An approach based on electrochemistry to differentiate between phosphorylated and nonphosphorylated amino acid analogues adsorbed on gold is presented. Analogues of serine, threonine, and tyrosine, containing thiohexadecyl headgroups, were synthesized and assembled on gold, and the surface capacitance was evaluated using electrochemical impedance spectroscopy. A procedure for deprotection of tert-butyl phosphate protecting groups, on the monolayer, is also described. Characterizations of the assembled analogues by cyclic voltammetry, infrared spectroscopy, and ellipsometry are used to confirm the insulating properties of the monolayers and the outcome of surface modifications. The results from cyclic voltammetry show good insulating properties for the monolayers even after phosphate deprotection. The infrared measurements reveal well-ordered monolayers, and the thickness from ellipsometry is in good agreement with expectations from molecular modeling. The impedance experiments show a capacitance increase up to 0.6 μF/cm² as phosphate groups are introduced. The results in this study indicate the possibility of using a surface chemical and impedance spectroscopy approach to detect the kinase/phosphatase activity and kinetics involved in phosphorylation reactions.

The synthesis of a series of thiols containing phosphorylated and non-phosphorylated serine, threonine, and tyrosine amino acid residues is described. The synthesized molecules, based on 3-mercaptopropionic acid, were assembled onto gold and subsequently characterized using infrared reflection-absorption spectroscopy, ellipsometry, X-ray photoelectron spectroscopy, and contact angle goniometry. The ellipsometric analysis indicates that they form densely packed and well-oriented monolayers on gold, with thicknesses that are in good agreement with estimated values from space-filling models. The bulky and space-demanding phosphorylated threonine analog was, however, found to be an exception. The increase in layer thickness when adding a phosphate group to the threonine is only 35% of that observed for the two other analogs. A detailed infrared examination of the influence of cation coordination to the phosphorylated serine analog using calcium and magnesium reveals structural similarities to those of the inorganic phosphate compound calcium hydroxy apatite. We furthermore discuss the application of these monolayers as soft templates for biomineralization.

Electrochemical impedance spectroscopy has been used to evaluate the change in interracial capacitance upon calcium and magnesium coordination to a phosphate-modified electrode. The phosphate electrode was prepared via immobilization of phosphorylated, thiol-containing, serine analogues onto gold. Upon subjection to calcium and magnesium, a substantial drop in capacitance was observed. Magnesium displayed the largest influence on the capacitance: a 27% capacitance drop was observed upon introduction of a 1 mM solution of magnesium ions. The lowered capacitance is a result of a change in the potential and charge distribution at the film/electrolyte interface as the cations coordinate to the phosphate groups. Moreover, the relationship between electrode potential and capacitance has been investigated and reveals a significant difference between monovalent and divalent cations. As complementary information, infrared reflection absorption spectra of the phosphorylated monolayer having different counterions are presented. The results reported in this paper indicate that the phosphorylated amino acid analogue monolayers could be used in investigations of the biochemically important coordination of calcium and magnesium to phosphates and phosphorylated amino acids.

Strategies to form supported lipid assemblies on organophosphate- and organosulfate-monolayer-modified gold surfaces are described. By varying surface treatment and the Mg2+ (Ca2+) content in a solution containing phosphatidylcholine vesicles, we demonstrate (i) efficient formation of supported phosphatidylcholine bilayers (SPBs), (ii) formation of supported nonruptured phosphatidylcholine vesicles, and (iii) reduced phosphatidylcholine vesicle adsorption. Thus, by simply varying the solution conditions, the system can be tuned to controlled formation of either a SPB, supported nonruptured vesicles, or a surface with fairly low coverage of nonruptured vesicles. The profound effects induced on the system by Mg2+ and Ca2+ are assigned to a combination of ion-coordination to the surface, ion-association to the lipid headgroups, and osmotic pressure.

New efficient routes to enantiopure phospholipids, starting from (S)-glycidol, are described. Lysophosphatidic acids and phosphatidic acids were obtained in good overall yields from (S)-glycidol, in only three and four steps, respectively. Moreover, the strategy can also be used to produce phosphatidylcholines in three steps. Using dialkylphosphoramidites, (S)-glycidol was phosphorylated to give (R)-1-O-glycidyl dialkyl phosphates. Regiospecific epoxide opening, using hexadecanol or cesium palmitate, followed by phosphate deprotection, provided lysophosphatidic acids. 2-O-Esterification prior to phosphate deprotection provided 1,2-O-diacyl and 1-O-alkyl-2-O-acyl phosphatidic acids. Phosphorylation of (S)-glycidol using phosphorus oxychloride followed by in situ treatment with choline tosylate produced (R)-glycidyl phosphocholine. Subsequent nucleophilic opening of the epoxide using cesium palmitate produced 1-O palmitoyl-sn-glycero-3-phosphocholine, which has been used in syntheses of phosphatidylcholines.