Report on the preparation of deuterium-labelled aconitine and mesaconitine and their application to the analysis of these alkaloids from body fluids as internal standard

Improved analysis of aconitine and mesaconitine, highly toxic compounds from Aconitum species, in body fluids by gas chromatography–selected ion monitoring with their deuterium-labelled analogues as internal standards (I.S.s) is described. Deuterium-labelled analogues of aconitine and mesaconitine were synthesized by the substitution of the N-alkyl group for a deuterium-labelled one. The mass spectra of the derivatives of the deuterium labels closely resembled that of the nonlabelled compounds except for an obvious mass shift produced by substitution of the deuterium atoms at N-alkyl groups. Using these deuterium-labelled compounds as I.S.s, the standard curves for aconitine and mesaconitine were linear (r²=0.999 each) in the concentration range of 50 pg to 50 ng, respectively. The detection limit of the alkaloids was 10 pg each per injection. The recovery, accuracy and precision of the analysis were evaluated with three different concentration of spiked human blood and urine (n=5 each). The recovery rates ranged from 97.6% to 101.3% and the standard deviations of the interseries ranged from 2.1% to 3.9%. These I.S.s give us a more precise analysis and may be useful in examining the behavior of these alkaloids in the human body.     

Synthesis of ring- and side-chain-substituted m-iodobenzylguanidine analogues

With the goal of developing MIBG analogues with improved targeting properties especially for oncologic applications, several radioiodinated ring- and side-chain-substituted MIBG analogues were synthesized. Except for 3-[(131)I]iodo-4-nitrobenzylguanidine and N-hydroxy-3-[(131)I]iodobenzylguanidine, the radioiodinated analogues were prepared at no-carrier-added levels from their respective tin precursors. The radiochemical yields generally were in the range of 70-90% except for 3-amino-5-[(131)I]iodobenzylguanidine for which a radiochemical yield of about 40% was obtained. While the silicon precursor N(1),N(2)-bis(tert-butyloxycarbonyl)-N(1)-(4-nitro-3-trimethylsilylbenzyl)guanidine did not yield 3-[(131)I]iodo-4-nitrobenzylguanidine, its deprotected derivative, N(1)-(4-nitro-3-trimethylsilylbenzyl)guanidine was radioiodinated in a modest yield of 20% providing 3-[(131)I]iodo-4-nitrobenzylguanidine. Exchange radioiodination of 3-iodo-4-nitrobenzylguanidine gave 3-[(131)I]iodo-4-nitrobenzylguanidine in 80% radiochemical yield. No-carrier-added [(131)I]NHIBG was prepared from its silicon precursor N(1)-hydroxy-N(3)-(3-trimethylsilylbenzyl)guanidine in 85% radiochemical yield.     

New insight for fluoroquinophenoxazine derivatives as possibly new potent topoisomerase I inhibitor

Fluoroquinolones, represented by ciproxacin and norfloxacin, are well-known clinical antimicrobial agents, and their phenyl ring expanded quinophenoxazines are reported as possible antitumor active compounds. These quinophenoxazines are known to inhibit DNA topoisomerase II essential for cell replication cycle. But there were no reports for topoisomerase I inhibition study for these compounds. In this report, we have prepared a few quinophenoxazine analogues and tested their topoisomerases I and II inhibitory activities and cytotoxicity. From the result, we found that quinophenoxazine analogues possessed strong topoisomerase I inhibitory capacity as well as topoisomerase II inhibition. Among the compounds prepared, A-62176 analogues showed strong topoisomerases I and II inhibitory activities. Interestingly, compound 8 missing the 3-aminopyrrolidine moiety at C2 position has similar potent inhibitory capacity against topoisomerases I and II at higher concentrations (20 and 10 microM, respectively). But compound 8 inhibited topoisomerase I function more selectively at lower concentration, 2 microM. Our observation might strongly implicate that fluoroquinophenoxazines can be developed as efficient topoisomerase I inhibitor with the elaborate modification.     

