In a previous report we demonstrated that the blood compatibility of poly(ether urethane) (PEU) was improved by grafting phosphorylcholine (PC) groups on the surface. The improved blood compatibility was indicated by decreased platelet adsorption/activation and reduced thrombin formation at the polymer surface in experiments in which the surfaces were contacted with platelet-rich plasma in vitro. In the present study, we investigated the effect of grafted PC groups at a PEU surface on protein and phospholipid adsorption. Adsorption of human fibrinogen (Fg), human serum albumin (Alb), human high-molecular-weight kininogen (HMWK), and dioleoyl phosphatidylcholine (DOPC) vesicles was measured by ellipsometry. For this purpose, thin PEU films were cast on silicon wafers. The polymer film was photochemically modified with a PC-containing aryl azide. The presence of PC groups on the polymer surface was demonstrated by ESCA (Electron Spectroscopy for Chemical Analysis). The hydrophilicity of the polymer surface increased by the surface modification, as indicated by a decrease of the contact angle from 59 degrees before to 43 degrees after modification. Our data show that the presence of PC groups has little effect on the adsorption of proteins to a PEU surface. The highest adsorption was observed for Fg (0.49 microgram/cm2 on PC-modified PEU and 0.50 microgram/cm2 on PEU), followed by HMWK (0.28 microgram/cm2 on both PC-modified PEU and PEU), and Alb (0.16 microgram/cm2 on PC-modified PEU and 0.18 microgram/cm2 on PEU). Protein adsorption was further studied on a "biomembrane-like" DOPC bilayer formed on hydrophilic silicon. We found no protein adsorption on this DOPC bilayer. The adsorption of small unilamellar DOPC vesicles on the polymer surfaces amounted to about 0.06 microgram/cm2 (corresponding to circa 30% of monolayer coverage) and was similar for PC-modified PEU and PEU. Despite this partial surface coverage, preadsorbed DOPC on the polymer surface diminished the subsequent adsorption of proteins considerably. These results show that the mere presence of phosphorylcholine groups on a PEU surface is insufficient to suppress protein adsorption. The highly ordered structure of natural phospholipid bilayers seems to be required to suppress protein adsorption effectively.

Phosphorylcholine groups attached to polymer surfaces are known to improve hemocompatibility. A photochemical method is presented to couple phosphorylcholine-containing aryl azides to poly(etherurethane) surfaces (PEUs). Two aryl azides that consist of a photoactivatable 4-azidobenzoyl group, a short spacer chain, and a phosphorylcholine endgroup were synthesized. The two compounds differ only in the type of spacer used: triethylene glycol for compound 1 and hexanediol for compound 2. These compounds were physically adsorbed to PEU surfaces. Upon UV irradiation, reactive intermediates are formed that react with nucleophilic groups on the polymer surface. The modified surfaces showed decreased underwater contact angles, indicating that hydrophilic phosphorylcholine groups are present at the surface. ESCA measurements showed the presence of phosphorus and positively charged nitrogen atoms in the outermost polymer layers (analyzed depth about 50 A), which is a strong indication of the presence of phosphorylcholine groups. Hemocompatibility in vitro was tested with thrombin generation assays and platelet adhesion tests. In thrombin generation assays the clotting time of platelet-rich plasma in contact with the polymer surface is determined. Clotting times were clearly prolonged for the modified surfaces. Surfaces modified with compound 2 showed slightly higher clotting times than those modified with compound 1. Repeated surface modification with compound 2 further increased the clotting time. For the tested surfaces an increase in the clotting time corresponds to an increase in the concentration of phosphorylcholine groups at the surface (as measured by ESCA and contact angle). Platelet adhesion studies with scanning electron microscopy demonstrated that fewer platelets (showing less activation) adhered to the modified surfaces than to the unmodified polyurethane.

A fluorescent aryl azide [N-methyl-N-(2-hydroxyethyl)-5-(dimethylamino)naphthalene-1-sulfonamide 4-azidobenzoate] (2) was synthesized as a model compound for aryl azides that are used for the photochemical surface modification of poly(ether urethanes) (PEUs). We are using this method of surface modification in our search for artificial surfaces with optimized blood compatibility. As a part of this work, two pivotal questions are now addressed: (i) what is the surface density of coupled molecules that can be reached by this method and (ii) is binding to the surface really covalent? After the photochemical coupling of 2 to a PEU surface, the ester bonds of the coupled molecules were selectively hydrolyzed with 0.1 M NaOMe/MeOH and the concentration of fluorescent molecules in solution determined spectrofluorometrically. Since not all of the fluorescent molecules were removed by this procedure, an additional experiment was performed (in which the polymer was completely dissolved). It was found that 25 nmol/cm 2 had coupled to the outermost polymer layers and 7.7 nmol/cm 2 to polymer layers more within the material (unreachable for the NaOMe/MeOH reagent). In another experiment a modified PEU sheet was dissolved in THF, after which MeOH was added to precipitate it again. From the fact that no fluorescence was detected in solution it could be concluded that all of the fluorescent molecules were indeed covalently coupled to the polymer surface.

