An acid-labile anchoring linkage for solid-phase synthesis of C-terminal peptide amides under mild conditions

A series of polymer-supported benzylamides substituted with one to three alkoxy groups in the ring positions were prepared and shown to give carboxamides upon treatment with acid. Based on the initial screening, the bis(o-methoxy)-p-alkoxybenzylamide anchoring linkage was selected for a detailed evaluation of its suitability for solid-phase synthesis of C-terminal peptide amides. The handle derivative 5-[(2' or 4')-Fmoc-aminomethyl-3',5'-dimethoxyphenoxy]valeric acid (1) was prepared in seven facile steps [purification of intermediates unnecessary; overall yield 15% for crystalline product, which is a mixture of positional isomers], and was quantitatively coupled onto amino group-containing supports by use of N,N'-dicyclohexylcarbodiimide plus 1-hydroxybenzotriazole in N,N-dimethylformamide. Stepwise elaboration of peptide chains proceeded smoothly with both N alpha-9-fluorenyl-methyloxycarbonyl (Fmoc) and N alpha-dithiasuccinoyl (Dts) amino acids, and final cleavage of tert.-butyl side-chain protecting groups and of the anchoring linkage occurred readily in trifluoroacetic acid-dichloromethane (7:3) at 25 degrees. The methodology was demonstrated by the syntheses of H-Trp-Asp-Met-Phe-NH2 (tetragastrin) and H-Tyr-Gly-Gly-Phe-Met-NH2 (methionine-enkephalinamide), both with high yields and purities.     

Suppression of epimerization by cupric (II) salts in peptide synthesis using free amino acids as amino components.

An epimerization-free system for coupling N-protected peptides with free amino acids was developed. A number of inorganic substances were tested as epimerization suppressant additives during the coupling by various methods (carbodiimide plus additives, uronium salts, Woodward's reagent-K, isobutyl-chloroformate, etc.). Some of them (ZnCl2, RbClO4, LiCl, SnCl4, AlCl3, etc.) in combination with some coupling methods can guarantee coupling with minimal epimerization (D-epimer < 1%). But only a simultaneous use of 1-hydroxybenzotriazole and Cu2+ ions as additives in carbodiimide-mediated peptide couplings appeared to give a standard result (D-epimer < 0.1%). There was no epimerization even in the case when N-methyl amino acid (sarcosine) was used as an amino component, while in the absence of Cu2+ ions an unacceptable level of epimerization was observed (D-epimer, 22% for carbodiimide with the 1-hydroxybenzotriazole method). So far it has been considered that Cu2+ ions prevent obtaining peptides in high yields (< 90%) by various coupling methods. We have found that the use of 1-hydroxybenzotriazole, CuCl2 and 1-ethyl-3-(3-dimethylaminopropyl) carbodiimide instead of dicyclohexylcarbodiimide provides a possible method for obtaining the desired peptides in 90-99% yields without epimerization. All these results were shown by employing several model peptide couplings with free amino acids as amino components dissolved in an effective solvent system which readily dissolved them.     

Azoindoxyl methods for the histochemical investigation of hydrolases. I. Lactase (lactase-beta-glucosidase complex) (author's transl)]

An azoindozyl method for the histochemical demonstration of lactase (lactase-beta-glucosidase complex) is described. The incubation medium consists of 5 mg 5-Br-4-Cl-3-indolyl-beta-D-fucoside (dissolved in 0.5 ml N,N-dimethylformamide) and 0.02 ml hexazotized prosaniline in 10 ml 0.1 M citric acid phosphate buffer, pH 6-6.5. By means of this method lactase can be exactly localized in the brush border of the enterozytes in the jejunum of suckling rats. Compared to the corresponding indigogenic method the azoindoxyl reaction proceeds faster and the reaction product is often precipitated more precisely.     

Synthesis of the sequential polypeptide poly[Cys(Acm)-Ser-Phe-Glu-Glu] as a model for hydrolytic enzymes.

