3=Hydroxyproline, a New Amino Acid of Collagen*enzymatic hydrolysates of collagen, a peptide was isolated which contained an unidentified component (1, 2). This peptide was shown to

THE JOURNAL OF Bxo~oaxcn~ CHE~IIISTRY Vol. 237, No. 12, December 1962

Printed in U.S.A.

3=Hydroxyproline, a New Amino Acid of Collagen*

JAMES D. OGLE,~ RALPH B. ARLINGHAUS,~ AND MILAN A. LOGAN

From the Department of Biological Chemistry, University of Cincinnati College of Medicine, Cincinnati 19, Ohio

(Received for publication, May 28, 1962)

During amino acid sequence studies of peptides obtained from enzymatic hydrolysates of collagen, a peptide was isolated which contained an unidentified component (1, 2). This peptide was shown to be Gly-X-Hypro, where X refers to the unknown residue. Since the amino acid sequence of the peptide was determined by the phenylthiohydantoin degradation procedure (3), it was presumed that the middle component of the peptide was indeed an amino acid. The new amino acid was then obtained from complete acid hydrolysates of collagen and was tentatively identified as 3-hydroxyproline (1). This paper deals with the method of isolation, proof of structure as 3-hydroxyproline, and synthesis of this new amino acid.

EXPERIMENTAL PROCEDURE

Preparation of Collagen-The collagen was prepared from Achilles tendons of cattle as described by Neuman (4).

Acid Hydrolysates of Cohen-Collagen was hydrolyzed as 3 y. solutions in 6 N HCI. The hydrolyses were carried out either in sealed tubes at 105” or in capped flasks in the autoclave at 15 pounds of pressure (120”) for 8 to 10 hours. The HCl was removed by repeated evaporation and by treatment with Amber- lite IR-4B.

Amino Acid Analysis-The new amino acid and the other amino acids involved in this study were determined on ion exchange analytical columns (5). The ion exchange resins employed were either Dowex 50-X4 (200 to 400 mesh) or Amberlite IR-120 (chromatographic grade). Columns were 1 X 100 cm, operated with sodium phosphate buffers in the manner described by Schrohenloher, Ogle, and Logan (6)) except that the temperature was 48” and flow rate was 10 to 12 ml per hour. Column fractions were analyzed by the calorimetric ninhydrin procedure of Moore and Stein (7).

Isolation of New Amino Acid-Procedure A: The new amino acid was isolated from a 3-g hydrolysate of collagen by one pass- age over a column of Amberlite IR-120 in the ammonium form (8 x 100 cm) operated at 37”. The column was eluted with 0.2 M ammonium formate, pH 3.1, at a flow rate of 90 ml per hour. The new amino acid emerged in a pure state in Fractions 300 to 340 (2Oml fractions). It was detected in the fractions by acid ninhydrin method B of Piez, Irreverre, and WolfI (8). The am-

* A preliminary report was made at the 1961 Annual Meeting of the American Society of Biological Chemists (1). Taken in part from a dissertation for the degree of Doctor of Philosophy nresented bv Ralnh B. Arlinzhaus to the Graduate School of the ijniversitv of Cincinnati. -

t Fellow of the Charles Fleischmann Endowment. t Predoctoral Research Fellow (GF-10.335) of the Division of

General Medical Sciences, United ‘States’ Public Health Service. Present address, Department of Biological Chemistry, University of Kentucky College of Medicine, Lexington, Kentucky.

monium formate was removed by lyophilization, yielding 7 mg of the compound. Because only small amounts of hydrolysate could be employed in this method, however, Procedure B was used.

