Synthesis and Anticancer Activity of Pentafluorobenzenesulfonamide Derivatives as Caspase-Dependent Apoptosis-Inducing Agents.

Arylsulfonamides are ubiquitous in a number of anticancer agents, and fluorine substitution on aromatic rings often improves drug profile. Herein, a series of novel pentafluorobenzenesulfonamide derivatives with different molecular scaffolds were readily synthesized and assessed for their antitumor activities against multiple cancer cell lines, including A549, HepG2, HuCCA-1, and MOLT-3. Dihydroimidazoline-containing analogue and its Diels-Alder cycloadducts exhibited enhanced cytotoxicity at micromolar range while the incorporation of other heterocyclic cores via nucleophilic substitution reaction resulted in diminished potency. Selected analogues were shown to induce the accumulation of cleaved forms of Casp-9, Casp-7 and PARP in cancer cells, indicating intrinsic apoptosis via a caspase-dependent process.

Sequential post-polymerization modification of a pentafluorophenyl ester-containing homopolymer: a convenient route to effective pH-responsive nanocarriers for anticancer drugs.

Recently, pH-responsive polymeric micelles have gained significant attention as effective carriers for anti-cancer drug delivery. Herein, pH-responsive polymeric micelles were constructed by a simple post-polymerization modification of a single homopolymer, poly(pentafluorophenyl acrylate) (PPFPA). The PPFPA was first subjected to modification with 1-amino-2-propanol yielding the amphiphilic copolymer of poly(pentafluorophenyl acrylate)-ran-poly(N-(2-hydroxypropyl acrylamide)). A series of amphiphilic random copolymers of different compositions could self-assemble into spherical micelles with a unimodal size distribution in aqueous solution. Then, 1-(3-aminopropyl)imidazole (API), a reagent to introduce charge conversional entities, was reacted with the remaining PPFPA segment in the micellar core resulting in API-modified micelles which can encapsulate doxorubicin (DOX), a hydrophobic anti-cancer drug. As monitored by dynamic light scattering, the API-modified micelles underwent disintegration upon pH switching from 7.4 to 5.0, presumably due to imidazolyl group protonation. This pH-responsiveness of the API-modified micelles was responsible for the faster and greater in vitro DOX release in an acidic environment than neutral pH. Cellular uptake studies revealed that the developed carriers were internalized into MDA-MB-231 cells within 30 min via endocytosis and exhibited cytotoxicity in a dose-dependent manner.

Reactive Conjugated Polymers for the Modulation of Islet Amyloid Polypeptide Assembly.

Misfolding and abnormal assembly of proteins cause many intractable diseases. The modulation of the assembly process of these proteins could contribute to understanding and controlling amyloid protein aggregation. Previous works focused mainly on the inhibition of the assembly process. To broaden the interaction modality of modulators with proteins for developing new modulators, in this work, we designed and synthesized two reactive poly ( p-phenylene vinylene) polymers, respectively, functionalized with N-hydroxysuccinimide ester (PPV-NHS) and pentafluorophenol ester (PPV-PFP), which exhibited the prevention or co-assembly effect on the aggregation process of islet amyloid polypeptide (IAPP). Cell assays demonstrated that both of the two polymers could effectively eliminate the cytotoxicity of IAPP. Moreover, PPV-NHS also could irreversibly disrupt preformed IAPP fibrils. We envision that PPV-NHS and PPV-PFP might offer a new design method for the modulation of protein assembly.

Single-Cell Imaging Using Radioluminescence Microscopy Reveals Unexpected Binding Target for [18F]HFB.