To construct a possible model of hydrolytic enzymes, the sequential polypeptide H[Cys(Acm)-Ser-Phe-Glu-Glu]nOH, n = 3-110, was prepared by polycondensation of H-Cys(Acm)-Ser(But)-Phe-Glu(OBut)-Glu(OBut)-O Su/1-hydroxybenzotriazole. In a solid state the polypeptide material showed beta-conformation both before and after cleavage of t-butyl protecting groups. The pentapeptide unit was synthetized by the Merrifield method utilizing modifications as follows: Gel phase synthesis on less than 0.5% cross-linked copolystyrene (quasi dissolved state). Centrifugal reactor. Ddz-amino acids, deprotected by 5% trifluoroacetic acid/dichloromethane/15 min. 3-Nitrophthalic anhydride to suppress formation of false sequences. Continuous photometric control of the completion of all operations during synthesis and transesterfication, yielding Ddz-Cys(Acm)-Ser(But)-Phe-Glu-(OBut)-Glu(OBut)-OMe (0.827 g; 53%, m.p. 180-182 degrees).     

Synthesis and 13C-NMR spectra of the N-terminal decapeptide sequence of human lymphoblast interferon

The N-terminal sequence 1-10 of interferon HuIFN-alpha(Ly) from human lymphoblasts Ser-Asp-Leu-Pro-Gln-Thr-His-Ser-Leu-Gly (LIF[1-10]) was synthesized by the Merrifield method. N-tert-Butyloxycarbonylglycin was esterified via its cesium salt with a chloro-methylated polystyrene-1% divinylbenzene support yielding a loading of 0.3 mmol/g. Double couplings, each with a five-fold excess of N-protected amino acid, were performed with N,N'-dicyclohexylcarbodiimide and 1-hydroxybenzotriazole, followed by an acetylation step. N-tert-Butyloxycarbonyl-L-amino acids with O-benzyl protection for serine, threonine, and Nim-2,4-dinitrophenyl protection for histidine, and N-fluorenylmethyloxycarbonylaspartic acid beta-tert-butyl ester were used. N-tert-Butyloxycarbonyl-glutamine was coupled as 4-nitrophenyl ester in the presence of 1-hydroxybenzotriazole. The butyloxycarbonyl groups of the residues 3 to 10 were removed with trifluoroacetic acid in dichloromethane; the 9-fluorenylmethyloxycarbonyl group was split off with diethylamine. After quantitative hydrazinolysis in dimethylformamide, chromatography on Sephadex LH-20 with methanol and reversed-phase chromatography on silica gel RP-8 with methanol/water 9:1, the decapeptide hydrazide Boc-Ser(Bzl)-Asp(But)-Leu-Pro-Gln-Thr(Bzl)-His-Ser(Bzl)-Leu-Gly-NH-HN2 was isolated in pure state. The partially protected decapeptide was characterized by 13C-NMR spectroscopy, analysed, and linked with poly(L-lysine) (molecular mass 37 300) via its azide and also using m-xylylene diisocyanate. After a deprotection step the polylysine-LIF[1-10] antigens were dialyzed and lyophilized. Furthermore the free decapeptide LIF[1-10] was split-off from the resin using HBr/CF3CO2H, followed by mercaptoethanol treatment. After purification on Sephadex G-15 with 0.1 M acetic acid and on the reversed-phase silicagel RP-8 with methanol/water 9:1 water soluble LIF-[1-10] was obtained in pure state as shown by thin-layer-chromatography, electrophoreses amino acid analysis and 13C-NMR spectroscopy.     

Derivatives of Aminoquinones with N-protected Amino Acids

The synthesis of derivatives of aminoquinones with N-protected amino acids is reported here. 2-Amino-1,4-benzoquinone and 2-amino-1,4-naphthoquinone, prepared by the azide method in yields of 60 and 95% respectively, were coupled with N-Boc-protected amino acids including glycine, serine, proline and tyrosine, to give the correspondening derivatives. N,N'-Diisopropylcarbodiimide/1-hydroxybenzotriazole or N,N'-dicyclohexylcarbodiimide/HOBt used as coupling reagents provided the expected products in satisfactory yields and purities as supported by TLC, HPLC and spectral analysis.     