Synthesis of modified fragments of fibrinogen and their effect on the activity of proteolytic enzymes

New analogues of the Gly-Pro-Arg and Arg-Gly-Asp fragments of fibrinogen were synthesized: Gly-Pro-Arg-Pro (I), Gly-Pro-Arg-Pro-Met-OMe (II), Gly-Pro-Arg-Pro-Phe (III), Gly-Pro-Arg-Pro-Asp (IV), Gly-Pro-Arg-Pro-Glu (V), and Arg-Asn-Trp-Asp (VI). Their effect on the activity of proteases of various types was studied with the method of lysis of fibrin plates. All the peptides were found to inhibit plasmin activity (by 60-85%) and the gamma-subunit of nerve growth factor (by 55-93%). Tetrapeptide (VI) proved to be an effective inhibitor of tissue activator of plasminogen and the gamma-subunit of nerve growth factor (by 96 and 93%, respectively). The peptides exerted practically no effect on the activity of urokinase and moderately inhibited the activity of streptokinase [(III), (IV), and (VI)], papain [(I), (II), (IV), and (VI)], subtilisin [(V) and (VI)], alpha-chymotrypsin [(III), (V), and VI)], and Bacillus subtilis metalloprotease (VI). They inhibit trypsin [except for (I) and (III)] when applied on fibrin plates at a concentration of 1 x 10(-2) M, while, at a concentration of 1 x 10(-3) M, (I) and (II) induced an increase in proteolytic activity by 35 and 47%, respectively.     

Synthesis and biological profile of new 1,2,3,4-tetrahydroisoquinolines as selective carbonic anhydrase inhibitors

In a previous paper we identified several 1-aryl-6,7-dimethoxy-1,2,3,4-tetrahydroisoquinoline-2-sulfonamides that displayed inhibitory effects toward selected carbonic anhydrase isozymes at micromolar concentration. In order to deepen the structure-activity relationships (SARs) and identify novel compounds with improved activity, we synthesized a series of monomethoxy analogues of the previously investigated dimethoxy derivatives. The evaluation of biological profile has been focused on in vitro effects against several CA isoforms. The new monomethoxy derivatives showed higher hCA inhibitory effects against several isoforms compared to the dimethoxy analogues. Particularly, some of these compounds (e.g., 1b and 1h) showed low nanomolar K(I) values and excellent selectivity for hCA IX and hCA XIV versus hCA I and II inhibition.     

Synthesis and biological activities of angiotensin II and Sarmesin analogues containing cyclohexylalanine

Analogues of angiotensin II with cyclohexylalanine (Cha) at position 4 or 8, and analogues of the competitive (type II) angiotensin antagonist [Sar1,Tyr(Me)4]ANG II (Sarmesin) with Cha at position 8, have been prepared by the solid phase method and purified by reversed-phase HPLC. Analogues of ANG II with Cha at position 8 in which the position 1 residue was substituted with sarcosine (Sar) or amino-isobutyric acid (Aib) or was deleted (Des), were slowly reversing (Type I) antagonists with "pA2" values in the rat isolated uterus assay of approximately 8.5. The additional substitution of Tyr(Me) for Tyr at position 4 of these peptides gave reversible competitive (Type I/II) antagonists with pA2 values of 6.7, 5.8, and less than 5, while substitution of Phe for Tyr gave pA2 values of 7.4, 6.7, and less than 5, respectively. All 19 peptides synthesized in this study had low intrinsic agonist activity in the rat isolated uterus assay except for the type I antagonists [Sar1, Cha8]ANG II (7%), [Aib1, Cha8]ANG II (12%) and [Des1, Cha8]ANG II (20%). These data illustrate that the substitution of Cha at position 8 of ANG II analogues produces potent antagonists; however, Type I antagonists retain significant agonist activity whereas Type I/II antagonists do not. In contrast, substitution of Cha at position 4 in a variety of ANG II analogues resulted in severely diminished biological activity, illustrating that the presence of an aromatic ring quadrupole at position 4 is obligatory for receptor binding and activity.     

The 3'-amido and 5'-amido analogues of adenosine 3':5'-monophosphate; interaction with cAMP-specific proteins.

