Synthesis of phospho-urodilatin by combination of global phosphorylation with the segment coupling approach

The chemical synthesis of biologically active phosphorylated urodilatin (CDD/ANP-95-126) was achieved by using a strategy of coupling protected peptide segments in solution. Three protected peptide segments corresponding to urodilatin (1-14) with side chain-unprotected Ser¹⁰, (15-24) and (25-32) were prepared manually using Fmoc chemistry on an aminopropyl polystyrene resin with the super acid-labile HMPB linker. For the coupling of segments, the carboxy group of the C-terminal segment (25-32) was converted into the tert-butyl ester by treatment with TBTA. The protected peptide segments were coupled in the presence of EDC/HOOBt or TBTU/HOBt to yield fully protected urodilatin with a free hydroxy function at Ser¹⁰. Introduction of the phosphate was performed with Et₂NP(OtBu)₂ and tetrazole followed by oxidation of the phosphite. Alternatively, a prephosphorylated protected segment (1-14) was used in the segment condensation. Our investigations indicate that both pathways, phosphorylation of protected urodilatin in solution and use of a prephosphorylated building block, are suitable methods to obtain a large phosphopeptide of high purity without formation of H-phosphonates or other by-products. © Munksgaard 1996.     

Carbonyldiimidazole/1-hydroxybenzotriazole activation in polymer phase synthesis of the arginine rich proalbumin hexapeptide extension

The synthesis on different polymer phases of Arg-Gly-Val-Phe-Arg-Arg, the proalbumin extension, is reported. The peptide was prepared both on a 0.5% cross-linked polystyrene gel containing 2-oxoethyl bromide anchor functions and on a 1% cross-linked chloromethyl polystyrene. For temporary blocking of the amino groups we utilized the 2-(3, 5-dimethoxyphenyl)propyl-(2)-oxycarbonyl (Ddz). The guanidino groups of the three arginine moieties were protected by the 4-toluenesulfonyl (Tos) group. As coupling procedure we used 1 equiv. carbonyldiimidazole (CDI)/2 equiv. 1-hydroxybenzotriazole (HOBt). The C-terminal activation of the Ddz-amino acids with CDI/HOBt made it possible to recover the excess Ddz-amino acids in 60–80% yield. We also investigated different procedures to cleave the 2-oxoethyl ester bond between the peptide and the polymer. This bond was completely stable against trifluoro-methanesulfonic acid and was split by 1 N triethylamine in methanol/dioxane 1:1 (v/v) + 1 vol% 1 N NaOH. The optical rotation and HPLC properties of Arg-Gly-Val-Phe-Arg-Arg from this synthesis are identical to the product from a different synthesis published earlier.     

(1H‐Benzotriazol‐1‐yloxy)‐N,N‐dimethylmethaniminium hexachloroantimonate (BOMI), a novel coupling reagent for solution and solid‐phase peptide synthesis

Abstract: A HOBt‐based immonium‐type compound,(1H‐benzotriazol‐1‐yloxy)‐N,N‐dimethyl methaniminium hexachloro‐antimonate (BOMI), was synthesized and successfully applied to the synthesis of various oligopeptides with good yields. The estimation of racemization and the influence of several reaction parameters such as solvents, bases and temperature were studied by HPLC using a model reaction. It was found that the reactivity of BOMI was much higher than that of HOBt‐based phosphonium‐ and uronium‐type coupling reagents. Moreover, its racemization was lower than that of other HOBt‐derived coupling reagents. The effectiveness of BOMI was also demonstrated by the synthesis of Leu‐enkephalin both in solution and in the solid‐phase.     

