PEPTIDE COMPOUND PRODUCTION METHOD

Abstract

A method of producing a polypeptide compound, wherein a peptide compound represented by formula (P) is obtained by inducing an amide formation reaction between an amino-protected lactam compound represented by formula (R1) and an amino acid ester or peptide ester compound represented by formula (R2).

##STR00001##

The definitions for the reference signs in formulae (R1), (R2), and (P) are as set forth in the claims.

Claims

1. A method for producing a polypeptide compound represented by formula (P), comprising causing amide forming reaction between an amino-protected lactam compound represented by formula (R1) and an amino acid ester or peptide ester compound represented by formula (R2). ##STR00089## In formula (R1), PG.sup.a represents C(?O)OR.sup.a, C(?O)R.sup.a, or S(?O).sub.2R.sup.a a protective group represented by (where R.sup.a represents a monovalent substituent.), A.sup.11 and A.sup.12 represents, independently of each other, a bivalent aliphatic hydrocarbon group having 1 to 5 carbon atoms which may optionally have one or more substituents, p11 and p12 represents, independently of each other, 0 or 1, L.sup.1 represents a bivalent linking group, q1 represents 0 or 1, provided that the ring containing (A.sup.11), (L.sup.1), and (A.sup.12) is a 4- to 17-membered ring, wherein the bivalent linking group L.sup.1 may be combined with A.sup.11 and/or A.sup.12 to form a 4- to 8-membered heterocyclic ring that fuses with the lactam ring of formula (R1). ##STR00090## In formula (R2), PG.sup.b represents a protective group for carboxyl groups, R.sup.21 and R.sup.22 represents, independently of each other, a hydrogen atom, halogen atom, hydroxyl group, carboxyl group, nitro group, cyano group, or thiol group, and which may optionally have one or more substituents, amino group, monovalent aliphatic hydrocarbon group, monovalent aromatic hydrocarbon group, or monovalent heterocyclic group, R.sup.23 represents a hydrogen atom, carboxyl group, hydroxyl group, and which may optionally have one or more substituents monovalent aliphatic hydrocarbon group, aromatic hydrocarbon group, or heterocyclic group, provided that when R.sup.23 is a monovalent aliphatic hydrocarbon group, aromatic hydrocarbon group, or heterocyclic group, R.sup.23 may be bound to the nitrogen atom via a linking group, or alternatively, R.sup.21 and R.sup.23 may bind to each other and, along with the carbon atom to which R.sup.21 binds and the nitrogen atom to which R.sup.23 binds, form a heterocyclic ring which may optionally have one or more substituents, A.sup.21 and A.sup.22 represents, independently of each other, a bivalent aliphatic hydrocarbon group having 1 to 3 carbon atoms which may optionally have one or more substituents, p21 and p22 represents, independently of each other, 0 or 1, and n.sup.2 represents an integer of 1 or higher corresponding to the number of the structure units parenthesized with [ ], provided that when is n.sup.2 equal to or greater than 2, then the two or more structure units in [ ] may be either identical to each other or different from each other. ##STR00091## In formula (P), PG.sup.b, A.sup.11, A.sup.12, L.sup.1, p11, p12, and q1 each represent the same definition as that of the same symbol in formula (R1), and PG.sup.b, R.sup.21, R.sup.22, R.sup.23, A.sup.21, A.sup.22, p21, p22, and n.sup.2 each represent the same definition as that of the same symbol in the formula (R2).

2. The method according to claim 1, wherein in formula (R1), PG.sup.a is a protective group selected from a tert-butoxy carbonyl group (Boc), benzyloxycarbonyl group (Cbz), 2,2,2-trichloroethoxy carbonyl (Troc) group, allyoxycarbonyl (Alloc) group, 9-fluorenyl methyloxycarbonyl (Fmoc) group, 2-(trimethylsilyl)ethoxy carbonyl (Teoc) group, benzoyl (Bz) group, phthaloyl (Phth) group, paramethoxybenzoyl group (PMPCO), cinnamoyl group, toluene sulfonyl (Ts) group, ands 2- or 4-nitrobenzene sulfonyl (Ns) group.

3. The method according to claim 1 or 2, wherein in formula (R1), p11 is 1, p12 and q1 are both 0, A.sup.11 represents a bivalent aliphatic hydrocarbon group having 2 to 6 carbon atoms which may optionally have one or more substituents.