The sensitivity for recognition of adenosine 3:5'-monophosphate (cAMP) by its coordinate proteins towards chemical changes in the six-membered cyclic phosphate ring has been investigated. A comparison of the interaction parameters of the 3' and 5'-amido analogues (I, II) and of unsubstituted cAMP has been made using two different protein kinases and the phosphodiesterase from bovine heart. Binding affinity and the capacity of the amido analogues to stimulate the phosphotransferase activity of the kinases is greatly reeuced relative to cAMP, the 3'-position being more sensitive towards the modification than the 5'-position. The coordinate noncyclic derivatives, 3'-deoxy-3'-amino-5'-AMP (IV) and 5'-deoxy-5'-amino-3'-amp (iii), were also tested. Surprisingly activity towards protein kinases was found to be considerable for the 5'-deoxy-5'-amino-3'-AMP (III), while the 3'-deoxy-3'-amino-5'-AMP (IV) is practically inactive. A possible reason for this is that the noncylic 5'-analogue (III) may be able to assume a cyclic structure maintained by internal salt formation. The phosphodiesterase splits both cyclic amido analogues but with reduced rates compared to that of natural cAMP. Kinetic data obtained from different methods reveal a stronger affinity for the 5'-analogue (I) than the 3'-analogue (II) for the active site, although the reaction rate at saturated substrate concentration is significantly higher with II than with I. The properties of the amido and the noncyclic amino analogues are discussed with available data from chemotaxis of the cellular slime moulds. Furthermore data of the respective methylene cyclic derivatives are used for a more comprehensive comparison. The above is interpreted in terms of the electronic features of the substitutions and of the changes in bond distances or angles upon replacement of O by NH or CH2 in the cyclic phosphate ring (obtained from X-ray work).     

Inhibition of P-glycoprotein by cyclosporin A analogues and metabolites.

The interaction between P-glycoprotein (P-gp) from membranes isolated from multidrug-resistant Chinese hamster ovary cells and cyclosporin A (CsA) analogues and its metabolites was characterized. Screening of these latter as chemosensitizers was performed using three different assays: (i) vinblastine uptake, (ii) photoaffinity labeling by [125I]iodoaryl azidoprazosin, and (iii) P-gp ATPase activity. Oxidation of the hydroxyl group at position I of CsA (200-096), CsG (215-834), or CsD (PSC-833) increased their inhibition of P-gp. CsA analogues (208-032, 208-183) modified at position 11 retained their ability to inhibit P-gp while analogues modified at position 2 (CsC and CsD) lost their efficiency. The inhibitions induced by metabolites of CsA were also compared to those obtained with CsG metabolites. From all the molecules tested, PSC-833 and 280-446 peptolide were the strongest inhibitors. Our results indicate that modifications of CsA analogues at position 1 and 2 are critical for their interaction with P-gp and that CsA metabolites retain a portion of the inhibitory activity of the parent drug.     

Stimulation of fatty acid oxidation in myocytes by phosphodiesterase inhibitors and adenosine analogues.

The effect of various phosphodiesterase inhibitors, and adenosine analogues on palmitate oxidation, were studied in isolated rat myocytes. Enoximone, milrinone, and dipyridamole, at a concentration of 250 microM, stimulated palmitate oxidation by 78%, 40%, and 43%, respectively. The specific A1-agonist, N6-cyclopentyladenosine, increased palmitate oxidation by 56%, at a concentration of 250 microM. Moreover, the nucleoside transport inhibitor, S-(P-Nitrobenzyl-)6-thioinosine, increased palmitate oxidation by 40%, at a concentration of 100 microM. These data suggest that the stimulation of palmitate oxidation by enoximone and adenosine analogues may be mediated via the inhibition of the uptake and/or the oxidation of glucose in myocytes.     

New carbocyclic analogues of netropsin: synthesis and inhibition of topoisomerases

A series of carbocyclic analogues of netropsin were synthesized and evaluated for their capacity to inhibit human topoisomerases I and II in vitro. The compounds are oligopeptides containing 1,4-di- and 1,2,5-trisubstituted benzene rings and unsubstituted N-terminal NH2 groups. Compounds 4-7 consist of two netropsin-like units linked by aliphatic (tetra- and hexamethylene) chains. In the topoisomerase I and II assay, the relaxation of pBR322 plasmid was inhibited by compounds 4-7 at 100 microM concentration.     

Inhibition of beta-glycosidases by acarbose analogues containing cellobiose and lactose structures

Acarbose analogues, containing cellobiose and lactose structures, were prepared by reaction of the two disaccharides with acarbose and Bacillus stearothermophilus maltogenic amylase. The kinetics for the inhibition by the two analogues was studied for beta-glucosidase, beta-galactosidase, cyclomaltodextrin glucanosyltransferase (CGTase), and alpha-glucosidase. Both analogues were potent competitive inhibitors for beta-glucosidase, with K(I) values in the range of 0.04-2.44 microM, and the lactose analogues were good uncompetitive inhibitors for beta-galactosidase, with K(I) values in the range of 159-415 microM, while acarbose was not an inhibitor for either enzyme at 10 and 5 mM, respectively. Both analogues were also potent mixed inhibitors for CGTase, with K(I) values in the range of 0.1-9.3 microM. The lactose analogue was a 6.4-fold better competitive inhibitor for alpha-glucosidase than was acarbose.     