Practical protocols for stepwise solid‐phase synthesis of cysteine‐containing peptides

Abstract: This study details a series of conditions that may be applied to ensure ‘safe’ incorporation of cysteine with minimal racemization during automated or manual solid‐phase peptide synthesis. Earlier studies from our laboratories [Han et al. (1997) J. Org. Chem. 62 , 4307–4312] showed that several common coupling methods, including those exploiting in situ activating agents such as N‐[(dimethylamino)‐1H‐1,2,3‐triazolo[4,5‐b]pyridin‐1‐ylmethylene]‐N‐methylmethanaminium hexafluorophosphate N‐oxide (HATU), N‐[1H‐benzotriazol‐1‐yl)‐(dimethylamino)methylene]‐N‐methylmethanaminium hexafluorophosphate N‐oxide (HBTU), and (benzotriazol‐1‐yl‐N‐oxy)tris(dimethylamino)phosphonium hexafluorophosphate (BOP) [all in the presence of N‐methylmorpholine (NMM) or N,N‐diisopropylethylamine (DIEA) as a tertiary amine base], give rise to unacceptable levels (i.e. 5–33%) of cysteine racemization. As demonstrated on the tripeptide model H‐Gly‐Cys‐Phe‐NH₂, and on the nonapeptide dihydrooxytocin, the following methods are recommended: O‐pentafluorophenyl (O‐Pfp) ester in DMF; O‐Pfp ester/1‐hydroxybenzotriazole (HOBt) in DMF; N,N′‐diisopropylcarbodiimide (DIPCDI)/HOBt in DMF; HBTU/HOBt/2,4,6‐trimethylpyridine (TMP) in DMF (preactivation time 3.5–7.0 min in all of these cases); and HBTU/HOBt/TMP in CH₂Cl₂/DMF (1:1) with no preactivation. In fact, several of the aforementioned methods are now used routinely in our laboratory during the automated synthesis of analogs of the 58‐residue protein bovine pancreatic trypsin inhibitor (BPTI). In addition, several highly hindered bases such as 2,6‐dimethylpyridine (lutidine), 2,3,5,6‐tetramethylpyridine (TEMP), octahydroacridine (OHA), and 2,6‐di‐tert‐butyl‐4‐(dimethylamino)pyridine (DB[DMAP]) may be used in place of the usual DIEA or NMM to minimize cysteine racemization even with the in situ coupling protocols.     

Racemization during peptide coupling with N,N'-bis(2-oxo-3-oxazolidinyl) phosphinic chloride (BOP-Cl)

The extent of racemization under various circumstances is reported for the coupling of the N-protected model dipeptide Z-Gly-Phe to Val-OMe, mediated by N,N'-bis(2-oxo-3-oxazolidinyl)phosphinic chloride (BOC-Cl). BOP-Cl can be used for peptide syntheses either in a one-pot reaction or with preactivation. Maximum yields are obtained with 1.2 equiv. BOP-Cl and 1.2–1.5 equiv. amino-nucleophile and 2.2 equiv. mediatorial base in tetrahydrofuran, but the amount of stereomutation is only tolerable with the use of suppressors (4–6%). BOP-Cl/ HOBt (88% yield, 0% racemization) and BOP-Cl/imidazole (96% yield, 1.6% racemization) are the best choices in apolar solvents and HOBt is to be preferred in DMF (89% yield, 2.4% racemization). Most other additives, HOSu, NP, Pfp, ZnCl₂, DMAP, etc., are not good suppressors.     

Coupling in the absence of tertiary amines

In order to avoid base catalyzed side reactions during coupling, attempts were made to render superfluous the addition of tertiary amines to the reaction mixture. Weak acids were applied for the removal of acid labile protecting groups. Acetic acid and other carboxylic acids were considered unsuitable for this purpose because the resulting salts could be the cause of chain termination in the next coupling step. Pentachlorophenol and 2,4-dinitrophenol cleaved the Bpoc, Nps and Trt groups but more practical rates were reached with solutions of 1-hydroxybenzotriazole (HOBt) in trifluoroethanol, in acetic acid, or in a mixture of phenol and p-cresol. In addition to acidolysis, HOBt salts of amino components could also be obtained through hydrogenolysis of the Z group or thiolysis of the Nps group in the presence of HOBt, or by the displacement of acetic acid from acetate salts with HOBt. Acylation of HOBt salts of amino components with symmetrical or mixed anhydrides or with active esters did not require the addition of tertiary amine.     