4. The method according to claim 1 or 2, wherein in formula (R1), p11, p12, and q1 are all 1, A.sup.11 and A.sup.12 are, independently of each other, a bivalent aliphatic hydrocarbon group having 1 to 3 carbon atoms which may optionally have one or more substituents, and L.sup.1 is C(?O)N.

5. The method according to any one of claims 1 to 4, wherein the molar ratio of the lactam compound of formula (R1) to the amino acid ester or peptide ester compound of formula (R2) used is from 1:5 to 5:1.

6. The method according to any one of claims 1 to 5, wherein the reaction is a batch reaction or a flow reaction.

Description

EXAMPLES

[0205] The present invention will be described in more detail below with reference to examples. However, the present invention should in no way be bound by the following examples, and can be implemented in any form within the scope that does not depart from the purpose of the invention. The percentage values in the following description are expressed as mass percentages unless otherwise stated. Yields are determined by GC (gas chromatography) analysis using octane as an internal standard, or by isolation using chromatography. Diastereoselectivity of the products was determined by .sup.1H NMR analysis. The products were identified by .sup.1H NMR analysis and liquid chromatogram mass spectrometry (LC/MS).

Example Group I: Peptide Synthesis Using Amino-Protected Lactams and Amino Acid Esters at Various Ratios

(1) Example I-1: Synthesis of a Peptide (Boc-?-HoGly-L-Ala-OtBu) Using an Amino-Protected Lactam (N-Boc-2-piperidone) With an Amino Acid Ester (H-L-Ala-OtBu) at a Molar Ratio of 1:1

[0206] ##STR00010##

[0207] A 5.0 mL screw-cap vial equipped with a stirrer bar (SmCo) was charged with N-Boc-2-piperidone (N-Boc-?-lactam) (99.625 mg, 0.50 mmol) and H-L-Ala-OtBu (72.6 mg, 0.50 mmol), and sealed with a screw cap. The mixture was caused to react with agitation at 30? C. for 6 hours. The reaction mixture was diluted with chloroform (CHCl.sub.3) (3.0 mL), and transferred to a silica (SiO.sub.2) gel column with a pipette. The vial and the pipette used was washed with chloroform (12 mL), and the wash liquid was added to the reaction mixture. The resulting mixture was purified with flash column chromatography (50 to 100% AcOEt/hexane) to thereby produce the title compound as colorless and transparent liquid (yield: 55%, >99:1 er).

(2) Example I-2: Synthesis of a Peptide (Boc-?-HoGly-L-Ala-OtBu) Using an Amino-Protected Lactam (N-Boc-2-piperidone) With an Amino Acid Ester (H-L-Ala-OtBu) at a Molar Ratio of 1:2

[0208] ##STR00011##

[0209] The same reaction procedure as Example I-1 was carried out except that the amount of H-L-Ala-OtBu used was changed to 145.2 mg (1.0 mmol), and the reaction time was changed to 12 hours, to thereby produce the title compound as colorless and transparent liquid (yield: >99%, >99:1 er).

(3) Example I-3: Synthesis of a Peptide (Boc-?-HoGly-L-Ala-OtBu) Using an Amino-Protected Lactam (N-Boc-2-piperidone) With an Amino Acid Ester (H-L-Ala-OtBu) at a Molar Ratio of 2:1

[0210] ##STR00012##

[0211] The same reaction procedure as Example I-1 was carried out except that the amount of N-Boc-2-piperidone used was changed to 199.25 mg (1.0 mmol), and the reaction time was changed to 12 hours, to thereby produce the title compound as colorless and transparent liquid (yield: 92%, >99:1 er).

Example Group II: Peptide Synthesis by Reacting Amino-Protected Lactams and Amino Acid Esters in Various Solvents

(1) Example II-1: Synthesis of a Peptide (Boc-?-HoGly-L-Ala-OtBu) by Reacting an Amino-Protected Lactam (N-Boc-2-piperidone) With an Amino Acid Ester (H-L-Ala-OtBu) in AcOEt

[0212] ##STR00013##

[0213] The same reaction procedure as Example I-1 was carried out except that the amount of H-L-Ala-OtBu used was changed to 145.2 mg (1.0 mmol), 0.25 mL of ethyl acetate (AcOEt) was added to the vial as a reaction solvent, and the reaction time was changed to 12 hours, to thereby produce the title compound as colorless and transparent liquid (yield: 53%, >99:1 er).