PURPOSE: Cell-based therapies are showing great promise for a variety of diseases, but remain hindered by the limited information available regarding the biological fate, migration routes and differentiation patterns of infused cells in trials. Previous studies have demonstrated the feasibility of using positron emission tomography (PET) to track single cells utilising an approach known as positron emission particle tracking (PEPT). The radiolabel hexadecyl-4-[18F]fluorobenzoate ([18F]HFB) was identified as a promising candidate for PEPT, due to its efficient and long-lasting labelling capabilities. The purpose of this work was to characterise the labelling efficiency of [18F]HFB in vitro at the single-cell level prior to in vivo studies. PROCEDURES: The binding efficiency of [18F]HFB to MDA-MB-231 and Jurkat cells was verified in vitro using bulk gamma counting. The measurements were subsequently repeated in single cells using a new method known as radioluminescence microscopy (RLM) and binding of the radiolabel to the single cells was correlated with various fluorescent dyes. RESULTS: Similar to previous reports, bulk cell labelling was significantly higher with [18F]HFB (18.75 ± 2.47 dpm/cell, n = 6) than 2-deoxy-2-[18F]fluoro-D-glucose ([18F]FDG) (7.59 ± 0.73 dpm/cell, n = 7; p ≤ 0.01). However, single-cell imaging using RLM revealed that [18F]HFB accumulation in live cells (8.35 ± 1.48 cpm/cell, n = 9) was not significantly higher than background levels (4.83 ± 0.52 cpm/cell, n = 12; p > 0.05) and was 1.7-fold lower than [18F]FDG uptake in the same cell line (14.09 ± 1.90 cpm/cell, n = 13; p < 0.01). Instead, [18F]HFB was found to bind significantly to fragmented membranes associated with dead cell nuclei, suggesting an alternative binding target for [18F]HFB. CONCLUSION: This study demonstrates that bulk analysis alone does not always accurately portray the labelling efficiency, therefore highlighting the need for more routine screening of radiolabels using RLM to identify heterogeneity at the single-cell level.

Radiosynthesis and 'click' conjugation of ethynyl-4-[(18)F]fluorobenzene--an improved [(18)F]synthon for indirect radiolabeling.

Reproducible methods for [(18)F]radiolabeling of biological vectors are essential for the development of new [(18)F]radiopharmaceuticals. Molecules such as carbohydrates, peptides and proteins are challenging substrates that often require multi-step indirect radiolabeling methods. With the goal of developing more robust, time saving, and less expensive procedures for indirect [(18)F]radiolabeling of such molecules, our group has synthesized ethynyl-4-[(18)F]fluorobenzene ([(18)F]2, [(18)F]EYFB) in a single step (14 ± 2% non-decay corrected radiochemical yield (ndc RCY)) from a readily synthesized, shelf stable, inexpensive precursor. The alkyne-functionalized synthon [(18)F]2 was then conjugated to two azido-functionalized vector molecules via CuAAC reactions. The first 'proof of principle' conjugation of [(18)F]2 to 1-azido-1-deoxy-ß-D-glucopyranoside (3) gave the desired radiolabeled product [(18)F]4 in excellent radiochemical yield (76 ± 4% ndc RCY (11% overall)). As a second example, the conjugation of [(18)F]2 to matrix-metalloproteinase inhibitor (5), which has potential in tumor imaging, gave the radiolabeled product [(18)F]6 in very good radiochemical yield (56 ± 12% ndc RCY (8% overall)). Total preparation time for [(18)F]4 and [(18)F]6 including [(18)F]F(-) drying, two-step reaction (nucleophilic substitution and CuAAC conjugation), two HPLC purifications, and two solid phase extractions did not exceed 70 min. The radiochemical purity of synthon [(18)F]2 and the conjugated products, [(18)F]4 and [(18)F]6, were all greater than 98%. The specific activities of [(18)F]2 and [(18)F]6 were low, 5.97 and 0.17 MBq nmol(-1), respectively.

A practical, automated synthesis of meta-[(18)F]fluorobenzylguanidine for clinical use.

Many neuroendocrine tumors, such as neuroblastoma (NB), arise from neural crest cells of the sympathetic nervous system. This nerve-like phenotype has been exploited for functional imaging using radioactive probes originally designed for neuronal and adrenal medullary applications. NB imaging with meta-[(123)I]iodobenzylguanidine ([(123)I]MIBG) is limited by the emissions of (123)I, which lead to poor image resolution and challenges in quantification of its accumulation in tumors. meta-[(18)F]Fluorobenzylguanidine ([(18)F]MFBG) is a promising alternative to [(123)I]MIBG that could change the standard of practice for imaging neuroendocrine tumors, but interest in this PET radiotracer has suffered due to its complex and inefficient radiosynthesis. Here we report a two-step, automated method for the routine production of [(18)F]MFBG by thermolysis of a diaryliodonium fluoride and subsequent acid deprotection. The synthesis was adapted for use on a commercially available synthesizer for routine production. Full characterization of [(18)F]MFBG produced by this route demonstrated the tracer's suitability for human use. [(18)F]MFBG was prepared in almost 3-fold higher yield than previously reported (31% corrected to end of bombardment, n = 9) in a synthesis time of 56 min with >99.9% radiochemical purity. Other than pH adjustment and dilution of the final product, no reformulation was necessary after purification. This method permits the automated production of multidose batches of clinical grade [(18)F]MFBG. Moreover, if ongoing clinical imaging trials of [(18)F]MFBG are successful, this methodology is suitable for rapid commercialization and can be easily adapted for use on most commercial automated radiosynthesis equipment.