3-Dimethylphosphinothioyl-2(3H)-oxazolone (MPTO), a promising new reagent for racemization-free couplings

3-Dimethylphosphinothioyl-2(3H)-oxazolone (MPTO) was synthesized, and its ability to effect racemization-free couplings and cyclization of a peptide and its C-terminal epimer was examined. MPTO showed good reactivity in aprotic polar solvents such as N,N-dimethylformamide (DMF) and N-methylpyrrolidone. In reactivity MPTO resembles DPPA and dimethylphosphinothioyl azide (MPTA) previously developed by us, but it is much better than these reagents because the side reactions specific to the azide method could be avoided. In coupling of Z-Gly-Val-OH with H-Val-OMe in DMF at 0°C, no racemization was observed without use of racemization-suppressing additives. Slight racemization observed at room temperature could be completely suppressed by addition of HOBt but not by HOSu. The utility of MPTO was demonstrated by the synthesis of cyclo-(d -Trp-d -Glu(OBzl)-Ala-D-Val-Leu), an intermediate for an endothelin-binding inhibitor BE 18257A. In a comparative study using DPPA, MPTA and MPTO, no racemization was observed for MPTA or MPTO, while DPPA caused considerable racemization. When MPTO was used in the presence of HOBt rapid cyclization (3 h at RT) occurred to give the optically pure cyclic product.     

4-Sulfobenzyl ester – its use in peptide synthesis

Amino acid 4-sulfobenzyl esters were employed for the synthesis of peptides in solution. They significantly increase the hydrophilicity of protected intermediates as shown by analytical reversed-phase high performance liquid chromatography and counter-current distribution. General methods for working up are described which permit simple and standardized isolations of products. The use of several coupling methods was demonstrated in the synthesis of Z-Val-Gly-OBzl-SO₃NH₄. Ion-exchange chromatography was introduced as a selective purification procedure for protected peptide 4-sulfobenzyl esters. A step-wise synthesis of [Leu⁵]-enkephalin was performed, starting from leucine 4-sulfobenzyl ester. Removal of N-terminal protecting groups produced zwitterionic peptide 4-sulfobenzyl esters. These were readily purified by crystallization from slightly acidic media; no further purification was necessary. The biologically fully active pentapeptide Tyr-Gly-Gly-Phe-Leu was obtained in an overall yield of 30.2%.     

Synthesis of hydrophobic enkephalin amides

The important role of the C-terminal residue of enkephalins in bringing about selectivity for the specific opioid receptors and the possibility that certain hydrophobic modifications could enhance the ability of the molecules to cross biological barriers, prompted us to synthesize a series of amides: Tyr-D-Met-Gly-Phe-Pro-NH-(CH₂)_n-CH₃ where n is 7, 9, 11 and 13 respectively, derived from the very potent (D-Met², Pro-NH₂⁵) enkephalin analogue. The synthesis has been carried out by the solution phase procedures with isolation and characterization of intermediates and final products. Mixed anhydrides and DCC/HOBt have been used as condensation agents. Homogeneity of final products was checked by t.l.c. and reversed phase HPLC under programmed gradient elution. A considerable decrease in the chemical yields in the last coupling step for the C₁₂ and C₁₄ derivatives has been observed, probably due to the steric hindrance of the aliphatic chain at the C-terminal position.     

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^α-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: CF₃ COOH—CH₂ Cl₂—β-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 (350nm) in toluene—CF₃ CH₂ OH (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 > 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.     