(2) Example II-2: Synthesis of a Peptide (Boc-?-HoGly-L-Ala-OtBu) by Reacting an Amino-Protected Lactam (N-Boc-2-piperidone) With an Amino Acid Ester (H-L-Ala-OtBu) in CPME

[0214] ##STR00014##

[0215] The same reaction procedure as Example II-1 was carried out except that the solvent was changed to cyclopentyl methyl ether (CPME) 0.25 mL, and the reaction time was changed to 24 hours, to thereby produce the title compound as colorless and transparent liquid (yield: 88%, >99:1 er).

(3) Example II-3: Synthesis of a Peptide (Boc-?-HoGly-L-Ala-OtBu) by Reacting an Amino-Protected Lactam (N-Boc-2-piperidone) With an Amino Acid Ester (H-L-Ala-OtBu) in DME

[0216] ##STR00015##

[0217] The same reaction procedure as Example II-1 was carried out except that the reaction solvent was changed to 0.25 mL of 1,2-dimethoxy ethane (DME), and the reaction time was changed to 24 hours, to thereby produce the title compound as colorless and transparent liquid (yield: 58%, >99:1 er).

(4) Example II-4: Synthesis of a Peptide (Boc-?-HoGly-L-Ala-OtBu) by Reacting an Amino-Protected Lactam (N-Boc-2-piperidone) With an Amino Acid Ester (H-L-Ala-OtBu) in DMF

[0218] ##STR00016##

[0219] The same reaction procedure as Example II-1 was carried out except that the reaction solvent was changed to 0.25 mL of N,N-dimethyl formamide (DMF), and the reaction time was changed to 24 hours, to thereby produce the title compound as colorless and transparent liquid (yield: 52%, >99:1 er).

(5) Example II-5: Synthesis of a Peptide (Boc-?-HoGly-L-Ala-OtBu) by Reacting an Amino-Protected Lactam (N-Boc-2-piperidone) With an Amino Acid Ester (H-L-Ala-OtBu) in tBuOH

[0220] ##STR00017##

[0221] The same reaction procedure as Example II-1 was carried out except that the reaction solvent was changed to 0.25 mL of tert-butyl alcohol (tBuOH), and the reaction time was changed to 24 hours, to thereby produce the title compound as colorless and transparent liquid (yield: 55%, >99:1 er).

(6) Example II-6: Synthesis of a Peptide (Boc-?-HoGly-L-Ala-OtBu) by Reacting an Amino-Protected Lactam (N-Boc-2-piperidone) With an Amino Acid Ester (H-L-Ala-OtBu) in iPr.SUB.2.OH

[0222] ##STR00018##

[0223] The same reaction procedure as Example II-1 was carried out except that the reaction solvent was changed to 0.25 mL of diisopropyl ether (iPr.sub.2O), and the reaction time was changed to 24 hours, to thereby produce the title compound as colorless and transparent liquid (yield: 81%, >99:1 er).

(7) Example II-7: Synthesis of a Peptide (Boc-?-HoGly-L-Ala-OtBu) by Reacting an Amino-Protected Lactam (N-Boc-2-piperidone) With an Amino Acid Ester (H-L-Ala-OtBu) in 2-Me-THF

[0224] ##STR00019##

[0225] The same reaction procedure as Example II-1 was carried out except that the reaction solvent was changed to 0.25 mL of 2-methyl tetrahydrofuran (2-Me-THF), and the reaction time was changed to 24 hours, to thereby produce the title compound as colorless and transparent liquid (yield: 69%, >99:1 er).

(8) Example II-8: Synthesis of a Peptide (Boc-?-HoGly-L-Ala-OtBu) by Reacting an Amino-Protected Lactam (N-Boc-2-piperidone) With an Amino Acid Ester (H-L-Ala-OtBu) in TBME

[0226] ##STR00020##

[0227] The same reaction procedure as Example II-1 was carried out except that the reaction solvent was changed to 0.25 mL of tert-butyl methyl ether (TBME), and the reaction time was changed to 24 hours, to thereby produce the title compound as colorless and transparent liquid (yield: 76%, >99:1 er).

