NMR spectroscopic and molecular dynamics (MD) studies were performed to support these observations. The influence of chirality and side chain is also discussed.     

Rethinking Cysteine Protective Groups: S‐Alkylsulfonyl‐l‐Cysteines for Chemoselective Disulfide Formation

The ability to reversibly cross‐link proteins and peptides grants the amino acid cysteine its unique role in nature as well as in peptide chemistry. We report a novel class of S‐alkylsulfonyl‐l ‐cysteines and N‐carboxy anhydrides (NCA) thereof for peptide synthesis. The S‐alkylsulfonyl group is stable against amines and thus enables its use under Fmoc chemistry conditions and the controlled polymerization of the corresponding NCAs yielding well‐defined homo‐ as well as block co‐polymers. Yet, thiols react immediately with the S‐alkylsulfonyl group forming asymmetric disulfides. Therefore, we introduce the first reactive cysteine derivative for efficient and chemoselective disulfide formation in synthetic polypeptides, thus bypassing additional protective group cleavage steps.     

Application of HR‐MAS NMR in the solid‐phase synthesis of a glycopeptide using Sieber amide resin

The solid‐phase synthesis (SPS) of a structurally complex glycopeptide, using Sieber amide resin, was monitored by high resolution magic angle spinning NMR, demonstrating the further application of this technique. A synthetic peptidoglycan derivative, a precursor of a biologically active PGN, known to be involved in the cellular recognition, was prepared by SPS. The synthesis involved the preparation of an N‐alloc glucosamine moiety and the synthesis of a simple amino acid sequence L ‐Ala‐D ‐Glu‐L ‐Lys‐D ‐Ala‐D ‐Ala. Last step consisted the coupling, on solid‐phase, of the protected muramyl unit to the peptide chain. Proton spectra with good suppression of the polystyrene signals in swollen resin samples were obtained in DMF‐d₇ as a solvent and by using a nonselective 1D TOCSY/DIPSI‐2 scheme, thus allowing to follow the SPS without losses of compound and cleavage from the resin. The assignment of the proton spectra of the resin‐bound amino acid sequence and of the bound glycopeptide was achieved through the combination of MAS COSY, TOCSY and NOESY. Copyright © 2010 John Wiley & Sons, Ltd.     

The Propensity of α‐Aminoisobutyric Acid (=2‐Methylalanine; Aib) to Induce Helical Secondary Structure in an α‐Heptapeptide: A Computational Study

We present a molecular‐dynamics simulation study of an α‐heptapeptide containing an α‐aminoisobutyric acid (=2‐methylalanine; Aib) residue, Val¹‐Ala²‐Leu³‐Aib⁴‐Ile⁵‐Met⁶‐Phe⁷, and a quantum‐mechanical (QM) study of simplified models to investigate the propensity of the Aib residue to induce 3₁₀/α‐helical conformation. For comparison, we have also performed simulations of three analogues of the peptide with the Aib residue being replaced by L ‐Ala, D ‐Ala, and Gly, respectively, which provide information on the subtitution effect at C(α) (two Me groups for Aib, one for L ‐Ala and D ‐Ala, and zero for Gly). Our simulations suggest that, in MeOH, the heptapeptide hardly folds into canonical helical conformations, but appears to populate multiple conformations, i.e., C₇ and 3₁₀‐helical ones, which is in agreement with results from the QM calculations and NMR experiments. The populations of these conformations depend on the polarity of the solvent. Our study confirms that a short peptide, though with the presence of an Aib residue in the middle of the chain, does not have to fold to an α‐helical secondary structure. To generate a helical conformation for a linear peptide, several Aib residues should be present in the peptide, either sequentially or alternatively, to enhance the propensity of Aib‐containing peptides towards the helical conformation. A correction of a few of the published NMR data is reported.     

Differentiation of Boc‐protected α,δ‐/δ,α‐ and β,δ‐/δ,β‐hybrid peptide positional isomers by electrospray ionization tandem mass spectrometry