Chemical synthesis of urotensin II,a somatostatin-like peptide in the caudal neurosecretory system of fishes

In the goby, Gillichthys mirabilis, urotensin II (a bioactive neuropeptide present in the urophysis of teleost fish) has the dodecapeptide sequence, H₂N-AGTADC-FWKYCV-OH, which is homologous with mammalian somatostatin at positions 1, 2 and 7–9. The Merrifield solid phase synthesis of Gillichthys urotensin II (UII) was accomplished by stepwise assembly from the carboxy terminus using N^α-tert.-butyloxycarbonyl (Boc) amino acids containing benzyl-derived groups for protection of side-chain functionalities. Coupling of amino acids to the growing peptide was mediated by diisopropylcarbodiimide (DIC) in the presence of 1-hydroxybenzotriazole (HOBt). Residual α-amino groups remaining after coupling were blocked by acetylation with 1-acetylimidazole. Crude, synthetic UII was extracted from the HF-treated, protected peptide-resin product, reduced with dithiothreitol (DTT), reoxidized at high dilution with O₂, and separated into its components using a single, preparative, reverse-phase HPLC step. The pure, synthetic UII, obtained in 7.6% yield from oxidized crude UII, was indistinguishable from pure, native UII in specific bioactivity, amino acid sequence, and retention time in each of two different HPLC systems.     

Effect of tertiary amine on the carbodiimide-mediated peptide synthesis

The effect of tertiary amine (DIEA) on reaction rate and product purity of a carbodiimide/HOBt-mediated peptide synthesis was studied. It was found that very rapid activation can be achieved using carbodiimide/HOBt in non-polar solvents, such as DCM. Although the HOBt is poorly soluble in DCM, the activation proceeds within 2 min, probably forming the HOBt-ester. By such a preactivation followed by a coupling in the presence of DIEA the rate of coupling is comparable with other rapid methods using BOP or TBTU, and no racemization was found in a model coupling (< 0.1%). For comparison, syntheses of neurotensin by means of different coupling reagents (BOP, TBTU, OPfp-esters) and the DIEA-catalyzed coupling after carbodiimide/HOBt-activation under comparable conditions have shown that these procedures are of the same value in view of coupling efficiency and product purity.     

Solid-phase synthesis of neurokinin A antagonists

During the preparation of the NK-2 selective tachykinin antagonist MEN 10208 (Thr-Asp-Tyr-D-Trp-Val-D-Trp-D-Trp-Arg-NH₂) and its analogs by the solid-phase method employing the Boc strategy routinely used in our laboratory, we encountered difficulties in the coupling of hydrophobic amino acids D-Trp and Val. To study the coupling problems several syntheses of MEN 10208 and analogs were carried out with different activation strategies. These syntheses yielded considerable amounts of deletion sequences even though a negative Kaiser test was obtained after each coupling. Inaccessibility of the free amino group of the growing peptide due to steric hindrance of the hydrophobic residues during coupling, and for the ninhydrin complex during the Kaiser test, may account, at least in part, for the unsatisfactory synthetics results and for the false-negative ninhydrin tests. Repetition of each synthesis with the Fmoc strategy on a newly developed DOD resin for peptide amides using the DCC/HOBt chemistry gave superior results in terms of the yield and purity of the crude peptides. Therefore, the Fmoc strategy appears to offer advantages over the Boc method for the preparation of these peptides containing hydrophobic amino acids.     

Synthesis of peptide amides by Fmoc-solid-phase peptide synthesis and acid labile anchor groups

The preparation and use of new anchor groups for the synthesis of peptide amides by solid-phase peptide synthesis employing the Fmoc-method is described. Based on the structure of the 4,4′-dimethoxybenzhydryl group (Mbh) handles were developed, which could be cleaved by mild acid treatment to give carboxamides. The syntheses and application of Fmoc-amino-acid-(4-carboxylatomethyloxyphenyl-4′-methoxyphenyl) methyl amide and Fmoc-(4-carboxylatopropyloxyphenyl-4′-methoxyphenyl) methyl amide are described in detail. These handles were coupled to resins and a stepwise elongation of peptide chains proceeded smoothly with N^x-9-fluorenylmethoxycarbonyl (Fmoc) amino acid derivatives using a carbodiimide/HOBt mediated reaction. The final cleavage of side-chain protecting groups and the release of the C-terminal amide moiety was achieved by the treatment with trifluoroacetic acid, dichloromethane in the presence of scavengers. Various peptides, such as the Leu-enkephalin amide and Leu-Gly-Gly-Gly-Gln-Gly-Lys-Val-Leu-Gly-NH₂, which is a good substrate for F XIII, were prepared in high yields and purities.     

