An oral carbon monoxide-releasing molecule protects against acute hyperhemolysis in sickle cell disease.

ABSTRACT: Acute hyperhemolysis is a severe life-threatening complication in patients with sickle cell disease (SCD) that may occur during delayed hemolytic transfusion reaction (DHTR), or vaso-occlusive crises associated with multiorgan failure. Here, we developed in vitro and in vivo animal models to mimic endothelial damage during the early phase of hyperhemolysis in SCD. We then used the carbon monoxide (CO)-releasing molecule CORM-401 and examined its effects against endothelial activation, damage, and inflammation inflicted by hemolysates containing red blood cell membrane-derived particles. The in vitro results revealed that CORM-401: (1) prevented the upregulation of relevant proinflammatory and proadhesion markers controlled by the NF-κB enhancer of activated B cells, and (2) abolished the expression of the nuclear factor erythroid-2-related factor 2 (Nrf2) that regulates the inducible antioxidant cell machinery. We also show in SCD mice that CORM-401 protects against hemolysate-induced acute damage of target organs such as the lung, liver, and kidney through modulation of NF-κB proinflammatory and Nrf2 antioxidant pathways. Our data demonstrate the efficacy of CORM-401 as a novel therapeutic agent to counteract hemolysate-induced organ damage during hyperhemolysis in SCD. This approach might be considered as possible preventive treatment in high-risk situations such as patients with SCD with history of DHTR.

A synthetic porphyrin as an effective dual antidote against carbon monoxide and cyanide poisoning.

Simultaneous poisoning by carbon monoxide (CO) and hydrogen cyanide is the major cause of mortality in fire gas accidents. Here, we report on the invention of an injectable antidote against CO and cyanide (CN-) mixed poisoning. The solution contains four compounds: iron(III)porphyrin (FeIIITPPS, F), two methyl-ß-cyclodextrin (CD) dimers linked by pyridine (Py3CD, P) and imidazole (Im3CD, I), and a reducing agent (Na2S2O4, S). When these compounds are dissolved in saline, the solution contains two synthetic heme models including a complex of F with P (hemoCD-P) and another one of F with I (hemoCD-I), both in their iron(II) state. hemoCD-P is stable in its iron(II) state and captures CO more strongly than native hemoproteins, while hemoCD-I is readily autoxidized to its iron(III) state to scavenge CN- once injected into blood circulation. The mixed solution (hemoCD-Twins) exhibited remarkable protective effects against acute CO and CN- mixed poisoning in mice (~85% survival vs. 0% controls). In a model using rats, exposure to CO and CN- resulted in a significant decrease in heart rate and blood pressure, which were restored by hemoCD-Twins in association with decreased CO and CN- levels in blood. Pharmacokinetic data revealed a fast urinary excretion of hemoCD-Twins with an elimination half-life of 47 min. Finally, to simulate a fire accident and translate our findings to a real-life scenario, we confirmed that combustion gas from acrylic cloth caused severe toxicity to mice and that injection of hemoCD-Twins significantly improved the survival rate, leading to a rapid recovery from the physical incapacitation.

Inclusion Complex Formation with Tetra-PEGylated Tetraphenylporphyrin and Face-to-Face Cyclodextrin Dimer through Unprecedented Molecular Threading.

Herein, a host-guest inclusion complex formation between tetra-PEGylated tetraphenylporphyrin with a per-O-methylated cyclodextrin (CD) dimer through the molecular threading process that is physically unexpected to occur is described. Although the molecular size of the PEGylated porphyrin is much greater than that of the CD dimer, the sandwich-type porphyrin/CD dimer 1 : 1 inclusion complex was spontaneously formed in water. The ferrous porphyrin complex binds O2 reversibly in aqueous solution, which functions as an artificial O2 carrier in vivo. Pharmacokinetic study using rats revealed that the inclusion complex showed a long circulation in blood in contrast to the complex without PEG. We further demonstrate the unique host-guest exchange reaction from the PEGylated porphyrin/CD monomer 1/2 inclusion complex to the 1/1 complex with the CD dimer through the complete dissociation process of the CD monomers.

