Highly Branched Poly(l-lysine) - American Chemical Society

Biomacromolecules 2003, 4, 249-258

249

Highly Branched Poly(L-lysine) Juan Rodrı´guez-Herna´ ndez, Marco Gatti, and Harm-Anton Klok* Max Planck Institute for Polymer Research, Ackermannweg 10, D-55128 Mainz, Germany Received August 28, 2002; Revised Manuscript Received December 12, 2002

This paper describes the synthesis of several novel water-soluble highly branched polypeptides. The synthesis starts with the ring-opening polymerization of -benzyloxycarbonyl-L-lysine N-carboxyanhydride (Z-Lys NCA) or -trifluoroacetyl-L-lysine N-carboxyanhydride (TFA-Lys NCA), followed by end functionalization of the peptide chain with NR,N-di(9-fluorenylmethoxycarbonyl)-L-lysine (NR,N-diFmoc Lys). Deprotection of the NR,N-diFmoc Lys end group affords two new primary amine groups that can initiate the polymerization of a second generation of branches. Repetition of this ring-opening polymerization-end functionalization sequence affords highly branched poly(-benzyloxycarbonyl-L-lysine) (poly(Z-Lys)) and poly(-trifluoroacetylL-lysine) (poly(TFA-Lys)) in a small number of straightforward synthetic steps. Removal of the side-chain protective groups yields water-soluble and highly branched poly(L-lysine)s, which may be of potential interest for a variety of medical applications. Introduction Dendritic polypeptides have attracted much interest for a variety of (bio)medical applications,1 e.g., for the development of multiple-antigen peptides (MAPs),2 as scaffolds for the preparation of contrast agents for magnetic resonance imaging3 and as gene carriers.4 Generally, the synthesis of peptide dendrimers is a very laborious process that involves a large number of successive coupling and deprotection steps.2-5 Although this strategy affords products that are structurally uniform and perfectly monodisperse, it is not very attractive for the preparation of high molecular weight dendrimers on a large scale. In view of their potential in the medical field, it seems worthwhile to search for alternative synthetic routes that facilitate the preparation of dendritic polypeptides and which also allow access to high molecular weight polymers. An attractive alternative to stepwise peptide synthesis is the ring-opening polymerization of R-amino acid N-carboxyanhydrides (NCAs).6 This well-established method permits simple preparation of high molecular weight polypeptides in multigram quantities but shares some of the same problems as other polymerizations. Chain breaking, chain transfer, and termination can lead to polydispersity. As a consequence, the synthetic ease of the NCA ring-opening polymerization goes at the expense of chain length uniformity and structural perfection. Nevertheless, this method has been extensively used for the preparation of a wide range of polypeptide homopolymers, random copolypeptides, and (hybrid) block copolymers.6 To a limited extent, the NCA ring-opening polymerization has also been used to prepare more complex graft7 and star-shaped polypeptide architectures.8 In this contribution, the NCA ring-opening polymerization is explored for the synthesis of highly branched poly(L-lysine)s, * To whom correspondence should be addressed. Fax: ++ 49 6131 379 100. e-Mail: [email protected]

which may be attractive alternatives for perfect peptide dendrimers whose preparation involves a multistep synthetic pathway and extensive isolation and purification. The highly branched poly(L-lysine)s described in this contribution are prepared via a repetitive sequence of NCA ring-opening polymerization and end-coupling reactions. Following this strategy high molecular weight highly branched polypeptides can be prepared readily in multigram quantities, without the need for extensive isolation and/or purification procedures. Experimental Section Materials. -Trifluoroacetyl-L-lysine N-carboxyanhydride (TFA-Lys NCA) and -benzyloxycarbonyl-L-lysine N-carboxyanhydride (Z-Lys NCA) were synthesized according to the method described by Poche´ et al.9 Ethyl acetate (EtAc, Acros Organics, >99.5%) was dried over molecular sieves (4 Å). N,N′-Dimethylformamide (DMF, Riedel-de-Hae¨n, >99.5%) was distilled from CaH2 under reduced pressure and subsequently stored over molecular sieves (4 Å) under argon atmosphere. n-Hexylamine (Aldrich, > 99%) and 1,4diaminobutane (Aldrich, >99%) were distilled from CaH2 under reduced pressure and stored under argon atmosphere. All other solvents and reagents were purchased from commercial suppliers and were used as received. Physical and Analytical Methods. 1H NMR spectra were recorded on Bruker AMX250, AMX500, and WS700 spectrometers using the residual proton resonance of the deuterated solvent as the internal standard. Chemical shifts are reported in parts per million (ppm). When peak multiplicities are given, the following abbreviations are used: s, singlet; d, doublet; t, triplet; m, multiplet; b, broad. Matrix-assisted laser desorption ionization time-of-flight (MALDI-TOF) mass spectra were recorded on a Bruker “Reflex II” spectrometer with a LSI-N2 laser. Samples for MALDI-TOF mass spectrometry were prepared from DMF solution using dithranol as the matrix and sodium trifluoroacetate as