Two new series of Boc‐N‐α,δ‐/δ,α‐ and β,δ‐/δ,β‐hybrid peptides containing repeats of L ‐Ala‐δ⁵‐Caa/δ⁵‐Caa‐L ‐Ala and β³‐Caa‐δ⁵‐Caa/δ⁵‐Caa‐β³‐Caa (L ‐Ala = L ‐alanine, Caa = C‐linked carbo amino acid derived from D ‐xylose) have been differentiated by both positive and negative ion electrospray ionization (ESI) ion trap tandem mass spectrometry (MS/MS). MSⁿ spectra of protonated isomeric peptides produce characteristic fragmentation involving the peptide backbone, the Boc‐group, and the side chain. The dipeptide positional isomers are differentiated by the collision‐induced dissociation (CID) of the protonated peptides. The loss of 2‐methylprop‐1‐ene is more pronounced for Boc‐NH‐L ‐Ala‐δ‐Caa‐OCH₃ (1), whereas it is totally absent for its positional isomer Boc‐NH‐δ‐Caa‐L ‐Ala‐OCH₃ (7), instead it shows significant loss of t‐butanol. On the other hand, second isomeric pair shows significant loss of t‐butanol and loss of acetone for Boc‐NH‐δ‐Caa‐β‐Caa‐OCH₃ (18), whereas these are insignificant for its positional isomer Boc‐NH‐β‐Caa‐δ‐Caa‐OCH₃ (13). The tetra‐ and hexapeptide positional isomers also show significant differences in MS² and MS³ CID spectra. It is observed that ‘b’ ions are abundant when oxazolone structures are formed through five‐membered cyclic transition state and cyclization process for larger ‘b’ ions led to its insignificant abundance. However, b₁⁺ ion is formed in case of δ,α‐dipeptide that may have a six‐membered substituted piperidone ion structure. Furthermore, ESI negative ion MS/MS has also been found to be useful for differentiating these isomeric peptide acids. Thus, the results of MS/MS of pairs of di‐, tetra‐, and hexapeptide positional isomers provide peptide sequencing information and distinguish the positional isomers. Copyright © 2010 John Wiley & Sons, Ltd.     

Identification of (6R)‐5,6,7,8‐Tetrahydro‐D‐monapterin (=(6R)‐2‐Amino‐5,6,7,8‐tetrahydro‐6‐[(1R,2R)‐1,2,3 ‐trihydroxypropyl]pteridin‐4(3H)‐one) as the Native Pteridine in Tetrahymena pyriformis

The structure of the native pteridine in Tetrahymena pyriformis was determined as (6R)‐5,6,7,8‐tetrahydro‐D ‐monapterin (=(6R)‐2‐amino‐5,6,7,8‐tetrahydro‐6‐[(1R,2R)‐1,2,3‐trihydroxypropyl]pteridin‐4(3H)‐one; 4 ). First, the configuration of the 1,2,3‐trihydroxypropyl side chain was confirmed as D ‐threo by the fluorescence‐detected circular dichroism (FDCD) spectrum of its aromatic pterin derivative 2 obtained by I₂ oxidation (Fig. 1). The configuration at the 6‐position of 4 was determined as (R) by comparison of its hexaacetyl derivative 6 with authentic (6R)‐ and (6S)‐hexaacetyl‐5,6,7,8‐tetrahydro‐D ‐monapterins 6 and 7 , respectively, in the HPLC, LC/MS, and LC‐MS/MS (Figs. 3 – 6). (6R)‐5,6,7,8‐Tetrahydro‐D ‐monapterin ( 4 ) is a newly discovered natural tetrahydropterin.     

Tripeptides of the Type H‐D‐Pro‐Pro‐Xaa‐NH2 as Catalysts for Asymmetric 1,4‐Addition Reactions: Structural Requirements for High Catalytic Efficiency

Analysis of the structural and functional requirements within the asymmetric peptidic catalyst H‐D ‐Pro‐Pro‐Asp‐NH₂ led to the development of the closely related peptide H‐D ‐Pro‐Pro‐Glu‐NH₂ as an even more efficient catalyst for asymmetric conjugate addition reactions of aldehydes to nitroolefins. In the presence of as little as 1 mol % of H‐D ‐Pro‐Pro‐Glu‐NH₂, a broad range of aldehydes and nitroolefins react readily with each other. The resulting γ‐nitroaldehydes were obtained in excellent yields and stereoselectivities at room temperature. Within the structure of the peptidic catalysts, the D ‐Pro‐Pro motif is the major contributor to the high stereoselectivities. The C‐terminal amide and the spacer to the carboxylic acid in the side‐chain of the C‐terminal amino acid are responsible for the fine‐tuning of the stereoselectivity. The peptidic catalysts not only allow for highly effective asymmetric catalysis under mild conditions, but also function in the absence of additives.     

Preparation of N‐Fmoc‐Protected (S)‐5‐Amino‐4,4‐difluoro‐7‐methyloctanoic Acid,a Possible Dipeptide Isostere

The title compound 1 was prepared from L ‐leucine. The key steps include a Grignard addition to Bn₂‐leucinal, a CO/CF₂ replacement with Et₂NSF₃ (DAST) and use of a Ph group as synthetic equivalent of a COOH group. The difluoro‐δ‐amino acid 1 was incorporated into a peptide 8 ; tests with various proteases showed no inhibition by this particular peptide.     