Development of carbon monoxide-releasing molecules conjugated to polysaccharides (glyco-CORMs) for delivering CO during obesity.

Metal carbonyls have been developed as carbon monoxide-releasing molecules (CO-RMs) to deliver CO for therapeutic purposes. The manganese-based CORM-401 has been recently reported to exert beneficial effects in obese animals by reducing body weight gain, improving glucose metabolism and reprogramming adipose tissue towards a healthy phenotype. Here, we report on the synthesis and characterization of glyco-CORMs, obtained by grafting manganese carbonyls on dextrans (70 and 40 kDa), based on the fact that polysaccharides facilitate the targeting of drugs to adipose tissue. We found that glyco-CORMs efficiently deliver CO to cells in vitro with higher CO accumulation in adipocytes compared to other cell types. Oral administration of two selected glyco-CORMs (5b and 6b) resulted in CO accumulation in various organs, including adipose tissue. In addition, glyco-CORM 6b administered for eight weeks elicited anti-obesity and positive metabolic effects in mice fed a high fat diet. Our study highlights the feasibility of creating carriers with multiple functionalized CO-RMs.

Carbon Monoxide, a Retrograde Messenger Generated in Postsynaptic Mushroom Body Neurons, Evokes Noncanonical Dopamine Release.

Dopaminergic neurons innervate extensive areas of the brain and release dopamine (DA) onto a wide range of target neurons. However, DA release is also precisely regulated. In Drosophila melanogaster brain explant preparations, DA is released specifically onto α3/α'3 compartments of mushroom body (MB) neurons that have been coincidentally activated by cholinergic and glutamatergic inputs. The mechanism for this precise release has been unclear. Here we found that coincidentally activated MB neurons generate carbon monoxide (CO), which functions as a retrograde signal evoking local DA release from presynaptic terminals. CO production depends on activity of heme oxygenase in postsynaptic MB neurons, and CO-evoked DA release requires Ca2+ efflux through ryanodine receptors in DA terminals. CO is only produced in MB areas receiving coincident activation, and removal of CO using scavengers blocks DA release. We propose that DA neurons use two distinct modes of transmission to produce global and local DA signaling.SIGNIFICANCE STATEMENT Dopamine (DA) is needed for various higher brain functions, including memory formation. However, DA neurons form extensive synaptic connections, while memory formation requires highly specific and localized DA release. Here we identify a mechanism through which DA release from presynaptic terminals is controlled by postsynaptic activity. Postsynaptic neurons activated by cholinergic and glutamatergic inputs generate carbon monoxide, which acts as a retrograde messenger inducing presynaptic DA release. Released DA is required for memory-associated plasticity. Our work identifies a novel mechanism that restricts DA release to the specific postsynaptic sites that require DA during memory formation.

FRET-Based In-Cell Detection of Highly Selective Supramolecular Complexes of meso-Tetraarylporphyrin with Peptide/BODIPY-Modified Per-O-Methyl-ß-Cyclodextrins.

Artificial supramolecular systems capable of self-assembly and that precisely function in biological media are in high demand. Herein, we demonstrate a highly specific host-guest-pair system that functions in living cells. A per-O-methyl-ß-cyclodextrin derivative (R8-B-CDMe ) bearing both an octaarginine peptide chain and a BODIPY dye was synthesized as a fluorescent intracellular delivery tool. R8-B-CDMe was efficiently taken up by HeLa cells through both endocytosis and direct transmembrane pathways. R8-B-CDMe formed a 2 : 1 inclusion complex with tetrakis(4-sulfonatophenyl)porphyrin (TPPS) as a guest molecule in water, from which fluorescence resonance energy transfer (FRET) from R8-B-CDMe to TPPS was observed. The FRET phenomenon was clearly detected in living cells using confocal microscopy techniques, which revealed that the formed supramolecular R8-B-CDMe /TPPS complex was maintained within the cells. The R8-B-CDMe cytotoxicity assay revealed that the addition of TPPS counteracts the strong cytotoxicity (IC50 =16 µM) of the CD cavity due to complexation within the cells. A series of experiments demonstrated the bio-orthogonality of the supramolecular per-O-methyl-ß-CD/tetraarylporphyrin host-guest pair in living cells.

