Dipeptides detection

FIGURE 8.5 SEC of aromatic amino acids and dipeptides. Column Same as Fig. 8.1. Flow rate 0.6 ml/min. Mobile phase 50 m/VI formic acid. Detection Ajj, = 0.5 AUFS. 

Our initial attempts with this design using the NDA/CN" system were encouraging far beyond our expectations. Our enthusiasm was sustained when we found that dipeptide-CBI derivatives could be detected at the femtomole level (Tables IV and V) and that such limits of detection with our purified derivatives under less-than-... 

Weber, S. G., Tsai, H., and Sandberg, M., Electrochemical detection of dipeptides with selectivity against amino acids, ]. Chromatogr., 638, 1, 1993. 

The intestinal mucosal peptidases are distributed in the brush border and cytosol of the absorptive cell. There are, however, distinct differences between the brush border and cytosolic peptidases [75], The tetrapeptidase activity is associated exclusively with the brush border enzyme. Furthermore, brush border peptidases exhibit more activity against tripeptides than dipeptides, whereas the cytosolic enzymes show greater activity against dipeptides. Studies have demonstrated that more than 50% of dipeptidase activity was detected in the cytosol [76] and just 10% in the brush border membrane [77]. The brush border enzymes include... 

Using experimental conditions similar to those described above, Huber and Wachtershauser were able to detect the formation of peptide bonds in reactions involving three amino acids the main product, however, was only a dipeptide. 

FIGURE 1.28 Chromatograms of the HPLC enantiomer separation of Z-protected a-aminophosphinic acids (a,b) and phosphinic acid-ilr-dipeptide (c) on (a and b) a 0-9-(tert-butylcarbamoyl)quinidine CSP and (c) corresponding 0-9-(fcrr-butylcarbamoyl)qninine-CSP, respectively. Experimental conditions Column dimensions, 150 mm x 4 mm ID mobile phase, methanol-50 mM sodium phosphate buffer (80 20 v/v) (pHa 5.6) temperature, 40°C flow rate, 1 mLmin detection, UV at 250 nm and optical rotation detection (ORD). (Reproduced from M. Lammerhofer et ah, Tetrahedron Asymmetry, 14 2557 (2003). With permission.)... 

FIGURE 1.30 Micro-HPLC separation of all 4 stereoisomers of the dipeptide alanyl-alanine as FMOC derivatives (a) and DNP-derivatives (b), respectively, on a 0-9-(tert-butylcarbamoyl)quinine-based CSP. Experimental conditions Column dimension, 150 X 0.5 mm ID mobile phase (a) acetonitrile-methanol (80 20 v/v) containing 400 mM acetic acid and 4 mM triethylamine, and (b) methanol-0.5 M ammonium acetate buffer (80 20 v/v) (pHa 6.0) flow rate, 10 ixLmin temperature, 25 C injection volume, 250 nL detection, UV at 250 nm. (Reproduced fromC. Czerwenka et al., J. Pharm. Biomed. Anal., 30 1789 (2003). With permission.)... 

Gagne and coworkers utilized this combination to discover enantioselec-tive receptors for (-)-adenosine [12]. A racemic dipeptide hydrazone [( )-pro-aib] generated a stereochemically diverse DCL of n-mer. The dimers were composed of two chiral (DD/LL) and one achiral isomer (DL), the four trimers (DDD, LLL, DDL, and LLD), the tetramers of four chiral and two achiral isomers, etc. Two techniques were used to measure the enan-tio-imbalance that was caused by the enantioselective binding of the chiral analyte to the enantiomeric receptors (Fig. 5.11). Since the unperturbed library is optically inactive, the optical enrichment of each library component could be measured by a combined HPLC optical rotation detection scheme (laser polarimeter, LP). LP detection differentiated unselective binding (amplification but not optical enrichment) from enantioselective recognition of the analyte (amplification and optical enrichment). In this manner the LL dimer (SS) of the dipeptide was amplified and identified as the enantioselective match for (-)-adenosine. 

Production of ROS is associated with deafness in animals and humans. It appears that carnosine can suppress loss of hearing induced by antibiotics and other agents, although it is uncertain as to the precise mechanisms involved (Zhuravskii et ah, 2004a,b). Early studies had shown, however, that carnosine exhibited excitatory activity to the afferent fibers in the lateral line organ of frogs (Mroz and Sewell, 1989 Panzanelli et ah, 1994) which may indicate an evolutionary role of the dipeptide in sound detection. 

