[Transformation of [5'-3H]riboflavin into 5,6-dimethylbenzimidazole]. / Umwandlung von [5'-3H]Riboflavin in 5,6-Dimethylbenzimidazol.

The 5,6-dimethylbenzimidazole unit of vitamin B12 is formed from riboflavin via FMN in aerobic and some aerotolerant bacteria. Thereby C-1' of the ribityl side chain gets C-2 of 5,6-dimethylbenzimidazole. Experiments with homogenates of Propionibacterium shermanii on the fate of C-atoms 2' to 5' of the ribityl side chain of riboflavin in this transformation are reported. It was found that [5'-3H]riboflavin leads to radioactive sugar phosphates. These were isolated, dephosphorylated, and separated. Thus 3H-D-fructose and 3H-D-glucose were detected. The degradation of 3H-D-glucose revealed that 14 per cent of the total radioactivity was located in C-1, and 86 per cent in C-6. This indicates that during 5,6-dimethylbenzimidazole biosynthesis a three carbon unit is formed from the ribityl side chain of riboflavin.

Biological evaluation of substance P antagonists.

Five undeca- and six C-terminal heptapeptide substance P (SP) analogues were tested for their capacity to block the contractile effect of SP on the guinea-pig isolated taenia coli. They had one feature in common, namely substitutions in positions 7 and 9 in the SP molecule. In the majority of analogues D-tryptophan was used for these substitutions. All analogues tested were found to be competitive antagonists to exogenous SP and to be capable of blocking the electrically induced non-cholinergic, non-adrenergic neuronal contraction of the taenia. Of the undecapeptides, (D-Arg1, D-Pro2, D-Trp7,9, Leu11) SP and (D-Arg1, D-Trp7,9, Leu11) SP (Spantide) had the highest pA2 value, 7.1-7.2, and the lowest IC50 value, 10(-6) M. The pA2 values of the heptapeptides were generally lower. Three of the most potent antagonists were tested for specificity and found to block the smooth muscle contraction induced by SP, physalaemin, eledoisin and bombesin but not that induced by bradykinin, carbachol, 5-hydroxytryptamine, histamine, prostaglandins and vasopressin. The SP antagonists were also tested for spasmogenic effect on the taenia and for their capacity to release histamine from rat isolated peritoneal mast cells. The spasmogenic activity displayed by most of the SP antagonists tested is likely to be related to their ability to release histamine since the contractile response was reduced by mepyramine, a histamine H1-receptor antagonist. (D-Arg1, D-Trp7,9, Leu11) SP was notable for combining a high antagonistic potency with a weak spasmogenic effect (and poor histamine releasing effect).

Structure-activity relationship of C-terminal hexa- and heptapeptide substance P antagonists as studied in the guinea-pig ileum.

The 14 C-terminal heptapeptide analogues and one hexapeptide analogue of substance P (SP) were synthesized on the basis of the SP antagonist [D-Pro2,D-Trp7,9]SP-(1-11). They were tested in the guinea-pig ileum preparation for spasmogenic and antagonistic activities. All analogues except two had antagonistic activity. Spasmogenic activity was observed in three heptapeptide SP antagonists: [Arg5,D-Trp7,D-pCl-Phe9]SP-(5-11), [Arg5,D-Trp7,9,p-Cl-Phe8]SP-(5-11) and [Arg5,D-Trp7,9,Nle11]SP-(5-11). However, this effect became greatly reduced upon successive applications in almost all ileum preparations. For antagonistic potency D-Trp turned out to be of greater importance in position 9 than in position 7 of the SP molecule. The presence of a free amino group at the N-terminal of the peptide was also of significant importance for antagonistic potency. Exchange of Met11 for Nle resulted in a considerable increase of antagonistic potency, while other substitutions in this position were ineffective or slightly reduced the antagonistic effect in the ileum preparation.

Long-term administration of a substance P antagonist, (D-Pro2, D-Trp7,9)-SP, abolishes the response to ocular trauma.

The long-term effect of ocular administration of a substance P (SP) antagonist, (D-Pro2, D-Trp7,9)-SP, was studied in the rabbit. After 2-3 months of topical administration of the antagonist twice daily, a mild trauma was applied in the form of infrared irradiation of the iris. The control eye responded with disruption of the blood-aqueous barrier, the eye treated with the antagonist did not. Two days after termination of treatment, the response to ocular injury was still reduced. Another 2 days later, ocular injury evoked a normal response, which shows that the protection was reversible. No adverse reaction to the SP antagonist was noted.

Ocular responses evoked by capsaicin and prostaglandin E2 are inhibited by a substance P antagonist.

Injection of capsaicin or prostaglandin E2 into the vitreous chamber of the rabbit eye resulted in miosis and breakdown of the blood-aqueous barrier, manifested in aqueous flare. Pretreatment with the neuronal blocker tetrodotoxin or the substance P antagonist (D-Pro, D-Trp)-SP greatly reduced the ocular responses to capsaicin and prostaglandin E2. The results suggest a role for neuronal substance P in the ocular response to injury.

Bradykinin contracts the pupillary sphincter and evokes ocular inflammation through release of neuronal substance P.