Functional Myoglobin Model Composed of a Strapped Porphyrin/Cyclodextrin Supramolecular Complex with an Overhanging COOH That Increases O2/CO Binding Selectivity in Aqueous Solution.

A water-soluble strapped iron(III)tetraarylporphyrin (FeIIIPor-1) bearing two propylpyridinium groups at the side chains and a carboxylic acid group at the overhanging position of the strap was synthesized to mimic the function of myoglobin with the distal polar functionality in aqueous solution. FeIIIPor-1 forms a stable 1:1 inclusion complex with a per-O-methylated ß-cyclodextrin dimer having a pyridine linker (Py3OCD), providing a hydrophobic environment and a proximal fifth ligand to stabilize the O2-complex. The ferrous complex (FeIIPorCD-1) binds both O2 and CO in aqueous solution. The O2 and CO binding affinities (P1/2O2 and P1/2CO) and half-life time (t1/2) of the O2 complex of FeIIPorCD-1 are 6.3 and 0.021 Torr, and 7 h, respectively, at pH 7 and 25 °C. The control compound without the strap structure (FeIIPorCD-2) has similar oxygen binding characteristics (P1/2O2 = 8.0 Torr), but much higher CO binding affinity (P1/2CO = 3.8 × 10-4 Torr), and longer t1/2 (30 h). The O2 and CO kinetics indicate that the strapped structure in FeIIPorCD-1 inhibits the entrance of these gaseous ligands into the iron(II) center, as evidenced by lower konO2 and konCO values. Interestingly, the CO complex of FeIIPorCD-1 is significantly destabilized (relatively larger koffCO), while the koffO2 value is much smaller than that of FeIIPorCD-2, resulting in significantly increased O2/CO selectivity (reduced M value, where M = P1/2O2/P1/2CO = 320) in FeIIPorCD-1 compared to FeIIPorCD-2 (M = 21000).

PEGylated carboxyhemoglobin bovine (SANGUINATE) ameliorates myocardial infarction in a rat model.

Artificial oxygen (O2 ) carriers were reported to be protective in ischemia/reperfusion (I/R) in various organs including the heart. In the current study, 20 rats underwent ligation (MI) of the left anterior descending artery, were treated with 10 mL/kg of PEGylated carboxyhemoglobin bovine (SANGUINATE, S+, n = 10) or saline (S-, n = 10) 10 minutes after MI and daily thereafter for 3 days, and were followed by weekly echocardiography for 4 weeks, when they had left ventricular pressure volume relationship (PVR) analyses followed by necropsy. Echocardiography showed an increase in end-systolic dimension rather than end-diastolic dimension, preserved fractional shortening (36 vs. 26%, P < .01), and milder mitral regurgitation in S+ compared with S- rats. PVR revealed a milder increase in end-systolic volume, larger stroke volume (101 vs. 74 µL, P < .005) and cardiac output (33.4 vs. 23.8 mL/min, P = .004) in S+ rats in actual determination and under a wide range of standardized loading conditions 4 weeks after MI. Excised heart showed significantly limited area of MI (8.9 vs. 13.3%, P = .028). The results suggest that SANGUINATE in short-term repeated doses may accelerate weight recovery, preserving the myocardium, mitral competence, and cardiac function after MI. The mechanism of action and optimal treatment for MI remain to be studied.

Detection and Removal of Endogenous Carbon Monoxide by Selective and Cell-Permeable Hemoprotein Model Complexes.

