H-bond cooperativity: polarisation effects on secondary amides.

Formation of a H-bond with an amide carbonyl oxygen atom increases the strength of subsequent H-bonds formed by the amide NH, due to polarisation of the bond. The magnitude of this effect has been quantified by measuring association constants for the formation of 1 : 1 complexes of 2-hydroxylbenzamides with tri-n-butyl phosphine oxide. In 2-hydroxybenzamides, there is an intramolecular H-bond between the phenol OH group and the carbonyl oxygen atom. Comparison of the association constants measured for compounds with and without the 2-hydroxy group allows direct quantification of the effect of the intramolecular H-bond on the H-bond donor properties of the amide NH group. Substituents were used to modulate the strength of the intramolecular and intermolecular H-bonds. The presence of an intramolecular H-bond increases the strength of the intermolecular H-bond by more than one order of magnitude in n-octane solution. The increase in the H-bond donor parameter used to describe the amide NH group is directly proportional to the H-bond donor parameter of the phenol OH group that makes the intramolecular H-bond. These polarisation effects will lead to substantial cooperativity in complex systems that feature networks of non-covalent interactions, and the measurements described here provide a quantitative basis for understanding such phenomena.

Polarisation effects on the solvation properties of alcohols.

Alcohol solvents are significantly more polar than expected based on the measured H-bonding properties of monomeric alcohols in dilute solution. Self-association of alcohols leads to formation of cyclic aggregates and linear polymeric chains that have a different polarity from the alcohol monomer. Cyclic aggregates are less polar than the monomer, and the chain ends of linear polymers are more polar. The solvation properties of alcohols therefore depend on the interplay of these self-association equilibria and the equilibria involving interactions with solutes. Twenty-one different molecular recognition probes of varying polarity were used to probe the solvation properties of alkane-alcohol mixtures across a wide range of different solvent compositions. The results allow dissection of the complex equilibria present in these systems. Formation of a H-bond between two alcohol molecules leads to polarisation of the hydroxyl groups, resulting in an increase in binding affinity for subsequent interactions with the unbound donor and acceptor sites. The H-bond donor parameter (α) for these sites increases from 2.7 to 3.5, and the H-bond acceptor parameter (ß) increases from 5.3 to 6.9. Polarisation is a short range effect limited to the first H-bond in a chain, and formation of subsequent H-bonds in longer chains does not further increase the polarity of chain ends. H-bond donor sites involved in a H-bond are unavailable for further interactions, because the formation of a bifurcated three-centre H-bond is three orders of magnitude less favourable than formation of a conventional two-centre H-bond. These findings are reproduced by quantum chemical calculations of the molecular electrostatic potential surfaces of alcohol aggregates. Thus, the overall solvation properties of alcohols depend on the speciation of different aggregates, the polarities of these species and the polarities of the solutes. At low alcohol concentrations, polar solutes are solvated by alcohol monomers, and at higher alcohol concentrations, solutes are solvated by the more polar chain ends of linear polymers. The less polar cyclic aggregates are less important for interactions with solutes. Similar behavior was found for ten different alcohol solvents. Tertiary alcohols are marginally less polar solvents than primary alcohols, due to steric interactions that destabilises the formation of polymeric aggregates leading to lower concentrations of polar chain ends. One alcohol with an electron-withdrawing substituent was studied, and this solvent showed slightly different behavior, because the H-bond donor and acceptor properties are different.

Enhanced Chelate Cooperativity in Polar Solvents.

High-throughput UV-vis titrations in combination with chemical double-mutant cycles (DMCs) have been used to study the competition of a polar solvent for formation of intramolecular H-bonds. Twenty-four different zinc porphyrin-pyridine complexes were investigated in mixtures of toluene and phenol. DMCs were used to determine effective molarities (EM) for the formation of intramolecular phenol-amide H-bonds as a function of solvent composition. The values of EM increase by an order of magnitude with increasing concentrations of the more polar solvent, phenol. Phenol solvates the amide groups on the ligands strongly, increasing the steric bulk and destabilizing the complexes. These adverse steric interactions are removed when intramolecular H-bonds are formed and therefore provide an increased driving force for formation of cooperative interactions. The result is that the effects of competitive interactions with polar solvents that reduce binding affinity are attenuated to a significant extent by a corresponding increase in EM in multivalent complexes.