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Biomacromolecules, Vol. 4, No. 2, 2003

cationizing agent. Samples were prepared using matrix, analyte, and salt in a ratio of ∼500:1:0,1. Gel permeation chromatography (GPC) was performed at 60°C with a setup consisting of a Waters 510 pump and a series of three styrene-divinylbenzene (SDV) columns (300 × 8 mm) with pores sizes of 500, 105, and 106 Å (Polymer Standards Service, Mainz, Germany). A 1 g/L solution of LiBr in DMF was used as the mobile phase, and the elution of the samples was monitored with refractive index (RI) detection. Elution times were converted to molecular weights with a calibration curve that was constructed from narrow polydispersity poly(ethylene oxide) standards. Procedures r-Amino Acid N-Carboxyanhydride Polymerizations. A Schlenk flask fitted with a drying tube was charged with a solution of the appropriate NCA in dry DMF (ca. 0.2 g/mL). Then, the initiator was added, and the reaction mixture was stirred at room temperature. n-Hexylamine and 1,4-diaminobutane, which are liquids, were added neat, all polymeric initiators were added as a solution in DMF. The amount of initiator depends on the desired length of the peptide branches, which can be controlled via the molar ratio of NCA and initiator. After 5 days, the solution was slowly added to a 20-fold excess of water. The precipitated polymer was filtered and freeze-dried. Below, the 1H NMR chemical shifts for the two types of highly branched polypeptides that have been investigated are listed, n refers to the number average degree of polymerization. Poly(E-benzyloxycarbonyl-L-lysine). 1H NMR (250 MHz, DMSO-d6) δ ) 7.3-7.2 (b, ArH, 5H × n), 5.0 (b, ArCH2, 2H × n), 4.2-3.9 (b, RCH, 1H × n), 2.9 (b, -CH2CH2NH(CdO)- + CH3(CH2)4CH2NH(CdO)-, 2H × n + 2H), 1.8-1.2 (b, -CH(CH2)3CH2NH- + CH3(CH2)4CH2NH(Cd O)-, 6H × n + 8H), 0.8 (t, CH3(CH2)4CH2NH(CdO)-, 3H). Poly(E-trifluoroacetyl-L-lysine). 1H NMR (250 MHz, DMSO-d6) δ ) 4.2-3.8 (b, RCH, 1H × n), 3.1 (b, -CH2CH2NH(CdO)- + CH2(CH2)4CH2NH(CdO)-, 2H × n + 2H), 1.9-1.2 (b, -CH(CH2)3CH2NH- + CH3(CH2)4CH2NH(CdO)-, 6H × n + 8H), 0.8 (t, CH3(CH2)4CH2NH(CdO)-, 3H). Endcoupling of Nr,NE-Di(9-fluorenylmethoxycarbonylL-lysine). A round-bottom flask containing a DMF solution of the appropriate polypeptide was charged with 4 equiv of NR,N-di(9-fluorenylmethoxycarbonyl-L-lysine), 4 equiv of 2-(1H-benzo-triazole-1-yl)-oxy-1,1,3,3-tetramethyluronium hexafluorophosphate (HBTU), and 12 equiv of 1-hydroxybenzotriazole (HOBt). Then, 10 equiv of N,N-diisopropylethylamine (DIPEA) was added, and the reaction was allowed to proceed at room temperature for 3 days. After that, the mixture was precipitated in water. After freeze-drying, the polymer was washed several times with Et2O and vacuum-dried. Poly(E-benzyloxycarbonyl-L -lysine)-N r ,N E -diFmocLys. 1H NMR (250 MHz, DMSO-d6) δ ) 7.9 (b, Ar′H, 4H), 7.7 (b, Ar′H, 4H), 7.3-7.2 (b, ArH + Ar′H, 5H × n + 8H), 5.0 (b, ArCH2, 2H × n), 4.2-3.9 (b, RCH + FmocCHCH2+ FmocCHCH2-, 1H × n + 7H), 2.9 (b, -CH2CH2NH(CdO)- + CH3(CH2)4CH2NH(CdO)-, 2H × n + 4H),

Rodrı´guez-Herna´ ndez et al.