Carbon monoxide (CO) is produced in mammalian cells during heme metabolism and serves as an important signaling messenger. Here we report the bioactive properties of selective CO scavengers, hemoCD1 and its derivative R8-hemoCD1, which have the ability to detect and remove endogenous CO in cells. HemoCD1 is a supramolecular hemoprotein-model complex composed of 5,10,15,20-tetrakis(4-sulfonatophenyl)porphinatoiron(II) and a per-O-methylated ß-cyclodextrin dimer having an pyridine linker. We demonstrate that hemoCD1 can be used effectively to quantify endogenous CO in cell lysates by a simple spectrophotometric method. The hemoCD1 assay detected ca. 260 pmol of CO in 106 hepatocytes, which was well-correlated with the amount of intracellular bilirubin, the final breakdown product of heme metabolism. We then covalently attached an octaarginine peptide to a maleimide-appended hemoCD1 to synthesize R8-hemoCD1, a cell-permeable CO scavenger. Indeed, R8-hemoCD1 was taken up by intact cells and captured intracellular CO with high efficiency. Moreover, we revealed that removal of endogenous CO by R8-hemoCD1 in cultured macrophages led to a significant increase (ca. 2.5-fold) in reactive oxygen species production and exacerbation of inflammation after challenge with lipopolysaccharide. Thus, R8-hemoCD1 represents a powerful expedient for exploring specific and still unidentified biological functions of CO in cells.

HemoCD as a Totally Synthetic Artificial Oxygen Carrier: Improvements in the Synthesis and O2 /CO Discrimination.

HemoCD, which is composed of an iron(II)porphyrin such as 5,10,15,20-tetrakis(4-sulfonatophenyl)porphinatoiron(II) (Fe(II)TPPS) and a cyclodextrin (CD) dimer having a pyridine linker, represents a synthetic hemoglobin (Hb) model compound that exhibits reversible oxygen (O2 ) binding ability in aqueous solution at an ambient temperature. Therefore, hemoCD has the potential to be used as a totally synthetic artificial oxygen carrier. In this article, we describe the improvements of hemoCD related to its synthesis and O2 /CO selectivity. The synthesis procedure of the CD dimer of hemoCD was re-examined, and the CD dimer was successively synthesized from inexpensive ß-CD with a 38% yield (three-steps), which enabled us to obtain the CD dimer in gram-quantities. The O2 /CO selectivity of hemoCD was also markedly improved using an iron(II)porphyrin having a carboxylate group at the distal site of hemoCD.

Iron(II)porphyrin-Cyclodextrin Supramolecular Complex as a Carbon Monoxide-Depleting Agent in Living Organisms.

The specific intermolecular interaction between an anionic tetraarylporphyrin and per-O-methylated ß-cyclodextrin (TMe-ß-CD) paved the way to produce a functional supramolecule that works as a strong carbon monoxide (CO)-depleting agent in living organisms. The supramolecular complex, hemoCD, that is composed of meso-tetrakis(4-sulfonatophenyl)porphinatoiron(II) and a TMe-ß-CD dimer linked by a pyridine linker, captured internal CO from carboxyhemoglobin during its circulation in the blood of animals. HemoCD thus produced the pseudo-knockdown (loss-of-functional) state of endogenous CO in the animals. This unique property led us to investigate the biological function of endogenous CO as a gaseous signal mediator in living systems. In this paper, we introduce our recent study on the hemoCD complex as a biological CO-depleting agent.

Esterification of 24S-OHC induces formation of atypical lipid droplet-like structures, leading to neuronal cell death.

The 24(S)-hydroxycholesterol (24S-OHC), which plays an important role in maintaining brain cholesterol homeostasis, has been shown to possess neurotoxicity. We have previously reported that 24S-OHC esterification by ACAT1 and the resulting lipid droplet (LD) formation are responsible for 24S-OHC-induced cell death. In the present study, we investigate the functional roles of 24S-OHC esters and LD formation in 24S-OHC-induced cell death, and we identify four long-chain unsaturated fatty acids (oleic acid, linoleic acid, arachidonic acid, and DHA) with which 24S-OHC is esterified in human neuroblastoma SH-SY5Y cells treated with 24S-OHC. Here, we find that cotreatment of cells with 24S-OHC and each of these four unsaturated fatty acids increases prevalence of the corresponding 24S-OHC ester and exacerbates induction of cell death as compared with cell death induced by treatment with 24S-OHC alone. Using electron microscopy, we find in the present study that 24S-OHC induces formation of LD-like structures coupled with enlarged endoplasmic reticulum (ER) lumina, and that these effects are suppressed by treatment with ACAT inhibitor. Collectively, these results illustrate that ACAT1-catalyzed esterification of 24S-OHC with long-chain unsaturated fatty acid followed by formation of atypical LD-like structures at the ER membrane is a critical requirement for 24S-OHC-induced cell death.