Inhibition of N-type calcium channels by fluorophenoxyanilide derivatives.

A set of fluorophenoxyanilides, designed to be simplified analogues of previously reported ω-conotoxin GVIA mimetics, were prepared and tested for N-type calcium channel inhibition in a SH-SY5Y neuroblastoma FLIPR assay. N-type or Cav2.2 channel is a validated target for the treatment of refractory chronic pain. Despite being significantly less complex than the originally designed mimetics, up to a seven-fold improvement in activity was observed.

Synthesis and evaluation of 18F-labeled benzylguanidine analogs for targeting the human norepinephrine transporter.

PURPOSE: Both (131)I- and (123)I-labeled meta-iodobenzylguanidine (MIBG) have been widely used in the clinic for targeted imaging of the norepinephrine transporter (NET). The human NET (hNET) gene has been imaged successfully with (124)I-MIBG positron emission tomography (PET) at time points of >24 h post-injection (p.i.). (18)F-labeled MIBG analogs may be ideal to image hNET expression at time points of <8 h p.i. We developed improved methods for the synthesis of known MIBG analogs, [(18)F]MFBG and [(18)F]PFBG and evaluated them in hNET reporter gene-transduced C6 rat glioma cells and xenografts. METHODS: [(18)F]MFBG and [(18)F]PFBG were synthesized manually using a three-step synthetic scheme. Wild-type and hNET reporter gene-transduced C6 rat glioma cells and xenografts were used to comparatively evaluate the (18)F-labeled analogs with [(123)I]/[(124)I]MIBG. RESULTS: The fluorination efficacy on benzonitrile was predominantly determined by the position of the trimethylammonium group. The para-isomer afforded higher yields (75 ± 7%) than meta-isomer (21 ± 5%). The reaction of [(18)F]fluorobenzylamine with 1H-pyrazole-1-carboximidamide was more efficient than with 2-methyl-2-thiopseudourea. The overall radiochemical yields (decay-corrected) were 11 ± 2% (n = 12) for [(18)F]MFBG and 41 ± 12% (n = 5) for [(18)F]PFBG, respectively. The specific uptakes of [(18)F]MFBG and [(18)F]PFBG were similar in C6-hNET cells, but 4-fold less than that of [(123)I]/[(124)I]MIBG. However, in vivo [(18)F]MFBG accumulation in C6-hNET tumors was 1.6-fold higher than that of [(18)F]PFBG at 1 h p.i., whereas their uptakes were similar at 4 h. Despite [(18)F]MFBG having a 2.8-fold lower affinity to hNET and approximately 4-fold lower cell uptake in vitro compared to [(123)I]/[(124)I]MIBG, PET imaging demonstrated that [(18)F]MFBG was able to visualize C6-hNET xenografts better than [(124)I]MIBG. Biodistribution studies showed [(18)F]MFBG and (123)I-MIBG had a similar tumor accumulation, which was lower than that of no-carrier-added [(124)I]MIBG, but [(18)F]MFBG showed a significantly more rapid body clearance and lower uptake in most non-targeting organs. CONCLUSION: [(18)F]MFBG and [(18)F]PFBG were synthesized in reasonable radiochemical yields under milder conditions. [(18)F]MFBG is a better PET ligand to image hNET expression in vivo at 1-4 h p.i. than both [(18)F]PFBG and [(123)I]/[(124)I]MIBG.

Synthesis, characterization and DFT calculation of 4-fluorophenyl substituted tris(8-hydroxyquinoline)aluminum(III) complexes.

New 4-fluorophenyl substituted 8-hydroxyquinoline derivatives, 5-(4-fluorophenyl)quinolin-8-ol and 5,7-bis(4-fluorophenyl)quinolin-8-ol, were synthesized and characterized by spectroscopic methods. The aluminum complexes of 5-(4-fluorophenyl)quinolin-8-ol (AlQF) and of 5,7-bis(4-fluorophenyl)quinolin-8-ol (AlQF2) exhibit strong fluorescence emission centered at 525 nm and 530 nm respectively. The quantum yield of both complexes were enhanced compared to the parent tris(8-hydroxyquinolinato)aluminum(III) complex. Electronic structures and photophysical properties of the new complexes were investigated theoretically by ab initio and density functional theory (DFT) and time dependent DFT (TD-DFT). Geometries of the ground state (S0) and the first excited state (S1) of the new complexes were optimized at the B3LYP/6-31G(d) functional and configuration interaction singles (CIS) method respectively. The aryl substituents were found to contribute significantly to the frontier molecular orbitals (FMOs). We have observed that in both cases the lowest occupied molecular orbital (LUMO) energy decreases while the energy of the highest occupied molecular orbital is slightly increased. The most significant increase was observed for AlQF2.