1.8-1.2 (b, -CH(CH2)3CH2NH- + CH3(CH2)4CH2NH(Cd O)-, 6H × n + 14H), 0.8 (t, CH3(CH2)4CH2NH(CdO)-, 3H). Poly(E-trifluoroacetyl-L-lysine)-Nr,NE-diFmoc-Lys. 1H NMR (250 MHz, DMSO-d6) δ ) 7.9 (m, Ar′H, 4H), 7.7 (m, Ar′H, 4H), 7.3 (b, Ar′H, 8H), 4.2-3.8 (b, RCH + FmocCHCH2- + FmocCHCH2-, 1H × n + 7H), 3.1 (b, -CH2CH2NH(CdO)- + CH2(CH2)4CH2NH(CdO)-, 2H × n + 4H), 1.9-1.2 (b, -CH(CH2)3CH2NH- + CH3(CH2)4CH2NH(CdO)-, 6H × n + 14H), 0.8 (t, CH3(CH2)4CH2NH(CdO)-, 3H). Selective Removal of the Fmoc-Protective Groups of the End-Functionalized Polypeptides. A round-bottom flask was charged with a solution of the NR,N-diFmoc-Lys end-functionalized polypeptide in DMF. Then, 20% (v/v) piperidine was added. The reaction mixture was stirred for 1 h at room temperature and subsequently precipitated in water. The precipitated polymers were washed several times with Et2O and finally vacuum-dried to afford the product in quantitative yield. Poly(E-benzyloxycarbonyl-L-lysine)-L-lysine. 1H NMR (250 MHz, DMSO-d6) δ ) 7.3-7.2 (b, ArH, 5H × n), 5.0 (b, ArCH2-, 2H × n), 4.2-3.9 (b, RCH, 1H × n + 1 H), 2.9 (b, -CH2CH2NH(CdO)- + -CH2CH2NH2 + CH3(CH2)4CH2NH(CdO)-, 2H × n + 4H), 1.8-1.2 (b, -CH(CH2)3CH2NH- + -CH(CH2)3CH2NH2 + CH3(CH2)4CH2NH(Cd O)-, 6H × n + 14H), 0.8 (t, CH3(CH2)4CH2NH(CdO)-, 3H). Poly(E-trifluoroacetyl-L-lysine)-L-lysine. 1H NMR (250 MHz, DMSO-d6) δ ) 4.2-3.8 (b, RCH, 1H × n + 1H), 3,1 (b, -CH2CH2NH(CdO)- + -CH2CH2NH2 + CH3(CH2)4CH2NH(CdO)-, 2H × n + 4H), 1.9-1.2 (b, -CH(CH2)3CH2NH- + -CH(CH2)3CH2NH2 + CH3(CH2)4CH2NH(Cd O)-, 6H × n + 14H), 0.8 (t, CH3(CH2)4CH2NH(CdO)-, 3H). Total Deprotection of Highly Branched Poly(Z-Llysine). A round-bottom flask was charged with a solution of the appropriate highly branched poly(Z-L-lysine) in CF3COOH (∼100 mg/3 mL). Then, a 4-fold molar excess of a 33 wt % solution of HBr in AcOH was added, and the reaction mixture was stirred for 1 h at room temperature. Finally, the reaction mixture was precipitated in Et2O and the product isolated in quantitative yield after filtration and vacuum-drying. 1H NMR (700 MHz, D2O) δ ) 4.44 (b, RCH, 1H × (n - 2)), 4.31 (b, C-terminal RCH, 1H), 4.20 (b, N-terminal RCH, 1H), 3.1 (b, -CH2CH2NH2 + CH3(CH2)4CH2NH(CdO)-, 2H × n + 2H), 1.90-1.70 (b, -CHCH2CH2CH2CH2NH2, 4H × n), 1.65-1.44 (b, -CHCH2CH2CH2CH2NH2, 2H × n), 1.44-1.30 (b, CH3(CH2)4CH2NH(CdO)-, 8H), 0.8 (t, CH3(CH2)4CH2NH(CdO)-, 3H). Total Deprotection of Highly Branched Poly(TFA-Llysine). A round-bottom flask was charged with ∼200 mg of the protected polypeptide in 30 mL of a 1:20 (v/v) mixture of H2O and MeOH. The suspension was heated to reflux, and 250 mg of K2CO3 was added to the reaction mixture. During the course of the reaction, the initial suspension turns into a homogeneous solution. After 4 h, the reaction mixture was cooled to room temperature and solvents were removed under vacuum. The solid residue was dissolved in water.

Highly Branched Poly(L-lysine)

Biomacromolecules, Vol. 4, No. 2, 2003 251

Figure 1. Schematic representation of highly branched polypeptides obtained using (a) a monofunctional and (b) a bifunctional primary amine initiator.

Subsequently, the polypeptide was precipitated from the aqueous solution by the addition of NaOH (aq). Filtration and freeze-drying afforded the deprotected polymer in quantitative yield. 1H NMR (700 MHz, D2O) δ ) 4.44 (b, RCH, 1H × (n - 2)), 4.31 (b, C-terminal RCH, 1H), 4.20 (b, N-terminal RCH, 1H), 3.1 (b, -CH2CH2NH2 + CH3(CH2)4CH2NH(CdO)-, 2H × n + 2H), 1.90-1.70 (b, -CHCH2CH2CH2CH2NH2, 4H × n), 1.65-1.44 (b, -CHCH2CH2CH2CH2NH2, 2H × n), 1.44-1.30 (b, CH3(CH2)4CH2NH(CdO)-, 8H), 0,8 (t, CH3(CH2)4CH2NH(CdO)-, 3H). Results and Discussion A schematic representation of the highly branched polypeptides that will be discussed in this contribution is presented in Figure 1. The polypeptides were prepared following a synthetic strategy similar to that reported earlier by Birchall and North10 and involves a repetitive sequence of NCA ringopening polymerization and end-functionalization/deprotection reactions. The synthesis is illustrated in Scheme 1 for the case where a monofunctional primary amine is used to start the NCA ring-opening polymerization. This results in highly branched polypeptides with structures as illustrated in Figure 1a. In addition to simple monofunctional primary amines, we have also explored the use of diamines as initiators, which yield the highly branched polypeptides depicted in Figure 1b. In their paper, Birchall and North exclusively reported the synthesis of highly branched block

