Experimental Shock Chemistry of Aqueous Amino Acid Solutions and the Cometary Delivery of Prebiotic Compounds

A series of shock experiments were conducted to assess thefeasibility of the delivery of organic compounds to theEarth via cometary impacts. Aqueous solutions containingnear-saturation levels of amino acids (lysine, norvaline,aminobutyric acid, proline, and phenylalanine) were sealedinside stainless steel capsules and shocked by ballisticimpact with a steel projectile plate accelerated along a12-m-long gun barrel to velocities of 0.5–1.9 km sec^-1. Pressure-temperature-time histories of the shocked fluidswere calculated using 1D hydrodynamical simulations. Maximum conditions experienced by the solutions lasted0.85–2.7 s and ranged from 5.1–21 GPa and 412–870 K. Recovered sample capsules were milled open and liquid wasextracted. Samples were analyzed using high performanceliquid chromatography (HPLC) and mass spectrometry (MS). In all experiments, a large fraction of the amino acidssurvived. We observed differences in kinetic behavior andthe degree of survivability among the amino acids. Aminobutyricacid appeared to be the least reactive, and phenylalanine appeared to be the most reactive of the amino acids. The impact process resulted in the formation of peptide bonds; new compounds included amino acid dimers and cyclic diketopiperazines. In our experiments, and in certain naturally occurring impacts, pressure has a greater influencethan temperature in determining reaction pathways. Our resultssupport the hypothesis that significant concentrations of organic material could survive a natural impact process.     

Engineering an ATP-dependent <Emphasis Type="SmallCaps">d</Emphasis>-Ala:<Emphasis Type="SmallCaps">d</Emphasis>-Ala ligase for synthesizing amino acid amides from amino acids

We successfully engineered a new enzyme that catalyzes the formation of d-Ala amide (d-AlaNH₂) from d-Ala by modifying ATP-dependent d-Ala:d-Ala ligase (EC 6.3.2.4) from Thermus thermophilus, which catalyzes the formation of d-Ala-d-Ala from two molecules of d-Ala. The new enzyme was created by the replacement of the Ser293 residue with acidic amino acids, as it was speculated to bind to the second d-Ala of d-Ala-d-Ala. In addition, a replacement of the position with Glu performed better than that with Asp with regards to specificity for d-AlaNH₂ production. The S293E variant, which was selected as the best enzyme for d-AlaNH₂ production, exhibited an optimal activity at pH 9.0 and 40 °C for d-AlaNH₂ production. The apparent K _m values of this variant for d-Ala and NH₃ were 7.35 mM and 1.58 M, respectively. The S293E variant could catalyze the synthesis of 9.3 and 35.7 mM of d-AlaNH₂ from 10 and 50 mM d-Ala and 3 M NH₄Cl with conversion yields of 93 and 71.4 %, respectively. This is the first report showing the enzymatic formation of amino acid amides from amino acids.     

Mediation of growth hormone-enhanced expression of the cartilage phenotype in vitro by the availability of the essential amino acid valine

Without increasing cell number, ovine growth hormone was shown to stimulate the incorporation of ²⁵SO₄ by cultured chick embryo chondrocytes into chondroitin sulfate. Since the stimulation of sulfation by growth hormone was abolished when the amino acid concentrations in the medium were doubled, the relationship between amino acids and growth hormone in promoting the synthesis of acid mucopolysaccharides was investigated. Comparison of the incorporation of various labeled amino acids into trichloroacetic acid-soluble and insoluble material revealed that growth hormone promoted the incorporation of only valine into trichloroacetic acid-insoluble material. Furthermore, growth hormone stimulated valine incorporation into both extracellular and intracellular protein, rather than preferentially into extracellular chondromucoprotein. Growth hormone gave a 4-fold stimulation of valine incorporation into collagen without stimulating collagen synthesis. That growth hormone enhances sulfation by stimulating valine availability was further supported by the observations: (a) doubling only the valine concentration in the medium enhanced sulfation; (b) in medium with twice the normal valine concentration, sulfation failed to be further stimulated with the addition of growth hormone; and (c) in medium with all the other amino acids except valine at twice normal concentrations, growth hormone enhanced sulfation. In addition the temporal relationships and synthetic events occurring between growth hormonealtered valine availability and enhanced chondromucoprotein synthesis were studied. It was found that growth hormone-promoted valine incorporation into acid-insoluble material is a rapid effect that can be detected by 10 min after hormone addition and does not require RNA synthesis. Increased valine availability is rapidly reversed after growth hormone removal ( ). On the other hand, growth hormone- and valine-enhanced chondromucoprotein synthesis are slower responses, taking over 24 hr of treatment for a maximal stimulation, and are mediated by RNA synthesis, as indicated by actinomycin D sensitivity. Enhanced chondromucoprotein synthesis is also relatively stable after removal of growth hormone or valine ( ).The evidence suggests that the availability of a single amino acid, valine, plays a regulatory role in the synthesis of a specialized cellular product and that growth hormone acts at some level to alter the availability of this essential amino acid.     