Bradykinin contracts the isolated rabbit sphincter pupillae muscle. The contraction produced by 10(-8) M bradykinin was resistant to atropine but not to tetrodotoxin, suggesting a non-cholinergic nervous mechanism. The contraction was blocked by specific substance P (SP) antagonists, suggesting the involvement of SP. The SP antagonists tested were [D-Pro2,D-Trp7,9]SP-(1-11) and [Arg5,D-Trp7,9]SP-(5-11). The bradykinin-induced contraction exhibited marked tachyphylaxis in contrast to that induced by SP. It appears that the tachyphylaxis reflects the depletion of a bradykinin-sensitive neuronal pool of SP. Injection of bradykinin into the vitreous chamber of the rabbit eye caused miosis and disruption of the blood-aqueous barrier (manifested as aqueous flare). A second administration of bradykinin a few hours after the first injection evoked a reduced response; the response to SP upon repeated administration was unchanged. Atropine was without effect on the response to bradykinin whereas tetrodotoxin and the SP antagonists reduced the response. The results suggest that bradykinin causes miosis and aqueous flare at least partly through local release of neuronal SP.

The mechanism of action of a substance P antagonist (D-Pro2, D-Trp7,9)-SP.

1 A newly synthesized substance P (SP) analogue, (D-Pro2, D-Trp7,9)-SP, specifically antagonizes the contractile effects of SP on the guinea-pig isolated taenia coli. In addition, previous studies had indicated that the SP analogue per se is capable of contracting this preparation. The results of the present study on the guinea-pig taenia suggest that the smooth muscle contractions produced by the SP analogue are due to histamine release. No contractions were observed following blockade of histamine H1-receptors by mepyramine or following pretreatment with the histamine liberating agent, compound 48/80. 2 Analysis of the inhibition of SP-induced contraction by the analogue suggests that the inhibition is of the competitive type; pA2 was calculated to be 6.1. 3 We conclude that (D-Pro2, D-Trp7,9)-SP is a competitive SP antagonist with histamine-releasing properties.

Biosynthesis of vitamin B12. Different pathways in some aerobic and anaerobic microorganisms.

Radioactivity from [1'-14C]riboflavin was incorporated into the 5,6-dimethylbenzimidazole moiety of Vitamin B12 in the aerobes Bacillus megaterium, Nocardia rugosa and Streptomyces sp. as well as in the aerotolerant anaerobe Propionibacterium freudenreichii, but not in the anaerobe Eubacterium limosum. As recently published for E. limosum, also in the anaerobe Clostridium barkeri radioactivity from [1-14C]glycine and [2-14C]glycine was found in the 5,6-dimethylbenzimidazole moiety, but not in the corrin moiety. The addition of L-[methyl-14C]methionine to C. barkeri led to the labeling of the corrin moiety and the 5,6-dimethylbenzimidazole moiety, showing that the seven "extra" methyl groups in the corrin ring as well as the two methyl groups of the base part originate from this precursor. In Clostridium thermoaceticum, forming the vitamin B12 analog 5-methoxybenzimidazolylcobamide, [1-14C]glycine and [2-14C]glycine were also incorporated into the 5-methoxybenzimidazole moiety, but not into the corrin ring. In E. limosum L-[U-14C]glutamate led to the labeling of the corrin ring of vitamin B12, but not of its base moiety. These results together with data from the literature indicate that a common biosynthetic pathway might exist for the corrinoid biosynthesis in aerobic microorganisms, and in those aerotolerant anaerobes like the Propionibacteria, which form the 5,6-dimethylbenzimidazole moiety of vitamin B12 only under aerobic conditions. They also show that this pathway differs from the pathway found in anaerobic bacteria.

Design and synthesis of effective antagonists of substance P.

The agonist/antagonist activities of four background analogs of substance P (SP) facilitated design and synthesis of 12 new analogs to achieve effective antagonists. (D-Pro2, D-Phe7, D-Trp9)-SP, (D-Pro2, D-Trp7,9)-SP and (D-Arg1, D-Phe7, D-Trp9)-SP showed no agonist activity; 9 analogs showed weak agonist activity of SP. (D-Pro2, D-Trp7,9)-SP was the most potent antagonist which at a concentration of 10(-5) required a 3-fold increase in SP to allow a 50% response by SP. (D-Pro2, Lys6, D-Phe7)-SP and (D-Pro2, D-pClPhe7, D-Trp9)-SP were also potent, and the antagonism was competitive. For specific pairs of peptides, Lys6 is a promising substituent. D-Trp7,9 was as effective as Lys6, D-Phe7. D-pClPhe7 was three times as effective as D-Phe7. D-Dln6 was 1.33-fold better than D-Gln5. D-Pro2 and D-Pro4 were equally effective. D-Pro2 was 1.5 times as effective as D-Lys3. D-Pro2 may not be important. D-pClPhe9 and D-Trp9 were equally effective.

Sensory and motor functions of spinal cord substance P.

Low doses of D-Pro2-D-Phe7-D-Trp9-substance P, as specific substance P antagonist, depressed the scratching and biting behaviors elicited by intrathecal injections of substance P, and cutaneous application of algesic substances. Higher antagonist doses caused hindlimb paralysis. This suggests that substance P is a neurotransmitter for primary nociceptor afferents and may also have an important function in motor control.

