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Synthesis and MALDI-Tof Characterization of Dendronized Brazilian Journal of Pharmaceutical Sciences vol. 49, special issue, 2013 Article Synthesis and MALDI-ToF characterization of dendronized poly(ethylene glycol)s Brittany K. Myers, Joanna E. Lapucha, Scott M. Grayson Department of Chemistry, Tulane University, New Orleans, LA, United States of America Brittany K. Myers earned her BS in Biochemistry in 2010 from Baylor Well-defined hybrids of linear poly(ethylene glycol)s (PEGs) and dendritic polyesters were University in Waco, TX, USA where she worked in the lab of Professor Robert prepared via the dendronization of the alcohol end groups of the mono and difunctional Kane and studied the use of 4-amino- linear PEGs. Though useful for rudimentary product characterization, GPC and NMR 1,8-naphthalimides in tissue bonding and meniscal repair. She is currently pursuing could not verify the overall structural purity of these linear-dendritic hybrids. On the other a Ph.D. in Chemistry at Tulane University hand, the detailed data provided by MALDI-ToF mass spectrometry enabled confirmation under the guidance of Professor Scott M. Grayson. Her research is focused on of the high structural purity of the dendronized PEGs at each step of the dendronization the exploration and synthesis of novel polymer architectures using bis-MPA-based dendritic scaffolding. procedure. The well-defined number of functionalities on these dendronized PEGs, renders them particularly useful for research in the biomedical sphere where functionality and purity Scott M. Grayson completed his PhD in Chemistry from the University of are of the utmost importance. The MALDI-ToF mass spectrometric approach described California, Berkeley (2002), studying herein represents a valuable technique for detailed monitoring of these dendronization the role of polymer architecture for drug delivery under Jean M. J. Fréchet. reactions, as well as a variety of other polymer end group modifications. Following post-doctoral studies in the laboratories of C. Grant Willson at the University of Texas, he was appointed Uniterms: Poly(ethylene glycol). Dendrons. Dendrimers. Dendronized polymers. as a professor at the department of chemistry at Tulane University in New Orleans, Louisiana, USA. As an associate professor at Tulane, his present research explores Híbridos bem definidos de poli(etilenoglicol) lineares (PEGs) e poliésteres dendriméricos the synthesis and applications of well-defined, yet complex foram preparados via “dendronização” de álcool e grupos de PEGs lineares mono e macromolecular structures. His research group has recently focused on amphiphilic and cyclic polymers. bifuncionais. Embora úteis para a caracterização rudimentar de produtos, Cromatografia por Permeação em Gel e RMN podem não demonstrar a pureza estrutural global desses híbridos lineares dendríticos. Por outro lado, informações detalhadas provenientes de espectrometria de massas MALDI-ToF permitiram a confirmação de elevada pureza estrutural de PEGs “dendronizados” em cada passo do processo de “dendronização”. O número de funcionalidades bem definidas destes PEGs “dendronizados”, torna-os particularmente úteis para pesquisa na área biomédica, na qual funcionalidade e pureza são de grande importância. A abordagem de espectrometria de massas MALDI-ToF descrita aqui representa uma técnica valiosa para o monitoramento detalhado destas reações de “dendronização”, bem como diversas modificações de outros polímeros e grupos. Unitermos: Poli(etilenoglicol). Dendrons. Dendrímeros. Polímeros “dendronizados”. INTRODUCTION The search for functional, biocompatible polymeric materials has in- tensified in recent years and one class of macromolecules that has garnered particular interest is linear-dendritic hybrids (Gitsov, Frechet, 1993; Gitsov, 2008; Lundberg et al., 2011). These hybrid materials are of particular interest because they address some of the long-standing shortcomings of *Correspondence: S. M. Grayson. 2015 Percival Stern Hall, the more studied linear polymers, namely, limited chain end functionality. 