N,N'-bis(2-oxo-3-oxazolidinyl) phosphinic chloride (BOP-Cl); a superb reagent for coupling at and with iminoacid residues

Analytically pure dipeptides containing two iminoacids [Pro, MeAla, MePhe, MeVal and SPip (γ-thiapipecolinic acid)] were prepared with BOP-Cl [N,N]-bis(2-oxo-3-oxazolidinyl) phosphinic chloride] at normal temperature (0° — r.t.) in high yields (80–100%) and with excellent optical purity (below noise limit in ¹H n.m.r.). Maximum yields were obtained with Zervas' preactivation procedure, using, for example, an excess of BOP-Cl followed by the addition of (an equal excess of) the aminocomponent. Racemization suppressors (HOBt, imidazole, etc.) are of no use in these difficult couplings. Z-Me Val-Me Val-OBu^t (yield: 89%, without racemization (!)) and Z-SPip-SPip-OMe (yield: 96%, less than 5% stereomutation) were prepared for the first time, whereas all other coupling reagents have failed. Peptide acids with N-methylaminoacids as the COOH-terminal residues were coupled with excellent yields (70–90%), but unfortunately chiral integrity could not be preserved. Pronounced chiral induction was observed during the preparation of Z-Pro-MePhe-MeAla-OBu^t, and an influence of the penultimate protecting group was detected. In contrast, Z-MeVal-MeVal-MeVal-MeVal-OBu^t was formed with probably only 5% stereomutation (yield: 52%). Some side reactions with BOP-Cl are observed: e.g. symmetrical anhydride and DKP formation.     

Synthesis of phosphopeptides containing O-phosphoserine or O-phosphothreonine

Peptides containing phosphoserine or phosphothreonine were synthesized by solid phase methods. Phosphoserine and phosphothreonine were incorporated into peptides using Boc-diphenylphosphono esters of serine and threonine and standard DCC/HOBt coupling. The phenylphosphoesters were not removed when the peptides were cleaved from the resin by HF or by trifluoromethane sulfonic acid, but were subsequently removed by catalytic hydrogenation. Phosphopeptides were purified by HPLC and by Fe⁺³-Chelex chromatography and their identity verified by mass spectrometry. Two peptides, Leu-Arg-Arg-Ala-Ser(P)-Leu-Gly and Leu-Arg-Arg-Ala-Thr(P)-Leu-Gly, were prepared by both enzymatic and chemical methods and had identical properties.     

Solution synthesis of the glyco-hex a peptide sequence of the human oncofetal fibronectin defined by monoclonal antibody FDC-6

The glyco-hexapeptide sequence H-Val-(GalNAc-α)Thr-His-Pro-Gly-Tyr-OH, was synthesized in solution by the segment condensation procedure and the stepwise procedure. A peracetylated, O-galactosaminyl threonine derivative was used for incorporating the glycosylated amino acid residue into the peptide chain. A consistent racemization occurred during the acylation of H-His-Pro-Gly-Tyr(Bzl)-OBzl with Z-Val-[GalNAc(Ac)₃-α]Thr-OH by the BOP-HOBt procedure and the D-allothreonine containing glycohexapeptide was isolated in about 20% yield. Stepwise elongation of the C-terminal tetrapeptide with Frnoc-[GalNAc(Ac)₃-a]Thr-OH and Z-Val-OH, in the presence of the same coupling reagents, yielded the l -threonine containing diastereoisomer without detectable racemization. A side product, the N^im-ethoxy-carbonylated hexapeptide derivative, formed during the EEDQ-mediated condensation of Fmoc-[GalNAc(Ac)₃-α]Thr-OH with the C-terminal tetrapeptide, was isolated and characterized. Preliminary studies showed that the synthetic glycohexapeptide is a good competitive inhibitor of the binding of the FDC-6 monoclonal antibody to the oncofetal fibronectin, supporting the idea that it should represent the minimum essential structure required for the FDC-6 activity.     