Addition of HOXt (X = A or B) improves the efficiency of phenol-based coupling reagents during peptide synthesis

Summary In the course of comparing the effectiveness of HATU, HBTU, and phenol-based coupling reagents, such as the pentafluorophenyl, 2-nitrophenyl, and 2,4,5-trichlorophenyl uronium salts by (a) formation of Fmoc-Ala-Val-OtBu, (b) (2+1) segment coupling and (c) stepwise solid phase peptide assembly of typical model peptides such as the pentapeptide H-Tyr-Aib-Aib-Phe-Leu-NH₂ and ACP decapeptide (65–74), we found a striking improvement of the less effective phenol-based coupling reagents (HPyOPfp, HPyONp, and HPyOTcp), both with regard to reaction rate and extent of epimerization, when HOAt was added and a clear superiority of HAPyU (in the presence and absence of HOAt) relative to the compounds derived from HOBt, HOPfp, HONp, and HOTcp. Abbreviations: Aib, α-aminoisobutyric acid; DIEA, diisopropylethylamine; TMP, collidine, 2,4,6,-tri-methylpyridine; DMF, N, N-dimethylformamide; HOBt, 1-hydroxybenzotriazole; HOAt, 7-aza-1-hydroxybenzotriazole; HAPyU, 1-(1-pyrrolidinyl-1H-1,2,3-triazolo[4,5-b]pyridin-1-ylmethylene)-N-methylmethanaminium hexafluorophosphate N-oxide; HOPfp, pentafluorophenol; HONp, 2-nitrophenol; HOTcp, 2,4,5-trichlorophenol; HPyOPfp,bis(tetramethylene)pentafluorophenoxyformamidinium hexafluorophosphate; HpyONp,bis(tetramethylene)-2-nitrophenoxyformamidinium hexafluorophosphate; HPyOTcp,bis(tetramethylene)-2,4,5-trichlorophenoxyformamidinium hexafluorophosphate; BTCFHbis(tetramethylene)chloroformamidinium hexafluororophosphate. Amino acids and peptides are abbreviated and designated following the rules of the IUPAC-IUB Commission of Biochemical Nomenclature [J. Biol. Chem., 247 (1972) 977]     

A convenient general method for synthesis of N alpha- or N omega-dithiasuccinoyl (Dts) amino acids and dipeptides: application of polyethylene glycol as a carrier for functional purification

N alpha-Dithiasuccinoyl (Dts) amino acids needed for solid-phase peptide synthesis have been prepared in good yields and excellent purities by a new method that exploits the solubility properties of polyethylene glycol (PEG; bifunctional with average molecular weight 2000 was found to be optimal). Suitably side-chain protected amino acid derivatives are first reacted with a polymeric xanthate, following which the free alpha-carboxyl is blocked by silylation and the Dts heterocycle is elaborated in the same pot by reaction with chlorocarbonylsulfenyl chloride. Upon aqueous work-up, the polymeric carrier removes any urethane blocked amino acids which arise during the process. Exaggerated conditions were explored to prove the power of this functional purification approach, and mechanisms of formation of polymer-bound urethanes are proposed and supported by solution model studies. The preparation and characterization of the companion N-(iso-propyldithio)carbonyl derivative of proline is also presented.     

The Use of a Chrome Alum-Gelatin (Subbing) Solution as a General Adhesive for Paraffin Sections

The adhesion obtained from a chrome alum-gelatin solution has been found far superior to results given by widely used general adhesives (Haupt's gelatin and Mayer's egg albumen) for paraffin sections. The subbing solution, which consists of 5.0 gin gelatin and 0.5 gm chrome alum per liter of water, is easier to apply and gives more consistent results. Sections affixed to subbed slides are resistant to removal by acids and bases: 1.0 and 0.1 N HCl or H₂SO₄, 1 M H₃PO₄, 5% oxalic and trichloroacetic acids, 1% and 10% lactic acid, 1.0 and 0.1 N NaOH or NH₄OH, and other fluids and solutions such as organic solvents, water, hypochlorite, KMnO₄ and thiosulfate. The applied adhesive is virtually unstained by many stains, including hematoxylin, eosin, fast green, safranin, PAS, Sudan IV and Mallory's triple stain. The only treatment yet found to detach affixed section in less than 6 hr is immersion in 5% trichloroacetic acid for 15 min at 100 C. The concentration of gelatin and chrome alum in the solution recommended is much lower than in previously described adhesives, but this does not seem to lessen its ability to affix the sections. If the concentrations of gelatin and chrome alum are decreased from those described, adhesive qualities are also decreased. An increase in the concentration of the ingredients causes the adhesive to become stained. The described solution therefore gives optimum adhesion and “resistance” to staining.     