Quantification of the effect of conformational restriction on supramolecular effective molarities.

The association constants for a family of 96 closely related zinc porphyrin-pyridine ligand complexes have been measured in two different solvents, toluene and 1,1,2,2-tetrachloroethane (TCE). The zinc porphyrin receptors are equipped with phenol side arms, which can form intramolecular H-bonds with ester or amide side arms on the pyridine ligands. These association constants were used to construct 64 chemical double mutant cycles, which measure the free energy contributions of intramolecular H-bonding interactions to the overall stability of the complexes. Measurement of association constants for the corresponding intermolecular H-bonding interactions allowed determination of the effective molarities (EM) for the intramolecular interactions. Comparison of ligands that feature amide H-bond acceptors and ester H-bonds at identical sites on the ligand framework show that the values of EM are practically identical. Similarly, the values of EM are practically identical in toluene and in TCE. However, comparison of two ligand series that differ by one degree of torsional freedom shows that the values of EM for the flexible ligands are an order of magnitude lower than for the corresponding rigid ligands. This observation holds for a range of different supramolecular architectures with different degrees of receptor-ligand complementarity and suggests that in general the cost of freezing a rotor in supramolecular complexes is of the order of 5 kJ/mol.

Steric desolation enhances the effective molarities of intramolecular H-bonding interactions.

Free energy contributions due to intramolecular phosphonate diester-phenol H-bonds have been measured for 20 different supramolecular architectures in cyclohexanone solution. High throughput UV/Vis titrations were used in combination with chemical double mutant cycles to dissect out the contributions of different functional group interactions to the stabilities of over 100 different zinc porphyrin-pyridine ligand complexes. These complexes have previously been characterised in toluene and in 1,1,2,2-tetrachloroethane (TCE) solution. Intramolecular ester-phenol H-bonds that were measured in these less polar solvents are too weak to be detected in cyclohexanone, which is a more competitive solvent. The stability of the intermolecular phosphonate diester-phenol H-bond in cyclohexanone is an order of magnitude lower than in TCE and two orders of magnitude lower than in toluene. As a consequence, only seven of the twenty intramolecular phosphonate diester-phenol interactions that were previously measured in toluene and TCE could be detected in cyclohexanone. The effective molarities (EM) for these intramolecular interactions are different in all three solvents. Determination of the EM accounts for solvent effects on the strengths of the individual H-bonding interactions and the zinc porphyrin-pyridine coordination bond, so the variation in EM with solvent implies that differences in the solvation shells make significant contributions to the overall stabilities of the complexes. The results suggest that steric effects lead to desolvation of bulky polar ligands. This increases the EM values measured in TCE, because ligands that fail to replace the strong interactions made with this solvent are unusually weakly bound compared with ligands that make intramolecular H-bonds.

Influence of H-bond strength on chelate cooperativity.

Intermolecular complexes formed between metalloporphyrins and pyridine ligands equipped with multiple H-bond donors and acceptors have been used to measure the free energy contributions due to intramolecular ether-phenol H-bonding in the 24 different supramolecular architectures using chemical double mutant cycles in toluene. The ether-phenol interactions are relatively weak, and there are significant populations of partially bound states where between zero and four intramolecular H-bonds are made in addition to the porphyrin-ligand coordination interaction. The complexes were analyzed as ensembles of partially bound states to determine the effective molarities for the intramolecular interactions by comparison with the corresponding intermolecular ether-phenol H-bonds. The properties of the ether-phenol interactions were compared with phosphonate diester-phenol interactions in a closely related ligand system, which has more powerful H-bond acceptor oxygens positioned at the same location on the ligand framework. This provides a comparison of the properties of weak and strong H-bonds embedded in the same 24 supramolecular architectures. When the product of the intermolecular association constant and the effective molarity KEM > 1, there is a linear increase in the free energy contribution due to H-bonding with log EM, because the intramolecular interactions contribute fully to the stability of the complex. When KEM < 1, the H-bonded state is not significantly populated, and there is no impact on the overall stability of the complex. Intermolecular phosphonate diester-phenol H-bonds are 2 orders of magnitude stronger than ether-phenol H-bonds in toluene, so for the phosphonate diester ligand system, 23 of the 24 supramolecular architectures make intramolecular H-bonds. However, only 8 of these architectures lead to detectable H-bonding in the ether ligand system. The other 15 complexes have a suitable geometry for formation of H-bonds, but the ether-phenol interaction is not strong enough to overcome the reorganization costs associated with making intramolecular contacts, i.e., KEM < 1 for the ether ligands, and KEM > 1 for the phosphonate diester ligands. The values of EM measured for two different types of H-bond acceptor are linearly correlated, which suggests that EM is a property of the supramolecular acrchitecture. However, the absolute value of EM for an intramolecular phosphonate diester H-bond is about 4 times lower than the corresponding value for an intramolecular ether-phenol interaction embedded in the same supramolecular framework, which suggests that there may be some interplay of K and EM.