Anticancer activities of some newly synthesized pyrazole and pyrimidine derivatives.

A series of pyrazolopyridine and pyridopyrimidine derivatives 2-6 were newly synthesized using 3,5-bisarylmethylene-1-methylpiperidone as the starting material. The anticancer activities of the synthesized compounds were evaluated using 59 different human tumor cell lines, representing cancers of CNS, ovary, renal, breast, colon, lung, leukemia, and melanoma, prostate as well as kidney. Some of the tested compounds, especially those with a fluorine substituent at the para-position in the phenyl ring and those with a pyridopyrimidine-2-thione with a free -NH or -SH, exhibited greater in vitro anti-tumor activities at low concentrations (log 10 [GI50] = -4.6) against the human tumor cell lines. Additionally, some of the compounds had moderate inhibitory effects on the growth of the cancer cell lines. The detailed synthesis, spectroscopic data and antitumor properties of the synthesized compounds are reported.

Efficient formation of heterodimers from peptides and proteins using unsymmetrical polyfluorophenyl esters of dicarboxylic acids.

An efficient method for the heteroconjugation of biomolecules carrying free amino groups was reported previously, where mixed polyfluorophenyl diesters of dicarboxylic acids with varied aliphatic chain length were shown to be efficient reagents for the conjugation of a variety of model biomolecules. The concept was based on the differential reactivity of the esters towards amines. The concept has now been further optimized, and a 2,6-difluorophenyl-pentafluorophenyl diester combination has been demonstrated to be the most efficient, both with respect to selectivity and to reaction rate. A pentafluorophenyl ester reacts faster with an amino group and requires a weaker base than a 2,6-difluorophenyl ester that requires a stronger base and longer reaction time. With the use of this combination of esters, we obtained considerably shortened reaction times compared with those reported previously, yet still retaining the desired selectivity in heteroconjugation. The increased reactivity of the bifunctional reagent allowed the construction of sophisticated peptide heteroconjugates from peptides, carbohydrates and proteins, showing a wide scope of applicability in the field of assembling functional bioconjugates.

Derivation of a retinoid X receptor scaffold from peroxisome proliferator-activated receptor gamma ligand 1-Di(1H-indol-3-yl)methyl-4-trifluoromethylbenzene.

PPARgamma agonist DIM-Ph-4-CF(3), a template for RXRalpha agonist (E)-3-[5-di(1-methyl-1H-indol-3-yl)methyl-2-thienyl] acrylic acid: DIM-Ph-CF(3) is reported to inhibit cancer growth independent of PPARgamma and to interact with NR4A1. As both receptors dimerize with RXR, and natural PPARgamma ligands activate RXR, DIM-Ph-4-CF(3) was investigated as an RXR ligand. It displaces 9-cis-retinoic acid from RXRalpha but does not activate RXRalpha. Structure-based direct design led to an RXRalpha agonist.1-Di(1H-indol-3-yl)methyl-4-trifluoromethylbenzene (DIM-Ph-4-CF(3)) is reported to inhibit cancer cell growth and to act as a transcriptional agonist of peroxisome proliferator-activated receptor gamma (PPARgamma) and nuclear receptor 4A subfamily member 1 (NR4A1). In addition, DIM-Ph-4-CF(3) exerts anticancer effects independent of these receptors because PPARgamma antagonists do not block its inhibition of cell growth, and the small pocket in the NR4A1 crystal structure suggests no ligand can bind. Because PPARgamma and NR4A1 heterodimerize with retinoid X receptor (RXR), and several PPARgamma ligands transcriptionally activate RXR, DIM-Ph-4-CF(3) was investigated as an RXR ligand. DIM-Ph-4-CF(3) displaces 9-cis-retinoic acid from RXRalpha but does not transactivate RXRalpha. Structure-based design using DIM-Ph-4-CF(3) as a template led to the RXRalpha transcriptional agonist (E)-3-[5-di(1-methyl-1H-indol-3-yl)methyl-2-thienyl]acrylic acid. Its docked pose in the RXRalpha ligand binding domain suggests that binding is stabilized by interactions of its carboxylate group with arginine 316, its indoles with cysteines 269 and 432, and its 1-methyl groups with hydrophobic residues lining the binding pocket. As is expected of a selective activator of RXRalpha, but not of RARs and PPARgamma, this RXRalpha agonist, unlike DIM-Ph-4-CF(3), does not appreciably decrease cancer cell growth or induce apoptosis at pharmacologically relevant concentrations.