Radiolysis,racemization, and the origin of optical activity

An investigation has been undertaken to determine whether ionizing radiation might engender racemization (radioracemization) of optically active amino acids, along with their well-known radiolysis. We have exposed a number of solid and dissolved optically active amino acids to the ionizing radiation from a 3000-Ci ⁶⁰Co γ-ray source for periods of time which would engender substantial, but not total radiolysis. γ-Ray doses which caused 55–68% radiolysis of solid amino acids typically engendered 2–5% racemization. Aqueous solutions of the sodium salts of amino acids which underwent 53–66% radiolysis typically showed 5–11% racemization. The corresponding hydrochloride salts in aqueous solution, however, underwent little or no racemization. In aqueous solution both percentage degradation and percentage racemization were approximately proportional to γ-ray dosage within the range employed (1–36 × 10⁶ rads). Mechanisms for the radioracemization of amino acids in the solid state and as dissolved sodium salts are proposed, and the absence of racemization for dissolved hydrochloride salts is rationalized. Implications of these observations with regard to the origin of optical activity by the Vester-Ulbricht β-decay mechanism are discussed, as are their implications regarding the use of diagenetic racemization rates of ancient amino acid samples as criteria for geochronological and geothermometric calculations.     

Chiral Discrimination by Ionic Liquids: Impact of Ionic Solutes

Chiral ionic liquids hold promise in many asymmetric applications. This study explores the impact of ionic solutes on the chiral discrimination of five amino acid methyl ester‐based ionic liquids, including L‐ and D‐alanine methyl ester, L‐proline methyl ester, L‐leucine methyl ester, and L‐valine methyl ester cations combined with bis(trifluoromethanesulfonimide) anion. Circularly polarized luminescence spectroscopy was used to study the chiral discrimination by measuring the racemization equilibrium of a dissymmetric europium complex, Eu(dpa)₃^3? (where dpa = 2,6‐pyridinedicarboxylate). The chiral discrimination measured was dependent on the concentration of Eu(dpa)₃^3? and this concentration‐dependence was different in each of the ionic liquids. Ionic liquids with L‐leucine methyl ester and L‐valine methyl ester even switched enantiomeric preference based on the solute concentration. Changing the cation of the Eu(dpa)₃^3? salt from tetrabutylammonium to tetramethylammonium ion also affected the chiral discrimination demonstrated by the ionic liquids. Chirality 27:320–325, 2015. © 2015 Wiley Periodicals, Inc.     

The effects of hypothyroidism and fasting on electrogenic amino acid transfer: Possible evidence for multiple neutral amino acid carrier systems in rat jejunum

The jejunal mechanisms for the electrogenic transfer of four neutral amino acids (alanine, leucine, methionine, valine) and for sarcosine were characterised by an electrical method in vitro. The values for apparent K_m obtained electrically agree well with those assessed by conventional chemical techniques. Hypothyroidism and/or fasting rats for 3 days induced differential changes in the apparent K_m and p.d._max for the various amino acids. These alterations were interpreted as indicating the presence of at least three mechanisms for neutral amino acid transfer and one for sarcosine.In euthyroid rats, only alanine showed changes in apparent K_m (decrease) and p.d._max (decrease) after fasting for 3 days. With hypothyroidism the kinetic parameters of electrogenic transfer for alanine, valine and sarcosine were significantly altered while those for leucine and methionine were unaffected.     