6400 Freret Street, Tulane University, 70118 - New Orleans Among traditional linear polymers, poly(ethylene glycol) has seen the most – LA, USA. E-mail: [email protected] widespread use for biomedical applications due to its high water solubility, 46 B. K. Myers, J. E. Lapucha, S. M. Grayson biocompatibility, low-toxicity, non-immunogenicity, and linear-dendritic hybrids. If flaws exist that truncate one or non-antigenic nature (Veronese, Pasut, 2005). As a result more of the expected branching points, this will yield a of this unique set of properties, PEG has been used for polymer with a different number of attachment points and, applications as diverse as improving the water solubility therefore, different biophysical properties. Additionally, in of drugs (Pasut, Veronese, 2010) and increasing the mo- NMR, the multiplicity of both linear and branched repeat lecular weight and therefore blood circulation of proteins units makes it difficult to confirm the structural purity, (Abuchowski et al., 1977). However, the applications of while GPC measures the hydrodynamic radius which is PEGs are limited by the simple fact that they bear only not likely to be affected substantially by minor structural one or two modifiable functionalities at the polymer chain impurities. Though important, these traditional character- ends. In this context, the ability to incorporate an increased ization methods must be complemented by a technique multiplicity of addressable functional groups through like matrix-assisted laser desorption/ionization time-of- dendronization is invaluable. However, as purity remains flight mass spectrometry (MALDI-ToF MS) that offers a a critical issue for biomedical materials applications, it is more complete picture in terms of composition, structure, necessary to establish the reproducible synthesis of well- number of end groups, and molecular weight distributions defined linear-dendritic hybrids as well as develop rigor- (Polce, Wesdemiotis, 2002). ous characterization methodologies to confirm their purity. Herein we report the synthesis and characterization Dendrimers are highly branched, monodisperse of linear-dendritic hybrids, and dendritic-linear-dendritic macromolecules with an exact multiplicity of functional “dumbbells” composed of a linear PEG block, and den- groups that increase exponentially in number as the dritic blocks based on the branched bis-MPA monomer. generation of the dendrimer increases (Frechet, 1994). While the synthesis of such hybrids has already been In this investigation, the dendritic component of linear reported (Ihre, Padilla de Jesus, Frechet, 2001), improve- dendritic hybrids is a divergently grown dendron, a ments in the resolution of MALDI mass spectrometers “wedge-like” dendritic segment. Although numerous enable a more detailed characterization, which will be de- dendritic syntheses have been reported (Frechet, Tomalia, scribed in this investigation. These compounds have been 2001), the polyester dendrimers and dendrons based on fully characterized using the standard methods employed 2,2-bis(hydroxymethyl)propionic acid (bis-MPA (Ihre et (NMR and GPC) but were also characterized extensively al., 1996;Ihre et al., 2001) are the most promising given by MALDI-ToF MS to verify their structural integrity. their ease of synthesis, exceptional structural purity, and biocompatible nature (Padilla de Jesus et al., 2002; MATERIAL AND METHODS Lundberg et al., 2011). The key advantage of coupling dendrons to linear Material polymers is the multivalent nature of the resulting mac- romolecule which allows for increased functionalization All reagents were purchased from Aldrich and used and can impart both increased solubility (Gitsov, Frechet, without further purification, unless otherwise noted. 1993) and increased strength of intermolecular interactions (Gitsov et al., 2000). These characteristics are particularly Characterization important when considering polymers for biomedical ap- Mass spectral data was acquired using a Bruker plications. One example of the useful modularity of the Autoflex III matrix-assisted laser desorption/ionization linear-dendritic hybrid system is that, while the linear time-of-flight mass spectrometer (MALDI-ToF MS) with PEG provides increased plasma half-life, low toxicity, and delayed extraction using both positive ion and reflector overall stability from enzymatic or hydrolytic degradation, detection modes. THF stock solutions of α-cyano-4- the dendrons provide a multiplicity of functional groups hydroxycinnamic acid or trans-2-[3-(4-tert-butylphenyl)- to increase the drug payload or targeting groups (Padilla 2-methyl-2-propenylidene]malononitrile were used as the de Jesus et al., 2002). matrix (10 mg mL-1) and sodium trifluoroacetate was used However, the use of linear-dendritic hybrids in the to provide a cation for ionization (1 mg mL-1). The den- biomedical realm necessitates rapid and reliable methods dronized PEG stock solutions were prepared at a 2 mg mL-1 of characterization to confirm their structural purity. Typi- concentration in THF. MALDI samples were prepared by cally, nuclear magnetic resonance spectroscopy (NMR) combining 4 µL of the dendronized PEG solution, 2 µL and gel permeation chromatography (GPC) are employed of the cation solution, and 2 µL of the matrix solution. to confirm the purity of macromolecules. However, these For data acquisition, the pulsed ion extraction delay was methods can provide vague data regarding the purity of set to 40 ns, with an ion
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