Aristoy, M. C. and Toldra, F. (2004). Histidine dipeptides HPLC-based test for the detection of mammalian origin proteins in feeds for ruminants. Meat Sci. 67, 211-217. 

Conversely, when 6 was extended N-terminally by a small portion of the prosequence, that is by the Lys-Arg dipeptide to give KR-ET-1 (7), and subjected to oxidation in 0.1 M NH4OAC buffer (pH 9.5) at 25 °C for 24 hours the ratio of native to nonnative-type disulfide isomer increases remarkably (88 12 vs 75 25 for 6), whilst isomer 3 is not detectable. In the presence of GSH/GSSG an additional increase to almost quantitative formation of the native isomer was observed (Table 2). 58 This improvement was completely abolished by substituting Asn for Asp at position 8 (D8N-KR-ET-1), whereas most of the increase was maintained with similar carboxamide analogues in positions 10 and 18 (Table 2). 

The classical methods for detection and quantitation of racemization require analysis of the chiral purity of the product of a peptide-bond-forming reaction. For example, the Anderson test is used to explore a variety of reaction conditions for the coupling of Z-Gly-Phe-OH to H-Gly-OEt (Scheme 6). 9 The two possible enantiomeric tripeptides are separable by fractional crystallization, so that gravimetric analysis furnishes the racemization data. This procedure has a detection limit of 1-2% of the epimerized tripeptide. A modification by Kemp,1"1 utilizing 14C-labeled carboxy components, extends the detection limit by two to three orders of magnitude by an isotopic dilution procedure. The Young test 11 addresses the coupling of Bz-Leu-OH to H-Gly-OEt, and the extent of epimerization is determined by measurement of the specific rotation of the dipeptide product. 

There are numerous studies of control of racemization for specific subsets of amino acids. For example, Benoiton has carried out extensive studies on racemization of Al-methyl amino acids. In particular, McDermott and Benoiton1261 demonstrated that the presence of tertiary amine salts in coupling reactions had a profound effect on activated TV-methyl amino acids in contrast to the nonmethylated form. In one example, when Z-Ala-MeLeu-OH was coupled to the tosylate salt of Gly-OBzl with ethyl 2-ethoxy-l,2-dihydroquinoline-l-carboxylate (EEDQ) in the presence of TEA, 15% of the l-d dipeptide was formed with a yield of 68%, compared to 0.5% for Z-Ala-Leu-OH with a yield of 78%. When the free base of Gly-OBzl was utilized in a DCC/HOSu coupling in the absence of tertiary amine, no l-d products were detected. The authors attributed this increased susceptibility to epimerization to an ox-azolonium intermediate, which can epimerize by proton abstraction or merely by tauto-merization (Scheme 11). 

Free dipeptides or their hydrobromide salts can be cyclized to the corresponding cyclodipeptides by heating in phenol (68JOC862). No detectable racemization takes place. The versatility of the method is shown by the synthesis of cyclo(Ser-Tyr), cyclo(Met-Tyr), cyclo(Gly-Trp), etc. Very facile cyclization (on warming in water) of sarcosyldehydrophenyl-alanine to l-methyl-3-fra/is-benzylidenepiperazine-2,5-dione (14) has been reported [70JCS(C)2530]. 

This type of disconnection is mainly used for the preparation of dipeptides of type Xaai >[ , CH=CH]Gly. It allows control of the stereochemistry of the Xaa residue by starting from chiral a-amino aldehydes. For the construction of the /ram -p,y-unsaturated carboxylic acid moiety, the use of the triphenylphosphonium salt 31 (Scheme 9) derived from 3-chloro-propanoic acid was not suitable.14 Instead, the trimethylsilylprop-2-ynyl phosphonium salt 33 serves as a three-carbon unit, which can be converted into the P,y-unsaturated acid by hydroboration and oxidation. The required Boc-protected a-amino aldehyde 32 can be prepared using virtually racemization-free procedures. 37 However, at the end of the reaction sequence, racemization has been detected, especially for Boc-Phet )[ , CH=CH]Gly-OH, but not for the Ala and Pro analogues. 63 A mixture of E- and Z-enynes 34 and 35 is formed (8 2 to 9 1), which can be separated by column chromatography. 4,48 50 53 64 65 ... 

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