Procedure B: The hydrolysate of 100 g of collagen, the HCI removed by evaporation and the pH adjusted to 2.2 by shaking with successive portions of Amberlite IR-4B, was placed on a column of Dowex 50-X4 (6 x 90 cm) prepared as follows. The resin was treated with excess NHhOH, washed, and resuspended in HzO. Oxalic acid, 0.5 N, was added until the pH of the super- natant fluid was 2.3, and the column was washed with HzO. The amino acids were eluted with HzO. Fractions of 20 ml each were collected. Every fifth fraction was spotted on paper and chromatographed in ethanol-water-ammonia (80 : 20 : 1) and stained with ninhydrin. The new amino acid emerged in the aspartic- glutamic acid fraction and in the first part of the 4-hydroxyproline fraction. Two cuts were made, one containing the acidic amino acids and the other containing approximately the first half of the 4-hydroxyproline. The first cut was concentrated by vacuum distillation, and much of the acidic amino acids crystallized out upon cooling. The second cut was concentrated to a small volume, and 95 To ethanol was added to crystallize out much of the 4-hydroxyproline. The filtrates and washings from these two crystallizations were combined and placed on a column of acid-treated alumina (3 x 95 cm) (2) and eluted with 76% ethanol, 0.15 N NH,OH. Again the fractions were chromatographed on paper. A small amount of unidentified material emerged from the column first, followed by the new amino acid, then by 4-hydroxyproline. The new amino acid peak tailed into the 4-hydroxyproline fraction. A cut was made to include as much of the new amino acid as possible without contamination with 4-hydroxyproline. This division was aided by scanning the chromatogram with an ultraviolet lamp (Mineralite, long wave). Traces of the 4-hydroxyproline-ninhydrin complex fluoresce red under ultraviolet light, whereas the new amino acid appears as a dark spot. The fractions containing the new amino acid seemed to contain some inorganic material from the alumina. The fractions were concentrated and passed through a column of Amberlite CG-50 in the hydrogen cycle (1.8 x 60 cm) and were eluted with water. The fractions containing the new amino acid, located by paper chromatography, appeared just after the anions, as indicated by the increase of the eluent to pH 3 to 4. These were evaporated to dryness, yielding 150 mg of the pure compound. The new ammo acid was crystallized as needles from a 0.8 % solution of hot 95 % ethanol.

Preparation of Esters of New Amino Acid--The new amino acid was esterified with ethanolic HCl at room temperature for 24 hours. The ester was crystallized from ether-petroleum ether. The new amino acid was acetylated under the conditions for

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3668 S-Hydroxyproline, a New Amino Acid of Collagen Vol. 237, No. 12

0-acetylation of Sakami and Toennies (9). The acetyl derivative was crystallized from the reaction mixture by the addition of ether-methyl Cellosolve (20:2.5). It was recrystallized from ethanol-ether.

Reduction with HI and Red P-The new amino acid, 7 mg, was placed in a Pyrex tube (18 X 150 mm) with 6 ml of HI (technical grade, 55 to 57%) and 100 mg of red P. The tube was sealed and placed in an oven at 150’ for 5 hours, The reaction mixture was cooled, and the contents were repeatedly evaporated to dryness. The residue was extracted with water and filtered. The filtrate was concentrated to a small volume and placed over a column of Dowex 50-X4 in the hydrogen form (3 x 60 cm). The amino acids (unreacted amino acid and proline) were eluted with 0.1 N NH40H. The tubes containing ninhydrin-positive material were pooled and concentrated. The solution was examined by paper chromatography, and the amino acids were determined on the Dowex 50 analytical column. Ninhydrin color values were determined at 420 mp.

Oxidation with Permanganate-The new amino acid, 3 mg, was dissolved in 3 ml of water, and the solution was heated to 70” in a water bath; 1.1 ml of a solution of 0.1 M KMn04 in 0.5 N

HzS04 acid then were added dropwise with shaking. The reaction mixture was cooled to room temperature, and Ba(OH) 2 was added to adjust the pH to 3. The suspension was filtered, and the precipitate was washed with small amounts of water. The reaction mixture was examined by paper chromatography, and the reaction product @-alanine) was determined on the Dowex 50 analytical column in the usual way, except that it was eluted with sodium phosphate buffer (0.2 N with respect to sodium ion), pH 4.3, at a temperature of 48”.