Feedback Response to Selective Depletion of Endogenous Carbon Monoxide in the Blood.

The physiological roles of endogenous carbon monoxide (CO) have not been fully understood because of the difficulty in preparing a loss-of-function phenotype of this molecule. Here, we have utilized in vivo CO receptors, hemoCDs, which are the supramolecular 1:1 inclusion complexes of meso-tetrakis(4-sulfonatophenyl)porphinatoiron(II) with per-O-methylated ß-cyclodextrin dimers. Three types of hemoCDs (hemoCD1, hemoCD2, and hemoCD3) that exhibit different CO-affinities have been tested as CO-depleting agents in vivo. Intraperitoneally administered hemoCD bound endogenous CO within the murine circulation, and was excreted in the urine along with CO in an affinity-dependent manner. The sufficient administration of hemoCD that has higher CO-affinity than hemoglobin (Hb) produced a pseudoknockdown state of CO in the mouse in which heme oxygenase-1 (HO-1) was markedly induced in the liver, causing the acceleration of endogenous CO production to maintain constant CO-Hb levels in the blood. The contents of free hemin and bilirubin in the blood plasma of the treated mice significantly increased upon removal of endogenous CO by hemoCD. Thus, a homeostatic feedback model for the CO/HO-1 system was proposed as follows: HemoCD primarily removes CO from cell-free CO-Hb. The resulting oxy-Hb is quickly oxidized to met-Hb by oxidant(s) such as hydrogen peroxide in the blood plasma. The met-Hb readily releases free hemin that directly induces HO-1 in the liver, which metabolizes the hemin into iron, biliverdin, and CO. The newly produced CO binds to ferrous Hb to form CO-Hb as an oxidation-resistant state. Overall, the present system revealed the regulatory role of CO for maintaining the ferrous/ferric balance of Hb in the blood.

Synthesis of 24(S)-hydroxycholesterol esters responsible for the induction of neuronal cell death.

We synthesized several candidates of 24(S)-hydroxycholesterol (24S-OHC) esters, which are involved in neuronal cell death, through catalysis with acyl-CoA:cholesterol acyltransferase-1 (ACAT-1). We studied the regioselectivity of the acylation of the secondary alcohol at the 3- or 24-position of 24S-OHC. The appropriate saturated and unsaturated long-chain fatty acids were esterified with the protected 24S-OHC and then de-protected to afford the desired esters at a satisfactory yield. We then confirmed by HPLC monitoring that the retention times of four esters of 24S-OHC, namely 3-oleate, 3-linoleate, 3-arachidonoate and 3-docosahexaenoate, were consistent with those of 24S-OHC esters observed in 24S-OHC-treated SH-SY5Y cells.

Intramolecular direct oxygen transfer from oxoferryl porphyrin to a sulfide bond.

A 1:1 supramolecular complex (met-hemoCD) of 5,10,15,20-tetrakis(4-sulfonatophenyl)porphyrinatoiron(III) (Fe(III)TPPS) with a per-O-methylated ß-cyclodextrin dimer having a -SCH2PyCH2S- (Py = pyridin-3,5-diyl) linker (Py3CD) reacted rapidly with hydrogen peroxide or cumene hydroperoxide in an aqueous solution forming two types of hydroperoxo or alkylperoxo intermediates, ROO-Fe(III)(OH(-))PCD and ROO-Fe(III)(Py)PCD, which underwent rapid homolysis to the corresponding ferryloxo species, namely, O═Fe(IV)(OH(-))PCD and O═Fe(IV)(Py)PCD, respectively. For the O═Fe(IV)(OH(-))PCD species, the iron-oxo oxygen facing the linker gradually transferred to the nearby sulfide bond on the linker, forming the sulfoxidized Py3CD (Py3CD-O)/Fe(II)TPPS complex, which then bound dioxygen in air forming an oxy-ferrous complex, O2-Fe(II)TPPS/Py3CD-O. In contrast, the O═Fe(IV)(Py)PCD species, in which the iron-oxo oxygen was located on the opposite side of the sulfide bond on the linker across the porphyrin ring, was reduced to the resting state (met-hemoCD) by the surroundings without any oxidation of the Py3CD linker.