Abiotic Racemization Kinetics of Amino Acids in Marine Sediments

The ratios of d- versus l-amino acids can be used to infer the sources and composition of sedimentary organic matter. Such inferences, however, rely on knowing the rates at which amino acids in sedimentary organic matter racemize abiotically between the d- and the l-forms. Based on a heating experiment, we report kinetic parameters for racemization of aspartic acid, glutamic acid, serine, and alanine in bulk sediment from Aarhus Bay, Denmark, taken from the surface, 30 cm, and 340 cm depth below seafloor. Extrapolation to a typical cold deep sea sediment temperature of 3°C suggests racemization rate constants of 0.50×10⁻⁵–11×10⁻⁵ yr⁻¹. These results can be used in conjunction with measurements of sediment age to predict the ratio of d:l amino acids due solely to abiotic racemization of the source material, deviations from which can indicate the abundance and turnover of active microbial populations.     

Amino Acid Metabolism of Lemna minor L. : I. Responses to Methionine Sulfoximine

When Lemna minor L. is supplied with the potent inhibitor of glutamine synthetase, methionine sulfoximine, rapid changes in free amino acid levels occur. Glutamine, glutamate, asparagine, aspartate, alanine, and serine levels decline concomitantly with ammonia accumulation. However, not all free amino acid pools deplete in response to this inhibitor. Several free amino acids including proline, valine, leucine, isoleucine, threonine, lysine, phenylalanine, tyrosine, histidine, and methionine exhibit severalfold accumulations within 24 hours of methionine sulfoximine treatment. To investigate whether these latter amino acid accumulations result from de novo synthesis via a methionine sulfoximine insensitive pathway of ammonia assimilation (e.g. glutamate dehydrogenase) or from protein turnover, fronds of Lemna minor were prelabeled with [¹⁵N]H₄⁺ prior to supplying the inhibitor. Analyses of the ¹⁵N abundance of free amino acids suggest that protein turnover is the major source of these methionine sulfoximine induced amino acid accumulations. Thus, the pools of valine, leucine, isoleucine, proline, and threonine accumulated in response to the inhibitor in the presence of [¹⁵N]H₄⁺, are ¹⁴N enriched and are not apparently derived from ¹⁵N-labeled precursors. To account for the selective accumulation of amino acids, such as valine, leucine, isoleucine, proline, and threonine, it is necessary to envisage that these free amino acids are relatively poorly catabolized in vivo. The amino acids which deplete in response to methionine sulfoximine (i.e. glutamate, glutamine, alanine, aspartate, asparagine, and serine) are all presumably rapidly catabolized to ammonia, either in the photorespiratory pathway or by alternative routes.     

The fate of organic matter during planetary accretion: Preliminary studies of the organic chemistry of experimentally shocked murchison meteorite

It is possible that Earth's biologic precursors were delivered by late-impacting asteroids or comets, and it is possible that these objects were a source of Earth's volatile inventory. To understand the behavior of organic matter in carbonaceous meteorites during hypervelocity impact (1–2 km s^–1), three samples of the Murchison (CM2) carbonaceous chondrite were shocked to 19, 20 and 36 GPa and analyzed by very sensitive thermal-desorption photoionization mass spectrometry (SALI). Thermal-desorption (25–800 °C) SALI mass spectra of unshocked Murchison reveal indigenous aliphatic, aromatic, sulfur and organosulfur compounds. Samples shocked to 20 GPa exhibit little or no loss of organic matter relative to the unshocked material. This is consistent with the earlier work of Tyburczyet al. (1986) which showed that incipient devolatilization of Murchison occurs at peak shock pressures near 20 GPa. The small amount of organic matter lost appears to have occurred by volatilization of elemental sulfur, amines and aliphatic compounds. In the sample shocked to 36 GPa, approximately 70% of the organic matter was volatilized as a result of impact. The residual organic matter desorbed at somewhat higher temperatures and displayed a different chemical signature. In particular, the shocked material has a lower alkene/alkane ratio than that of the starting material. The preliminary data suggest that it is unlikely that the indigenous organic matter in carbonaceous chondrite-like planetesimals could have survived impact on the Earth in the later stages of Earth's accretion. However, chemical reactions that produce organic compounds with greater thermal stabilities may occur during impact or subsequent to impact by condensation of the impact-produced vapor plume.     