(9) Example II-9: Synthesis of a Peptide (Boc-?-HoGly-L-Ala-OtBu) by Reacting an Amino-Protected Lactam (N-Boc-2-piperidone) With an Amino Acid Ester (H-L-Ala-OtBu) in Toluene

[0228] ##STR00021##

[0229] The same reaction procedure as Example II-1 was carried out except that the reaction solvent was changed to 0.25 mL of toluene, and the reaction time was changed to 24 hours, to thereby produce the title compound as colorless and transparent liquid (yield: 95%, >99:1 er).

Example Group III: Peptide Synthesis by Reacting Amino-Protected Lactams and Amino Acid Esters Having Various Structures

(1) Example III-1: Synthesis of a Peptide (Boc-?-HoGly-L-Ala-OtBu) by Reacting an Amino-Protected 4-Membered Ring Lactam (N-Boc-2-azetidinone) With an Amino Acid Ester (H-L-Ala-OtBu)

[0230] ##STR00022##

[0231] The same reaction procedure as Example I-1 was carried out except that 4-membered ring lactam N-Boc-2-azetidinone (N-Boc-?-lactam) (85.595 mg, 0.5 mmol) as used as an amino-protected lactam, the amount of H-L-Ala-OtBu used was changed to 145.2 mg (1.0 mmol), and the reaction time was changed to 12 hours, to thereby produce the title compound as colorless and transparent liquid (yield: 73%, >99:1 er).

(2) Example III-2: Synthesis of a Peptide (Boc-?-HoGly-L-Ala-OtBu) by Reacting an Amino-Protected 5-Membered Ring Lactam (N-Boc-2-pyrrolidinone) With an Amino Acid Ester (H-L-Ala-OtBu)

[0232] ##STR00023##

[0233] The same reaction procedure as Example III-1 was carried out except that 5-membered ring N-Boc-2-pyrrolidinone (N-Boc-?-lactam) (92.61 mg, 0.5 mmol) was used as an amino-protected lactam, to thereby produce the title compound as colorless and transparent liquid (yield: 46%, >99:1 er).

(3) Example III-3: Synthesis of a Peptide (Boc-?-HoGly-L-Ala-OtBu) by Reacting an Amino-Protected 7-Membered Ring Lactam (N-Boc-2-azacycloheptanone) With an Amino Acid Ester (H-L-Ala-OtBu)

[0234] ##STR00024##

[0235] The same reaction procedure as Example III-1 was carried out except that 7-membered ring N-Boc-2-azacycloheptanone (N-Boc-?-caprolactam) (106.64 mg, 0.5 mmol) was used as an amino-protected lactam, to thereby produce the title compound as colorless and transparent liquid (yield: 90%, >99:1 er).

(4) Example III-4: Synthesis of a Peptide (Boc-Gly-L-Ala-OtBu) by Reacting an Amino-Protected Lactam Having Two Endocyclic Peptide Structures (N-Boc-2,5-piperazinedione) With an Amino Acid Ester (H-L-Ala-OtBu)

[0236] ##STR00025##

[0237] The same reaction procedure as Example III-1 was carried out except that N-Boc-2,5-piperazinedione (107.12 mg, 0.5 mmol) was used as an amino-protected lactam having two endocyclic peptide structures, to thereby produce the title compound as white solid (yield: 91%, >99:1 er).

(5) Example III-5: Synthesis of a Peptide (Boc-L-Glu(L-Ala-OtBu)-OtBu) by Reacting an Amino-Protected Lactam Having a Carboxylate Ester Group (N-Boc-pyroglutamate t-butyl ester) With an Amino Acid Ester (H-L-Ala-OtBu)

[0238] ##STR00026##

[0239] The same reaction procedure as Example III-1 was carried out except that N-Boc-pyroglutamic acid (142.67 mg, 0.5 mmol) was used as an amino-protected lactam having a carboxylate ester group, the amount of H-L-Ala-OtBu used was changed to 217.8mg (1.5 mmol), the reaction temperature was changed to 60? C., and the reaction time was changed to 24 hours, to thereby produce the title compound as white solid (yield: 97%, >99:1 dr).