Synthesis of 3-Hydroxyproline

5-Phthalimido-d-pentenoic Acid-This compound was prepared essentially according to Method B of Baker et al. (lo), but in one-third to one-half the quantities. The trimethylbenzylam- monium hydroxide (Triton B) was added in three equal portions, and the temperature was maintained at 22-25” during the addi- tions. Upon addition of the malonic acid, the temperature rose to 50” and was kept at this point for 1 hour by warming. The product was crystallized from 95% ethanol; m.p., 201-203’; reported, 200-201”. The yield, based on phthalimide or malonic acid, was 40 To.

Methyl 5-Phthalimido-%pentenoate-The ester was prepared in 98% yield by Freudenberg esterification as described by Baker et al. (10). The ester was crystallized as described or by slow cooling of the reaction mixture; m.p., 90-92”; reported, 90-92’.

MethyE d-Chloro-S-methoxy-5-phthalimidovalerate-This method of methoxychlorination is essentially that of Jackson (11). Methyl 5-phthalimido-2-pentenoate, 10 g, was dissolved in 300 ml of absolute methanol, and dry Cl* was delivered into the solution at a rate of two bubbles per second at room temperature. The solution was frequently adjusted to the turning point of bromophenol blue (as judged by tests on aerated aliquots) by addition of methanolic sodium hydroxide. From time to time a 0.2-ml aliquot of the reaction mixture was withdrawn, evaporated to dryness, redissolved in methanol, and titrated with 0.1 N KMnO* to determine unsaturation. The gassing with Clz was continued until there was no longer a positive test for unsaturation. The reaction mixture was concentrated to a small volume (until bumping from salt precipitation started), and ether was added to precipitate the NaCl. The salt was filtered off and

washed with ether. This concentration and salt removal process was repeated, and the solution was evaporated to dryness. The product was dissolved in ether, the ether solution was evaporated to dryness, and the residue was dissolved in 50 ml of methanol and placed in the deep freeze. After 2 days, 5 g (40% yield) of crystals were obtained. The product was filtered and recrystallized as white prisms from warm methanol by cooling in the deep freeze; m.p., 88-90”.

Calculated: C 55.30, H 4.95, Cl 10.88, N 4.30, OCH3 19.05 Found: C 55.11, H 4.90, Cl 11.02, N 4.33, OCH3 18.62

The yield of the product was not improved by the use of AgNOs (12) or Ag&03 in the reaction mixture.

Methyl %Chloro-S-methoxy-Laminovalerate-Methyl 3-chloro- 3-methoxy-5-phthalimidovalerate, 3.3 g (0.01 mole), was dissolved in 50 ml of methanol with warming, and 0.5 ml (0.01 mole) of hydrazine hydrate was added (13). The solution was refluxed for 30 minutes, then evaporated to dryness. The residue was dissolved in 30 ml of water, and 2 ml of 8 N HzS04 were added. The solution was warmed to 65” for 10 minutes, then cooled to O”, and the phthalhydrazide was filtered off and washed with cold water. The filtrate and washings were concentrated to 35 ml and used directly for the following cyclization step.

Cyclization of Methyl 6-Chloro-S-methoxy-5-aminovalerate to 3- Methoxyproline-The solution of the above amino ester was made 1 N in NaOH and allowed to stand at room temperature for 1 day. Cyclization was followed by determining the amount of inorganic chloride formed.

Hydrolysis of S-Methoxyproline to S-Hyclroxyproline-The solution from the cyclization was neutralized with HBr and evaporated to dryness. The residue was dissolved in 48% HBr and refluxed for 3 hours. Considerable charring occurred during the refluxing. The solution was repeatedly evaporated to dryness to remove most of the HBr.

Purijication of Synthetic S-Hydroxyproline-When the pig- mented solution from the above hydrolysis was chromatographed on paper and stained with ninhydrin, several blue spots appeared on the chromatogram along with the yellow spot of 3-hydroxyproline. One-half of the solution was passed over a column of Dowex 50-X4 (2 x 65 cm) operated in the manner described above as Procedure B for isolation of the natural compound. Two adjacent peaks with RF values of the natural 3-hydroxyproline were obtained. The first peak was pooled, concentrated to a small volume, and passed through a column of acid-treated A1203 (1.5 x 70 cm) (2), then through a column of Amberlite CG-50 (1.5 x 60 cm) operated as previously described. The fractions containing the 3-hydroxyproline in chromatographically pure form were pooled and evaporated to dryness. The residue, 300 mg (46 y. yield), was dissolved in 30 ml of hot 95 y0 ethanol and crystallized in white needles upon cooling; m.p., 232” with decomposition.