copolypeptides based on water-insoluble hydrophobic R-amino acids.10 The solubility problems they describe may at least partly be due to the nature of the R-amino acids. In contrast, this contribution, will focus on highly branched polypeptides based on the water-soluble R-amino acid L-lysine. A consequence of the use of functionalized R-amino acids such as L-lysine, however, is that protection of the side chains is necessary to allow ring-opening polymerization of the corresponding NCAs. The highly branched polypeptides described in this contribution were obtained by ring-opening polymerization of -benzyloxycarbonyl-L-lysine N-carboxyanhydride (Z-Lys NCA) or -trifluoroacetyl-L-lysine Ncarboxyanhydride (TFA-Lys NCA). In addition to blocking the -NH2 groups, the Z and TFA groups are also expected to improve the solubility of the protected highly branched polypeptides in nonaqueous solutions. The presence of protective groups in the side chains of the R-amino acid repeat units also poses some restrictions on the choice of the R-amino acid which is used to end functionalize the peptide branches and which should act to initiate a subsequent NCA ring-opening polymerization step. Most importantly, the protective groups that block the -NH2 groups of this L-lysine derivative should be orthogonal to those present along the polypeptide backbone; i.e., it should be possible to remove these protective groups without affecting the Z and TFA groups. These requirements preclude the use of the Z-protected L-lysine derivative that was employed by Birchall and North.10 In this contribution, the branching

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Scheme 1

points will be based on NR,N-di(9-fluorenylmethoxycarbonyl)-L-lysine (NR,N-diFmoc Lys). These branching points will not be introduced in situ by adding an activated derivative to the polymerization mixture, as was described by Birchall and North,10 but will be attached in a separate coupling step. Although this means an additional synthetic step, the introduction of branching points via a separate coupling reaction allows a more careful monitoring and control of the degree of end functionalization. As shown in Scheme 1, the synthesis of the highly branched poly(L-lysine)s starts with the ring-opening polymerization of Z-Lys NCA or TFA-Lys NCA. A 10-fold molar excess of NCA monomer with respect to the primary amine initiator groups was used throughout the synthesis. In a subsequent reaction, the side-chain protected L-lysine oligomer is end functionalized with NR,N-diFmoc Lys under standard peptide coupling conditions.11 Finally, the Fmoc groups at the N-terminal L-lysine residue are removed by treating the polypeptide with a 20% (v/v) solution of piperidine in DMF, which generates two new terminal primary amine groups. These amine groups can serve as initiator for the addition of a next generation of branches. Repetition of the end-functionalization-deprotection-ringopening polymerization sequence affords the highly branched polypeptides schematically shown in Figure 1. The end functionalization of the side-chain protected L-lysine oligomers and the subsequent removal of the Fmoc protective groups can be followed with 1H NMR spectroscopy and MALDI-TOF mass spectrometry. As an example,

Figure 2 shows 1H NMR spectra of NR,N-diFmoc Lys, of a TFA protected oligo(L-lysine) core with a number-average degree of polymerization of 10 (poly(TFA-Lys)10), both before and after end functionalization with NR,N-diFmoc Lys, and of the same oligomer after removal of the Fmoc groups. Comparison of the integral of the triplet due to the terminal -CH3 group of the initiator moiety at ∼0.8 ppm with that of the aromatic protons of the Fmoc groups indicates almost quantitative end functionalization. The 1H NMR spectra also show that treatment of the NR,N-diFmoc Lys end-functionalized poly(TFA-Lys)10 with piperidine/ DMF results in complete removal of the Fmoc groups and does not change the number average degree of polymerization. Further support for the success of the end-functionalization was obtained from MALDI-TOF mass spectrometry. As a representative example, Figure 3 shows the MALDITOF mass spectrum of a poly(Z-Lys)10 core after end functionalization with NR,N-diFmoc Lys. The mass spectrum shows a single series of signals spaced at a distance of 262 Da, which corresponds to the molecular weight of a Z-Lys repeat unit. The mass of each of the peaks corresponds to that of the initiator moiety (n-hexylamine) plus an integer number of Z-Lys repeat units, the NR,N-diFmoc Lys end group, and Na+, which provides support for the success of the end functionalization. Since sodium trifluoroacetate was added during MALDI sample preparation, the signals in the mass spectrum represent the Na+-labeled molecular ions. The side-chain protected highly branched polypeptides were analyzed by gel permeation chromatography (GPC) and



Figure 2. 1H NMR spectra (DMSO-d6, 250 MHz) of: (a) NR,N-di(9-fluorenylmethoxycarbonyl)-L-lysine, (b) poly(-trifluoroacetyl-L-lysine)10, (c) NR,N-di(9-fluorenylmethoxycarbonyl)-L-lysine end-functionalized poly(-trifluoroacetyl-L-lysine)10, and (d) the same polypeptide after removal of the Fmoc protective groups. The poly(-trifluoroacetyl-L-lysine) was obtained using n-hexylamine as initiator. Peak assignments are given in the Experimental Section. 1

H NMR. GPC traces of different generations of each of the three classes of highly branched polypeptides are shown in Figure 4. 1H NMR spectra of four generations of highly branched poly(Z-Lys) prepared using n-hexylamine as the

initiator are shown in Figure 5. Unfortunately, most attempts to obtain useful MALDI-TOF mass spectra of highly branched poly(Z-Lys) samples were unsuccessful. An exception was the G0 highly branched poly(Z-Lys) that was

254


Figure 3. MALDI-TOF mass spectrum of an NR,N-diFmoc Lys endfunctionalized poly(Z-Lys)10 core. The poly(Z-lys) was prepared using n-hexylamine as initiator.