Assessment of the genetic effects of polymorphisms in the osteoprotegerin gene, TNFRSF11B, on serum osteoprotegerin levels and carotid plaque vulnerability.

BACKGROUND AND PURPOSE: Osteoprotegerin (OPG) is a secretory glycoprotein which belongs to the tumor necrosis factor receptor family. Various mechanisms have been suggested by which calcification might alter atherosclerotic plaque stability, but the significance of this intimal calcification is controversial. High concentrations of OPG have been associated with the presence of vascular and cardiovascular diseases. This study was designed to assess the association between gene polymorphisms of the OPG gene (TNFRSF11B), the serum OPG level, and plaque stability in patients with carotid atherosclerosis. METHODS: We studied 177 patients with internal carotid artery stenosis who underwent carotid endarterectomy and also 303 controls. Carotid endarterectomy samples removed from patients were assessed by immunohistochemistry. Concentrations of OPG were measured and gene polymorphisms were examined by polymerase chain reaction and restriction enzyme analysis and were compared, initially between patients with carotid atherosclerosis and controls, and subsequently between stable and unstable carotid plaques. RESULTS: We found that the GG genotype of the T245G polymorphism, the CC genotype of the T950C polymorphism, and the CC genotype of the G1181C polymorphism were significantly higher in patients with carotid plaque than in controls (21.5% versus 10.9% , P<0.01; 15.8% versus 7.6%, P<0.01; and 20.3% versus 10.9%, P<0.01, respectively) and that these polymorphisms were associated with high serum OPG levels (4.02 [3.07] versus 2.94 [1.81] pmol/L; P<0.01), which were significantly higher in patients with unstable atherosclerotic plaques (5.86 [4.02] versus 3.53 [1.87] pmol/L; P<0.01). CONCLUSIONS: The TNFRSF11B gene polymorphisms studied are associated with high serum OPG levels and might be potential markers for plaque instability.

Multiple thromboembolism with multiple causes in a 69-year-old woman: a case report.

INTRODUCTION: Aggressive, recurrent embolisms require accurate etiologic diagnosis. We describe the case of a 69-year-old Italian Caucasian woman with recurrent arterial embolisms in whom several sources and triggers of thrombosis were detected. CASE PRESENTATION: The patient, a 69-year-old Italian Caucasian woman, presented with a systemic embolism that was initially attributed to atrial fibrillation. The recurrence of embolisms despite anti-thrombotic therapy prompted a re-evaluation of the clinical presentation. New potential causes of thrombosis emerged in this patient, including thrombocytosis associated with the JAK2 V617F mutation and the very rare mural thrombosis of the descending aorta. A mural thrombus in the pulmonary artery was detected contiguous with the aortic mural thrombosis, raising the possibility of a clinically silent ductus Botalli as the initiating event. The patient was treated with warfarin, aspirin, hydroxyurea, and surgery. CONCLUSIONS: The diagnosis was achieved via systematic use of imaging procedures and reconsideration of blood tests performed to explore the diagnosis of thrombosis. This allowed a deeper and more detailed analysis of the case beyond the conventional approach, which would have aimed to identify one cause for the condition at hand, in this case, atrial fibrillation. The broader approach that we used resulted in the diagnosis of multiple embolisms from multiple sites and multiple causes.

Dissection of complex molecular recognition interfaces.

