Restricted dead‐end elimination: Protein redesign with a bounded number of residue mutations

Dead‐end elimination (DEE) has emerged as a powerful structure‐based, conformational search technique enabling computational protein redesign. Given a protein with n mutable residues, the DEE criteria guide the search toward identifying the sequence of amino acids with the global minimum energy conformation (GMEC). This approach does not restrict the number of permitted mutations and allows the identified GMEC to differ from the original sequence in up to n residues. In practice, redesigns containing a large number of mutations are often problematic when taken into the wet‐lab for creation via site‐directed mutagenesis. The large number of point mutations required for the redesigns makes the process difficult, and increases the risk of major unpredicted and undesirable conformational changes. Preselecting a limited subset of mutable residues is not a satisfactory solution because it is unclear how to select this set before the search has been performed. Therefore, the ideal approach is what we define as the κ‐restricted redesign problem in which any κ of the n residues are allowed to mutate. We introduce restricted dead‐end elimination (rDEE) as a solution of choice to efficiently identify the GMEC of the restricted redesign (the κGMEC). Whereas existing approaches require n‐choose‐κ individual runs to identify the κGMEC, the rDEE criteria can perform the redesign in a single search. We derive a number of extensions to rDEE and present a restricted form of the A* conformation search. We also demonstrate a 10‐fold speed‐up of rDEE over traditional DEE approaches on three different experimental systems. © 2009 Wiley Periodicals, Inc. J Comput Chem, 2010     

The minimized dead-end elimination criterion and its application to protein redesign in a hybrid scoring and search algorithm for computing partition functions over molecular ensembles

One of the main challenges for protein redesign is the efficient evaluation of a combinatorial number of candidate structures. The modeling of protein flexibility, typically by using a rotamer library of commonly-observed low-energy side-chain conformations, further increases the complexity of the redesign problem. A dominant algorithm for protein redesign is dead-end elimination (DEE), which prunes the majority of candidate conformations by eliminating rigid rotamers that provably are not part of the global minimum energy conformation (GMEC). The identified GMEC consists of rigid rotamers (i.e., rotamers that have not been energy-minimized) and is thus referred to as the rigid-GMEC. As a postprocessing step, the conformations that survive DEE may be energy-minimized. When energy minimization is performed after pruning with DEE, the combined protein design process becomes heuristic, and is no longer provably accurate: a conformation that is pruned using rigid-rotamer energies may subsequently minimize to a lower energy than the rigid-GMEC. That is, the rigid-GMEC and the conformation with the lowest energy among all energy-minimized conformations (the minimized-GMEC) are likely to be different. While the traditional DEE algorithm succeeds in not pruning rotamers that are part of the rigid-GMEC, it makes no guarantees regarding the identification of the minimized-GMEC. In this paper we derive a novel, provable, and efficient DEE-like algorithm, called minimized-DEE (MinDEE), that guarantees that rotamers belonging to the minimized-GMEC will not be pruned, while still pruning a combinatorial number of conformations. We show that MinDEE is useful not only in identifying the minimized-GMEC, but also as a filter in an ensemble-based scoring and search algorithm for protein redesign that exploits energy-minimized conformations. We compare our results both to our previous computational predictions of protein designs and to biological activity assays of predicted protein mutants. Our provable and efficient minimized-DEE algorithm is applicable in protein redesign, protein-ligand binding prediction, and computer-aided drug design.     

Preprocessing of rotamers for protein design calculations

We have developed a process that significantly reduces the number of rotamers in computational protein design calculations. This process, which we call Vegas, results in dramatic computational performance increases when used with algorithms based on the dead-end elimination (DEE) theorem. Vegas estimates the energy of each rotamer at each position by fixing each rotamer in turn and utilizing various search algorithms to optimize the remaining positions. Algorithms used for this context specific optimization can include Monte Carlo, self-consistent mean field, and the evaluation of an expression that generates a lower bound energy for the fixed rotamer. Rotamers with energies above a user-defined cutoff value are eliminated. We found that using Vegas to preprocess rotamers significantly reduced the calculation time of subsequent DEE-based algorithms while retaining the global minimum energy conformation. For a full boundary design of a 51 amino acid fragment of engrailed homeodomain, the total calculation time was reduced by 12-fold.     