obtained using n-hexylamine as initiator. The MALDI-TOF mass spectrum of this compound is shown in Figure 6. The GPC traces not only illustrate the increase in molecular weight with increasing generation but also provide indirect evidence for the success of the end functionalization with NR,N-diFmoc Lys. Most GPC traces are monomodal and do not contain any shoulders, suggesting that end functionalization proceeds almost quantitatively. Only for the G2 highly branched polypeptides prepared using n-hexylamine as initiator a shoulder was observed at lower molecular weights, pointing toward incomplete end functionalization and/or deprotection of the G1 precursor. From the 1H NMR spectra, the total number average degree of polymerization (DP) as well as the average chain length per branch (DParm) can be calculated. These data, together with the results from the GPC experiments are summarized in Tables 1-3. Since for the highly branched poly(Z-Lys) obtained using 1,4-diaminobutane as initiator no distinct signal of the initiator can be discerned, Table 3 only reports results from GPC analysis. At this point, it is important to note that in this case GPC only provides qualitative molecular weight information since the elution times of the samples were converted to molecular weights using a calibration curve constructed from standards that both had a different topology (linear vs branched) and chemical


composition (poly(ethylene oxide) vs poly(L-lysine)). Despite these limitations, the number-average molecular weights of highly branched poly(Z-Lys) obtained using n-hexylamine as initiator for the NCA polymerization as determined from 1H NMR and GPC are in fairly good agreement up to G1. Only for G2 the data from 1H NMR and GPC are significantly different. Although it is difficult to provide a final explanation, this difference may be attributed to the complex interplay of macromolecular architecture and chemical composition of the highly branched poly(Z-Lys) and the restricted quantitative character of the GPC experiments due to the use of linear poly(ethylene oxide) standards. Unfortunately, attempts to obtain complementary molecular weight information from static light scattering have not been successful so far. Nevertheless, the 1H NMR and GPC data demonstrate that the synthetic strategy outlined in Scheme 1 allows the preparation of highly branched polypeptides with number-average molecular weights of ∼33 kDa in a small number of straightforward reaction steps. More interestingly, Table 1 and Table 2 show that for the highly branched polypeptides obtained using n-hexylamine as initiator, the average chain length per branch in most cases remains constant around 10 R-amino acids, which would also be expected based on the monomer-initiator ratio (M/I). This is another indication that end functionalization with NR,NdiFmoc Lys and subsequent Fmoc removal proceed quantitatively. Only for the second generation highly branched polypeptides (G2) does the average chain length per branch deviate from M/I, which points to incomplete end coupling and/or deprotection. These observations are in agreement with the results from GPC analysis (Figure 4). The MALDI-TOF mass spectrum shown in Figure 6 contains a single series of peaks spaced at 262 Da, which is the molecular weight of the Z-Lys repeat units. The mass of each of the signals corresponds to the sum of the masses of an integer number of Z-Lys repeat units plus that of the n-hexylamine initiator moiety, one Lys branching point, and Na+ and, thus, confirms the chemical composition of the G0 poly(Z-Lys). Deprotection of highly branched poly(TFA-Lys) and poly(Z-Lys) affords the corresponding water-soluble poly(Llysine) homologues (Scheme 2). TFA groups were removed by treating the polypeptides with K2CO3 in a H2O/MeOH 1/20 (v/v) mixture.12 Deprotection of the highly branched

Figure 4. GPC chromatograms of different generations of protected highly branched polypeptides: (a) based on Z-Lys NCA using n-hexylamine as initiator; (b) based on TFA-Lys NCA using n-hexylamine as initiator; (c) based on Z-Lys NCA using 1,4-diaminobutane as initiator.



Figure 5. 1H NMR spectra (500 MHz, DMSO-d6, 333 K) of different generations of highly branched poly(Z-L-lysine) obtained using n-hexylamine as the initiator. Peak assignments are given in the Experimental Section.

poly(Z-Lys) was accomplished using a 33 wt % solution of HBr in AcOH.13 1H NMR spectra of the resulting highly branched poly(L-lysine)s indicated quantitative removal of the side-chain protective groups. In 1H NMR spectra of

highly branched poly(L-lysine)s obtained by n-hexylamine initiated NCA polymerization, the triplet of the terminal -CH3 group of the initiator species could be clearly discerned and allowed determination of the total number

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Rodrı´guez-Herna´ ndez et al. Table 2. Yields and Molecular Characteristics of Highly Branched Poly(Z-Lys) of Different Generations Obtained Using n-Hexylamine as the Initiator yield M nd Mn,the M nf Mwf (%)a DPb DParmc (g/mol) (g/mol) (g/mol) (g/mol) Mw/Mnf core G0 G1 G2

Figure 6. MALDI-TOF mass spectrum of a G0 poly(Z-Lys) obtained using n-hexylamine as initiator. Table 1. Yields and Molecular Characteristics of Highly Branched Poly(TFA-Lys) of Different Generations Obtained Using n-Hexylamine as the Initiator yield M nd Mn,the M nf Mwf (%)a DPb DParmc (g/mol) (g/mol) (g/mol) (g/mol) Mw/Mnf core G0 G1 G2