The synthesis of a family of zinc porphyrins and pyridine ligands equipped with peripheral H-bonding functionality has provided access to a wide range of closely related supramolecular complexes featuring between zero and four intramolecular H-bonds. An automated UV/vis titration system was used to characterize 120 different complexes, and these data were used to construct a large of number of different chemical double mutant cycles to quantify the intramolecular H-bonding interactions. The results probe the quantitative structure-activity relationship that governs cooperativity in the assembly of complex molecular recognition interfaces. Specifically, variations in the chemical structures of the complexes have allowed us to change the supramolecular architecture, conformational flexibility, geometric complementarity, the number and nature of the H-bond interactions, and the overall stability of the complex. The free energy contributions from individual H-bonds are additive, and there is remarkably little variation with architecture in the effective molarity for the formation of intramolecular interactions. Intramolecular H-bonds are not observed in complexes where they are geometrically impossible, but there are no cases where excellent geometric complementarity leads to very high affinities. Similarly, changes in conformational flexibility seem to have limited impact on the values of effective molarity (EM). The major variation that was found for all of the 48 intramolecular interactions that were examined using double mutant cycles is that the values of EM for intramolecular carboxylate ester-phenol H-bonds (200 mM) are an order of magnitude larger than those found for phosphonate diester-phenol H-bonds (30 mM). The corresponding intermolecular phosphonate diester-phenol H-bonds are 2 orders of magnitude more stable than carboxylate ester-phenol H-bonds, and the large differences in EM may be due to some kind of compensation effect, where the stronger H-bond is harder to make, because it imposes tighter constraints on the geometry of the complex.

Cooperativity in multiply H-bonded complexes.

The free energy of complexation of supramolecular complexes containing phenol-carbamate H-bonds is an additive function of the number of H-bonds, with a constant increment of 6 kJ mol(-1) per interaction in carbon tetrachloride.

White blood cell scintigraphy in the diagnosis of infection of endovascular prostheses within the first month after implantation.

BACKGROUND: 99mTc-HMPAO-labeled leukocyte scintigraphy (WBCs) is useful in the diagnosis of vascular graft infection. However, false positives can occur in the early postoperative period. There are no data concerning the usefulness of WBCs in detecting endovascular prosthesis infection during this period. The aim of our study was to assess the reliability of WBCs in the diagnosis of early infection after endovascular prosthesis implantation. MATERIAL/METHODS: 23 patients treated with endovascular grafts were submitted to preoperative diagnostic screening and to 3 WBCs (1 week before, 1 week after, and 1 month after surgery). After the last WBCs, all patients underwent a 14-month follow-up (range 6-37 months, median 12 months). RESULTS: Three patients died, at 3, 4 and 5 months after surgery respectively. At the end of the follow-up, the presence of infection was demonstrated in one patient. Of the 70 WBCs performed, 4 gave positive results. No positive scintigraphies were obtained in the preoperative period. A positive scintigraphy was found one week after surgery in a patient who showed lymphorrhage at the site of surgical access. In the same patient, the scintigraphic result returned to normal at 1-month scintigraphy. At 12-month follow-up the patient did not present signs of infection. Three positive scintigraphies were obtained in the same patient. In this patient clinical symptoms of graft infection became evident about 19 months after surgery. WBCs was repeated and showed extension of the infection. The infection was confirmed at surgery. CONCLUSIONS: WBCs do not provide false-positives in the first month after endovascular prosthesis implantation.

Aortoiliac stent graft infection: current problems and management.

