mr*" and "nr*" excitation energies calculated by the INDO/S method13 at these geometries (Table I) reflect the experimental trends correctly but are a little too low, as was also the case for other imines.5•6 The structure assignment of the nitrene 4 is based on its ESR signal at 0.8124 T (9.3 GHz; assuming E = 0, \D/hc\ = 1.65 cm™1),14 a sharp UV peak at 33 560 cm™1, whose intensity follows that of the ESR signal,15 and on photochemical trapping with CO in Ar at 36 K. The IR spectrum of this matrix, containing 2, 3, CO, and presumably 4, was unchanged for many hours in the dark, but on UV irradiation7' a weak band of 1-norbornyl isocyanate at 2263 cm™1 appeared16 (IR, MS, and GC comparison with an authentic sample).To our knowledge, the above results represent the first observation of geometrical isomerism at a strained bridgehead double bond. Taken together with prior work,2™6 they suggest that the C=N stretching frequencies are typically reduced by about 100 cm™1 in Zzvm-azacycloheptene rings and by about 200 cm™1 in iranr-azacyclohexene rings, respectively, relative to an unstrained imine.Acknowledgment. This work was supported by the National Science Foundation (CHE 81-21122).
A series ofderivatives for both the arthropod and mollusc hemocyanin biopolymers has been prepared; the derivatives contain a small fraction ofelectron paramagnetic resonancedetectable half-met [Cu(fl) Cu(I)] sites dispersed among the nondetectable oxy binuclear copper active sites. Upon deoxygenation, large changes in the electron paramagnetic resonance signal of these half-met spectral probe derivatives are observed, which are further adjusted by the heterotropic effectors Ca2+ and HW. The active site structural changes indicated by these spectral changes as the hemocyanins go from a relaxed to a tensed quaternary structure are then discussed.Hemocyanins, the oxygen transport proteins in the hemolymph of molluscs and arthropods, reversibly bind dioxygen with a stoichiometry ofone dioxygen per two copper ions. Under physiological conditions, the hemocyanins are present as highly aggregated biopolymers with a molecular architecture that differs dramatically between the two phyla. For the arthropods, a single subunit has a molecular weight of "'70,000 and contains one binuclear copper active site. At pH values below 8 and in the presence of Ca2+, these subunits aggregate into one, two, four, and in the case of Limulus polyphemus, up to eight hexamers.Alternatively, the smallest subunit in the molluscs contains about eight binuclear copper active sites (domains) covalently linked in a single polypeptide chain of "'400,000 daltons. In the mollusc Busycon canaliculatum, at pH less than 8, twenty chains aggregate to form a whole molecule that contains 160 active sites and appears as a cylindrical barrel under the electron microscope. When aggregated, the hemocyanins are highly cooperative in their oxygen binding, with Hill coefficients dependent on species, pH, and the presence of divalent cations (1,2).In general, current thermodynamic and kinetic theories of oxygen binding to the hemocyanins parallel [with small modifications (3, 4)] the allosteric model of hemoglobin. Deoxyhemocyanin has a "tensed" quaternary structure with a low oxygen affinity. Initial binding of oxygen to a limited number of active sites alters the successive oxygen affinities ofthe remaining sites by a ligand-induced conformational change of the protein (5) (i.e., oxygen is a homotropic effector); this results in the oxyhemocyanin having a "relaxed" quaternary structure with a high oxygen affinity. Protons and divalent cations act as heterotropic effectors, which, depending upon the species, stabilize either the tensed or relaxed quaternary structure.For hemoglobin, through both protein crystallography (6) and the detailed study of iron prophyrin model complexes (7), significant advances have been made in the development of a structural model that accounts for the active site changes related to these changes in oxygen affinity. Two basic spectroscopic approaches have been taken to relate this mechanical model to the hemoglobin tetramer. One approach involves the preparation of valence hybrids in which either the a or the (3 chains con...
Cyclophane 5H2 was prepared from 5H' and BNAH as described for 1H2 in 78% yield. 'H NMR CDC13) 6 1.95 (ArCH3), 2.36 (ArCH3), 2.48 (C(4)H8), 2.56 (C(4)HA), 2.8-4.2 (CH2CH2), 3.82 (OCH3), 4.60 (CH2Ph), 5.41 (NH), 6.62 (Ar H), 7.30 (C(2)H, C(6)H), 7.35 (PhH).(3.6) Deuterium Incorporation. The monodeuterated compounds nHD (n = 3-6) were prepared by reaction of the appropriate pyridinium ion ( I N ' ) with an equimolar amount of BNAH-4-d2. For n = 4-6 the same reaction conditions were applied as described above for the conversion 1H' -1H2. Under these conditions formation of 3HD gave no satisfactory results. In this case the reaction was performed at 20 OC during 4 days (yield 64%).(3.7) Oxidation of nHD by AcH+. Compounds 4HD, 5HD, and 6HD were subjected to oxidation by AcH' and the deuterium content of the 10-methylacridan thus obtained was determined by MS (field-ionization technique). To avoid isotopic exchange23 between acridan and unreacted AcH' the reactions were carried out with a twofold excess of nHD. Typically solutions of nHD (0.6 mmol) and AcH' (0.3 mmol) in dimethyl sulfoxide (2 mL each) were mixed and stirred for 1 h at 20 OC. The reaction mixture was than poured in ice water and the white precipitate of IO-methylacridan collected by filtration and purified by recrystallization from ethanol/water 2:l.Determination of the deuterium content was performed by MS using the field-ionization (FI) technique as described extensively before.22 MS-FI was found22 to avoid the problem of erratic M/M + 1 ratios, Acknowledgment. W e thank C. H. Stam (University of Amsterdam, Laboratory for Crystallography) for performance and discussion of the X-ray structure determinations. Measurement of the 360-MHz 'H NMR spectra by R. Kaptein and K. Dijkstra (University of Groningen) is gratefully acknowledged. W e thank C. Kruk et al. for extensive 250-MHz 'H NMR and NOE measurements. FI-MS measurements were realized by the courtesy of N. M. M. Nibbering, R. H. Fokkens, and J. J. Zwinselman. W e thank U. K. Pandit for stimulating discussions on the reactivity of NAD(H) models. The 360-MHz 'H NMR facilities were made available-Abstract: Eu3+ was substituted for Ca2' as an allosteric effector in hemocyanin, and its laser-induced f-f fluorescence was studied to probe the allosteric effects on the CaZ+ binding site induced by deoxygenation of the binuclear copper active site.The 'FO -sDo excitation spectrum of Eu3' was monitored to define two kinds of high-affinity Eu3' binding sites in the hemocyanin biopolymer, one of which is competitive with the Ca2+ ion. The fluorescence decay lifetimes of this Ca2'-competitive Eu" site were measured in D 2 0 and H 2 0 buffer which permitted an estimate of the coordinated water number at the Eu3+ ion. The results show that in the relaxed quaternary structure the Ca2' site is located in a very hydrophilic environment with -5
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