CJH~NO~

Calculated: C 45.81, H 6.92, N 10.69, 0 36.61 Found : C 45.97, H 6.93, N 10.61, 0 36.76

The over-all yield of 3-hydroxyproline, based on phthalimide or malonic acid, was 7.2%; based on the last characterized intermediate, methyl 2-chloro-3-methoxy-5-phthalimidovalerate, the yield was 46%.


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J. Il. Ogle, R. B. Arlinghaus, and M. A. Logan

Other Nethods of Synthesis of S-Ilydroxyproline

Nethod f--JIt:thyl 5-benzamido-2-pentonoate was prrpared according to the m&hod of r1bderhalden for the preparation of thr hescnoatc homologuc (14) or by the method of Baker et al. (10). The unsaturated c&cr was then treated with mercuric acetate in methanol followed by aqueous Iil<r and Iirz according to the method of Kcst and Carter (15) for the prrparation of threonine. The mc%hyl 2-acetos~mer~uri-3-metho~~-5-phthalimidovalerate was obtained in 970/, yield by cooling the reaction mixture. It tnrlted at 170” and gave the c&orrectt analysis for methosyl group. However, the nitrogen analysis was high.

CnHdgXh

Calculated: N 2.54, OCHn 11.24 Found : S 3.32, OCHI 11.20

In both casts, the bromine in the 2-bromo-3-methosy intermediate appcarcd to bc extremely labile and hydrolyzed so rap- idly upon addition of sodium hydrosidc that very little cyclization was obsctrved. When chlorination instead of bromination was used in this method, a 50yG yield of methyl 5-phthalimido-2- chloro-3-mcthosyvalerate was obtained. Howcvrr, this intcr- mediate has not yet been cyclizrd, and the amount of diasterco- isomer corresponding to the natural 3-hydrosyprolinc is not known.

.l/ethod 2---\n attempt was made to caarry out an aldol condensation of ,&~hloropropionaldchyde and glycinc catalyzed by CuSOA (IQ, or by treating the aldehydc with c’opptbr glyrinate in alkalim solution (17). Treatment of the condensation prod- uc+ with NaOH should have effectrxd ring closure> to product 3-hydrosyprolinc. The P-chloropropionaldchyd(: was purchasrd as the dic%hylacctal, which was trcatcd with dilutca HCI under a variety of caonditions for hydrolysis to the free aldtthyde. There was ncvcr any indication that thr free aldehyde was obtained b) hydrolysis, and this method failed in our hands.

Meth,od S-Ethyl acrylate was treated with (Ithy hippurate in a one-step Michael condensation and ring closure reaction catalyzed by sodium cathylate, according to the method of Sasaki (18). Reduction of the N-benzoyl-3-ketoproline formed with sodium borohydride followed by acid hydrolysis would produce 3-hyrlrosyproline. No imino acid was obtained by this method.

RESULTS AND DISCUSSION

The chromatographic behavior on Dowex 50 columns of the new amino acid from the tripeptide isolated from collagen has brcn reported (1). Since the unidentified residue was apparently bound in pcptide linkage in this tripeptide, and hence in collagen, a complctc a&d hydrolysate of collagen was ehromatographed on the Dowcs 50 analytical column. The separation of the new amino a&l from 4-hydrosyproline is shown in Fig. 1. Based on the extinction at 420 mp of the new amino acid-ninhydrin rom- pies, dctcrmined later with the crystalline compound, the content of the new amino acid in cattle 19chilles tendon collagen is 0.260/,.