Dead-end elimination for multistate protein design

Multistate protein design is the task of predicting the amino acid sequence that is best suited to selectively and stably fold to one state out of a set of competing structures. Computationally, it entails solving a challenging optimization problem. Therefore, notwithstanding the increased interest in multistate design, the only implementations reported are based on either genetic algorithms or Monte Carlo methods. The dead-end elimination (DEE) theorem cannot be readily transfered to multistate design problems despite its successful application to single-state protein design. In this article we propose a variant of the standard DEE, called type-dependent DEE. Our method reduces the size of the conformational space of the multistate design problem, while provably preserving the minimal energy conformational assignment for any choice of amino acid sequence. Type-dependent DEE can therefore be used as a preprocessing step in any computational multistate design scheme. We demonstrate the applicability of type-dependent DEE on a set of multistate design problems and discuss its strength and limitations.     

Comparing three stochastic search algorithms for computational protein design: Monte Carlo,replica exchange Monte Carlo,and a multistart,steepest‐descent heuristic

Computational protein design depends on an energy function and an algorithm to search the sequence/conformation space. We compare three stochastic search algorithms: a heuristic, Monte Carlo (MC), and a Replica Exchange Monte Carlo method (REMC). The heuristic performs a steepest‐descent minimization starting from thousands of random starting points. The methods are applied to nine test proteins from three structural families, with a fixed backbone structure, a molecular mechanics energy function, and with 1, 5, 10, 20, 30, or all amino acids allowed to mutate. Results are compared to an exact, “Cost Function Network” method that identifies the global minimum energy conformation (GMEC) in favorable cases. The designed sequences accurately reproduce experimental sequences in the hydrophobic core. The heuristic and REMC agree closely and reproduce the GMEC when it is known, with a few exceptions. Plain MC performs well for most cases, occasionally departing from the GMEC by 3–4 kcal/mol. With REMC, the diversity of the sequences sampled agrees with exact enumeration where the latter is possible: up to 2 kcal/mol above the GMEC. Beyond, room temperature replicas sample sequences up to 10 kcal/mol above the GMEC, providing thermal averages and a solution to the inverse protein folding problem. © 2016 Wiley Periodicals, Inc.     

Fast search algorithms for computational protein design

One of the main challenges in computational protein design (CPD) is the huge size of the protein sequence and conformational space that has to be computationally explored. Recently, we showed that state‐of‐the‐art combinatorial optimization technologies based on Cost Function Network (CFN) processing allow speeding up provable rigid backbone protein design methods by several orders of magnitudes. Building up on this, we improved and injected CFN technology into the well‐established CPD package Osprey to allow all Osprey CPD algorithms to benefit from associated speedups. Because Osprey fundamentally relies on the ability of to produce conformations in increasing order of energy, we defined new strategies combining CFN lower bounds, with new side‐chain positioning‐based branching scheme. Beyond the speedups obtained in the new ‐CFN combination, this novel branching scheme enables a much faster enumeration of suboptimal sequences, far beyond what is reachable without it. Together with the immediate and important speedups provided by CFN technology, these developments directly benefit to all the algorithms that previously relied on the DEE/ combination inside Osprey* and make it possible to solve larger CPD problems with provable algorithms. © 2016 Wiley Periodicals, Inc.     

Protein:Ligand binding free energies: A stringent test for computational protein design

A computational protein design method is extended to allow Monte Carlo simulations where two ligands are titrated into a protein binding pocket, yielding binding free energy differences. These provide a stringent test of the physical model, including the energy surface and sidechain rotamer definition. As a test, we consider tyrosyl‐tRNA synthetase (TyrRS), which has been extensively redesigned experimentally. We consider its specificity for its substrate l ‐tyrosine (l ‐Tyr), compared to the analogs d ‐Tyr, p‐acetyl‐, and p‐azido‐phenylalanine (ac‐Phe, az‐Phe). We simulate l ‐ and d ‐Tyr binding to TyrRS and six mutants, and compare the structures and binding free energies to a more rigorous “MD/GBSA” procedure: molecular dynamics with explicit solvent for structures and a Generalized Born + Surface Area model for binding free energies. Next, we consider l ‐Tyr, ac‐ and az‐Phe binding to six other TyrRS variants. The titration results are sensitive to the precise rotamer definition, which involves a short energy minimization for each sidechain pair to help relax bad contacts induced by the discrete rotamer set. However, when designed mutant structures are rescored with a standard GBSA energy model, results agree well with the more rigorous MD/GBSA. As a third test, we redesign three amino acid positions in the substrate coordination sphere, with either l ‐Tyr or d ‐Tyr as the ligand. For two, we obtain good agreement with experiment, recovering the wildtype residue when l ‐Tyr is the ligand and a d ‐Tyr specific mutant when d ‐Tyr is the ligand. For the third, we recover His with either ligand, instead of wildtype Gln. © 2015 Wiley Periodicals, Inc.     