Determination of methylated basic, 5-hydroxylysine, elastin crosslinks, other amino acids, and the amino sugars in proteins and tissues

Analytical single-column chromatographic methods have been developed for determining all methylated basic amino acids, isodesmosine, desmosine, the amino sugars glucosamine and galactosamine, the diastereoisomers of 5-hydroxylysine, and related compounds at picomole levels in protein and tissue hydrolysates. Complete resolution of all these unique basic amino acids as discrete peaks was achieved in 5.4 on a 50 X 0.28-cm microcolumn of Dionex type DC-4A spherical resin (9.0 +/- 0.5 micron) using updated instrumentation commonly available for amino acid analysis. The column was operated at 5.65 ml/h with two 0.35 M sodium citrate buffers (pH 5.700 and 4.501), at two temperatures (31.5 and 73 degrees C). Excellent resolution of all omega-N-methylarginines and related compounds was also achieved in 3 h using a 17.5 X 0.28-cm microcolumn of Dionex DC-5A resin (sized to 6.0 +/- 0.5 microns), two citrate buffers (0.21 M Na+, pH 5.125; 0.35 M Na+, pH 5.700), a buffer flow rate of 5.75 ml/h, and a temperature of 52 degrees C. Complete separation of all other amino acids found in protein or tissue hydrolysates including S-carboxymethyl cysteine, 4-hydroxyproline, methionine S,S-dioxide, and the amino sugars was also carried out in 95 min using a 23.5 X 0.28-cm microcolumn of Dionex DC-5A resin. The use of purified microcolumn buffers gave smooth baselines without interference from artifacts or minor hydrolysate components. The major advantages of these methods are: first, their high resolving power; second, their high sensitivity which is comparable and in some aspects superior to the newer instruments; and third, their high reproducibility (100 +/- 2.5%) and low operating costs. These methods should be especially valuable for determining myosin, actin, and elastin in tissue hydrolysates from the amounts of N tau-methylhistidine, desmosine, or isodesmosine present, respectively, and for studying protein methylation, hydroxylation, cross-linking formation, and the turnover rates of contractile and connective tissue proteins in biological systems.     

Orthogonal solid-phase synthesis of human gastrin-I under mild conditions

An approach to the solid-phase segment condensation synthesis of the 17-peptide amide human gastrin-I has been developed. N alpha-amino and side-chain protection were provided by 9-fluorenylmethyloxycarbonyl (Fmoc) and tert.-butyl groups, and a series of anchors cleavable under mild conditions were used. The N-terminal pentapeptide pGlu-Gly-Pro-Trp-Leu-OH was prepared using a p-alkoxybenzyl ester linkage made by a preformed handle strategy. Cleavage, in 65% yield, was with the new Reagent M: CF3COOH-CH2Cl2-beta-mercaptoethanol--anisole (70:30:2:1), which was optimized to preserve the labile tryptophan residue. A new preformed handle procedure expedited solid-phase synthesis of the protected "middle" hexapeptide, Fmoc-(Glu(OtBu]5-Ala-OH, anchored as an o-nitrobenzyl ester. Chains were not lost during this assembly, and final photolytic cleavage (350 nm) in toluene--CF3CH2OH (4:1) occurred in 59% yield. Both protected intermediates were purified by simple gel filtration, whereupon they were shown to be pure by analytical HPLC, and gave satisfactory NMR and FABMS spectra. Last, the C-terminal hexapeptide, Tyr(tBu)-Gly-Trp-Met-Asp(OtBu)-Phe, was assembled on a tris(alkoxy)benzylamide "PAL" support. For the polymer-supported segment condensation, the middle and N-terminal pieces were added respectively in greater than 98% and 89% yields (judged by amino acid analysis and solid-phase sequencing), by overnight couplings in N,N-dimethylformamide (DMF) mediated by benzotriazolyl N-oxytrisdimethylaminophosphonium hexafluorophosphate (BOP) in the presence of 1-hydroxybenzotriazole (HOBt) and N-methylmorpholine (NMM). Racemization was 4% and 11% respectively at Ala and Leu. Cleavage with Reagent M followed by reversed-phase chromatography gave pure gastrin-I in an overall 30% isolated yield. These results compare favorably with those from a stepwise assembly.     