The new amino acid was isolated in 150-mg amounts from each 100-g batch of complete acid hydrolysate of collagen and was crystallizad as white needles from hot 95% ethanol. A photomicrograph of the crystals is shown in Fig. 2.

The absorbanoy at 420 rnp of the ninhydrin coml)les (7) of the new amino acid suggested that it might bo an imino acid. The compound also reacted with ninhydrin in glacial acetic arid

or in glacial acetic acid-nhosuhoric acid (19) to form an am-

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t-7

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E 2 0 0.50 - z i3 $0.25- New amino z z

Tube number

FIG. 1. Dowex 50-54 chromatogram of a 4G.5-mg acid hydrolysate of collagen. Amino acid- and hydroxyproline-ninhydiin complexes were read at 420 rnp. The column was 1 X 100 cm, operated with sodium phosphate buffer, pH 3.1, at 48” and a flow rate of 10 ml per hour.

FIG. 2. Photomicrograph of crystals of new amino acid (from hot 95y0 ethanol).

her-colored complex with an absorbancy at 420 mp. I’rolinc reacts under these conditions to form a pink-colored complex (absorbancy at 515 mp), and 4-hydrosyprolinc does not react. The new rompound gavcb a positive test for imino nitrogen with nitroprusside and acetaldchyde (20).

After oxidation with Hz02 in alkaline &SO4 solution (21), the rompound gave a positive test for the pyrrolinc> ring with o-amino- brnzaldehydc (22) (Table I), which suggrstcd the presence of a pyrrolidinr structure. However, in clontast to the 4-hydroxyprolines, the new amino acid gave no (solor with p-dimethylamino- henzaldrhydr according to the methods of Neuman and Logan (21) and Htrgrtnann (23) for the analysis of these imino acids (Table IT). Roth the normal and allo-4-hydrosyprolines (24) give positive results under these conditions. The compound cave a nelrative test for a carbonvl eroun with azobenzcne~)hcnrl-


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3-Hydrozyproline, a New Amino Acid of Collagen Vol. 237, No. 12

TABLE I

Pyrroline ring formation as indicated by reaction with o-amino- benzaldehyde after oxidation with H&z in alkaline copper

sulfate solution

Tubes were read versus a water blank.

Amino acid Amount

/moles

Absorbancy at 420 mp

Proline..................... Proline*...................... 4-Hydroxyproline. 4-Hydroxyproline*. New amino acid. New amino acid*. Alanine...... .._......

* No H&Z was added.

10 0.222 10 0

10 0.444 10 0 8t 0.201 8t 0

10 0

t Assuming a molecular weight of 131.

TABLE II Reaction with p-dimethylaminobenzaldehyde after oxidation with

chloramine-7

Amino acid Amount

/moles

4-Hydroxyproline. 0.2 Proline. 0.2 New amino acid. 0.2* New amino acid. 0.4* New amino acid. 15.0%

* Assuming a molecular weight of 131.

Absorbancy at 540 rn~

0.446

0 0 0

0.372t

t This value would correspond to a 1% impurity of 4-hydroxyproline in the new amino acid.

TABLE III Compound 1

h’lemental analysis and melting points of new amino acid and its

esters

Values for 4.hydroxyproline are given for comparison. New amino acid ..................................... 4-Hydroxyproline .................................. New amino acid ethyl ester. ........................ 4.Hydroxyproline ethyl ester ....................... 0-Acetyl derivative of new amino acid. ............. 4-Acetoxyproline. ............................... N-Acetyl-4-hydroxyproline . .........................

Compound

New amino acid

4-Hydroxyproline New amino acid ethyl es-

ter-HCl 4-Hydroxyproline ethyl

ester-HCl 0-Acetyl derivative of

new amino acid 4-Acetoxyproline

* With decomposition.

C

44.8 6.78 44.6 7.00 45.8 6.86 42.7 7.17 42.6 7.03 43.0 7.2

48.7 6.44

48.7 6.23 48.5 6.36

H

0.7 6.78

6.89 7.2

36.9

37.0 36.6

1 Melting point (uncorrected)

232-233”*

270* 125-130

149-153

205*

173*

hydrazinesulfonic acid (25)) and its chromatographic behavior on the Dowex 50 analytical column was unaltered after treatment with sodium borohydride. The new amino acid exhibited no absorbancy in the range of 240 to 360 rnp.