Simultaneous separation of three isomeric sennosides from senna leaf (Cassia acutifolia) using counter‐current chromatography

Senna leaf is widely consumed as tea to treat constipation or to aid in weight loss. Sennoside A, A₁, and B are dirheinanthrone glucosides that are abundant and the bioactive constituents in the plant. They are isomers that refer to the (R*R*), (S*S*), and (R*S*) forms of protons on C‐10 and C‐10′ centers and it is difficult to refine them individually due to their structural similarities. The new separation method using counter‐current chromatography successfully purified sennoside A, A₁, and B from senna leaf (Cassia acutifolia) while reversed‐phase medium‐pressure liquid chromatography yielded sennoside A only. n‐Butanol/isopropanol/water (5:1:6, v/v/v) was selected as the solvent system for counter‐current chromatography operation, and the partition coefficients were carefully determined by adding different concentrations of formic acid. High‐resolution mass spectrometry and NMR spectroscopy were performed to verify the chemical properties of the compounds.     

Oligonucleotide Analogues with Integrated Bases and Backbone. Part 17

The formation of cyclic duplexes (pairing) of known oxymethylene‐linked self‐complementary U*[o]A⁽*⁾ dinucleosides contrasts with the absence of pairing of the ethylene‐linked U*[c_a]A⁽*⁾ analogues. The origin of this difference, and the expected association of U*[x]A⁽*⁾ and A*[x]U⁽*⁾ dinucleosides with x=CH₂, O, or S was analysed. According to this analysis, pairing occurs via constitutionally isomeric Watson–Crick, reverse Watson–Crick, Hoogsteen, or reverse Hoogsteen H‐bonded linear duplexes. Each one of them may give rise to three diastereoisomeric cyclic duplexes, and each one of them can adopt three main conformations. The relative stability of all conformers with x=CH₂, O, or S were analysed. U*[x]A⁽*⁾ dinucleosides with x=CH₂ do not form stable cyclic duplexes, dinucleosides with x=O may form cyclic duplexes with a gg‐conformation about the C(4′)? C(5′) bond, and dinucleosides with x=S may form cyclic duplexes with a gt‐conformation about this bond. The temperature dependence of the chemical shift of H? N(3) of the self‐complementary, oxymethylene‐linked U*[o]A⁽*⁾ dinucleosides 1 – 6 in CDCl₃ in the concentration range of 0.4–50 mM evidences equilibria between the monoplex, mainly linear duplexes, and higher associates for 3 , between the monoplex and cyclic duplexes for 6 , and between the monoplex, linear, and cyclic duplexes as well as higher associates for 1, 2, 4 , and 5 . The self‐complementary, thiomethylene‐linked U*[s]A⁽*⁾ dinucleosides 27 – 32 and the sequence isomeric A*[s]U⁽*⁾ analogues 33 – 38 were prepared by S‐alkylation of the 6‐(mesyloxymethyl)uridine 12 and the 8‐(bromomethyl)adenosine 22 . The required thiolates were prepared in situ from the C(5′)‐acetylthio derivatives 9, 15, 19 , and 25 . The association in CHCl₃ of the thiomethylene‐linked dinucleoside analogues was studied by ¹H‐NMR and CD spectroscopy, and by vapour‐pressure osmometric determination of the apparent molecular mass. The U*[s]A⁽*⁾ alcohols 28, 30 , and 31 form cyclic duplexes connected by Watson–Crick H‐bonds, while the fully protected dimers 27 and 29 form mainly linear duplexes and higher associates. The diol 32 forms mainly cyclic duplexes in solution and corrugated ribbons in the solid state. The nucleobases of crystalline 32 form reverse Hoogsteen H‐bonds, and the resulting ribbons are cross‐linked by H‐bonds between HOCH₂? C(8/I) and N(3/I). Among the A*[s]U⁽*⁾ dimers, only the C(8/I)‐hydroxymethylated 37 forms (mainly) a cyclic duplex, characterized by reverse Hoogsteen base pairing. The dimers 34 – 36 form mainly linear duplexes and higher associates. Dimers 34 and particularly 38 gelate CHCl₃. Temperature‐dependent CD spectra of 28, 30, 31 , and 37 evidence π‐stacking in the cyclic duplexes. Base stacking in the particularly strongly associating diol 32 in CHCl₃ solution is evidenced by a melting temperature of ca. 2°.     

