**2.1 Polyamine-dialkyl phosphate conjugates via a synthetic intermediate 1**

Our preliminary idea for the syntheses of polyamine-dicetyl phosphate (DCP) conjugates was to use the bromoethylated compound, **2**, as a synthetic intermediate via condensation of DCP and 2-bromoethanol (Scheme 1) by using 1,3,5-triisopropylbenzenesulfonyl chloride, TPSCl (Uragami, Miyake & Regen, 2000; Tanaka, et al., 1987). Instead of the expected adduct, **2**, however, we obtained another product having high *Rf* value, 0.97 (*Rf* = 0.56 for DCP by eluting CHCl3/MeOH/H2O = 13/6/1, v/v/v). The resulting product is stable enough to be isolated by column chromatography. SIMS indicates almost double molecular mass, 1075.4 (546.9 for DCP). The observed IR absorption band at 952 cm-1 is assignable to the P–O–P stretching mode. The 31P NMR spectrum of the product, whose signals appear at δ = -12.27 and -0.13 ppm, is clearly distinguishable from that of DCP having a signal at δ = 2.15 ppm. This evidence, taken together, leaves no doubt that the product is dimerized DCP in anhydrous form, **1**, [(C64H133O7P2)+ calcd 1075.9] connected via P–O–P bonding. There are some prior examples in the synthesis of pyrophosphate derivatives bearing small alkyl moieties, methyl, ethyl, propyl, and butyl (Cullis, Kaye & Trippett, 1987), however, the dimerized anhydride, **1**, bearing phospholipid-like long alkyl chains is an unprecedented compound. This compound is a convenient synthetic intermediate for formimg phosphoramidate bonds, as described below.

The anhydride **1** readily react with amines, *e.g.,* spermidine, spermine, and even polymer, PEI(1800), to form the phosphoramidate, P–N bond, providing the corresponding adducts shown in Scheme 1. The reactivity toward these nucleophiles indicates that the anhydrous compound **1** is potentially a good synthetic intermediate for making polyamine-dialkyl phosphate conjugates via the P–N bond. When reacted with spermine, for example, the adduct **DCP-spm** readily forms, concomitantly with the loss of DCP. The 31P NMR signal of compound **DCP-spm** is shifted downfield, to 9.76 ppm compared with that of DCP at 2.15 ppm, indicating the formation of the P–N bond in the compound. The down-field-shifted signal is attributed to the lower electronegativity of the nitrogen atom in spermine relative to the oxygen atom in DCP. Anhydride **1** is a useful synthetic intermediate because it is (1) stable yet reactive with amino groups, (2) very simple and easy to prepare, and (3) produced in high yield (~90 %). Furthermore, modification on the hydrophobic chain is easy when various dialkyl phosphates were used. These compounds could hence provide new category of polycationic lipids. Gall et al. have developed cationic lipid derivatives bearing P-N linkage, which possess gene transfer activity (Gall et al., 2010).

#### **2.2 Polyamine-diacylphosphatidic acid conjugates via a synthetic intermediate 3**

To diacylphosphatidic acid can be attached 2-bromoethanol with TPSCl, giving an intermediate **3**. When this reacted with an amino group of polyamine compounds via nucleophilic substitution reaction, a corresponding polyamine conjugate was formed as shown in Scheme 1 (Dewa et al., 2004b). Purified products can be afforded by column chromatography using an amino group-modified silicagel.

compounds can be prepared via two-step reactions without such protection/deprotection reactions. The synthetic schemes are shown in Scheme 1, where two types of polyaminelipid conjugate; dialkyl phosphate- and diacylphosphatidic acid-based compounds, are described. In this section, we showed a synthetic strategy for polyamine-lipid conjugates via

Our preliminary idea for the syntheses of polyamine-dicetyl phosphate (DCP) conjugates was to use the bromoethylated compound, **2**, as a synthetic intermediate via condensation of DCP and 2-bromoethanol (Scheme 1) by using 1,3,5-triisopropylbenzenesulfonyl chloride, TPSCl (Uragami, Miyake & Regen, 2000; Tanaka, et al., 1987). Instead of the expected adduct, **2**, however, we obtained another product having high *Rf* value, 0.97 (*Rf* = 0.56 for DCP by eluting CHCl3/MeOH/H2O = 13/6/1, v/v/v). The resulting product is stable enough to be isolated by column chromatography. SIMS indicates almost double molecular mass, 1075.4 (546.9 for DCP). The observed IR absorption band at 952 cm-1 is assignable to the P–O–P stretching mode. The 31P NMR spectrum of the product, whose signals appear at

= -12.27 and -0.13 ppm, is clearly distinguishable from that of DCP having a signal at

2.15 ppm. This evidence, taken together, leaves no doubt that the product is dimerized DCP in anhydrous form, **1**, [(C64H133O7P2)+ calcd 1075.9] connected via P–O–P bonding. There are some prior examples in the synthesis of pyrophosphate derivatives bearing small alkyl moieties, methyl, ethyl, propyl, and butyl (Cullis, Kaye & Trippett, 1987), however, the dimerized anhydride, **1**, bearing phospholipid-like long alkyl chains is an unprecedented compound. This compound is a convenient synthetic intermediate for formimg

The anhydride **1** readily react with amines, *e.g.,* spermidine, spermine, and even polymer, PEI(1800), to form the phosphoramidate, P–N bond, providing the corresponding adducts shown in Scheme 1. The reactivity toward these nucleophiles indicates that the anhydrous compound **1** is potentially a good synthetic intermediate for making polyamine-dialkyl phosphate conjugates via the P–N bond. When reacted with spermine, for example, the adduct **DCP-spm** readily forms, concomitantly with the loss of DCP. The 31P NMR signal of compound **DCP-spm** is shifted downfield, to 9.76 ppm compared with that of DCP at 2.15 ppm, indicating the formation of the P–N bond in the compound. The down-field-shifted signal is attributed to the lower electronegativity of the nitrogen atom in spermine relative to the oxygen atom in DCP. Anhydride **1** is a useful synthetic intermediate because it is (1) stable yet reactive with amino groups, (2) very simple and easy to prepare, and (3) produced in high yield (~90 %). Furthermore, modification on the hydrophobic chain is easy when various dialkyl phosphates were used. These compounds could hence provide new category of polycationic lipids. Gall et al. have developed cationic lipid derivatives bearing P-N

**2.2 Polyamine-diacylphosphatidic acid conjugates via a synthetic intermediate 3**  To diacylphosphatidic acid can be attached 2-bromoethanol with TPSCl, giving an intermediate **3**. When this reacted with an amino group of polyamine compounds via nucleophilic substitution reaction, a corresponding polyamine conjugate was formed as shown in Scheme 1 (Dewa et al., 2004b). Purified products can be afforded by column

δ=

**2.1 Polyamine-dialkyl phosphate conjugates via a synthetic intermediate 1** 

facile routes (Dewa et al., 2004a,b, 2010).

phosphoramidate bonds, as described below.

linkage, which possess gene transfer activity (Gall et al., 2010).

chromatography using an amino group-modified silicagel.

δ

Fig. 1. Transfection efficacy of polyamine-DCP (A and B) and polyamine-DPPA conjugates (C) on VSMC, in the absence (A and C) and the presence of 20% of FBS (B). The ratios of compound/β-galactosidase plasmid DNA (w/w) were 1.5/1, 3/1, and 6/1, respectively.