The alkaline hydrolysis of the P-chiral ds-nucleoside 3',5'-cyclic aryl [180] monophosphates 4a-c and of the unlabeled analogs 3a-c was studied. Hydrolysis of the 180-labeled phosphate triesters 4a-c yielded three products: 3',5'-cyclic [180]phosphate diester, S'-acyclic and [180]phosphate diester, and 3'-acyclic aryl [180]phosphate diester. The stereochemistry of the formation of the 3',5'-cyclic [180] phosphate diester was determined by means of methylating the hydrolysis products with methyl iodide. The formation of the 3',5'-cyclic [180] phosphate diester during hydrolysis of compounds 4a and 4c proceeds with 17% inversion of configuration at phosphorus, whereas 40% inversion is found during hydrolysis of 4b. Inversion of configuration indicates the existence of a PV-TBP with a diequatorially located dioxaphosphorinane ring. Retention of configuration (83% for 4a and 4c, and 60% for 4b) can be explained in terms of Berry pseudorotation. The formation of the S'-acyclic aryl [180]phosphate diester during hydrolysis of compounds 4a and 4c proceeds with about 50% inversion of configuration at phosphorus, whereas formation of the 3'-acyclic aryl [180]phosphate diester proceeds with an inversion/retention ratio of 88:12 or 12:88 for 4a and 79:21 or 21:79 for 4c. It is clear that Berry pseudorotation takes place during hydrolysis of the 3',5'-cyclic phosphate triesters 4a-c. This is in contrast with earlier hydrolysis studies on 3',5'-cyclic phosphate diesters proceeding without Berry pseudorotation, leading to complete inversion of configuration at phosphorus. Because of the very small amounts of 3'and S'-acyclic aryl [180] phosphate diesters formed during the hydrolysis reaction of compound 4b, the stereochemistry could not be determined. The hydrolysis reactions, which have been studied on the unlabeled compounds 3a-c, obey second-order kinetics. Changing the ribose ring to a deoxyribose ring or changing the adenine base to thymine in the 3',5'-cyclic phosphate triester does not dramatically influence the second-order reaction rate constant. However, the nature of the P-OR substituent significantly influences the reaction rate. The 3',5'-cyclic phosphate triester with p-nitrophenoxy as substituent hydrolyzes approximately 18 times ( = 294 K) faster than the corresponding triester with phenoxy as substituent.

Chem. 71, 855 (1993). This study is focussed on 2'-0-methyl-cis-adenosine 3',5'-cyclic methyl monophosphate (cis-4; cis describes the relationship between OMe (bound to phosphorus) and the adenine base), which is regarded a model for enzyme-bound cyclic adenosine monophosphate (CAMP). In this complex the negative phosphate Eharge is shielded in part via complexation with cationic sites on the enzyme surface. We report synthesis, crystal structure, solution conformation and N6-H...N7 hydrogen bonds in such a way that infinite one-dimensional chains are formed. This hydrogen bond scheme is very similar to that observed in the shucture of 2'-deoxy-3',5'-di-0-acetyl adenosine. An additional hydrogen bond is formed between methanol, incorporated in the crystal structure, and the adenine base. The conformational preferences of the cis-4 dissolved in methanol have been determined with 400 MHz 'H NMR. It is found that the conformations observed in the solid and solution states are practically the same. Hydrolysis of the title compound yields two acyclic phosphate diesters (a 3'-and a 5'-phosphate diester). 'The intermediates formed during the hydrolysis reaction are most likely five-coordinated phosphorus (P") compounds with a trigonal bipyramidal geometry and an equatorial-axial located 3'3'-dioxaphosphorinane ring.NIEK L.H.L. BROEDERS, ARTHUR P. VAN 855 (1993).La prksente etude est orientte vers le monophosphate cyclique-3',5' de methyle de la 2'-0-methyl-cis-adenosine (cis-4; cis se refkre 2 la relation entre le OMe (lie au phosphore) et la base adenine) qui est consider6 comme un modble pour le monophosphate cyclique de l'adenosine liee a un enzyme (CAMP). Dans ce complexe, la charge negative du phosphate est masqute en partie par une complexation avec les sites cationiques sur la surface de l'enzyme. On rapporte la synthese et des etudes sur la determination de la structure, de la conformation en solution (400 MHz, RMN du 'H) et des aspects cinetique-mecanisme de l'hydrolyse alcaline du produit cis-4. La produit de solvatation de cis-4 avec du methanol cristallise dans le Froupe d'espace orthorhombique P2,2,2, avec a = 8,170(2), b = 9,249 (1) d'une fagon telle qu'il y a formation de chaines unidimensionnelles infinies. Cette arrangement par liaisons hydrogbnes est trks semblable 2 celui observe dans la structure de la 2'-dCsoxy-3',5'-di-0-acetyl adenosine. Une liaison hydrogbne additionnelle se forme avec le methanol, incorpore dans la structure cristalline, et la base adenine. Faisant appel a la RMN du 'H, a 400 MHz, on a determine les preferences conformationnelles du produit cis-4 en solution dans le methanol. On a trouve que les conformations observees en phase solide et en solution sont les m&mes. L'hydrolyse du compose mentionnk dans le titre fournit deux esters acycliques de l'acide phosphorique (un diester 3'-et un diester 5'-phosphate). Ces intermediaires qui se forment durant la reaction d'hydrolyse sont probablement des composCs pentacoordines du phosphore (P") de geometric bipyramidale trigonale et...