W2466.48 Opens a Gate for a Continuous Intrinsic Water Pathway during Activation of the Adenosine A2A Receptor

The question how G‐protein‐coupled receptors transduce an extracellular signal by a sequence of transmembrane conformational transitions into an intracellular response remains to be solved at molecular detail. Herein, we use molecular dynamics simulations to reveal distinct conformational transitions of the adenosine A_2A receptor, and we found that the conserved W246^6.48 residue in transmembrane helix TM6 performs a key rotamer toggle switch. Agonist binding induces the sidechain of W246^6.48 to fluctuate between two distinct conformations enabling the diffusion of water molecules from the bulk into the center of the receptor. After passing the W246^6.48 gate, the internal water molecules induce another conserved residue, Y288^7.53, to switch to a distinct rotamer conformation establishing a continuous transmembrane water pathway. Further, structural changes of TM6 and TM7 induce local structural changes of the adjacent lipid bilayer.     

Oligonucleotide Analogues with Integrated Bases and Backbone. Part 15

The self‐complementary (Z)‐configured U*[c_e]A⁽*⁾ dinucleotide analogues 6, 8, 10, 12, 14 , and 16 , and the A*[c_e]U⁽*⁾ dimers 19, 21, 23, 25, 27 , and 29 were prepared by partial hydrogenation of the corresponding ethynylene linked dimers. Photolysis of 14 led to the (E)‐alkene 17 . These dinucleotide analogues associate in CDCl₃ solution, as evidenced by NMR and CD spectroscopy. The thermodynamic parameters of the duplexation were determined by van't Hoff analysis. The (Z)‐configured U*[c_e]A⁽*⁾ dimers 14 and 16 form cyclic duplexes connected by Watson–Crick H‐bonds, the (E)‐configured U*[c_e]A dimer 17 forms linear duplexes, and the U*[c_e]A⁽*⁾ allyl alcohols 6, 8, 10 , and 12 form mixtures of linear and cyclic duplexes. The C(6/I)‐unsubstituted A*[c_e]U allyl alcohols 19 and 23 form linear duplexes, whereas the C(6/I)‐substituted A*[c_e]U* allyl alcohols 21 and 25 , and the C(5′/I)‐deoxy A*[c_e]U⁽*⁾ dimers 27 and 29 also form minor amounts of cyclic duplexes. The influence of intra‐ and intermolecular H‐bonding of the allyl alcohols and the influence of the base sequence upon the formation of cyclic duplexes are discussed.     

Oligonucleotide Analogues with Integrated Bases and Backbone

The thiomethylene‐linked U*[s]U⁽*⁾ dimers 9 – 14 were synthesized by substitution of the 6‐[(mesyloxy)methyl]uridine 6 by the thiolate derived from the uridine‐5′‐thioacetates 7 and 8 followed by O‐deprotection. Similarly, the thiomethylene‐linked A*[s]A⁽*⁾ dimers 9 – 14 were obtained from the 8‐(bromomethyl)adenosine 15 and the adenosine‐5′‐thioacetates 16 and 17 . The concentration dependence of both H? N(3) of the U*[s]U⁽*⁾ dimers 9 – 14 evidences the formation of linear and cyclic duplexes, and of linear higher associates, C(8 or 6)CH₂OH and/or C(5′/II)OH groups favouring the formation of cyclic duplexes. The concentration dependence of the chemical shift for both H₂N? C(6) of the A*[s]A⁽*⁾ dimers 18 – 23 evidences the formation of mainly linear associates. The heteroassociation of U*[s]U⁽*⁾ to A*[s]A⁽*⁾ dimers is stronger than the homoassociation of U*[s]U⁽*⁾ dimers, as evidenced by diluting equimolar mixtures of 11 / 20 and 13 / 22 . A 1 : 1 stoichiometry of the heteroassociation is evidenced by a Job's plot for 11 / 20 , and by mole ratio plots for 9 / 18, 10 / 19, 12 / 21, 13 / 22 , and 14 / 23 .     