Aortic stent graft infection is uncommon. Most cases have been described anecdotaly in single-case reports. After observing one case in our experience, we decided to review the literature and contact centers performing endovascular aortic repair to determine the frequency, risk factors, and current treatment of stent graft infection. The literature was reviewed and the authors of identified articles were contacted for further information. In addition, 40 centers specializing in endovascular treatment were contacted by means a dedicated questionnaire. A total of 65 aortic stent graft infections were identified, including 43 reported cases and 22 previously unpublished cases that were observed at specialized centers. Stent grafts were implanted in the aorta in 50 cases and in the iliac artery in 15 cases. The frequency of infection was 0.43%. The gender ratio was 4:1 (M:F). Twenty-three percent of patients had immunodeficiency factors. Placement was performed in an interventional radiology suite in 62.5% of cases and in a sterile operating theater in 37.5%. Also, 35.5% of patients underwent other vascular procedures during the course of study and 29.2% stent grafts benefited from adjuvant endovascular procedures. Infection was classified as low grade in 35.4% of patients and high grade in 64.6%. Thirty-one percent of infections were associated with aortoenteric fistula. The offending microorganism was Staphylococcus aureus in 54.5% of cases. Treatment was conservative in 18% of cases and surgical in 82%. Surgical treatment consisted of stent graft removal followed by either extraanatomical bypass (59.5%) or in situ prosthetic reconstruction (40.5%). Mortality was 18% overall, 36.4% after conservative treatment and 14% after surgical treatment ( p = 0.083). Mortality was 16% after surgical treatment with extraanatomical bypass vs. 5.8% surgical treatment with in situ reconstruction. From these results we conclude that stent graft infection is an uncommon occurrence associated with poorly defined risk factors. Surgical treatment with complete excision of the infected stent graft followed by in situ reconstruction provides the best outcome. Establishment of a multicenter register to record such complications is needed to confirm the findings of this study.

Vitamin E supplementation in patients with carotid atherosclerosis: reversal of altered oxidative stress status in plasma but not in plaque.

OBJECTIVE: Oxidative stress is believed to play a pivotal role in the initiation and progression of atherosclerosis. We analyzed whether vitamin E supplementation influences oxidative stress in plasma and atherosclerotic plaques of patients with severe atherosclerosis. METHODS AND RESULTS: In 16 patients who were candidates for carotid endarterectomy and in 32 age- and sex-matched controls, plasma levels of 7beta-hydroxycholesterol, 7-ketocholesterol, cholesterol, and vitamin E were measured. Patients were randomly allocated to standard treatment with or without 900 mg/d vitamin E. After 6 weeks of treatment, the reported variables were measured in plasma and plaques. The plasma vitamin E/cholesterol ratio was significantly lower in patients than in controls (3.05+/-0.6 versus 6.3+/-1.7 micromol/mmol cholesterol, P<0.001). Plasma 7beta-hydroxycholesterol was significantly higher in patients than in controls (5.0+/-1.04 versus 4.4+/-0.6 ng/mL, P<0.05). Patients who were given vitamin E supplementation showed a significant increase of plasma vitamin E with concomitant decrease of 7beta-hydroxycholesterol. Conversely, no treatment dependence was observed in oxysterol or vitamin E content of plaques. CONCLUSIONS: An imbalance between oxidative stress and antioxidant status is present in patients with advanced atherosclerosis. Vitamin E supplementation improves this imbalance in plasma but not in plaques.

Endovascular graft infection: preliminary results of an international enquiry.

PURPOSE: To investigate the frequency of aortoiliac endovascular graft infections and seek the main factors influencing their development. METHODS: To augment personal experience (1 case), a questionnaire was sent to 40 international centers of vascular and endovascular surgery. The literature was also reviewed to collect data on infections developing in endovascular grafts. RESULTS: The survey (85% response rate) and literature review identified 62 cases of infected endovascular grafts (0.4% frequency of endograft infection). In 22 (35%) patients, the infection manifested initially with vague symptoms only, but 41 (65%) patients eventually presented with abdominal abscess, groin fistula, and septic embolization. Common bacteria, such as Staphylococcus aureus, were identified as the cause of most infections (54.5%). The majority (49, 79%) of the 62 patients were treated surgically; 11 (17.7%) patients received conservative therapy (no therapeutic data in 2 patients). Overall mortality was 27.4% (17/62), and operative mortality was 16.3% (8/49). Conservative treatment led to a mortality rate of 36.4% (4/11). The mean follow-up for all patients was 47.8 weeks. Possible factors influencing the development of an infection were secondary adjunctive procedures, immunosuppression, treatment of false aneurysms, and infected central lines. CONCLUSIONS: Infected endovascular grafts are an urgent problem that has been heretofore underestimated and will probably increase as follow-up lengthens. New techniques should be sought to expedite the diagnosis, and an international registry should be set up to provide validated data.