(6) Example III-6: Synthesis of a Peptide (Boc-L-Ala-L-Ala-L-Ala-OtBu) by Reacting an Amino-Protected Lactam Having Two Endocyclic Peptide Structures and Two Methyl Groups (N-Boc-cyclo(L-Ala-L-Ala)) With an Amino Acid Ester (H-L-Ala-OtBu)

[0240] ##STR00027##

[0241] The same reaction procedure as Example III-1 was carried out except that N-Boc-cyclo(L-Ala-L-Ala) (121.14 mg, 0.5 mmol) was used as an amino-protected lactam having two endocyclic peptide structures and two methyl groups, the amount of H-L-Ala-OtBu used was changed to 217.8 mg (1.5 mmol), and the reaction time was changed to 24 hours, to thereby produce the title compound as white solid (yield: 80%, >99:1 dr).

(7) Example III-7: Synthesis of a Peptide (Boc-Gly-L-Pro-L-Ala-OtBu) by Reacting an Amino-Protected Lactam Having a Condensed Ring Structure (Boc-cyclo(Gly-L-Pro)) With an Amino Acid Ester (H-L-Ala-OtBu)

[0242] ##STR00028##

[0243] The same reaction procedure as Example III-1 was carried out except that Boc-cyclo(Gly-L-Pro) (63.57 mg, 0.25 mmol) was used as an amino-protected lactam having a condensed ring structure, and the amount of H-L-Ala-OtBu used was changed to 72.6 mg (0.50 mmol), to thereby produce the title compound as white solid (yield: 96%, >99:1 r).

(8) Example III-8: Synthesis of a Peptide (Boc-??-HoGly-L-Thr(tBu)-OtBu) by Reacting an Amino-Protected Lactam (N-Boc-2-piperidone) With Another Amino Acid Ester (H-L-Thr(tBu)-OtBu)

[0244] ##STR00029##

[0245] The same reaction procedure as Example III-1 was carried out except that N-Boc-2-piperidone (N-Boc-?-lactam) (99.625 mg, 0.50 mmol) was used as an amino-protected lactam, H-L-Thr(tBu)-OtBu (231.3 mg, 1.00 mmol) was used as an amino acid ester, and the reaction was carried out at 50? C. for 24 hours, to thereby produce the title compound as white solid colorless and transparent liquid (yield: >99%, >99:1 dr).

(9) Example III-9: Synthesis of a Peptide (Boc-??-HoGly-L-Ile-OtBu) by Reacting an Amino-Protected Lactam (N-Boc-2-piperidone) With Another Amino Acid Ester (H-L-Ile-OtBu)

[0246] ##STR00030##

[0247] The same reaction procedure as Example III-1 was carried out except that N-Boc-2-piperidone (N-Boc-?-lactam) (99.625 mg, 0.50 mmol) was used as an amino-protected lactam, H-L-Ile-OtBu (187.28 mg, 1.00 mmol) was used as an amino acid ester, and the reaction was carried out at 50? C. for 24 hours, to thereby produce the title compound as white solid to colorless and transparent liquid (yield: >99%, >99:1 dr).

(10) Example III-10: Synthesis of a Peptide (Boc-Gly-L-Pro-L-AlaSer(tBu)-OtBu) by Reacting an Amino-Protected Lactam Having a Condensed Ring Structure (Boc-cyclo(Gly-L-Pro)) With Another Amino Acid Ester (H-L-AlaSer(tBu)-OtBu)

[0248] ##STR00031##

[0249] The same reaction procedure as Example III-1 was carried out except that Boc-cyclo(Gly-L-Pro) (63.57 mg, 0.25 mmol) was used as an amino-protected lactam having a condensed ring structure, H-L-Ser(tBu)-OtBu (108.66 mg, 0.50 mmol) was used as an amino acid ester, and the reaction was carried out at 50? C. for 24 hours, to thereby produce the title compound as white solid (yield: >99%, >99:1 dr).

Example Group IV: Peptide Synthesis by Reacting Amino-Protected Lactams Having Various Amino Protective Groups With Amino Acid Esters

(1) Example IV-1: Synthesis of a Peptide (Boc-Gly-L-Ala-O-tB) by Reacting a Lactam Having Two Amino Protective Groups (N,N-di-Boc-2,5-piperazinedione) With an Amino Acid Ester (H-L-Ala-OtBu)

[0250] ##STR00032##

[0251] The same reaction procedure as Example I-1 was carried out except that N,N-di-Boc-2,5-piperazinedione (157.17 mg, 0.5 mmol) was used as an amino-protected lactam having two Boc groups, and the amount of H-L-Ala-OtBu used was changed to 290.4 mg (2.0 mmol), to produce Boc-Gly-L-Ala-OtBu as colorless and transparent liquid (yield: 98%, >99:1 er).