Selectivity in vitamin B-6 model reactions: Selective α or β deuteration of amino acids under transamination or racemization conditions

Al(III)-catalyzed reactions of vitamin B-6 (pyridoxal)-amino acid schiff bases have been studied in ²H₂O. By using excess of the amino acid and varying conditions, amino acids selectively deuterated in the α-position, the β-position, or in both α- and β-positions are isolated. Reaction conditions are those of model systems in which amino acids are known to be reversibly transaminated and racemized by pyridoxal and Al(III). The racemization reaction leads to α-deuteration of the amino acid while transamination followed by its reverse leads to both α- and β-deuteration. The two reactions are viewed as passing through a common dihydropyridine intermediate. The Al(III) serves as an interesting model for the enzyme in that it not only catalyzes transamination and racemization but also can be made to select which of these reactions predominates. This selective catalysis of these reactions is attributed to strong and different pH dependence of the reactivity of various sites of the dihydropyridine intermediate for vitamin B-6 catalysis when incorporated in an Al(III) complex. The biochemical importance of this selectivity and the practical extension of the method of deuteration to other amino acids is discussed.     

Effects of heat shock on amino Acid metabolism of cowpea cells

When cowpea (Vigna unguiculata) cells maintained at 26°C are transferred to 42°C, rapid accumulation of γ-aminobutyrate (>10-fold) is induced. Several other amino acids (including β-alanine, alanine, and proline) are also accumulated, but less extensively than γ-aminobutyrate. Total free amino acid levels are increased approximately 1.5-fold after 24 hours at 42°C. Heat shock also leads to release of amino acids into the medium, indicating heat shock damage to the integrity of the plasmalemma. Some of the changes in metabolic rates associated with heat shock were estimated by monitoring the ¹⁵N labeling kinetics of free intracellular, extracellular and protein-bound amino acids of cultures supplied with ¹⁵NH₄⁺, and analyzing the labeling data by computer simulation. Preliminary computer simulation models of nitrogen flux suggest that heat shock induces an increase in the γ-aminobutyrate synthesis rate from 12.5 nanomoles per hour per gram fresh weight in control cells maintained at 26°C, to as high as 800 nanomoles per hour per gram fresh weight within the first 2 hours of heat shock. This 64-fold increase in the γ-aminobutyrate synthesis rate greatly exceeds the expected (Q₁₀) change of metabolic rate of 2.5- to 3-fold due to a 16°C increase in temperature. We suggest that this metabolic response may in part involve an activation of glutamate decarboxylase in vivo, perhaps mediated by a transient cytoplasmic acidification. Proline appears to be synthesized from glutamate and not from ornithine in cowpea cells. Proline became severalfold more heavily labeled than ornithine, citrulline and arginine in both control and heat-shocked cultures. Proline synthesis rate was increased 2.7-fold by heat shock. Alanine, β-alanine, valine, leucine, and isoleucine synthesis rates were increased 1.6-, 3.5-, 2.0-, 5.0-, and 6.0-fold, respectively, by heat shock. In contrast, the phenylalanine synthesis rate was decreased by 50% in response to heat shock. The differential effects of heat stress on metabolic rates lead to flux and pool size redistributions throughout the entire network of amino acid metabolism.     

The effect of sugar,amino acid,metal ion,and NaCl on model Maillard reaction under pH control

Summary. The color intensities was determined of Maillard reaction products (MRPs) prepared by heating each of five sugars (maltose, fructose, glucose, arabinose, and xylose) with each of 12 amino acids (aspartic acid, glutamic acid, alanine, leucine, isoleucine, valine, proline, serine, cysteine, phenylalanine, arginine, and lysine). The remaining percentages of glucose and rate of change of color intensity due to the addition of a metal ion and NaCl were monitored for nine MRPs that had been formed between glucose and each of nine amino acids (aspartic acid, glutamic acid, alanine, valine, serine, cysteine, phenylalanine, arginine, and lysine). Model MRPs were prepared in a block heater at 100°C for 1–12h with the pH value controlled at 6.5. The resulting color intensity of each MRPs formed from the basic amino acids was greater due to the higher reactivity than those from the acidic amino acids. The remaining percentage of glucose in each MRPs from the basic amino acids was lower than those from the acidic amino acids. The MRPs from the nonpolar amino acids showed an intermediate color intensity and remaining percentages of glucose between those formed from the basic and acidic amino acids. Browning tended to be accelerated in the presence of metal ions, especially Fe²⁺ and Cu²⁺, although it was affected by the property of the amino acid and heating time as well as by the type of metal ion. On the other hand, browning was greatly inhibited by a high concentration of NaCl.     