Derivatives of Aminoquinones with N-protected Amino Acids

Summary The synthesis of derivatives of aminoquinones with N-protected amino acids is reported here. 2-Amino-1,4-benzoquinone and 2-amino-1,4-naphthoquinone, prepared by the azide method in yields of 60 and 95% respectively, were coupled with N-Boc-protected amino acids including glycine, serine, proline and tyrosine, to give the correspondening derivatives.N, N′-Diisopropylcarbodiimide/1-hydroxybenzotriazole orN, N′-dicyclohexylcarbodiimide/HOBt used as coupling reagents provided the expected products in satisfactory yields and purities as supported by TLC, HPLC and spectral analysis.     

Interaction of hydrated electron with dietary flavonoids and phenolic acids: rate constants and transient spectra studied by pulse radiolysis.

The reaction rate constants and transient spectra of 11 flavonoids and 4 phenolic acids reacting with e(aq)- at neutral pH were measured. Absorption bands of the transients of e(aq)- reacting with the above compounds all located at a wavelength shorter than 400 nm. The e(aq)- scavenging abilities were divided into three groups: (+)catechin ((1.2 +/-0.1) x 10(8) M(-1)s(-1)) < 4-chromanol ((4.4 +/- 0.4) x 10(8) M(-1)s(-1)) < genistein ((6.2+/-0.4) x 10(9) M (-1) s(-1) approximately genistin ((8 +/- 1) x 10(9) M(-1)s(-1)) approximately rutin ((7.6 +/- 0.4) x M(-1)s(-1) approximately caffeic acid ((8.3 +/- 0.5) x 10(9)M(-1)s(-1)) < transcinnamic acid((1.1 +/- 0.1) x 10(10) M(-1)s(-1)) approximately p-coumaric acid ((1.1 +/- 0.1) x 10(10) M(-1)s(-1) approximately 2,4,6-trihydroxylbenzoic acid((1.1 +/- 0.1) x 10(10) M(-1)s(-1)) approximately baicalein ((1.1 +/- 0.5) x 10(10) M(-1)s(-1)) approximately baicalin((1.3 + 0.1) X 10(10) M(-1)s(-1)) approximately naringenin ((1.2 +/- 0.1) x 10(10) M(-1)s(-1)) approximately naringin ((1.0 +/- 0.1) x 10(10) M(-1)s(-1)) approximately gossypin((1.2 +/- 0.1) x 10(10) M(-1)s(-1)) approximately quercetin((1.3 +/- 0.5) x 10(10) M(-1)s(-1)). These results suggested that C4 keto group is the active site for e(aq)- to attack on flavonoids and phenolic acids, whereas the o-dihydroxy structure in B ring, the C2,3 double bond, the C3-OH group, and glucosylation, which are key structures that influence the antioxidant activities of flavonoids and phenolic acids, have little effects on the e(aq)- scavenging activities.     