(2) Example IV-2: Synthesis of a Peptide (Boc-Gly-L-Val-OtBu) by Reacting a Lactam Having Two Amino Protective Groups (N,N-di-Boc-2,5-piperazinedione) With an Amino Acid Ester (H-L-Val-OtBu)

[0252] ##STR00033##

[0253] The same reaction procedure as Example IV-1 was carried out except that H-L-Val-OtBu (346.6 mg, 2.0 mmol) was used as an amino acid ester, and the reaction time was changed to 24 hours, to thereby produce the title compound as colorless and transparent liquid (yield: 91%, >99:1 er).

(3) Example IV-3: Synthesis of a Peptide (Cbz-?-HoGly-L-Ala-OtBu) by Reacting a Lactam Having Cbz as an Amino Protective Group (N-Cbz-2-piperidone (N-Cbz-?-lactam)) With an Amino Acid Ester (H-L-Ala-OtBu)

[0254] ##STR00034##

[0255] The same reaction procedure as Example IV-1 was carried out except that N-Cbz-2-piperidone (N-Cbz-?-lactam) (116.63 mg, 0.50 mmol) was used as an amino-protected lactam having Cbz, and the reaction time was changed to 12 hours, to thereby produce the title compound as white solid (yield: 43%, >99:1 er).

(4) Example IV-4: Synthesis of a Peptide (Alloc-Gly-L-Thr(tBu)-OtBu) by Reacting a Lactam Having Two Alloc Groups as Amino Protective Groups (N,N-di-Alloc-2,5-piperazinedione) With an Amino Acid Ester (H-L-Thr(tBu)-OtBu)

[0256] ##STR00035##

[0257] The same reaction procedure as Example IV-1 was carried out except that N,N-di-Alloc-2,5-piperazinedione (141.1 mg, 0.50 mmol) was used as an amino-protected lactam having two Alloc groups, H-L-Thr(tBu)-OtBu (462.68 mg, 2.0 mmol) was used as an amino acid ester, and the reaction time was changed to 24 hours, to thereby produce the title compound as colorless and transparent liquid (yield: 91%, >99:1dr).

Example Group V: Other Synthesis Examples

[0258] Peptides were synthesized by following each of the reaction procedures in Example Groups I to IV above, with making various changes in reaction conditions, such as the selection of the amino-protected lactam and/or the amino acid ester, reaction temperature, reaction time, the presence and absence and type of the solvent, and the presence and absence and selection of the catalyst. The reactions and results are summarized in the tables below.

TABLE-US-00004 TABLE 3-1 No. Reactions and results 1 [00036] 2 [00037] 3 [00038] 4 [00039] 5 [00040] 6 [00041] 7 [00042] 8 [00043]

TABLE-US-00005 TABLE 3-2 No. Reactions and results 9 [00044] 10 [00045] 11 [00046] 12 [00047] 13 [00048] 14 [00049] 15 [00050] 16 [00051] 17 [00052]

TABLE-US-00006 TABLE 3-3 No. Reactions and results 18 [00053] 19 [00054] 20 [00055] 21 [00056] 22 [00057] 23 [00058] 24 [00059]

TABLE-US-00007 TABLE 3-4 No. Reactions and results 25 [00060] 26 [00061] 27 [00062] 28 [00063] 29 [00064] 30 [00065]

TABLE-US-00008 TABLE 3-5 No. Reactions and results 31 [00066] 32 [00067] 33 [00068] 34 [00069] 35 [00070] 36 [00071] 37 [00072] 38 [00073]

TABLE-US-00009 TABLE 3-6 No. Reactions and results 39 [00074] 40 [00075] 41 [00076] 42 [00077] 43 [00078]

TABLE-US-00010 TABLE 3-7 No. Reactions and results 44 [00079] 45 [00080] 46 [00081]

TABLE-US-00011 TABLE 3-8 No. Reactions and results 47 [00082] 48 [00083] 49 [00084] 50 [00085]

TABLE-US-00012 TABLE 3-9 No. Reactions and results 51 [00086] 52 [00087] 53 [00088]