79 63 55 66

10 30 75 107

10 10 11 4

2400 6800 16400 23500

2350 6730 15510 33060

n.d.g

n.d.g

5030 7040 18300 23700 68700 100000

n.d.g 1.4 1.3 1.5

a

Isolated yields determined gravimetrically after precipitation, filtration, and freeze-drying of the polypeptides. b Total number average degree of polymerization as determined by 1H NMR using the signal between 1.2 and 1.9 ppm, which is due to the methylene protons of the CH(CH2)3CH2side chains of the L-lysine repeats and the CH3(CH2)4CH2NH(CdO)initiator moiety, relative to that of the triplet of the methyl group of the initiator moiety at 0.8 ppm. c Number average degree of polymerization of the polypeptide arms added during the last NCA ring-opening polymerization step, determined using 1H NMR. d Number average molecular weight of the polypeptides calculated from the results of the 1H NMR experiments. e Expected number average molecular weight of the polypeptides, calculated assuming a number average degree of polymerization of 10 repeat units per branch and quantitative yields during NR,N-diFmoc Lys end functionalization and Fmoc deprotection. f Number average molecular weight (Mn), weight average molecular weight (Mw), and polydispersity (Mw/Mn) determined by GPC. g Not determined. The molecular weight of this sample was outside the range covered by the PEO calibration curve.

average degree of polymerization (DP) as well as the average number of R-amino acids per branch (DParm). These data are summarized in Table 4 and Table 5, respectively. The total number average degree of polymerization of the highly branched polypeptides (DP) and lengths of the branches (DParm) listed in Table 4 and Table 5 are in fairly good agreement with the data presented in Table 1 and Table 2. This indicates that removal of the side chain protective groups does not lead to significant side reactions. Interestingly, 1H NMR spectra of the poly(L-lysine)s not only give molecular weight information but also provide evidence for their branched structure. Figure 7 shows part of the 1H NMR spectrum of a highly branched poly(L-lysine) obtained by n-hexylamine initiated ring-opening polymerization of Z-Lys NCA. In the spectra, four different methine protons can be distinguished, which are labeled Ha-Hd in Figure 7.14 The integrals corresponding to each of these peaks, determined relative to that of the terminal -CH3 group of the initiator moiety, are also listed in Table 4 and Table 5. For a linear polypeptide, the ratio [I(Ha) + I(Hb) + I(Hc) + I(Hd)]/Hc is

65 89 58 70

11 25 77 132

11 7 13 7

2900 6600 19800 33800

2720 7830 18040 38470

n.d.g n.d.g 4660 6800 14500 19000 82500 105000

n.d.g 1.5 1.3 1.3

a Isolated yields determined gravimetrically after precipitation, filtration, and freeze-drying of the polypeptides. b Total number average degree of polymerization as determined by 1H NMR using the signal between 1.2 and 1.8 ppm, which is due to the methylene protons of the CH(CH2)3CH2side chains of the L-lysine repeats and the CH3(CH2)4CH2NH(CdO)initiator moiety, relative to that of the triplet of the methyl group of the initiator moiety at 0.8 ppm. c Number average degree of polymerization of the polypeptide arms added during the last NCA ring-opening polymerization step, determined using 1H NMR. d Number average molecular weight of the polypeptides calculated from the results of the 1H NMR experiments. e Expected number average molecular weight of the polypeptides, calculated assuming a number average degree of polymerization of 10 repeat units per branch and quantitative yields during NR,N-diFmoc Lys end functionalization and Fmoc deprotection. f Number average molecular weight (Mn), weight average molecular weight (Mw), and polydispersity (Mw/Mn) determined by GPC. g Not determined. The molecular weight of this sample was outside the range covered by the PEO calibration curve.

Table 3. Yields and Molecular Characteristics of Highly Branched Poly(Z-Lys) of Different Generations Obtained Using 1,4-Diaminobutane as the Initiator

core G0 G1 G2

yield (%)a

Mn (g/mol)b

Mw (g/mol)b

Mw/Mnb

89 60 95 79

2270 9780 18200 62300

2660 13900 22400 79200

1.2 1.4 1.2 1.3

a Isolated yields determined gravimetrically after precipitation, filtration, and freeze-drying of the polypeptides. b Number average molecular weight (Mn), weight average molecular weight (Mw), and polydispersity (Mw/Mn) determined by GPC.

Scheme 2

equal to the number average degree of polymerization. Table 4 and Table 5, however, show that for the polypeptides discussed in this contribution, this ratio is much smaller than the total number average degree of polymerization and has a value around 20, independent of generation. This reflects the large number of end groups present in the polypeptides and is an indication for their highly branched topology.



Table 4. Results of the 1H NMR Characterization of Highly Branched Poly(L-lysine)s Prepared via n-Hexylamine Initiated Ring-Opening Polymerization of TFA-Lys NCA and Subsequent Removal of the TFA Protective Groups 1H

core G0 G1 G2

I(Ha) + I(Hb) 10.9 40.4 87.6 119.7

NMR integralsa

I(Hc)

I(Hc,th)b

I(Hd)

1.0 2.5 3.5 7.0

1 2 4 8

1.0

[I(Ha) + I(Hb) + I(Hc) + I(Hd)]/I(Hc) 12.9 17.2 26.0 18.1

DPc

Mnd (g/mol)

DParme

13 43 91 127

1940 6360 13400 18600

13 15 13 4

a 1H NMR integrals are reported relative to that of the signal of the methyl group of the initiator moiety at 0.97 ppm, which was set equal to 3. b Theoretical value, assuming quantitative NR,N-diFmoc Lys end functionalization and Fmoc deprotection. c Total number average degree of polymerization calculated from 1H NMR results. d Total number average molecular weight calculated from the 1H NMR results. e Number average degree of polymerization of the polypeptide arms added during the last NCA ring-opening polymerization step.