The structural parameters for antimicrobial activity, human epithelial cell cytotoxicity and killing mechanism of synthetic monomer and dimer analogues derived from hBD3 C-terminal region

Understanding the molecular mechanisms of antimicrobial peptide?Cmembrane interactions is crucial in predicting the design of useful synthetic antimicrobial peptide analogues. Defensins are small (3?C5?kDa) cysteine-rich cationic proteins which constitute the front line of host innate immunity. In this study, a series of eight 10 AA C-terminal analogues of hBD3 [sequence: RGRKXXRRKK, X?=?W, F, Y, V, L, I, H, C(Acm); net charge?=?+7, coded as W2, F2, Y2, V2, L2, I2, H2, and C2] and covalent V2-dimer [(RGRKVVRR)₂KK] (18 AA, net charge?=?+11) were synthesized using solid phase peptide synthesis (SPPS) in Fmoc chemistry. Wild-type hBD3 was used as a control in all analyses. W2, V2, and especially Y2 showed high activity selectively against Gram-negative bacteria Pseudomonas aeruginosa in the concentration range of 4.3?C9.7???M. The covalent dimeric form of V2-monomer, V2-dimer, showed increased antibacterial killing compared to the monomeric form, V2-monomer. Cytotoxicity assays on a human conjunctival epithelial cell line (IOBA-NHC cells) showed that no change in viable cell number 24?h after constant exposure to all the eight peptide analogues even at concentrations up to 200???g/ml. Fluorescence correlation spectroscopy (FCS) was used to study the interaction of these peptides against POPC vesicles (neutral; mammalian cell membrane mimic) and POPG vesicles (negatively charged; bacterial cell membrane mimic). Using FCS, significant aggregation and some leakage of Rhodamine dye were observed with POPG with Y2, W2 and V2 at the concentration of 5?C10???M and no significant aggregation or disruption of vesicles was observed for all peptide analogues tested against POPC. V2-dimer induced more leakage and aggregation than the monomeric form. Overall, V2-dimer is the most effective antimicrobial peptide, with aggregation of POPG vesicles observed at concentrations as low as 1???M. The concentration of 5?C10???M for Y2 from FCS correlated with the concentration of 5???M (6.25???g/ml), at which Y2 showed a cooperative increase in the activity. This suggests a structural transition of Y2 in the 2.5?C5???M concentration range resulting in the correlated increased antimicrobial activity. These results and the FCS together with previous NMR and molecular dynamics (MD) suggested that the charge density-based binding affinity, stable covalent dimerization, the ability to dimerize or even oligomerize and adopt a well-defined structure are important physicochemical properties distinguishing more effective cationic antimicrobial peptides.     

Effect of alkylating derivatives of cyclic adenosine-3' ,5'-monophosphate on proliferation of mouse bone marrow stem cells

The effect of the physiological concentration of cyclic adenosine-3' ,5'-monophosphate (cAMP) analogues on the proliferation of mouse bone marrow stem hemopoietic cells (CFUs) was examined. The stimulating effect was estimated from the decrease in CFUs expressed in the percentage derived from comparing the number of spleen colonies in the control and experimental groups treated with hydroxyurea 10(-3) M (incubation with hydroxyurea resuted in the cell death in S-phase). Cyclic AMP stimulted the proliferation of CFUs by 60%, while its analogues such as 8-(N-chloroacetylaminoethylamino)-cAMP, 1-(N-chloroacetylaminoethyoxy)-cAMP and 1-(N-(p-fluorosulfonyl)-benzoylaminoethoxy)-cAMP stimulated the proliferation by 39.2%, 32.4% and 21.9%, respectively. Therefore, the synthetic analogues of cAMP were not only far from inhibiting the proliferation of CFUs but, on the contrary, exerted a stimualting effect unlike most antineoplastic alkylating drugs that depress hemopoiesis up to its total aplasia.     