Control of the Electronic Ground State on an Electron‐Transfer Copper Site by Second‐Sphere Perturbations

The Cu_A center is a dinuclear copper site that serves as an optimized hub for long‐range electron transfer in heme–copper terminal oxidases. Its electronic structure can be described in terms of a σ_u* ground‐state wavefunction with an alternative, less populated ground state of π_u symmetry, which is thermally accessible. It is now shown that second‐sphere mutations in the Cu_A containing subunit of Thermus thermophilus ba₃ oxidase perturb the electronic structure, which leads to a substantial increase in the population of the π_u state, as shown by different spectroscopic methods. This perturbation does not affect the redox potential of the metal site, and despite an increase in the reorganization energy, it is not detrimental to the electron‐transfer kinetics. The mutations were achieved by replacing the loops that are involved in protein–protein interactions with cytochrome c, suggesting that transient protein binding could also elicit ground‐state switching in the oxidase, which enables alternative electron‐transfer pathways.     

Exact rotamer optimization for protein design

Computational methods play a central role in the rational design of novel proteins. The present work describes a new hybrid exact rotamer optimization (HERO) method that builds on previous dead-end elimination algorithms to yield dramatic performance enhancements. Measured on experimentally validated physical models, these improvements make it possible to perform previously intractable designs of entire protein core, surface, or boundary regions. Computational demonstrations include a full core design of the variable domains of the light and heavy chains of catalytic antibody 48G7 FAB with 74 residues and 10(128) conformations, a full core/boundary design of the beta1 domain of protein G with 25 residues and 10(53) conformations, and a full surface design of the beta1 domain of protein G with 27 residues and 10(60) conformations. In addition, a full sequence design of the beta1 domain of protein G is used to demonstrate the strong dependence of algorithm performance on the exact form of the potential function and the fidelity of the rotamer library. These results emphasize that search algorithm performance for protein design can only be meaningfully evaluated on physical models that have been subjected to experimental scrutiny. The new algorithm greatly facilitates ongoing efforts to engineer increasingly complex protein features.     

Oligonucleotide Analogues with Integrated Bases and Backbone. Part 16

The self‐complementary, ethylene‐linked U*[c_a]A⁽*⁾ dinucleotide analogues 8, 10, 12, 14, 16 , and 18 , and the sequence‐isomeric A*[c_a]U⁽*⁾ analogues 20, 22, 24, 26, 28 , and 30 were obtained by Pd/C‐catalyzed hydrogenation of the corresponding, known ethynylene‐linked dimers. The association of the ethylene‐linked dimers was investigated by NMR and CD spectroscopy. The U*[c_a]A⁽*⁾ dimers form linear duplexes and higher associates (K between 29 and 114M ^?1). The A*[c_a]U⁽*⁾ dimers, while associating more strongly (K between 88 and 345M ^?1), lead mostly to linear duplexes and higher associates; they form only minor amounts of cyclic duplexes. The enthalpy–entropy compensation characterizing the association of the U*[c_x]A⁽*⁾ and A*[c_x]U⁽*⁾ dimers (x=y, e, and a) is discussed.     

1,4‐Dilithio‐1,1,4,4‐tetraphenylbutane in diethyl ether: An effective initiator for the living anionic polymerization of dienes

The anionic polymerization of butadiene initiated with 1,4‐dilithio‐1,1,4,4‐tetraphenylbutane (LiTPB) in diethyl ether (DEE) gives polybutadiene (PBD) with high 1,2 content (>70%), narrow polydispersities (1.04 < M_w/M_n < 1.20), and predicted molecular weights. In THF, this polymerization does not work very well. After removal of DEE and addition of THF, the PBD dianion is end capped quantitatively by addition of 1,1‐diphenylethylene (DPE) to give the diphenylalkyl end capped PBD dianion. Subsequent addition of methyl methacrylate at low temperatures results in the formation of well‐defined PMMA‐b‐PBD‐b‐PMMA triblock copolymers. The results are accounted for by taking into account the effects of Li ion solvation on the BD initiation and end capping of the PBD anion by DPE. © 2009 Wiley Periodicals, Inc. J Polym Sci Part A: Polym Chem 47: 2198–2206, 2009     

Anomalous behaviour in the SmA*–SmCA* pre‐transitional regime of a chiral swallow‐tailed antiferroelectric liquid crystal

