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Logo of nihpaAbout Author manuscriptsSubmit a manuscriptHHS Public Access; Author Manuscript; Accepted for publication in peer reviewed journal;
ACS Macro Lett. Author manuscript; available in PMC 2013 April 17.
Published in final edited form as:
ACS Macro Lett. 2012 April 17; 1(4): 441–444.
doi:  10.1021/mz200165c
PMCID: PMC3555137

Synthesis of water-soluble poly(α-hydroxy acids) from living ring-opening polymerization of O-benzyl-L-serine carboxyanhydrides


O-benzyl-L-serine carboxyanhydrides were synthesized via diazotization of O-benzyl-L-serine with sodium nitrite in aqueous sulfuric acid solution followed by cyclization of the resulting serine-based α-hydroxy acid with phosgene. Degradable, water-soluble poly(α-hydroxy acids) bearing pendant hydroxyl groups were readily prepared under mild conditions via ring-opening polymerization of O-benzyl-L-serine carboxyanhydrides followed by removal of the benzyl group and showed excellent cell compatibility, suggesting their potential being used as novel materials in constructing drug delivery systems and as hydrogel scaffolds for tissue engineering applications.

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Poly(α-hydroxy acids) (PAHAs), such as poly(lactic acid) (PLA), poly(glycolic acid) and poly(lactic-co-glycolic acid), are widely used in controlled release, drug delivery and tissue engineering because of their excellent biocompatibility and biodegradability.1 One drawback of conventional PAHAs is their lack of side-chain functionalities, which has prevented structural alteration via side-chain modifications and thus limited PAHA applications, particularly in situations requiring postmodification of PAHA side chains. Over the past decades, tremendous effort has been devoted to the development of side chain functionalized PAHAs via the introduction of pendant functional groups to modulate the physicochemical and biological properties of PAHAs,2 in particular via the synthesis and polymerization of various substituted 1,4-dioxane-2,5-diones or morpholine-2,5-diones.2o,3,4 However, the approach and the level of difficulty for the synthesis of these monomers vary from case to case. It has been of great interest to develop a general strategy for the synthesis of PAHAs with a variety of pendant functional groups.

Ring-opening polymerization (ROP) of O-carboxyanhydrides (OCAs),5 a class of five-membered ring compounds derived from amino acids, has recently emerged as a viable method to prepare side-chain functionalized PAHA.3b,6 OCAs were found to have excellent polymerization activities; the ROP of lactide-OCA (Lac-OCA, derived from alanine, Scheme 1) in the presence of 4-dimethylaminopyridine (DMAP) finished in hours and afforded PLA with controlled molecular weights in quantitative yields. In conjunction with our efforts of developing PLA-based nanomedicines,7 we have been interested in utilizing ROP of OCAs to synthesize a class of hydrophilic PAHAs for drug delivery applications. Most of the prior work on ROP of OCAs has focused on developing hydrophilic, charged polymers.2r,5c However, when used in vivo, charged, hydrophilic polymers tend to show non-specific tissue interaction and reduced circulation half-lives. Hydrophilic, non-charged polymers are often more useful materials in drug delivery, exemplified by polyethylene glycol8 and cyclodextrin-based polymers.9 In this study, we report the design and synthesis of Ser-PAHA, a class of non-charged, water-soluble PAHA with pendant hydroxy groups, via the living ROP of O-benzyl-L-serine carboxyanhydrides (Ser(Bn)-OCA), a new OCA monomer derived from serine (Scheme 1).

Scheme 1
Synthesis and polymerization of Ser(Bn)-OCA. The molecular structure of Ser(Bn)-OCA obtained by X-ray diffraction is shown in the dotted frame.

The functionalized monomer Ser(Bn)-OCA was prepared via diazotization of O-benzyl-L-serine with sodium nitrite in aqueous sulfuric acid solution followed by cyclization of the resulting serine-based α-hydroxy acid with phosgene (Scheme 1). Recrystallization from diisopropyl ether and dichloromethane afforded grams to tens of grams of colorless crystalline Ser(Bn)-OCA in 50% yield. The ROP of Ser(Bn)-OCA was evaluated with DMAP as the catalyst and isobutanol (IB) as the initiator at various monomer-to-initiator (M/I) ratios in dichloromethane (DCM; Fig. 1a) under conditions similar to those reported by Bourissou and coworkers.5a DMAP/IB showed remarkable control for the ROP of Ser(Bn)-OCA and gave Ser(Bn)-PAHA with the expected molecular weights (MWs) and very narrow molecular weight distributions (MWDs, around 1.05–1.15) over a broad range of M/I ratios (50–800; Fig. 1a). The Mn values of Ser(Bn)-PAHA at M/I ratios of 50 and 800 were 9.4 × 103 g/mol and 1.41 × 105 g/mol (entries 1–2, Table 1), respectively, both of which were in excellent agreement with the calculated Mn values (9.0 × 103 g/mol and 1.42 × 105 g/mol, respectively). Gel permeation chromatography (GPC) analysis of the Ser(Bn)-PAHAs obtained at various M/I ratios showed monomodal distributions (Fig. 1b). Analysis of a plot of Ln([M]0/[M]) versus polymerization time for Ser(Bn)-OCA polymerization mediated by DMAP/IB revealed that the reaction was first order with respect to the Ser(Bn)-OCA monomer concentration (Fig. 1c).