The new amino acid was esterified with ethanol and HCl and was also acetylated under the conditions for 0-acetylation of Sakami and Toennies (9). The elemental analyses and melting

points of the new compound and its esters are shown in Table III.

From these analyses, the empirical formula of the new amino acid is C,H9N03, which is the formula of hydroxyproline.

To establish that the acetyl group was on the oxygen and not, on the nitrogen, the hydrosamic acid test (26) was employed. The results are given in Table IV. N-Acetyl-4-hydroxyproline, prepared according to the method of Robinson and Greenstein (27)) gave a negative test, whereas the 0-acetyl derivative of the new amino acid and of 4-hydroxyproline gave positive results. The ethyl ester of the new amino acid also gave a positive test.

The neutralization equivalents of the new amino acid and of 4-hydrosyproline were determined from the partial titration curves shown in Fig. 3. The neutralization of 1 meq of base from pH 8 to 12 by 1 mmole (I31 mg) of 4-hydroxyproline and by 131 mg of the new amino acid established 131 as the equivalent weight of the new acid.

When the new amino acid (2.6 mg) was allowed to react with ninhydrin according to the method of Van Slyke, MacFadyen, and Hamilton (%), 0.02 mmole of CO2 was liberated. This is in good agreement with the equivalent weight of 131 obtained from the titration curve of the amino acid. No NHB was produced in the ninhydrin reaction (29), which is characteristic of the action of imino acids with this reagent.

When the new amino acid was reduced with HI and red I’, as described under “Experimental Procedure,” proline was obtained in 63% yield. The proline was determined on the Dowex 50 analytical column and confirmed by chromatographing the reaction product of the reduction with authentic proline as marker. In Fig. 4 the first, peak corresponds to some unreacted new amino

acid. One peak in the range of proline was obtained. The two peaks accounted for the total ninhydrin-positive material put

TABLE IV ConJrmation of ester group as indicated by hydroxamic acid test

Hydroxamic acid test

I I I I I I 1

1.0 -

0.75. 4-Hydroxyproline

.-I %

0.25 -

5 6 7 8 9 IO II I2 PH

FIG. 3. The partial titrat)ion curves of the new amino acid (131 mg) and 4-hydroxyproline (1 mmole, 131 mg).


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December 1962 J. D. Ogle, R. B. Arlinghaus, and M. A. Logan 3671

over the column. Proline was also identified by paper chromatography.

The Rp values of the new amino acid and of 4-hydroxyproline, chromatographed on paper in four different solvent systems, are shown in Table V. The new amino acid has exactly the same Rp

values as 4-hydroxyproline in these solvent systems. However, 4-hydroxyproline stains pink with the ninhydrin reagent, whereas the new amino acid stains yellow. The 4-hydroxyproline-ninhydrin spot on paper fluoresces red under the ultraviolet lamp (Mineral&e, long wave), but the new amino acid-ninhydrin spot appears as a dark spot.

The results given thus far indicate that the new amino acid is a hydroxyproline but not one of the 4-hydroxyprolines. In order to establish the location of the hydroxyl group, the compound was oxidized with KMn04 in acid solution as described in “Ex- perimental Procedure.” The reaction product was chromatographed on paper in four different solvent systems, and a spot appea,red at the RF and with the distinctive color of p-alanine

t; 2 24

0.75 -

f5 v) 0.50 -

$ Unreocted

new

4

amino acid

0.25-

2 E 0

.k

g 80 I 90 1 , y I I I

100 140 150 160 170 0.75

c, r 1

80 90 100 140 150 160 170

Tube number

FIG. 4. The Dowex 50 chromatograms of the reaction mixture from the HI and red P reduction of the amino acid. Upper curve, an aliquot of the reaction mixture alone; lower curve, smaller aliquot of the reaction mixture with added proline. Amino acid-ninhydrin complexes were read at 420 rnp.