Synthesis of poly(lactide‐ran‐MOHEL) and its biodegradation with proteinase K

Homopoly(L ‐lactide) and homopoly(D,L ‐lactide) were almost inert for biodegradation with tricine buffer or normal enzymes such as bromelain, pronase, and cholesterol esterase but biodegradable with proteinase K. Significantly enhanced biodegradation was observed when an optically active (R)‐ or (S)‐3‐methyl‐4‐oxa‐6‐hexanolide (MOHEL) unit was introduced into poly(L ‐lactide) [poly(L ‐LA)] or poly(D,L ‐lactide) [poly(D,L ‐LA)] sequences. Poly[L ‐LA‐ran‐(R)‐MOHEL] in molar ratios of 86/14 to 43/57 showed good biodegradability that was independent of crystallinity. The biodegradation of polymers with proteinase K increased in the following order: poly[D,L ‐LA‐ran‐(R)‐MOHEL] > poly[L ‐LA‐ran‐(R)‐MOHEL] > poly[D,L ‐LA‐ran‐(S)‐MOHEL] > poly[L ‐LA‐ran‐(S)‐MOHEL] > poly(R)‐MOHEL > poly(D,L ‐LA). The number‐average molecular weight, molecular weight distribution, glass‐transition temperature, and melting temperature did not change before and after the biodegradation of poly[L ‐LA‐ran‐(R)‐MOHEL], indicating that the degradation occurred from the polymer surface. © 2001 John Wiley & Sons, Inc. J Polym Sci A: Polym Chem 39: 1374–1381, 2001     

Cucurbit[7]uril: A High‐Affinity Host for Encapsulation of Amino Saccharides and Supramolecular Stabilization of Their α‐Anomers in Water

Cucurbit[7]uril (CB[7]), an uncharged and water‐soluble macrocyclic host, binds protonated amino saccharides (D ‐glucosamine, D ‐galactosamine, D ‐mannosamine and 6‐amino‐6‐deoxy‐D ‐glucose) with excellent affinity (K_a=10³ to 10⁴ M ^?1). The host–guest complexation was confirmed by NMR spectroscopy, isothermal titration calorimetry (ITC), and MALDI‐TOF mass spectral analyses. NMR analyses revealed that the amino saccharides, except D ‐mannosamine, are bound as α‐anomers within the CB[7] cavity. ITC analyses reveal that CB[7] has excellent affinity for binding amino saccharides in water. The maximum affinity was observed for D ‐galactosamine hydrochloride (K_a=1.6×10⁴ M ^?1). Such a strong affinity for any saccharide in water using a synthetic receptor is unprecedented, as is the supramolecular stabilization of an α‐anomer by the host.     

Synthesis of star‐shaped poly(D,L‐lactic acid‐alt‐glycolic acid)‐b‐poly(L‐lactic acid) with the poly(D,L‐lactic acid‐alt‐glycolic acid) macroinitiator and stannous octoate catalyst

Two types of three‐arm and four‐arm, star‐shaped poly(D,L ‐lactic acid‐alt‐glycolic acid)‐b‐poly(L ‐lactic acid) (D,L ‐PLGA50‐b‐PLLA) were successfully synthesized via the sequential ring‐opening polymerization of D,L ‐3‐methylglycolide (MG) and L ‐lactide (L ‐LA) with a multifunctional initiator, such as trimethylolpropane and pentaerythritol, and stannous octoate (SnOct₂) as a catalyst. Star‐shaped, hydroxy‐terminated poly(D,L ‐lactic acid‐alt‐glycolic acid) (D,L ‐PLGA50) obtained from the polymerization of MG was used as a macroinitiator to initiate the block polymerization of L ‐LA with the SnOct₂ catalyst in bulk at 130 °C. For the polymerization of L ‐LA with the three‐arm, star‐shaped D,L ‐PLGA50 macroinitiator (number‐average molecular weight = 6800) and the SnOct₂ catalyst, the molecular weight of the resulting D,L ‐PLGA50‐b‐PLLA polymer linearly increased from 12,600 to 27,400 with the increasing molar ratio (1:1 to 3:1) of L ‐LA to MG, and the molecular weight distribution was rather narrow (weight‐average molecular weight/number‐average molecular weight = 1.09–1.15). The ¹H NMR spectrum of the D,L ‐PLGA50‐b‐PLLA block copolymer showed that the molecular weight and unit composition of the block copolymer were controlled by the molar ratio of L ‐LA to the macroinitiator. The ¹³C NMR spectrum of the block copolymer clearly showed its diblock structures, that is, D,L ‐PLGA50 as the first block and poly(L ‐lactic acid) as the second block. © 2001 John Wiley & Sons, Inc. J Polym Sci Part A: Polym Chem 40: 409–415, 2002