Influence of the aminoacyl-tRNA synthetase inhibitors and the diadenosine-5'-tetraphosphate phosphonate analogues on the catalysis of diadenosyl oligophosphates formation

Well-known aminoacyl-tRNA synthetase (ARSase) inhibitors, namely the analogues of amino acids and aminoacyl adenylates (aminoalkyl- and aminophosphonyl adenylates with Ki congruent to 0.1 microM) as well as the diadenosine 5',5'-p1,p4-tetraphosphate (Ap4A) phosphonoanalogues, were for the first time used for the Ap4A biosynthesis regulation. Effects of a set of such compounds on lysyl-, phenylalanyl- and alanyl-tRNA synthetases from E. coli, capable of synthesizing Ap4A in the presence of Zn2+ ions and pyrophosphatase, have been studied. The adenylate analogues were found to inhibit the Ap4A and Ap3A formation (I50 congruent to 6 mM). Aminophosphonic and aminophosphonous acids are not involved in Ap3A and Ap4A biosynthesis and inhibited it at high concentrations. The Ap4A phosphoanalogues slightly inhibited the major reactions of ARSases, as well as the biosynthesis of Ap3A and Ap4A, at a concentration of 5 mM.     

In vitro and in vivo inhibition of LPS-induced tumor necrosis factor-alpha production by dimeric gallotannin analogues.

Designed dimeric gallotannin analogues featuring two tetragalloylglucopyranose cores connected by various hydrocarbon linkers inhibit tumor necrosis factor-alpha secretion from lipopolysaccharide-stimulated human peripheral blood mononuclear cells by up to 53% (5-24 microM concentration range) compared to control. Comparable suppression of tumor necrosis factor-alpha levels (approximately 50% vs control) was observed in the plasma of rats co-treated with lipopolysaccharide and specific tannin analogues selected for their lack of interleukin 1-beta stimulating activity.     

B型烟粉虱对三种寄主植物及其挥发物的行为反应

利用Y型嗅觉仪,测定了B型烟粉虱Bemisia tabaci（Gennadius）雌虫对3种寄主植物、挥发物提取液、挥发物标样以及寄主植物挥发物模拟样的行为反应,并比较鉴定寄主植物挥发物的组分和含量,以期明确植物挥发物在B型烟粉虱寄主定向行为中的作用。结果表明：1）番茄植株和甘蓝植株及其相应的挥发物提取物对烟粉虱雌成虫均具有显著的引诱作用,而辣椒植株和挥发物提取物的引诱作用不明显;3种寄主植物和挥发物提取物分别两两之间比较时,烟粉虱的选择行为均极显著地表现为番茄 > 甘蓝 > 辣椒。2）番茄、甘蓝和辣椒具有不同的挥发物化学图谱,且挥发物组分的相对百分含量差异很大;番茄挥发物总量远远大于甘蓝和辣椒;从组分来看,番茄挥发物中主要为萜烯类（89.8%）,甘蓝挥发物中主要为烷烃类（53.0%）。3）8种寄主植物挥发物标样（10-¹～10^-6 μL/μL）中,除1,8-桉树脑始终对烟粉虱具有引诱作用外,丁子香酚、苎烯、里那醇和月桂烯则是分别在10^-4～10^-6,10^-1～10^-4,10^-1～10^-4和10^-1～10^-4μL/μL时,才具有引诱作用;α-蒎烯、顺-3-已烯-1醇则在高浓度下（10^-1和10^-2 μL/μL）对烟粉虱具有排斥作用,低浓度下对烟粉虱没有任何影响;正十二烷在任何浓度下对烟粉虱均没有任何作用。4）在辣椒上分别喷施番茄挥发物模拟样和甘蓝挥发物模拟样,均显著增加对烟粉虱的引诱作用,而在番茄上喷施辣椒挥发物模拟样则无明显增效作用。综合分析认为,挥发性物质在B型烟粉虱对不同寄主植物的选择偏好时具有重要的行为导向作用。     

Cyclic nucleotides modulate the release of [3H] adenosine cyclic 3',5'-phosphate bound to the regulatory moiety of protein kinase I by the catalytic subunit of the kinase

