Most early reports on brush and branched polypeptides utilized conventional NCA polymerization techniques. The first graft polypeptides were published in 1956 by Sela et al.⁶¹ Using poly(l-lysine) or poly(d/l-ornithine) as a multifunctional initiator for the ROP of a number of different NCAs, these authors prepared a variety of graft polypeptides, which they termed multichain poly(amino acids) (eqn [18]). Since the NCA polymerizations were carried out in aqueous dioxane, the synthesis of the graft polypeptides was accompanied by the formation of short linear polypeptides. These by-products, however, could be removed by dialysis. For the graft polymerization, multifunctional poly(l-lysine) or poly(d/l-ornithine) initiators with degrees of polymerization between 20 and 200 were used. The number of amino acid residues per graft was determined by chromatographic analysis of the hydrolyzed product or by end-group titrations. Depending on the length of the poly(l-lysine) or poly(d/l-ornithine) initiator, the number of amino acids per graft varied from 3 to 25. In a subsequent paper, Sela et al.⁶² used this procedure to prepare a family of multichain copolymers with grafts containing l-tyrosine, l-glutamic acid, and l-alanine residues. The interest in these polymers was due to their potential application as synthetic polypeptide antigens. In this case, in addition to poly(l-lysine) homopolymer, copolymers of d/l-alanine and l-lysine were also used as multifunctional initiators for the NCA graft copolymerization. Furthermore, the grafts of the multichain copolymers were not only simple homopolypeptides, but, in most cases, block-type sequences.

[18]

More complex, higher branched polypeptide architectures can be obtained when functional groups in the side chains of the polypeptide grafts are used to initiate a subsequent NCA ROP step. Repetition of this graft-on-graft strategy leads to highly branched, so-called dendrigraft, polypeptides.⁶⁸ This is illustrated in Scheme 3, which shows the synthesis of dendrigraft polylysine by a repetitive sequence of ROP and deprotection steps using two orthogonally protected l-lysine NCA derivatives. Following this strategy, dendrigraft polylysines containing up to ∼ 160 amino acids, corresponding to a number-average molecular weight of ∼ 40 kDa, could be prepared in just four ring-opening copolymerization–deprotection cycles.⁶⁸

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Scheme 3. Preparation of dendrigraft polylysine in a stepwise process.

Another approach to prepare polylysine dendrigrafts was reported by Tsogas et al.⁶⁹ Here, TFA-Lys NCA was oligomerized in aqueous buffer, followed by removal of the TFA groups at pH 11 to give an oligolysine macroinitiator. This process was then repeated by adding more TFA-Lys NCA to the macroinitator to give a second-generation branched polymer. This cycle was then repeated to give third-generation dendrigraft polylysine (Scheme 4). Simultaneous NCA polymerization and side-chain deprotection have also been used to prepare hyperbranched copolypeptides. Vlasov et al.⁷⁰ reported the ROP of Z-Lys NCA in the presence of H₂/activated Pd, which removes the side-chain protecting groups during polymerization, allowing simultaneous branching and chain growth (Scheme 5). Here, the side-chain amine groups that are revealed after hydrogenation serve as polymerization initiation sites. Hyperbranched copolypeptides of lysine containing alanine and glutamic acid were also prepared using this process. Although neither of these processes are controlled polymerizations, branched copolymers with properties substantially different from the linear chains could be prepared.

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Scheme 4. Preparation of dendrigraft polylysine using in situ base deprotection.

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Scheme 5. Preparation of dendrigraft polylysine using in situ hydrogenation deprotection.

In addition to the graft-on-graft strategies discussed above, highly branched polypeptides can also be prepared by an iterative sequence of NCA ROP and end-functionalization reactions as illustrated in Scheme 6.⁷¹ Each NCA ROP step is followed by an end-functionalization reaction with an appropriate N^α,N^ε-diprotected lysine derivative. Deprotection of the lysine amine groups doubles the number of end groups that can be used to initiate a subsequent NCA ROP step and leads to branching of the polymer architecture. The strategy outlined in Scheme 6 has been used to prepare highly branched polylysines with number-average molecular weights of up to 33 kDa in only a small number of reaction steps. Birchall and North⁷² have used a related approach to prepare highly branched block copolypeptides. In this case, however, relatively hydrophobic water-insoluble amino acids such as alanine, leucine, and phenylalanine were used, which made it necessary to keep the polymer chains relatively short (∼ 5 to 10 amino acid repeat units) in order to avoid solubility problems.

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Scheme 6. Preparation of chain-end branched polylysine in a stepwise process.