Fig. 1
(a) Plot of Mn (■) and Mw/Mn (▲) of Ser(Bn)-PAHA versus M/I for polymerization with DMAP as the catalyst and IB as the initiator in DCM ([IB]0 = [DMAP]0 = 0.001 M, room temperature). (b) Overlay of GPC curves (obtained with multi-angle ...
Table 1
Polymerization of Ser(Bn)-OCAa

We also investigated the DMAP-catalyzed ROP of Ser(Bn)-OCA with other initiators. DMAP/pyrenemethanol (Pyr) in DCM solution gave similarly well controlled polymerization (entries 3–4, Table 1). The polymerizations mediated by DMAP/Pyr in tetrahydrofuran (THF) and toluene also gave Ser(Bn)-PAHAs with the expected MWs, although the polydispersity indices of the Ser(Bn)-PAHAs obtained in THF were slightly higher than those obtained in toluene (entries 5–8, Table 1). When methoxypoly(ethylene glycol) (mPEG) was used as the initiator, mPEG-Ser(Bn)-PAHA copolymers were obtained with the expected MWs and low polydispersity indexes (entries 9–10, Table 1).

Mn was linearly correlated with monomer conversion throughout the polymerization, suggesting that the ROP of Ser(Bn)-OCA with DMAP as catalyst might have proceeded in a living fashion (Fig. 1d). To further study if this was a living polymerization, we sequentially added Ser(Bn)-OCA monomers during polymerization. The first block of Ser(Bn)-PAHA had a monomodal GPC curve, an Mn of 1.95 × 104 g/mol and an Mw/Mn of 1.15 at a Ser(Bn)-OCA/IB/DMAP ratio of 100:1:1. Subsequent addition of a second portion of 100 equivalents of Ser(Bn)-OCA afforded Ser(Bn)-PAHA with a well-maintained monomodal GPC curve that was shifted toward higher MW compared to the MW observed for the first block; Mn was 3.81 × 104 g/mol, and Mw/Mn was 1.09. We also investigated copolymerization of two different OCA monomers. Block PAHA copolymers, such as Ser(Bn)-PAHA-b-Phe-PAHA and Ser(Bn)-PAHA-b-Lac-PAHA (entries 11–14, Table 1), were readily prepared with predictable MWs and narrow MW distributions by sequential addition of Ser(Bn)-OCA and Phe-OCA or Ser(Bn)-OCA and Lac-OCA to the catalyst and initiator solution (Scheme 1). The ROP of Ser(Bn)-OCA likely proceeded via the similar mechanism suggested by Bourissou for the ROP of Lac-OCA.5a

The pendant benzyl ether groups of Ser(Bn)-PAHA were removed via catalytic hydrogenolysis under hydrogen in THF/methanol (3/1 v/v) for 24 hours in the presence of Degussa-type Pd/C catalyst (30%, Scheme 1). 1H NMR analysis of the deprotected polymer (Ser-PAHA) showed no detectable aromatic signals at 7.2 ppm, indicating the complete removal of the benzyl group. Pd/C mediated deprotection of polyester side chains is known to be mild enough that does not lead to degradation of polyester backbones even at elevated temperature.5d After hydrogenolysis, a 50-mer Ser(Bn)-PAHA with an Mn value of 11.6 × 103 g/mol resulted in a deprotected polymer Ser-PAHA with an Mn value of 6.3 × 103 g/mol (Figure S7), which is in excellent agreement with the expected Mn (5.7 × 103 g/mol). To further evaluate whether the catalytic hydrogenolysis condition has deleterious effect on PAHA, we went on to treat the Ser-PAHA for an additional 24 hours in the identical condition used for deprotection. No degradation was observed based on GPC analysis of the further treated Ser-PAHA (Figure S7). Another strong evidence of successful deprotection of side chain benzyl group of Ser(Bn)-PAHA is the drastically changed solvent solubility of Ser(Bn)-PAHA. Ser(Bn)-PAHA is very soluble in THF but is insoluble in water and methanol. However, Ser-PAHA, the deprotected polymer shows completely opposite solubility in those solvents; it is highly soluble in water and methanol but insoluble in THF.

The cytotoxicity of Ser-PAHA was evaluated in HeLa cells using the MTT assay. Ser-PAHA showed no cytotoxicity (p > 0.05 v.s. the control) at a concentration up to 200 μg/mL (60 μM) following both short-term (4 h) and long-term (24 h) incubation (Fig. 2a). To demonstrate the utility of this new water-soluble material, we prepared a Ser-PAHA-coated surface by spin coating and found that HeLa cells adhered strongly to the surface with no difference with the control (Fig. 2b)

Fig. 2
(a) Viability of HeLa cells as determined by the MTT assay following treatment with water-soluble Ser-PAHA100 for 4 and 24 h, respectively. (b) Microscopy images showing proliferation of HeLa cells on a Ser-PAHA100 film after 24-h incubation (left) as ...

Given the broad interest of PAHA, and other related degradable10 and hydroxyl-containing, water-soluble polymers,9a,11 the excellent cell compatibility along with its polyfunctionality makes Ser-PAHA a useful material for the construction of a drug delivery system and for hydrogel scaffolds for tissue engineering applications. We have demonstrated that OCAs could be successfully used as functional monomers for drug-initiated, controlled ROP, a technique we recently developed for the synthesis of drug-polyester conjugates and nanoconjugates.7e,7f The application of Ser-PAHA in drug delivery will be reported separately.

Supplementary Material



This work is supported by National Science Foundation (Career Program DMR-0748834), the National Institute of Health (NIH Director’s New Innovator Award 1DP2OD007246-01; 1R21EB013379), and UIUC from NIH National Cancer Institute Alliance for Nanotechnology in Cancer ‘Midwest Cancer Nanotechnology Training Center’ Grant R25 CA154015A.


The authors declare no competing financial interest.

Supporting Information. Experimental section, NMR spectra and GPC results for all compound tested. This material is available free of charge via the Internet at


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