The rate of release of bound c[3H]AMP from the two types (A and B) of cAMP binding sites on the regulatory subunit dimer (R2I) of rabbit muscle protein kinase I was studied in the presence of the catalytic (C) subunit of protein kinase. Rebinding of released c[3H]AMP was avoided by using highly diluted reactants or adding unlabeled cAMP or its analogues. No significant C-induced dissociation of R2I-(c[3H]AMP)4 occurred in the absence of Mg2+-ATP. Of the two options that one or two molecules of C are required to induce the release of c[3H]AMP bound to R2I, only the first one was compatible with the first-order dependence on [C] of the rate of release of c[3H]AMP observed over a wide range of C concentrations. In the absence of added unlabeled cyclic nucleotide, the rate of the C-induced release of c[3H]AMP was the same from site A and site B. The apparent second-order rate constant for the association of C to R2I(c[3H]AMP)4 was 6 X 10(6) M-1 s-1 (37 degrees C, 0.15 M KCl). Raising the concentration of unlabeled cAMP in the medium up to 1 microM decreased by up to 50% the rate of the C-induced release of bound c[3H]AMP from both sites. This is explained by assuming that the association of one molecule of C to R2I(c-[3H]AMP)4 leads to the release of c[3H]AMP first from one R subunit and subsequently, by a process that can be blocked by about 1 microM cAMP, from the other R subunit. A further rise of the cAMP concentration decreased the rate of release from site B only, so that the C-induced release of c[3H]AMP occurred almost exclusively from site A at very high concentrations of cAMP. This suggests that c[3H]AMP is released first from site A and that this vacant site by interacting with cAMP inhibits the release of c[3H]AMP from site B of the same R subunit. The role of site A in controlling the C-induced release was further supported by the finding that several cAMP analogues inhibited the release with potencies correlating with their affinities for site A. The C-induced release of c[3H]AMP from aged R2I was about 10 times slower than that from fresh R2I. No significant C-induced release of c[3H]AMP was observed from the monomeric fragment obtained by limited trypsin treatment of R2(1).     

Retro-inverso peptide analogues of Trypanosoma cruzi B13 protein epitopes fail to be recognized by human sera and peripheral blood mononuclear cells

Retro inverso (RI) analogues of antigenic synthetic peptides, which are made of D-amino acids with a reversed sequence, may mimic the side chain conformation of natural all-L peptides. RI analogues were cross-reactively recognized by antibodies and CD4+ T cells reactive against natural all-L synthetic peptides or native proteins in animal models. Since peptides containing D-amino acids are highly resistant to proteolytic digestion, cross-reactive RI analogues may be ideal for in vivo administration to humans as synthetic peptide vaccines or immunomodulators. B13 is an immunodominant tandemly repetitive protein from Trypanosoma cruzi, a protozoan parasite that is the causative antigen of Chagas' disease. In order to test whether RI peptides can be recognized by human antibody and T cells, we synthesized two all-L peptides containing the immunodominant B (S12) and T (S15.7) cell epitopes of B13 protein from T. cruzi and their retro (R, made of all-L amino acids with reversed sequence), inverso (I, made of all-D amino acids) and RI analogues. Recognition of peptides S12, S12-R, S12-I and S12-RI by anti-B13 antibodies in sera from T. cruzi-infected patients was tested in competitive ELISA assay with recombinant B13 protein as the solid phase antigen. Peptides S15.7 and its topological analogues were tested at the 10-50 microM range in proliferation assays on peripheral blood mononuclear cells (PBMC) from S15.7-responder individuals. The median percentage inhibition of B13 ELISA for peptide S12 was 94%, while those of the RI analogue or the other topological analogues were below 12%. While peptide S15.7 was recognized by PBMC from all subjects tested, none recognized the RI analogue of the S15.7 T cell epitope. Our results indicate that cross-reactivity with natural epitopes is not an universal property of RI analogues. This may limit the general applicability of the use of cross-reactive RI analogues as human vaccines and immunotherapeutic agents.     

Semi-synthetic aequorins with improved sensitivity to Ca2+ ions.

Thirty-seven coelenterazine analogues were synthesized and incorporated into apo-aequorin, yielding 30 semi-synthetic aequorins that have the capacity to emit a significant amount of light in the presence of Ca2+. The properties of resultant photoproteins were investigated. The most prominent feature of those photoproteins was the wide range in their sensitivities to Ca2+ concentration. The relative intensity of Ca2+-triggered luminescence of the photoproteins ranged from 0.01 to 190 when compared with natural aequorin (relative intensity 1.0) at pCa 6 for the cases where the relative intensity is less than 1 and at pCa 7 for the cases where the relative intensity is higher than 1. Eight of the semi-synthetic aequorins belonged to the class of e-aequorin. With two of those photoproteins, the degree of dependence of the luminescence intensity ratio I400/I465 on pCa was greater than that with e-aequorin, suggesting that these two photoproteins are possibly superior to e-aequorin in measuring Ca2+ concentration by the ratio method.