TABLE V

Paper chromatography of new amino acid compared with Q-hydroxyproline

The amino acid spot was located by 0.2yo ninhydrin in water- saturated n-butanol.

Solvent New amino acid 4ez$iy

RF RF

Ethanol-water-ammonia (80:20:1). 0.42 0.41

Phenol-water*. 0.68 0.69

Pyridine-water (65:35). 0.67 0.65

Butanol-acetic acid-water (80:20:20). 0.22 0.24

* Phenol is saturated with an aqueous 6.3% sodium citrate and 3.770 sodium dihydrogen phosphate solution.

TABLE VI Paper chromatography of product of KMnOa oxidation of new amino

acid and of @-alanine

Methanol-water (95:5). Ethanol-water-ammonia (80:20:1). Pyridine-water (65:35). Butanol-water-ethanol (80:20:20),

RF RF

0.38 0.38 0.41 0.41 0.51 0.53 0.10 0.11

% 0.75W b 2 g o-50- 0

2 Q 0.25- I

2

d Ol50 1 I I

> 160 170 180 190

-150 160 170 180 I90 TUBE NUMBER

FIG. 5. The Dowex 50 chromatogram of the reaction mixture from the KMn04 oxidation of the new amino acid. Upper curve, an aliquot of the reaction mixture alone; lower curve, a smaller aliquot of the reaction mixture with added fl-alanine. The amino acid-ninhydrin complex was read at 565 mp. The column was eluted with 0.2 N sodium phosphate, pH 4.3, at 48”.

(Table VI). The identity of &alanine was confirmed, and the amount was determined on the Dowex 50 analytical column. The yield was 31%. The reaction product of the oxidation was chromatographed alone and with authentic @alanine as marker. The results (Fig. 5) show that one peak was obtained which accounted for the total ninhydrin-positive material put over the column. It is difficult to conceive how @alanine could arise

from the oxidation of the compound unless the hydroxyl group is on carbon atom 3, as indicated in the reaction shown in Scheme 1.

H

KMnOa

KMnO,L+ H,C- COOH

+ co, H,C

‘N

+ H,C

H2 p-alanine

SCHEMEI


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3672 S-Hydroxyproline, a New Amino Acid of Collagen Vol. 237, No. 12

COOH

‘ii c 2

c\” \

NH + CHz=CH-CHO ---f N-CHz-CH2-CHO COOH

C/o

“\” COOH

cl -co2

N-CHz-CHZ-CH- H -H20

C/o dH \

COOH 1 “\”

N-CHs-CHZ-CH=CH-COOCH, :&OH

C/o (CH,OClj

CH,OH CH&oC1 )

c? HsN-NH2 N-CHY-CH2-CH--CC-COOOH -

C/o ACH, Al

HsN-CHZ-CHZ-CH--CH-COOCHS

d CH, bl NaOH HBr --OH

-COOH - I/J- COOH

:

SCHEME 2

I I I I

0.40 -

0.35 - l Natural 3-hydroxyproline

plus 4-hydroxyproline o Synthetic 3-hydroxyproline

plus 4-hydroxyproline

P 85 90 95 too 105 110 II5

E 0.50-

2 0.45 - 8 z 0.40-

E 0.35- Synthetic 3-hydroxyproline _

9 plus natural 3-hydroxyproline g 0.30’

z 0.25-

0.20-

O.l5-

0.10.

0.05 -

O- I I I I I \ 85 90 95 100 105 II0 II5

TUBE NUMBER

FIG. 6. Dowex 50 chromatograms of natural and synthetic 3-hydroxyproline and 4-hydroxyproline.

The results of thii oxidation reaction would indicate that the new amino acid is 3-hydroxyproliie.

The identity of the new amino acid as 3-hydroxyproline was confirmed by synthesis of this compound in 7.2% over-all yield by the following scheme (Scheme 2).’ The yield based on the last characterized intermediate was 46 %.

cedure.” Either these methods failed completely in our hands, or the over-all yields of the imino acid were too small to be prac- tical or to be considered as good proof of structure. Further- more, no intermediates could be obtained in pure enollgh form to 1 Attempts were made to synthesize 3-hydroxyproline by sev-

eral other methods, which are described in “Experimental Pro- be characterized.