Application of tryptic condensation strategy for the synthesis of α-melanocyte stimulating hormone

The use of trypsin in 1,1,1,3,3,3-hexafluoro-2-propanol (HFIP) paired with N,N-dimethylformamide (DMF) has been proposed for the enzymatic condensation of peptides. The tryptic condensation strategy was applied to the synthesis of the 13-peptide, α-melanocyte stimulating hormone (α-MSH), which contains two susceptible points to trypsin, in a low-water-containing solvent system, 4% H₂O in HFIP/DMF (1/1, v/v). The N-terminal 8-peptide segment (S1) ending with Arg and C-terminal 5-peptide with the side-chain protections at Lys and Trp (S2) were prepared by the stepwise coupling on p-nitrobenzophenone oxime resin and by a conventional method, respectively. Both segments were condensed by the aid of trypsin. The acid component was converted into the partially protected α-MSH at as high as 95% conversion determined by reversed-phase HPLC. When the side-chain of Lys at the 11-position was not protected, α-MSH was obtained only in 35% yield, and was contaminated with products of secondary hydrolysis. Although the Lys¹¹-Pro¹² sequence was very poorly susceptible to trypsin, the side-chain of Lys had to be protected in order to be inert to trypsin under the synthetic conditions. HFIP is demonstrated as a good solvent with DMF to allow the efficient tryptic condensation of peptides. The strategy increased the value of the enzymatic condensation as practical method by avoidance of secondary hydrolysis, high dissolution of peptides and retention of activity of enzyme.     

Unexpected racemization of proline or hydroxy-proline phenacyl ester during coupling reactions with Boc-amino acids

When L-proline or O-benzyl-trans-4–hydroxy-L-proline phenacyl ester was coupled with Boc-amino acids in dimethylformamide using water-soluble carbodiimide (WSCI) in the presence of anhydrous 1-hydroxybenzotriazole (HOBt) as coupling reagents, extensive racemization was observed at the Cα of the proline or hydroxy-proline residue. The extent of racemization was measured by HPLC after the coupling with Boc-L-Leu-OH in the presence or absence of HOBt. The extent of racemization increased when HOBt was added to the reaction mixture, but greatly decreased when it was not, indicating that HOBt was needed for inducing racemization. Almost no racemization was observed when the coupling reaction was carried out by the mixed anhydride procedure in tetrahydrofuran or by the carbodiimide method in dichloromethane without using HOBt. In the case of coupling reactions with ordinary L-amino acid phenacyl esters, no racemization was observed. Examination of some model systems yielded sufficient evidence to prove that HOBt is an efficient catalyst for racemizing proline or hydroxy-proline phenacyl ester not only in the stage of cyclic intermediate formation but also in the opening of the ring structure. Thus, the racemization reaction was found to be closely related to the formation of the cyclic carbinol-amine derivative.     

Influence of reaction conditions on syntheses of sweetener precursors catalyzed by thermolysin in tert-amyl alcohol

Abstract: The activity of enzymes to form a peptide bond in organic solvent was greatly influenced by observed pH and water content. The precursors of two sweeteners, P-Asp-Xaa-OR (P = Z or For, Xaa-OR = Phe-OMe or Ala-OcHex), were synthesized by enzyme, and the reaction conditions were studied systematically. Z-Asp-OH was coupled with H-Phe-OMe or H-Ala-OcHex by thermolysin in tert-amyl alcohol. The best coupling results were obtained when the optimized observed pH was 8 or 9, and the water content was about 6% (V/V). The protecting group Z is better than For under the reaction conditions and H-Phe-OMe is a better nucleophile than H-Ala-OcHex. The expected optically pure peptides were obtained when the racemic amino acids were used as amino components in the starting materials. The physical constants of P-Asp-Xaa-OR synthesized by thermolysin are identical with those of peptides synthesized by chemical method.