CCXXXIX. EXPERIMENTS ON AMINO-ACIDS
`IV. THE METHYL ETHERS OF SOME N-ACETYL-
`HYDROXYAMINO-ACIDS
`
`BY RICHARD LAURENCE MILLINGTON SYNGE
`From the Biochemical Laboratory, Cambridge
`(Received 5 October 1939)
`IN Paper III of this series the isolation of a "hydroxyamino-acid fraction " from
`protein hydrolysates is described, and it is shown that the components of this
`fraction are not readily separable from one another by direct crystallization. In
`view of the possibility of extractional fractionation of amino-acids in the form of
`their N-acyl derivatives outlined in Papers I and II of this series, it seemed
`desirable to search for a series of derivatives of the hydroxyamino-acids which
`would show a similar " spread " in their partition coefficients between a suitable
`pair of immiscible solvents.
`The possibility of using the N-benzoyl derivatives was first investigated, al-
`though there are theoretical objections to introducing so heavy and fat-soluble
`a group. These objections were confirmed by experiment.
`The ratio of the solubilities of N-benzoyl-dl-serine [S0rensen & Andersen,
`1908] in water and ethyl acetate was determined. This may be taken as a
`measure of the partition, and can be compared with the same figures obtained
`for N-benzoyl-dl-threonine and N-benzoyl-dl-allothreonine by West & Carter
`[1937]. This is done in Table I. It seems unlikely from this that the N-benzoyl
`derivatives would be suitable for the extractional fractionation ofhydroxyamino-
`acids.
`
`Table I. Solubilities of N-benzoyl-hydroxyamino-acids in
`ethyl acetate and water
`Solubility in
`Solubility in
`Temp. EtOAcinmg./ml. H20 in mg./ml.
`B/A
`Compound
`0 C.
`A
`B
`1-2
`20-9
`25-5
`N-Benzoyl-dl-serine
`19
`2-0
`10-2
`N-Benzoyl-dl-threonine
`20-6
`25
`3-5
`8-5
`2-4
`N-Benzoyl-dl-allothreonine
`25
`The partition of the N-acetyl derivatives between ethyl acetate and water
`phases was next investigated. In this case the determination was carried out as
`in Paper I of this series, and the symbols P and c have the same meaning here
`as there. The "N-acetylthreonine" was prepared by acetylation in solution
`without working up. A mixture of dl-threonine and dl-allothreonine (the gift of
`Prof. A. C. Chibnall, to whom I express my thanks) was used as starting material.
`The aqueous phase for this particular determination was 0-3N NaCl and 0-IN
`acetic acid. The results are shown in Table II.
`From this, the N-acetyl series seemed as unsuitable for the purpose as the
`N-benzoyl series. It seemed likely that the properties of the molecule were
`dominated by the presence of a free hydroxyl group, and it was therefore decided
`to investigate the possibilities of masking this group. Methylation, as introducing
`only a small group into the molecule, seemed the most hopeful line of approach.
`It seemed likely that the original amino-acid could be regenerated from its
`1931
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`1932
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`RI
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`L. M. SYNGE
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`Table II. Partition of N-acetyl-hydroxyamino-acids between ethyl acetate and water
`Temp.
`Compound
`P
`°
`c
`N-Acetyl-dl-serine
`20
`75
`6
`" N-Acetylthreonine"
`20
`45
`4
`N-Acetyl-l-hydroxyproline
`20
`50
`5
`N-acetyl-O-methyl derivative by refluxing with HBr, as in the preparation by
`West & Carter [1937] of very good yields of threonine isomerides in one step
`from their N-formyl-O-methyl derivatives.
`N-Acetyl-O-methyl-l-tyrosine has been prepared from N-acetyl-l-tyrosine
`by methylation with aqueous NaOH and dimethyl sulphate [Karrer et at. 1922;
`Behr & Clarke, 1932]. If this method were applied to the methylation of a
`mixture of N-acetyl-hydroxyamino-acids, obvious difficulties would arise in
`freeing the product from excess reagents and salt. A possible alternative seemed
`to be the use of Purdie's reagents (silver oxide and methyl iodide). This would be
`expected to yield the methyl esters of the N-acetyl-O-methyl compounds, and,
`in view of the work of Cherbuliez et al. [1929; 1930] on the distillation of the ethyl
`esters of acetamino-acids, these should be distillable from any non-volatile
`reaction products. The possibility of N-methylation by the reagents had to be
`borne in mind, but since I had in the past observed that N-acetyl-O-trimethyl-
`,B-methylglucosaminide [Cutler et al. 1937] results from the direct action of
`Purdie's reagents on N-acetyl-glucosamine, it seemed worth while to ascertain
`their effect on N-acetyl-hydroxyamino-acids.