The phthalimidopentenoic acid and its methyl ester were obtained in crystalline form and have been reported previously (10). The methyl 2-chloro-3-methoxy-5-phthalimidovalerate was obtained in crystalline form, and the elemental and methoxyl analyses were in excellent agreement with the theoretical values (see “Experimental Procedure”). After cyclization, followed by HBr hydrolysis of the methoxyl group, the reaction mixture was passed over columns of Dowex 50, alumina, and Amberlite CG-50 for purification. The purification was followed by paper chromatography of the fractions. Two adjacent peaks having the same RF values on paper as the new amino acid were obtained. The first peak was pooled, evaporated to dryness, and crystallized from 95% ethanol. The elemental analysis was in excellent agreement for 3..hydroxyproline (“Experimental Procedure”), and the over-all yield of the synthetic compound was based on the amount of this material. The second peak has not been studied, but it may be the diastereoisomer, since theoretically a mixture of both normal and allo-3-hydroxyproline could be obtained by this method of synthesis. The synthetic 3-hydroxyproline was chromatographed on the Dowex 50 analytical column alone and with the natural product as marker. The results are shown in Fig. 6. The emergence of the synthetic material with the natural product would indicate that the former was the same diastereoisomer as the natural 3-hydroxyproline isomer (if it is assumed that the analytical column would separate the diastereo- isomers). The synthetic 3-hydroxyproline chromatographed on paper in ethanol-water-ammonia (80 : 20 : 1) and stained with ninhydrin exactly like the natural compound.

The optical rotation and absolute configuration of the natural 3-hydroxyproline have yet to be determined. Also resolution of the synthetic product and isolation and characterization of the diastereoisomer have to be accomplished. These studies are now in progress.

SUMMARY

A new amino acid isolated from cattle Achilles tendon collagen has been shown to be 3-hydroxyproline. The amount present was found to be 0.26%.


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December 1962 J. D. Ogle, R. B. Arlinghaus, and M. A. Logan 3673

The major evidence showing that this new amino acid is 3-hydroxyproline includes elemental analysis, formation of an 0-acetyl derivative, reduction to proline, oxidation to /3-alanine, and synthesis. In the synthesis, two compounds with the empirical formula of hydroxyproline were obtained. One of these was identical with the natural compound upon analytical ion exchange chromatography.

Acknowledgment-We are indebted to Dr. Paul B. Hamilton of the Alfred I. du Pont Institute for extensive testing by his chromatographic procedure (30) of the natural amino acid that we have isolated from collagen and the two peaks that we obtained by our preparative columns used for purification of the synthetic preparations. These results have come to hand since the paper was submitted for publication.

Dr. Hamilton’s results show that Peak I of the synthetic amino acid obtained by the first two methods described in the text and the natural amino acid from collagen, when added together, come off as a single peak with no resolution. The amino acids came off at the same point (immediately ahead of methionine sulfox- ide) when tested singly or two at a time and when mixed with various other amino acids. These results appear to afford strong additional evidence that Peak I corresponds to the natural 3-hydroxyproline.

The elemental analysis of the second peak has been obtained and corresponds to that of a hydroxyproline, and reduction of the compound yields proline. Dr. Hamilton’s results show that this peak is separated from Peak I by essentially the same distance by which 4-allohydroxyproline is separated from 4-hydroxyproline. These results now make it appear likely that the second peak is in fact the allo compound.

Details of these new findings will appear in a publication of the studies of the absolute configuration.

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James D. Ogle, Ralph B. Arlinghaus and Milan A. Logan3-Hydroxyproline, a New Amino Acid of Collagen

1962, 237:3667-3673.J. Biol. Chem.

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3=Hydroxyproline, a New Amino Acid of Collagen*enzymatic hydrolysates of collagen, a peptide was isolated which contained an unidentified component (1, 2). This peptide was shown to