Determination of D-amino acids labeled with fluorescent chiral reagents, R(-)- and S(+)-4-(3-isothiocyanatopyrrolidin-1-yl)-7-(N, N-dimethylaminosulfonyl)-2,1,3-benzoxadiazoles, in biological and food samples by liquid chromatography

D-Amino acids in food and biological samples labeled with R(-)- and S(+)-4-(3-isothiocyanatopyrrolidin-1-yl)-7-(N, N-dimethylaminosulfonyl)-2,1,3-benzoxadiazoles (DBD-PyNCS) were separated by reversed-phase chromatography and detected fluorometrically at 550 nm (excitation at 460 nm). DL-Amino acids were efficiently labeled at 55 degrees C for 20 min in basic medium. The resulting thiocarbamoyl-amino acids were resolved by an isocratic elution using water:30% methanol in acetonitrile (72:28) containing 0.1% trifluoracetic acid as mobile phase for hydrophilic amino acids and gradient elutions using sodium acetate buffer (pH 5. 2)/acetonitrile as gradient solvent mixture for hydrophobic amino acids, respectively. The detection limits (S/N = 3) of DL-amino acids tested were in the range of 0.16-0.75 pmol. The proposed method was applied to determine the D-amino acid(s) in milk, cream, fermented dairy products (yogurt and yakult), tomato products (juice, puree, and catchup), fermented beverages (beer and red wine), and human urine. The existence of D-amino acid(s) was demonstrated in all the samples tested. Furthermore, the identification of the D-amino acid(s) was performed using both isomers of DBD-PyNCS and by on-line HPLC-electrospray ionization-MS.     

A new method for extraction of iron-molybdenum cofactor (FeMoco) from nitrogenase adsorbed to DEAE-cellulose. 1. Effects of anions, cations, and preextraction treatments

A convenient and rapid method of obtaining the cofactor of nitrogenase (FeMoco) with a low and apparently limiting Fe/Mo ratio has been developed. FeMoco can be extracted from the MoFe protein bound to DEAE-cellulose. The cofactor is eluted in either N-methylformamide (NMF), N,N-dimethylformamide (DMF), or mixtures of these solvents by use of salts such as Et4NBr,Bu4NBr,Ph4PCl, and Ph4AsCl. The method is simple, is rapid (45 min), yields concentrated cofactor, and, unlike the original method [Shah, V. K., & Brill, W. J. (1977) Proc. Natl. Acad. Sci. U.S.A. 74, 3249-3253] which requires anaerobic centrifugation, is easily scaled up. Furthermore, it gives yields of cofactor in excess of 70%. Its disadvantages are a high Fe:Mo ratio when DMF is the extracting solvent and a high salt concentration in the resultant FeMoco solution. These disadvantages are easily overcome by removing excess Fe by pretreating the cofactor with bipyridyl while still on the column. This gives Fe:Mo ratios of (6 +/- 1):1 (11 trials) with specific activities ranging from 170 to 220 nmol of C2H4/[min.(nmol of Mo)]. Chromatography on Sephadex LH-20 removes ca. 99% of the excess salt. The adsorption of MoFe protein to DEAE-cellulose seems to facilitate denaturation by organic solvents so that pretreatment of the protein with acid, used in earlier methods, is unnecessary. There is an apparent dependence on the charge density of the anion employed for elution of FeMoco bound to DEAE-cellulose, such that Cl- greater than Br- much greater than I-, PF6- is the order of effectiveness of the Bu4N+ salts of these anions.(ABSTRACT TRUNCATED AT 250 WORDS)     

Synthesis of a selectively protected trisaccharide building block of the capsular polysaccharide of Streptococcus pneumoniae types 6A and 6B

4-Methoxybenzyl 2,4-di-O-benzyl-3-O-[2,4,6-tri-O-benzyl-3-O-(3,4,6-tri-O-benzyl-alpha-D- galactopyranosyl)-alpha-D-glucopyranosyl]-alpha-L-rhamnopyranoside (22), a building block for the alpha-D-Galp-(1----3)-alpha-D-Glcp-(1----3)-alpha-L-Rhap fragment of the capsular polysaccharides of Streptococcus pneumoniae types 6A and 6B [----2)-alpha-D-Galp-(1----3)-alpha-D-Glcp-(1----3)-alpha-L-Rhap-( 1----X)-D- RibOH-(5-P----]n (6A, X = 3; 6B, X = 4) has been synthesised. Ethyl 3-O-allyl-2,4,6-tri-O-benzyl-1-thio-beta-D-glucopyranoside was coupled with 4-methoxybenzyl 2,4-di-O-benzyl-alpha-L-rhamnopyranoside in ether, using methyl triflate as promoter. The resulting alpha-D-Glcp-(1----3)-alpha-L-Rhap derivative was deallylated with KOBut in N,N-dimethylformamide followed by 0.1M HCl in 9:1 acetone-water. The product was coupled with 3,4,6-tri-O-acetyl-2-O-allyl-alpha,beta-D-galactopyranosyl trichloroacetimidate in ether, using trimethylsilyl triflate, to yield 19. Deacetylation, benzylation, and deallylation then gave 22.     