Biosynthesis of vitamin B12. Experiments with the anaerobe Eubacterium limosum and some labelled substrates.

1. The anaerobic microorganism Eubacterium limosum (DSM 20 402), grown in the presence of L-[methyl-14C]methionine, produces vitamin B12 not only labeled in the corrin ring, but also in the 5,6-dimethylbenzimidazole moiety. The vitamin B12 obtained in a similar experiment with L-[methyl-13C]methionine showed nine signals in the 13CV NMR spectrum. Seven of these signals are due to the seven 'extra' methyl groups of the corrin ring, two signals originate from the two methyl groups of the 5,6-dimethylbenzimidazole moiety. This result is in contrast to previous experiments with aerobic and aerotolerant organisms, where L-methionine is only the precursor of the seven corrin methyl groups, and the 5,6-dimethylbenzimidazole is formed from riboflavin. 2. E. limosum transforms exogenously added benzimidazole into benzimidazolylcobamide, but with [2-14C]benzimidazole it was shown that benzimidazole is not a precursor of the 5,6-dimethylbenzimidazole moiety of vitamin B12. 3. Radioactivity from [2-14C]glycine is almost exclusively incorporated into the 5,6-dimethylbenzimidazole moiety of vitamin B12, but not into the corrin moiety. This demonstrates that in E. limosum delta-aminolevulinic acid, the precursor of the corrin moiety, is not formed as in many other organisms from succinyl-CoA and glycine.

Biosynthesis of vitamin B12. Some properties of the 5,6-dimethylbenzimidazole-forming system of Propionibacterium freudenreichii and Propionibacterium shermanii.

1. Homogenates of Propionibacterium freudenreichii transform riboflavin into 5,6-dimethylbenzimidazole. This process is stimulated by nicotinamide. Homogenates of Propionibacterium shermanii form only small amounts of 5,6-dimethylbenzimidazole from riboflavin in the absence of nicotinamide, but also form appreciable amounts in the presence of nicotinamide. 2. The stimulation of the 5,6-dimethylbenzimidazole-forming system by nicotinamide shows a lag phase which is abolished by preincubation of the homogenate with nicotinamide. Since no lag phase is observed when nicotinamide is replaced by nicotinate, nicotinate seems to be the true stimulating agent. These observations are in agreement with the fact that nicotinamide is rapidly split to nicotinate in homogenates of P. freudenreichii. 3. The 5,6-dimethylbenzimidazole-forming homogenate system is only active at a high buffer concentration (0.3--0.5 M) and in the presence of oxygen. The system has a pronounced oxygen optimum. 4. Flavin mononucleotide and flavin-adenine dinucleotide are better substrates for the 5,6-dimethylbenzimidazole-forming homogenate system than riboflavin. But with [1'-14C]riboflavin as substrate the specific radioactivity of 5,6-dimethylbenzimidazole is higher than the specific radioactivity of flavin--adenine dinucleotide and lower than the specific radioactivie substrate for the formation of 5,6-dimethylbenzimidazole. 5. A tentative reaction sequence for the transformation of flavin mononucleotide into 5,6-dimethylbenzimidazole is discussed.

Nonenzymatic transformation of riboflavin into 5,6-dimethylbenzimidazole.

Riboflavin, the biosynthetic precursor of the 5,6-dimethylbenzimidazole moiety of vitamin B12, is transformed non-enzymatically into 5,6-dimethylbenzimidazole in small yield on treatment with 1 N or 5 N NaOH at 100 degrees C. Besides 5,6-dimethylbenzimidazole 1,2-diamino-4,5-dimethylbenzene, 1,2-dihydro-6,7-dimethyl-2-keto-1-D-ribityl-3-quinoxaline carboxylic acid and N-1-D-ribitylamino-2-amino-4,5-dimethylbenzene can be detected. When [1 degree -14C]riboflavin is used the 5,6-dimethylbenzimidazole contains about 75 per cent of the specific radioactivity of riboflavin. N-1-D-ribityl-amino-2-amino-4,5-dimethylbenzene is transformed into 5,6-dimethylbenzimidazole more efficiently than riboflavin. Oxygen enhances the yield of 5,6-dimethylbenzimidazole and 1,2-diamino-4,5-dimethylbenzene from riboflavin as well as from N-1-D-ribitylamino-2-amino-4,5-dimethylbenzene. 1,2-diamino-4,5-dimethylbenzene reacts together with formaldehyde but not with formate to form 5,6-dimethylbenzimidazole under alkaline conditions at 100 degrees C. It is therefore suggested that the nonenzymatic reaction of riboflavin proceeds via N-1-D-ribitylamino-2-amino-4,5-dimethylbenzene and 1,2-diamino-4,5-dimethylbenzene, and that the latter reacts with formaldehyde preferably formed by oxidative degradation of C-1 degree of the ribitly side chain to form 5,6-dimethylbenzimidazole via its unstable imidazoline derivative. The possible relevance of these results for the enzymatic process is discussed.