Shape-persistent macrocycles comprising perfluorinated benzene subunits: synthesis, aggregation behaviour and unexpected mu-rod formation.

The synthesis of a series of shape-persistent macrocycles (SPMs) (1-4 and 6) comprising different numbers and/or spatial arrangement of meta-substituted tetrafluorobenzene and benzene subunits interlinked with diacetylenes is described. To increase their solubility, all five SPMs were functionalized by four peripheral hexyl chains. These SPMs were assembled from common diacetylene building blocks by a modular synthetic strategy based on palladium and/or copper catalyzed versions of acetylene coupling reactions (oxidative acetylene coupling and Cadiot-Chodkiewicz coupling). The aggregation properties in chloroform of SPMs 1-6 were investigated by concentration- and temperature-dependent 1H-NMR investigations and by vapour pressure osmometry studies. Aggregation constants and thermodynamic data of the process were obtained by least-squares fitting of the NMR data and by van't Hoff analysis respectively. Aggregation was only observed for SPMs 2-6 comprising electron deficient tetrafluorobenzene corner units. While dimerization was the major aggregation process for SPMs 3-6, the formation of larger aggregates in solution was only observed for SPM 2. The formation of aggregates is in all cases enthalpically driven. As the largest and the smallest enthalpic contribution and entropic loss in the series of aggregating SPMs were found for the two SPMs 3 and 4, each comprising two fluorinated corner units, the spatial arrangement of these subunits within the macrocycle seems to be at least equally important as the ratio of tetrafluorobenzene and benzene moieties. Interestingly, micro-scaled hexagonal rods were formed from SPM 3 upon heating in toluene, presumably consisting of mixtures of oligomers arising from covalently interlinked macrocycles.

Novel high-affinity photoactivatable antagonists of corticotropin-releasing factor (CRF) photoaffinity labeling studies on CRF receptor, type 1 (CRFR1).

Novel photoactivatable antagonists of human/rat corticotropin-releasing factor (h/rCRF) have been synthesized and characterized. The N-terminal amino acid D-phenylalanine in astressin ¿cyclo(30-33) [D-Phe12, Nle21,38, Glu30, Lys33]h/rCRF-(12-41)¿, a potent CRF peptide antagonist, was replaced by a phenyldiazirine, the 4-(1-azi-2,2,2-trifluoroethyl)benzoyl (ATB) residue. Additionally, His32 of astressin was substituted by either alanine or tyrosine for specific radioactive labeling with 125I at either His13 or Tyr32, respectively. The photoactivatable CRF antagonists were tested for their ability to displace 125I-labeled Tyr0 ovine CRF ([125I-labeled Tyr0]oCRF) in binding experiments and to inhibit oCRF-stimulated adenylate cyclase activity in human embryonic kidney (HEK) 293 cells, permanently transfected with cDNA coding for rat CRF receptor, type 1 (rCRFR1) or human Y-79 retinoblastoma cells known to carry endogenous functional human CRFR1 (hCRFR1). ATB-cyclo(30-33)[Nle21,38, Glu30, Ala32, Lys33]h/rCRF-(13-41) (compound 1) was found to bind with higher affinity to rat or human CRFR1 when compared with ATB-cyclo(30-33)[Nle21,38, Glu30, Tyr32, Lys33]h/rCRF-(13-41) (compound 2) and exhibited higher inhibition of oCRF-stimulated cAMP accumulation in HEK 293 cells stably transfected with cDNA coding for rCRFR1 (HEK-rCRFR1 cells) or Y-79 cells. A highly glycosylated, 66-kDa protein was identified with SDS/PAGE, when the radioactively iodinated compounds 1 or 2 were covalently linked to rCRFR1. The specificity of the photoactivatable 125I-labeled CRF antagonists was demonstrated with SDS/PAGE by the finding that these analogs could be displaced from the receptor by their corresponding nonlabeled form, but not other unrelated peptides such as vasoactive intestinal peptide. The observed molecular size of the receptor was in agreement with the size of CRFR1 found in rat pituitary (66 kDa), but was significantly larger than the size of CRFR1 found in rat cerebellum and olfactory bulb (53 kDa).