Site of action of chlorsulfuron: inhibition of valine and isoleucine biosynthesis in plants

The sulfonylurea herbicide chlorsulfuron blocks the biosynthesis of the amino acids valine and isoleucine in plants. Addition of these two amino acids to excised pea root (Pisum sativum L. var Alaska) cultures incubated in the presence of chlorsulfuron completely alleviates herbicide-induced growth inhibition. The site of action of chlorsulfuron is the enzyme acetolactate synthase which catalyzes the first step in the biosynthesis of valine and isoleucine. This enzyme is extremely sensitive to inhibition by chlorsulfuron having I₅₀ values ranging from 18 to 36 nanomolar. In addition, acetolactate synthase from a wide variety of tolerant and sensitive plants species is highly sensitive to inhibition by chlorsulfuron.     

Updates on industrial production of amino acids using <Emphasis Type="Italic">Corynebacterium glutamicum</Emphasis>

l-Amino acids find various applications in biotechnology. l-Glutamic acid and its salts are used as flavor enhancers. Other l-amino acids are used as food or feed additives, in parenteral nutrition or as building blocks for the chemical and pharmaceutical industries. l-amino acids are synthesized from precursors of central carbon metabolism. Based on the knowledge of the biochemical pathways microbial fermentation processes of food, feed and pharma amino acids have been developed. Production strains of Corynebacterium glutamicum, which has been used safely for more than 50 years in food biotechnology, and Escherichia coli are constantly improved using metabolic engineering approaches. Research towards new processes is ongoing. Fermentative production of l-amino acids in the million-ton-scale has shaped modern biotechnology and its markets continue to grow steadily. This review focusses on recent achievements in strain development for amino acid production including the use of CRISPRi/dCas9, genome-reduced strains, biosensors and synthetic pathways to enable utilization of alternative carbon sources.     

Phloem amino acids and the host plant range of the polyphagous aphid, Aphis fabae

This study investigated the relationship between the essential amino acid requirement of the aphid Aphis fabae Scop. and the phloem sap amino acid composition of its host plants. The dietary amino acid requirement of A. fabae varied between clones. One or more of the eight clones of A. fabae tested displayed depressed larval survival, larval growth rate, or r_m on diets lacking histidine, methionine, threonine, and valine, but none of the other five essential amino acids. The required amino acids corresponded closely to the essential amino acids that varied in relative concentrations among 16 plant species tested: histidine, threonine, tryptophan, and valine. It is suggested that the interclonal variation in the dietary requirements of an aphid species may contribute to the intraspecific variation in plant utilisation patterns. The phloem sap amino acid composition and sucrose : amino acid ratio did not differ consistently between host plant species of A. fabae and non‐host species, indicating that phloem amino acid composition is not an important factor in determining the host plant range of this aphid species.     

Die Bildung höherer Alkohole bei Aminosäuremangelmutanten von Saccharomyces cerevisiae

1. The influence of varying amounts of amino acids on the uptake of threonine, isoleucine, valine and leucine and their degradation to higher alcohols was investigated using a mutant strain of Saccharomyces cerevisiae, mating type a, genetic markers ade2, hom2, thr4, ilv2, leu1.
2. The cell mass is increased by increasing concentrations of threonine, isoleucine, valine and leucine, the latter two resulting in a higher dry weight. The amino acids are completely utilised at low concentrations. At higher contents up to 20% of the amino acids remain in the medium. The uptake of threonine, isoleucine, valine and leucine depends on the relative amounts of the concentrations of these amino acids in the medium. A greater amount of an amino acid is taken up if its concentration is comparatively higher than those of the other amino acids. There is a competition between the amino acids for the uptake into the cells.

Higher amounts of intracellular isoleucine and leucine are converted to 2-and 3-methylbutanol when compared with the degradation of valine and threonine to isobutanol and n-propanol-1, isoleucine and leucine up to 90%, valine up to 24% and threonine up to 20%. There is a competition between the four amino acids for their degradation to the corresponding higher alcohols. This behaviour confirms the earlier assumption of a degradation of the four amino acids by unspecific enzymes.     