The temperature‐ and electric field‐dependent dielectric relaxation and polarisation of a new chiral swallow tailed antiferroelectric liquid crystal, i.e. 1‐ethylpropyl (S)‐2‐{6‐[4‐(4′‐decyloxyphenyl)benzoyloxy]‐2‐naphthyl}propionate (abbreviated as EP10PBNP), were investigated. The electric field‐induced dielectric loss spectra of EP10PBNP revealed electroclinic and anomalous dielectric behaviour in the chiral smectic A (SmA*)–chiral antiferroelectric smectic C (SmC_A*) pre‐transitional regime. From an analysis of thermal hysteresis of the dielectric constant, electric field‐induced polarisation and dielectric loss spectra, the appearance of a ferrielectric‐like mesophase is observed followed by an unstable SmC_A* phase in the SmA*–SmC_A* pre‐transitional regime.     

Blue light‐emitting hyperbranched polymers using fluorene‐co‐dibenzothiophene‐S,S‐dioxide as branches

A series of blue light‐emitting hyperbranched polymers comprising poly(fluorene‐co‐dibenzothiophene‐S,S‐dioxide) as the branch and benzene, triphenylamine, or triphenyltriazine as the core were synthesized by an “A₂ + A₂' + B₃” approach of Suzuki polymerization, respectively. All resulted copolymers exhibited quite comparable thermal properties with the glass transition temperatures in the range of 59–68 °C and relatively high decomposition temperatures over 420 °C. Photoluminescent spectra exhibited slight variation with the molar ratio of the dibenzothiophene‐S,S‐dioxide unit and the size of the core units. Polymer light‐emitting devices demonstrated blue emission with excellent stability of electroluminescence. Copolymers based on smaller core units of benzene and triphenylamine exhibited enhanced device performances regarding to that of triphenyltriazine. With the device configuration of ITO/PEDOT:PSS/polymer/CsF/Al, a maximum luminous efficiency of 4.5 cd A^?1 was obtained with Commission Internationale de L'.Eclairage (CIE) coordinates of (0.16, 0.19) for the copolymer PFSO15B. These results indicated that hyperbranched structure can be a promising strategy to attain spectrally stable blue‐light‐emitting polymers with high efficiency. © 2015 Wiley Periodicals, Inc. J. Polym. Sci., Part A: Polym. Chem. 2015 , 53, 1043–1051     

Conformational splitting: A more powerful criterion for dead‐end elimination

Dead‐end elimination (DEE) is a powerful theorem for selecting optimal protein side‐chain orientations from a large set of discrete conformations. The present work describes a new approach to dead‐end elimination that effectively splits conformational space into partitions to more efficiently eliminate dead‐ending rotamers. Split DEE makes it possible to complete protein design calculations that were previously intractable due to the combinatorial explosion of intermediate conformations generated during the convergence process. © 2000 John Wiley & Sons, Inc. J Comput Chem 21: 999–1009, 2000     

Synthesis and Characterization of Hexadecaaniline‐Grafted Comb‐like Poly(maleic acid‐alt‐1‐octadecene)

Highly soluble hexadecaaniline (A₁₆)‐grafted polyolefin derivatives poly(maleic acid‐hexadecaanilinamide‐alt‐1‐octadecene) (PMAO‐A₁₆) in a comb‐like configuration with alternate linear hexadecane and A₁₆ side‐chains were synthesized and characterized. The structure of PMAO‐A₁₆ was substantiated by infrared and UV‐Vis spectra showing high intensity of characteristic absorption peaks corresponding to a high degree of A₁₆ attachments. Covalent grafting of hexadecaanilines onto the polymer backbone of PMAO was confirmed by the detection of a new amide [–(C[dbnd]O)–NH–] absorption band appearing at 1661 cm^?1 accompanied with the full disappearance of anhydride carbonyl absorptions. Based on the comparison between TGA profiles of PMAO‐A₁₆ and hexadecaaniline, a 12.5% wt loss at 365–600°C was accounted for full elimination of aliphatic side‐chains that matches approximately with the weight percentage of total hexadecane arms (12.7%). The data revealed a nearly quantitative yield of A₁₆ grafting on anhydride subunits leading to complete conversion of PMAO into PMAO‐A₁₆. Furthermore, preliminary ¹H‐NMR study of PMAO‐A₁₆ indicated its capability to undergo molecular self‐assembly in DMSO where A₁₆s were dispersed in the solvent phase with hexadecane side‐chains located in a phase‐separated domain.