Table 5. Results of the 1H NMR Characterization of Highly Branched Poly(L-lysine)s Prepared via n-Hexylamine Initiated Ring-Opening Polymerization of Z-Lys NCA and Subsequent Removal of the Z Protective Groups 1H

core G0 G1 G2

NMR integralsa

I(Ha)

I(Hb)

I(Hc)

I(Hc,th)b

I(Hd)

10.1 38.6 72.6 138.1

1.0 1.9 3.7 7.7

1 2 4 8

1.0

2.3 4.0 11.5

[I(Ha) + I(Hb) + I(Hc) + I(Hd)]/I(Hc)

DPc

Mnd (g/mol)

DParme

12.1 22.5 21.7 20.4

12 43 80 157

1870 6350 11830 23070

12 15 9 10

a 1H NMR integrals are reported relative to that of the signal of the methyl group of the initiator moiety at 0.97 ppm, which was set equal to 3. Theoretical value, assuming quantitative NR,N-diFmoc Lys end functionalization and Fmoc deprotection. c Total number average degree of polymerization calculated from the 1H NMR results. d Total number average molecular weight calculated from the 1H NMR results. e Number average degree of polymerization of the polypeptide arms added during the last NCA ring-opening polymerization step.

b

Figure 7. Part of the 1H NMR (700 MHz, D2O, 298 K) of different highly branched poly(L-lysine)s obtained via n-hexylamine initiated ringopening polymerization of Z-Lys NCA. Peak assignments are given in the Experimental Section.

Another, simple way to deduce structural information from the 1H NMR spectra is to compare the value of the integral of the peak labeled Hc, which is identical to the number of end groups, with the theoretically expected value. These theoretically calculated numbers are also listed in Table 4 and Table 5. The relatively good agreement of the experimentally determined and theoretically calculated numbers also suggests a relatively defect-free highly branched topology. The possibility to observe the methine resonances of the N-terminal R-amino acid residues in the 1H NMR spectra of the highly branched poly(L-lysine)s also offers the

opportunity to estimate the total number average degree of polymerization and the average number of repeat units per branch for the polypeptides obtained using 1,4-diaminobutane as initiator. Under the assumption that each of the NR,NdiFmoc Lys couplings and subsequent Fmoc deprotections proceeds quantitatively, theoretical values for the integral of Hc are 2, 4, 8, and 16 for, respectively, the core, G0, G1, and G2 (Table 6). As was discussed (see, e.g., Figure 2 and Figure 3), these assumptions are not unreasonable. Comparison of the integrals of Hc with those of the residual methine protons (Ha + Hb) and taking into account the theoretical number of branches that would constitute a

258



Table 6. Results of the 1H NMR Characterization of Highly Branched Poly(L-lysine)s Prepared via 1,4-Diaminobutane Initiated Ring-Opening Polymerization of Z-Lys NCA 1H

NMR integralsa

[I(Ha)+I(Hb) + Mne I(Ha) I(Hb) I(Hc)b I(Hd)c I(Hc)+I(Hd)]/I(Hc) DPd (g/mol) DParmf core 20.4 G0 89 G1 164 G2 445

5 11 25

2 4 8 16

n.d. n.d. n.d. n.d.

11.21 24.62 22.87 30.38

22 3370 98 14410 180 26820 490 71060

11 20 11 19

a 1H

NMR integrals are reported relative to that of the methine protons of the N-terminal R-amino acids, which was set equal to the theoretical number assuming quantitative NR,N-diFmoc Lys coupling and Fmoc deprotection. b Theoretical value, assuming quantitative NR,N-diFmoc Lys coupling and Fmoc removal. c In contrast to the n-hexylamine based polypeptides the methine signals of the R-amino acids moieties adjacent to the initiator could not be identified when 1,4-diaminobutane was used as initiator. d Total number average degree of polymerization calculated from the 1H NMR results. e Total number average molecular weight calculated from the 1H NMR results. f Number average degree of polymerization of the peptide arms added during the last NCA polymerization step.

polypeptide of a particular generation allows an estimation of the total number average degree of polymerization and the average length per branch. These data, which are summarized in Table 6, also illustrate a rapid increase in molecular weight of these highly branched polypeptides with increasing generation. Also in this case, the average degree of polymerization per arm remains fairly constant throughout the synthesis, indicating the absence of major side-chain reactions and structural imperfections. Conclusions In this contribution, we have described the synthesis of several novel highly branched poly(L-lysine)s. The synthesis of these polypeptides is based on a strategy reported earlier by Birchall and North10 and involves a repetitive sequence of NCA ring-opening polymerization, end functionalization, and deprotection reactions. Following this strategy, highly branched poly(L-lysine)s with molecular weights up to ∼33 kDa could be prepared in a few straightforward steps. GPC and 1H NMR experiments provided proof for the structural integrity of the materials and supported the proposed highly branched topology. Although they are neither monodisperse nor structurally uniform, these highly branched poly(Llysine)s may be an attractive alternative to the perfect poly(L-lysine)s dendrimers whose synthesis involves a large number of reaction steps and laborious purification. Currently, we are exploring the potential of these polypeptides as synthetic vectors for gene therapy and as scaffolds for the generation of multiple antigen peptides. The central question in these studies will be to which extent the heterogeneous nature of the highly branched polypeptides affects their properties in comparison with perfect dendrimers.15