Coccidian parasites (Apicomplexa: Eimeriidae) of Microtus spp. (Rodentia: Arvicolidae) from the United States, Mexico, and Japan, with descriptions of five new species

Beginning in July 1980, 149 voles (Microtus spp.) representing 9 species and 14 subspecies collected in Japan, Mexico and the United States were examined for coccidia; 67 (45%) had oocysts in their feces. These included 1 of 3 (33%) M. californicus sactidiegi; 0 of 1 M. longicaudus longicaudus; 0 of 1 M. l. macrurus; 48 of 111 (43%) M. mexicanus including 11 of 26 (42%) M. m. fulviventer, 1 of 2 (50%) M. m. fundatus, 13 of 31 (42%) M. m. mexicanus, 1 of 4 (25%) M. m. mogollonensis and 22 of 48 (46%) M. m. subsimus; 5 of 8 (63%) M. montanus arizonensis; 6 of 6 M. montebelli montebelli; 2 of 4 (50%) M. oregoni oregoni; 5 of 13 (38%) M. pennsylvanicus pennsylvanicus; 0 of 1 M. quasiater and 0 of 1 M. townsendii townsendii. The following coccidians were identified from infected voles: Eimeria saxei n. sp. (syn. E. wenrichi "B") from M. c. sactidiegi; E. ochrogasteri, E. saxei, E. wenrichi (syn. E. wenrichi "A"), and Eimeria sp. from M. m. fulviventer, Eimeria sp. from M. m. fundatus; E. ochrogasteri, E. saxei, Eimeria tolucadensis n. sp., E. wenrichi, and Eimeria sp. from M. m. mexicanus; E. wenrichi from M. m. mogollonensis; Eimeria coahuiliensis n. sp., E. saxei, Eimeria subsimi n. sp., E. wenrichi, Eimeria sp., and Isospora mexicanasubsimi n. sp. from M. m. subsimus; E. tamiasciuri and E. wenrichi from M. m. arizonensis; Eimeria spp. from M. m. montebelli; E. saxei and E. wenrichi from M. o. oregoni; and E. ochrogasteri and E. wenrichi from M. p. pennsylvanicus. Sporulated oocytsts of Eimeria coahuiliensis n. sp. were ellipsoid, 29.6 X 19.6 (27-34 X 18-22) micron with ovoid sporocysts 14.4 X 8.9 (13-18 X 8-10) microns. Sporulated oocysts of Eimeria saxei n. sp. were subspheroid, 13.0 X 11.0 (11-14 X 10-12) micron with ovoid sporocysts 7.5 X 4.0 (6-9 X 4-5) micron. Sporulated oocysts of Eimeria subsimi n. sp. were ovoid/subspheroid, 25.1 X 18.7 (22-28 X 17-21) micron with ellipsoid sporocysts 13.9 X 7.4 (13-15 X 6-8) micron. Sporulated oocysts of Eimeria tolucadensis n. sp. were subspheroid, 25.4 X 20.3 (23-26 X 19-23) micron with ellipsoid sporocysts 11.3 X 7.8 (10-13 X 7-9) micron. Sporulated oocysts of Isospora mexicanasubsimi n. sp. were subspheroid, 23.7 X 23.1 (21-26 X 21-26) micron with ovoid sporocysts 14.9 X 10.8 (12-16 X 10-12) micron.(ABSTRACT TRUNCATED AT 400 WORDS)     

Synthesis of O-alpha-L-fucopyranosyl-(1----3)-O-beta-D-galactopyranosyl-(1----4)-2- acetamido-2-deoxy-D-glucopyranose (N-acetyl-3'-O-alpha-L-fucopyranosyllactosamine)