Synthesis and preliminary evaluation of para- and meta-[18F]fluorobenzylguanidine.

meta-[18F]Fluorobenzylguanidine ([18F]MFBG) and para-[18F]fluorobenzylguanidine ([18F]PFBG) were synthesized in three steps beginning with a fluoro for nitro exchange reaction on 3- and 4-nitrobenzonitrile, respectively. Overall radiochemical yields were 10-15% for [18F]MFBG and 50-55% for [18F]PFBG in a total synthesis time of 60 min. However, impurities interfered with the binding of the product to target cells. A new route was adopted for the synthesis of [18F]PFBG using 4-nitrilophenyl trimethylammonium trifluoromethanesulfonate (Q.S.) as the starting material. In addition to shortening the overall synthesis time by 10 min, this precursor also eliminated problems associated with the presence of small amounts of starting material in the preparation. In vitro binding of [18F]PFBG prepared by the Q.S. method to SK-N-SH, human neuroblastoma cells was 26.5 +/- 1.1%, compared to 16.9 +/- 1.6% when the nitro precursor was used. Selective uptake of both 18F-labeled isomers in the heart and adrenal was seen in mice. At 4 h, adrenal and heart uptake of [18F]PFBG prepared using Q.S. was 20.3 +/- 4.8 and 5.9 +/- 0.8% ID/g respectively, compared to 23.8 +/- 5.0 and 10.5 +/- 1.7% ID/g for [18F]MFBG. Based on the 5-fold higher radiochemical yields obtained with [18F]PFBG, this isomer would appear to be the more practical choice; however, in vitro and in vivo results suggest that [18F]MFBG exhibits greater similarities to MIBG.

Hydroxyperfluoroazobenzenes: novel inhibitors of enzymes of androgen biosynthesis.

In a search for inhibitors of 17 alpha-hydroxylase-C17,20-lyase and testosterone-5 alpha-reductase, target enzymes in the development of drugs to treat hormone-dependent prostatic cancer, we have identified certain compounds chemically derived by the hydrolysis of decafluoroazobenzene (4) as novel inhibitors for these two enzymes. Hydrolysis of 4 gave the known 4-hydroxynonafluoroazobenzene (1) and the novel 2-hydroxynonafluoroazobenzene (2). By AlI3 demethylation of 4,4'-dimethoxyoctafluoroazobenzene (5) or by hydrolysis of 4 under phase-transfer conditions 4,4'-dihydroxyoctafluoroazobenzene (3) was obtained. Compounds 1 and 2 were inhibitors of the hydroxylase (IC50 values, respectively, 30 and 63 microM) and of the lyase (IC50 values 33 and 16 microM) steps on the pathway of androgen biosynthesis. The 2-hydroxy compound 2 underwent spontaneous conversion into octafluorodibenz[b,f][1,4,5]oxadiazepine (6) which had IC50 values, respectively, of 50 and 15 microM for the hydroxylase and lyase steps and which contributed to the observed activity of 2. Effective inhibitors of the 5 alpha-reductase were 1 (Ki 10 microM) and 3 (Ki4 microM): the activities of 1 and 3 were markedly pH dependent, with respective IC50 values of 14 and 5 microM at pH 7.4 and of 2 and 0.8 microM at pH 6.6.

Motion at the active site of [(4-fluorophenyl)sulfonyl]chymotrypsin.

Fluorine and deuterium NMR relaxation studies have been used to examine the motion of the 4-fluorophenyl ring attached to the active site of [(4-fluorophenyl)sulfonyl]-alpha-chymotrypsin at pH 4. Analysis of the results indicates that rotation about the 2-fold axis of this ring is reasonably rapid, though not as fast as in tosylchymotrypsin. Two-dimensional (2D) nuclear Overhauser effects (NOEs) were used to suggest the shifts of those protons of the enzyme close enough to the fluorine nucleus to lead to relaxation; important proton-fluorine dipolar relaxation contributions arise from protons with shifts of 7.4 +/- 0.3 ppm and between 4.0 and 5.4 ppm. Specific deuteration permits the assignment of the first of these to the protons ortho to the fluorine while serine-189, cysteines-191 and -220, and methionine-192 are suggested as possible bearers of the other protons. The fluorine chemical shift effect observed for the native conformation of this protein is 9 ppm downfield of the shift observed with the denatured protein; this large shift may be the result of van der Waals interactions between the fluorine and one or more of the protons whose signals appear in the 2D NOE experiments.