Acknowledgment. This work was financially supported by the Fonds der Chemischen Industrie and the Deutsche Forschungsgemeinschaft (Emmy Noether-Program, KL 1049/ 2). The authors are grateful to Professor Klaus Mu¨llen for his generous support and continuous interest. References and Notes (1) For a recent review, see: Stiriba, S.-E.; Frey, H.; Haag, R. Angew. Chem., Int. Ed. 2002, 41, 1329-1334. (2) (a) Tam, J. P.; Spetzler, J. C. Methods Enzymol. 1997, 289, 612637. (b) Tam, J. P. J. Immunol. Methods 1996, 196, 17-32. (3) See e.g.: (a) Nicolle, G. M.; To´th, EÄ .; Schmitt-Willich, H.; Radu¨chel, B.; Merbach, A. E. Chem. Eur. J. 2002, 8, 1040-1048. (b) Jacques, V.; Desreux, J. F. Top. Curr. Chem 2002, 221, 123-164. (4) See e.g.: (a) Ohsaki, M.; Okuda, T.; Wada, A.; Hirayama, T.; Niidome, T.; Aoyagi, H. Bioconjugate Chem. 2002, 13, 510-517. (b) Choi, J. S.; Joo, D. K.; Kim, C. H.; Kim, K.; Park, J. S. J. Am. Chem. Soc. 2000, 122, 474-480. (5) The first report on peptide dendrimers was published by Denkewalter et al. and described the synthesis of peptide dendrimers by stepwise coupling and deprotection of NR,N-di-(tert-butoxycarbonyl)-Llysine: (a) Denkewalter, R. G.; Kolc, J. F.; Lukasavage, W. J. (Allied Corp.), U.S. US 4,410,688, 1983 [Chem. Abstr. 1984, 100, 103907p]. (b) Denkewalter, R. G.; Kolc, J. F.; Lukasavage, W. J. (Allied Corp.), U.S. US 4,289,872, 1981 [Chem. Abstr. 1985, 102, 79324q]. (c) Aharoni, S. M.; Murthy, N. S. Polym. Commun. 1983, 24, 132136. (6) For reviews on the synthesis and polymerization of R-amino acid N-carboxyanhydrides, see: (a) Kricheldorf, H. R. R-Amino acid N-carboxyanhydrides and related heterocycles; Springer-Verlag: Berlin, 1987. (b) Deming, T. J. J. Polym. Sci., Part A: Polym. Chem. 2000, 38, 3011-3018. (7) See e.g.: (a) Sela, M.; Fuchs, S.; Arnon, R. Biochem. J. 1962, 85, 223-235. (b) Yaron, A.; Berger, A. Biochim. Biophys. Acta 1965, 107, 307-332. (c) Sakamoto, M.; Kuroyanagi, Y. J. Polym. Sci.: Polym. Chem. Ed. 1978, 16, 1107-1122. (d) Sakamoto, M.; Kuroyanagi, Y.; Sakamoto, R. J. Polym. Sci.: Polym. Chem. Ed. 1978, 16, 2001-2017. (e) Mezo¨, G.; Kajta´r, J.; Szekerke, M.; Hudecz, F. Biopolymers 1997, 42, 719-730. (f) Mezo¨, G.; Reme´nyi, J.; Kajta´r, J.; Barna, K.; Gaa´l, D.; Hudecz, F. J. Controlled Release 2000, 63, 81-95. (8) (a) Aoi, K.; Tsutsumiuchi, K.; Yamamoto, A.; Okada, M. Tetrahedron 1997, 53, 15415-15427. (b) Aoi, K.; Hatanaka, A.; Tsusumiuchi, K.; Okada, M.; Imae, T. Macromol. Rapid Commun. 1999, 20, 378382. (b) Klok, H.-A.; Rodrı´guez Herna´ndez, J.; Becker, S.; Mu¨llen, K. J. Polym. Sci., Part A: Polym. Chem. 2001, 10, 1572-1583. (9) Poche´, D. S.; Moore, M. J.; Bowles, J. L. Synth. Commun. 1999, 29, 843-854. (10) Birchall, A. C.; North, M. Chem. Commun. 1998, 1335-1336. (11) (a) Fmoc solid-phase peptide synthesis; Chan, W. C., White, P. D., Eds.; Oxford University Press: Oxford, 2000. (b) Albericio, F.; Carpino, L. A. Methods Enzymol. 1997, 289, 104-126. (12) Bergeron, R. J.; McManis, J. S. J. Org. Chem. 1988, 53, 31083111. (13) Benishia, D.; Berger, A. J. Org. Chem. 1952, 17, 1564-1570. (14) 1H NMR resonances were assigned according to: Van Dijk-Wolthuis, W. N. E.; van de Water, L.; van de Wetering, P.; van Steenbergen, M. J.; Kettenes-van den Bosch, J. J.; Schuyl, W. J. W.; Hennink, W. E. Macromol. Chem. Phys. 1997, 198, 3893-3906. (15) For a recent paper that discusses the effect of macromolecular topology on the transfection efficiency of branched copolymers of histidine and lysine prepared via solid-phase synthesis, see: Chen, Q.-R.; Zhang, L.; Stass, S. A.; Mixson, A. J. Nucleic Acids Res. 2001, 29, 1334-1340.

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Highly Branched Poly(l-lysine) - American Chemical Society

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