Methyl 2-O-benzyl-beta-D-galactopyranoside (6) was obtained in five, good yielding steps from methyl beta-D-galactopyranoside (1). Treatment of 1 with tert-butylchlorodiphenylsilane in N,N-dimethylformamide in the presence of imidazole afforded a 6-(tert-butyldiphenylsilyl) ether, which was converted into its 3,4-O-isopropylidene derivative (3). Benzylation of 3 with benzyl bromide-silver oxide in N,N-dimethylformamide, and subsequent cleavage of its acetal and ether groups then afforded 6. On similar benzylation, followed by the same sequence of deprotection, benzyl 2-acetamido-3,6-di-O-benzyl-4-O-[6-O-(tert-butyldiphenylsilyl)-3,4 -O- isopropylidene-beta-D-galactopyranosyl]-2-deoxy-alpha-D-glucopyranoside gave the 2-O-benzyl derivative (10). Compound 10 was converted into its 4,6-O-benzylidene acetal (11). Glycosylation (catalyzed by halide-ion) of 11 with 2,3,4-tri-O-benzyl-alpha-L-fucopyranosyl bromide afforded the fully protected trisaccharide derivative (13). Cleavage of the benzylidene and then the benzyl groups of 13 furnished the title trisaccharide (16). The structure of 16 was established by 13C-n.m.r. spectroscopy.     

A chromatographic method for the quantitative analysis of the deprotection of dithiasuccinoyl (Dts) amino acids.

The dithiasuccinoyl (Dts)-protecting group for amino acids is revoved by thiols (2 equivalents) through the intermediacy of an open-chain carbamoyl disulfide. Starting materials, intermediates, and products can be separated from one another on the standard amino acid analyzer 0.9 × 54-cm column of sulfonated polystyrene resin with 0.2 n sodium citrate buffers. Compounds are detected with the standard ninhydrin-hydrindantin reagent because the hydrindantin acts as a reducing agent and the released amino acid reacts in situ with the ninhydrin to give a purple color. Elution times, integration constants, and the ratios of absorbances at 570 and 440 nm are tabulated. Quantitative conversion of dithiasuccinoyl amino acids to the parent amino acids can be achieved with 0.1 n sodium hydroxide, 0.01 m alcoholic sodium borohydride, 0.1 m triphenylphosphine or 2 m m tri-n-butylphosphine in dioxane-H₂O (9:1) or water, and with a variety of thiols under various conditions. The chromatographic methodology is applicable to the determination of rate constants of the pseudo-first-order reductive deprotection of dithiasuccinoyl amino acids.     

Utilization of 3-ethyl-1(N,N-dimethyl)aminopropylcarbodiimide (EDCI)/1-hydroxybenzotriazole (HOBt) as a polymerizing agent

The commonly used coupling reagents in peptide synthesessuch as dicyclohexylcabodiimide, diisopropylcarbodiimide and3-ethyl-1(N,N-dimethyl)aminopropylcarbodiimide with or without1-hydroxybenzotriazole or N-hydroxysuccinimide have been used as polymerizing agents in the synthesis of elastic/plastic protein-based polymers. It was found that 3-ethyl-1(N,N-dimethyl)aminopropylcarbodiimide with 1-hydroxybenzotriazole gave equally good polymers comparable toconventional p-nitrophenol approach. Further, we present here the polymerization and characterization of structural andfunctional properties of poly(Val-Pro-Gly-Val-Gly), which is themost striking repeating sequence in the bovine and porcine elastins. The polymers obtained by both p-nitrophenol and 3-ethyl-1(N,N-dimethyl)aminopropylcarbodiimide approach werecharacterized by carbon-13 and proton nuclear magnetic resonancespectroscopy, differential scanning calorimetry, circular dichroism and fourier transform infrared spectroscopy. These results conclude that poly(Val-Pro-Gly-Val-Gly) obtained by bothmethods were identical in all respects of physical and chemicalproperties indicates that 3-ethyl-1(N,N-dimethyl)aminopropylcarbodiimide with 1-hydroxybenzotriazole method can be conveniently employed to synthesize these polymers.