Clickable bisreactive small gold nanoclusters for preparing multifunctionalized nanomaterials: application to photouncaging of an anticancer molecule.

In this study, we successfully synthesized a small-sized gold nanocluster (2 nm) coated with homogeneous tripeptides bearing azido and amino groups that enable facile multifunctionalizations. Using sodium phenoxide to reduce tetrachloroauric(iii) acid in the presence of the cysteine-containing tripeptide, we efficiently prepared the gold nanoclusters without damaging the azido group. We then utilized this clickable bisreactive nanocluster as a versatile platform for synthesizing multifunctionalized gold nanomaterials. The resulting nanoclusters were conjugated with an anticancer compound connected to an indolizine moiety for photoinduced uncaging, a photodynamic therapy agent acting as a photosensitizer for uncaging, and a cyclic RGD peptide. The cytotoxicity of the multifunctionalized gold nanoclusters was demonstrated through red light irradiation of human lung cancer-derived A549 cells treated with the synthesized nanomaterials. The significant cytotoxicity exhibited by the cells underscores the potential utility of this method in advanced cancer therapies.

An orally active carbon monoxide-releasing molecule enhances beneficial gut microbial species to combat obesity in mice.

Carbon monoxide (CO), a gaseous signaling molecule, has shown promise in preventing body weight gain and metabolic dysfunction induced by high fat diet (HFD), but the mechanisms underlying these effects are largely unknown. An essential component in response to HFD is the gut microbiome, which is significantly altered during obesity and represents a target for developing new therapeutic interventions to fight metabolic diseases. Here, we show that CO delivered to the gut by oral administration with a CO-releasing molecule (CORM-401) accumulates in faeces and enriches a variety of microbial species that were perturbed by a HFD regimen. Notably, Akkermansia muciniphila, which exerts salutary metabolic effects in mice and humans, was strongly depleted by HFD but was the most abundant gut species detected after CORM-401 treatment. Analysis of bacterial transcripts revealed a restoration of microbial functional activity, with partial or full recovery of the Krebs cycle, ß-oxidation, respiratory chain and glycolysis. Mice treated with CORM-401 exhibited normalization of several plasma and fecal metabolites that were disrupted by HFD and are dependent on Akkermansia muciniphila's metabolic activity, including indoles and tryptophan derivatives. Finally, CORM-401 treatment led to an improvement in gut morphology as well as reduction of inflammatory markers in colon and cecum and restoration of metabolic profiles in these tissues. Our findings provide therapeutic insights on the efficacy of CO as a potential prebiotic to combat obesity, identifying the gut microbiota as a crucial target for CO-mediated pharmacological activities against metabolic disorders.

Cellular uptake of octaarginine-conjugated tetraarylporphyrin included by per-O-methylated ß-cyclodextrin.