`It was found that, on treating N-acetyl-l-tyrosine, N-acetyl-l-hydroxyproline
`and N-acetyl-dl-serine with Purdie's reagents, in each case a crystalline, distil-
`lable methyl ester of the N-acetyl-O-methyl-hydroxyamino-acid resulted in fair
`yield. In each case this could be converted quantitatively by saponification into
`the free acid.
`N-Acetyl-O-methyl-dl-serine was also prepared from O-methyl-dl-serine.
`N-Acetyl-O-methyl-dl-allothreonine was prepared from O-methyl-dl-allothreo-
`nine. Both these amino-acids were the gift of Dr Herbert E. Carter, to whom
`I express my thanks.
`The partition of the N-acetyl-O-methyl-hydroxyamino-acids between chloro-
`form and water was determined (see Paper I of this series). The results are shown
`in Table III.
`Table III. The partition of N-acetyl-O-methyl-hydroxyamino-acids
`between chloroform and water
`Temp.
`0 C.
`Compound
`c
`P
`N-Acetyl-0-methyl-dl-serine
`9
`20
`450
`N-Acetyl-O-methyl-dl-allothreonine
`20
`160
`7
`N-Acetyl-0-methyl-l-hydroxyproline
`20
`23
`6
`N-Acetyl-O-methyl-l-tyrosine
`20
`2.1
`2-4
`It will be seen that this series of derivatives is capable of providing the basis
`for extractional fractionation of the hydroxyamino-acids.
`No experiments have yet been carried out on mixtures; in order to prepare
`the "hydroxyamino-acid fraction" (see Paper III of this series) of a protein
`hydrolysate for such a fractionation, a possible line of approach would be to
`remove S04- eexactly with Ba from the aqueous layer after the second chloroform
`extraction had been carried out. The resulting solution would be evaporated to
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`0-METHYL ACETAMINO-ACIDS
`dryness, and methylated with Purdie's reagents. The product would be distilled.
`The distillate would be saponified by Ba(OH)2, and after exact removal of Ba
`by H2SO4, a solution of N-acetyl-O-methyl-hydroxyamino-acids suitable for
`extractional fractionation should result.
`
`1933
`
`EXPERIMENTAL
`N-Acetyl-O-methyl-l-tyroWine methyl ester (I)
`1 g. of N-acetyl-l-tyrosine was dissolved in 6 ml. of dry acetone and treated
`for 2 hr. at 370 with 6 ml. of methyl iodide and 6 g. Ag2O. The mixture was
`filtered, the insoluble material was well washed with acetone and the combined
`ifitrate and washings were evaporated to dryness in vacuo. The residue was again
`treated with 6 ml. of methyl iodide and 6 g. Ag2O at 370. The mixture was filtered
`and evaporated to dryness as before. On distillation, 0 9 g. of material distilled
`at 180-200o/0.05 mm. The product crystallized in the receiver, and recrystallisa-
`tion from ether yielded 0-6 g. M.P. 106-107'. [oc] ' + 26 3° (alcohol, I = 2, c =4.2).
`(Found: C, 61'9; H, 710; N, 5-64; OMe, 24.3%. C13H704N requires C, 62-1;
`H, 6'77; N, 5-58; OMe, 24.7%.)
`N-Acetyl-O-methyl-l-tyroWine (II)
`(I) was dissolved in excess of N NaOH and kept for 4 hr. at room tempera-
`ture. On acidifying with HCI, crystallization occurred, and the product on re-
`crystallization from water had M.P. 1510 (not depressed on admixture with a
`sample of N-acetyl-O-methyl-l-tyrosine provided by Prof. H. T. Clarke, to whom
`I express my thanks).
`[oc]2°+54.30, +54.40; [oc] +6590 (alcohol, 1=2, c=1-3). (Behr & Clarke
`[1932] record M.P. 150-151°; [oc]°l+67 60 (alcohol, c=5).) (Found: C, 60.5;
`H, 6-21; N, 5'84; OMe, 13.4%. Calc. for C12His04N: C, 60'8; H, 6*33; N, 5'90;
`OMe, 13-1 %.) Acid equiv. wt. Found: 231. Calc.: 237.
`N-Acetyl-O-methyl-l-hydroxyproline methyl eater (III)
`1-4 g. of N-acetyl-l-hydroxyproline (see Paper III of this series) were methy-
`lated as in the preparation of (I), using double the quantities of reagents. It was
`advisable to grind the starting material to a fine powder, as it is not very soluble
`1-24 g. of distillate (125-1500/0.05 mm.) were obtained, which
`in acetone.
`crystallized immediately. Recrystallization from ether yielded a product with
`M.P. 76-77°. [a]"'-81.00 (alcohol, 1=2, c=4-5). (Found: C, 54*1; H, 7'44;
`N, 6-91; OMe, 31.2%. C,H1504N requires C, 53-8; H, 7-46; N, 6'96; OMe,
`30*8 %.)