氨基酸的消旋研究

在纯乙酸溶剂中,以乙酸酐酰化消旋酪氨酸,消旋温度为90℃,L-酪氨酸和乙酸酐的摩尔比为12,每消旋1gL-酪氨酸时,溶剂乙酸的用量为5mL,效果较好。同样的方法应用于L-脯氨酸,L-苏氨酸,L-缬氨酸,L-赖氨酸,L-半胱氨酸等氨基酸,消旋率均可达到100%。     

Influence of valine and other amino acids on total diacetyl and 2,3-pentanedione levels during fermentation of brewer’s wort

Undesirable butter-tasting vicinal diketones are produced as by-products of valine and isoleucine biosynthesis during wort fermentation. One promising method of decreasing diacetyl production is through control of wort valine content since valine is involved in feedback inhibition of enzymes controlling the formation of diacetyl precursors. Here, the influence of valine supplementation, wort amino acid profile and free amino nitrogen content on diacetyl formation during wort fermentation with the lager yeast Saccharomyces pastorianus was investigated. Valine supplementation (100 to 300 mg L^?1) resulted in decreased maximum diacetyl concentrations (up to 37 % lower) and diacetyl concentrations at the end of fermentation (up to 33 % lower) in all trials. Composition of the amino acid spectrum of the wort also had an impact on diacetyl and 2,3-pentanedione production during fermentation. No direct correlation between the wort amino acid concentrations and diacetyl production was found, but rather a negative correlation between the uptake rate of valine (and also other branched-chain amino acids) and diacetyl production. Fermentation performance and yeast growth were unaffected by supplementations. Amino acid addition had a minor effect on higher alcohol and ester composition, suggesting that high levels of supplementation could affect the flavour profile of the beer. Modifying amino acid profile of wort, especially with respect to valine and the other branched-chain amino acids, may be an effective way of decreasing the amount of diacetyl formed during fermentation.     

DeltapH-Dependent Amino Acid Transport into Plasma Membrane Vesicles Isolated from Sugar Beet (Beta vulgaris L.) Leaves: II. Evidence for Multiple Aliphatic, Neutral Amino Acid Symports

Proton-coupled aliphatic, neutral amino acid transport was investigated in plasma membrane vesicles isolated from sugar beet (Beta vulgaris L., cv Great Western) leaves. Two neutral amino acid symport systems were resolved based on inter-amino acid transport competition and on large variations in the specific activity of each porter in different species. Competitive inhibition was observed for transport competition between alanine, methionine, glutamine, and leucine (the alanine group) and between isoleucine, valine, and threonine (the isoleucine group). The apparent K_m and K_i values were similar for transport competition among amino acids within the alanine group. In contrast, the kinetics of transport competition between these two groups of amino acids did not fit a simple competitive model. Furthermore, members of the isoleucine group were weak transport antagonists of the alanine group. These results are consistent with two independent neutral amino acid porters. In support of that conclusion, the ratio of the specific activity of alanine transport versus isoleucine transport varied from two- to 13-fold in plasma membrane vesicles isolated from different plant species. This ratio would be expected to remain relatively stable if these amino acids were moving through a single transport system and, indeed, the ratio of alanine to glutamine transport varied less than twofold. Analysis of the predicted structure of the aliphatic, neutral amino acids in solution shows that isoleucine, valine, and threonine contain a branched methyl or hydroxyl group at the β-carbon position that places a dense electron cloud close to the α-amino group. This does not occur for the unbranched amino acids or those that branch further away, e.g. leucine. We hypothesize that this structural feature of isoleucine, valine, and threonine results in unfavorable steric interactions with the alanine transport system that limits their flux through this porter. Hydrophobicity and hydrated volumes did not account for the observed differences in transport specificity.     

Large-scale production of peptides using the solid phase continuous flow method. Part 2: preparative synthesis of a 26-mer peptide thrombin inhibitor

A preparative method for the preparation of large peptides is described. An advantageous theoretical weight of peptide/weight of starting resin ratio (tP_w/R_w) of about 0.3 was successfully experimented. The esterification of the first amino acid was realized with a racemization of less than 1%. The study of the coupling conditions led to the use of a diluted acylating mixture that allowed a 56% consumption of the amino acid derivatives (percentage use of amino acids) introduced in the synthesis. The cost analysis of the synthesis showed that the recovery of the amino acid derivatives was not worthwhile. © 1998 European Peptide Society and John Wiley & Sons, Ltd.