This paper describes the synthesis, structural characterization and cellular uptake of a supramolecular 1 : 2 inclusion complex of meso-tetraphenylporphyrin having an octaarginine peptide chain (R8-TPP) and heptakis(2,3,6-tri-O-methyl)-ß-cyclodextrin (TMe-ß-CD). R8-TPP was synthesized by 2 approaches: (1) on-resin conjugation of the N-terminal of octaarginine with 5-(4-carboxyphenyl)-10,15,20-triphenylporphyrin, followed by cleavage from the resin, and (2) Michael addition reaction between 5-[4-(3-maleimidopropylamido)phenyl]-10,15,20-triphenylporphyrin and cysteine-octaarginine peptide (Cys-Arg8). The R8-TPP obtained from both the approaches formed stable inclusion complexes with TMe-ß-CD by which non-substituted phenyl groups at the 10- and 20-positions were included to form trans-type 1 : 2 inclusion complexes. The complexation prevented the self-aggregation of R8-TPP, which resulted in the solubilisation of R8-TPP in aqueous media. A cellular uptake study using HeLa cells showed that R8-TPP complexed with TMe-ß-CD in a serum-free medium was efficiently taken up by the cells and uniformly dispersed in the cytosol. In the serum-containing medium, the R8-TPP-TMe-ß-CD complex dissociated, and the serum protein bound R8-TPP. The R8-TPP-protein complex was localized in the endosomes of the cells. The cytosol-dispersed R8-TPP showed a higher photo-induced cytotoxicity than its endosome-trapped counterpart.

Spontaneous reduction of iron(III)porphyrin to iron(II)porphyrin-CO complex in mouse circulation.

Iron(II/III)porphyrin/cyclodextrin inclusion complexes serve as hemoprotein models in vivo. Here we showed the iron(III)porphyrin complex to be spontaneously reduced to its iron(II) state in mouse circulation. The reduced complex bound endogenous CO from carboxyhemoglobin, which was followed by urinary excretion. The natural reduction system was found to be effective for synthetic heme-model compounds.

Modification of a dioxygen carrier, hemoCD, with PEGylated dendrons for extension of circulation time in the bloodstream.

A supramolecular diatomic receptor, hemoCD, was modified with PEGylated dendrons to extend its circulation time in the bloodstream. The core component was 4-oxo-4-[[4-(10,15,20-tris(4-sulfonatophenyl)-21H,23H-porphin-5-yl)phenyl]amino]butanoic acid (Por-COOH). The building block of the dendrons was Fmoc-4-amino-4-(2-carboxyethyl)heptanedioic acid (FmocTA), which was condensed with α-amino-ω-methoxy-poly(ethylene glycol) (PEG(5000)-NH(2)) to yield an FmocG1-dendron. After deprotection, the G1-dendron was condensed with Por-COOH to yield G1-Por. A precursor (FmocNA) of an FmocG2-dendron was prepared via a condensation reaction of 4-amino-4-(2-t-butoxycarbonylethyl)heptanedioic acid di-t-butyl ester (TA-E) with FmocTA followed by hydrolysis of the resultant nona-carboxylic acid nona-t-butyl ester. Condensation of FmocNA with PEG(5000)-NH(2) yielded an FmocG2-dendron. After deprotection, the G2-dendron was condensed with Por-COOH to yield G2-Por. The ferrous complexes of G1- and G2-Pors formed stable 1:1 inclusion complexes with Py3CD, a per-O-methylated ß-cyclodextrin dimer with a pyridine linker, in aqueous solution yielding supramolecular complexes designated as G1-hemoCD and G2-hemoCD, respectively. Both G1- and G2-hemoCDs bound molecular oxygen, with the O(2) affinities (P(1/2)) of hemoCD, G1-, and G2-hemoCDs at pH 7.4 and 37 °C being 22, 20, and 20 Torr, respectively. The modification of hemoCD with the dendrons did not cause destabilization of the O(2) adducts via autoxidation, as indicated by their half-lives (t(1/2)) of 6.8, 6.1, and 5.5 h for hemoCD, G1-, and G2-hemoCDs, respectively. The blood concentration-time curves of G1- and G2-hemoCDs injected into the bloodstream of rats exhibited two phases, with the half-lives of the fast and slow decays being 0.45 and 5.3 h, respectively, for G1-hemoCD, and 0.20 and 12.8 h, respectively, for G2-hemoCD. The half-lives of hemoCD were 0.02 and 0.50 h, respectively. The circulation time of hemoCD was markedly extended by its modification with the PEGylated dendrons, which was very effective in protecting hemoCD against opsonization for uptake by the reticuloendothelial system.