`N-Acetyl-O-methyl-l-hydroxyproline (IV)
`1.0 g. of (III) was dissolved in 50 ml. N/3 Ba(OH)2 and was kept at room
`temperature for 3 hr. Ba was then removed exactly with H2SO4, and the filtrate
`from BaSO4 was evaporated to dryness in vacuo. The product was crystallized
`from chloroform by addition of ether, and was recrystallized from a minimum of
`M.P. 152-1530; [o]T - 104.3' (water, 1= 2
`water, in which it is very soluble.
`c=3). (Found: C, 515; H, 7 03; N, 7-47; OMe, 16.9%. C8HnO4N requires C,
`51*3; H, 6-95; N, 7-48; OMe, 16.6%.) Acid equiv. wt. Found: 184. Calc.: 187.
`N-Acetyl-O-methyl-dl-serine methyl eater (V)
`1-18 g. of N-acetyl-dl-serine (see Paper III of this series) were methylated
`with the same amounts of reagents as in the preparation of (III). As the starting
`material is a glass of low solubility in acetone, it was obtained in a finely divided
`Biochem. 1939 xxxiII
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`1934
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`R. L. M. SYNGE
`state by evaporating a strong aqueous solution with kieselguhr in a mortar in a
`vacuum desiccator. When dry, the product was thoroughly ground. The resulting
`powder was transferred to the reaction vessel and thoroughly dried in the
`After methylation, distillation (100-140o/0.05 mm. Hg) yielded
`desiccator.
`0-9 g. of a product which crystallized in the receiver on keeping overnight.
`Recrystallization from ether (in which the compound was rather soluble)
`yielded 0-4 g. M.P. 70-71°. (Found: C, 48-3; H, 7-25; N, 7-95; OMe, 35.0%.
`C7H1304N requires C, 48-0; H, 7-43; N, 8-00; OMe, 35.4%.)
`N-Acetyl-O-methyl-dl-serine (VI)
`This compound could be made by saponification of (V) but it was found that
`the crude distillate containing (V) on saponification yielded a contaminant which
`rendered the crystallization of (VI) rather difficult. This contaminant could be
`removed by prolonged extraction of an aqueous solution of the saponification
`product with chloroform. I am grateful to Mrs R. V. Pitt Rivers for carrying out
`this extraction. The compound could be more conveniently prepared by direct
`acetylation of O-methyl-dl-serine, using the procedure described in the Appendix
`to Paper I of this series. The compound crystallized very slowly from ethyl
`M.P. 108-109°. (Found: C, 43-8; H, 6-95; N, 8-42; OMe, 18-1 %.
`acetate.
`C6H,O4N requires C, 44-7; H, 6-82; N, 8-69; OMe, 19-2 %.) Acid equiv. wt.
`Found: 165. Calc.: 161.
`N-Acetyl-O-methyl-dl-allothreonine (VII)
`This compound was prepared in the same way as (VI) by direct acetylation of
`It was recrystallized from
`O-methyl-dl-allothreonine [West & Carter, 1937].
`acetone. M.P. 1510. (Found: C, 47-9; H, 7-77; N, 7-69; OMe, 18-1 %. C7H1304N
`requires C, 48-0; H, 7-43; N, 8-00; OMe, 17-7 %.) Acid equiv. wt. Found: 170.
`Calc.: 175.
`
`SUMMARY
`The preparation and properties of some N-acetyl-O-methyl-hydroxyamino-
`acids are described.
`The partition coefficients of these compounds between chloroform and water
`have been measured.
`It is suggested that, by means of these derivatives, extractional fractionation
`of the hydroxyamino-acids from protein hydrolysates might be accomplished.
`I wish in particular to thank Dr D. J. Bell, Prof. A. C. Chibnall, Prof. C. R.
`Harington, Prof. Sir F. G. Hopkins, Dr H. R. Marston, Dr A. J. P. Martin,
`Sir C. J. Martin, Dr A. Neuberger, Mr N. W. Pirie and Dr J. G. Sharp for their
`advice, interest, and encouragement in respect of the work described in these
`four papers.
`I am also particularly grateful to the International Wool Secretariat, who
`have maintained me with a Studentship, and have generously provided me with
`apparatus and materials.
`
`REFERENCES
`Behr & Clarke (1932). J. Amer. chem. Soc. 54, 1630.
`Cherbuliez & Plattner (1929). Helv. chim. Acta, 12, 317.
`Plattner & Ariel (1930). Helv. chim. Acta, 13, 1390.
`Cutler, Haworth & Peat (1937). J. chem. Soc. p. 1979.
`Karrer, Gisler, Horlacher, Locher, Mader & Thomann (1922). Helv. chim. Acta, 5, 469.
`S0rensen & Andersen (1908). Hoppe-Seyl. Z. 56, 250.
`West & Carter (1937). J. biol. Chem. 11i9, 109.
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