Int. J.

Peptide Protein Res. 8, 1976, 503-516

Published by Munkspaard Copenhagen, Denmark No part may be rcprodudd by any process without written permission from the authorb)

SYNTHESIS O F PEPTIDES AND DERIVATIVES O F 3- A N D 4- HYDROXYPROLINE ELIJAH

ADAMS

Department of'Biological Chemistry, University of Maryland School of Medicine, Baltimore, U.S.A.

Received 13 November 1975 Synthesis andproperties are reported .fora number of peptides and related derivatives of Ehydroxy-~-proIine and 4-hydroxy-~-proline.These were made for several different purposes, namely, synthesis of N-acetyl-O-tosyl-4-hydroxy-~-proline amide as a model for chemical reduction of peptide-bound hydroxyproline, synthesis of tripeptides with the sequence glycyl-4-hydroxy-~-prolyl,'A synthesis of the naturally occurring sequence, glycyl-3-hydroxy-~-prolyl-4-hydroxy-~-proline, and monomers for polymerization to yield the sequence -glycyl-prolyl-4-hydroxy-~-prolyl-.

Synthesis of the linear* hydroxyproline peptides (glycyl-4-hydroxy-L-prolylglycine, 4-hydroxy-Lprolylglycine, and related intermediates) was reported in 1944 (I). Other synthetic derivatives of 4-Hyp** (summarized till 1965 (2); and (3-12)) and additional di- and tripeptides (4,6,10-32) were subsequently described. Synthetic block polymers containing 4-Hyp, made either by solution methods (33,6,34,9) or solidphase methods (1,3) have been described. Synthetic peptides and intermediates of alto-4-Hyp (36,41,30) or 0-methyl-4-Hyp (36,37) have also *The synthesis of cyclo(4-Hyp-4-Hyp) had been described earlier (58) as noted below under preparation XXXI, Description of Compounds. While glycyl-4hydroxyproline and DL-leufyl-4-hydroxyproline were first reported by Abderhalden & Koppel (59), their purity has been questioned (1) because of the method of preparation. ** 4-Hyp refers to 4-hydroxy-L-proline and 3-Hyp to 3-hydroxy-~-proline.t.1.c. = thin layer chrornatography; DCC = dicyclohexyl carbodiimide; TEA = triethylamine; abbreviations of amino acids and peptides and generally used symbols are those listed in Biochem. J. (1973) 131, 1-20; abbreviations for N-blocking groups or esters of amino acids or peptides are those recommended by the IUPAC-IUB Commission on Biochemical Nomenclature (J. Biol. Chem. (1971) 247, 977-983). All amino acids, free or in peptides, are L except where specifically noted.

been reported, and synthetic G l y 3 - ~ ~ - H ywas p prepared, although not characterized, as a substrate for enzymatic resolution to yield 3-~-Hyp (42). The present paper describes the synthesis and properties of a number of previously unreported peptides and intermediates of both CHyp and 3-Hyp, made for several different purposes. N-Ac-4-Hyp(O-Tos)-NH2 was made to test methods for chemically reducing the hydroxyl carbon of peptide-bound hydroxyproline. Tripeptides with the sequence Gly-4-Hyp-X were prepared to determine if such sequences are present in vertebrate basement membrane collagen as they are in earthworm cuticle collagen (43,44). Peptides and intermediates containing 3-Hyp were made in connection with the identification of Gly-3-Hyp-CHyp isolated from swine kidney cortex (45). Finally, several compounds were made as intermediates toward synthetic polypeptides containing the internal sequence -Gly-Pr0-4-Hyp-. In view of the limited number of synthetic peptides containing 4-Hyp and especially of previously defined synthetic peptides of 3-Hyp, a consolidated description of all peptides and intermediates in this group seemed advisable. Most of the intermediates and peptides described are new; some compounds, reported here as the product of newer methods, were previously 503

ELIJAHADAMS made by older methods such as that of acyl chloride or azide coupling. MATERIALS AND METHODS

Starting materials obtained from commercial sources were carbobenzoxy chloride, N-carbobenzoxyglycine, glycine benzyl ester p-tosylate, glycine ethyl ester hydrochloride and alanine methyl ester hydrochloride (all from Sigma Chemical Co.), N-carbobenzoxy-L-glutamicacidy-benzyl ester (Cyclo Chemical Co.), N-hydroxysuccinimide (Eastman Kodak Co.), dicyclohexylcarbodiimide (Pierce Chemical Co.); Pd catalyst (10% in charcoal) was a product of Matheson, was Coleman 8c Bell. trans-3-Hydroxy-~-proline obtained as noted previously (45). Solvents were the best quality commercially obtainable, and in general were not redistilled or specially dried. Thin layer chromatography** was generally carried out on aluminum-backed silica gel F254 sheets (E. Merck); spots were made visible by U.V. absorption, by charring over a flame, by exposure to If vapor, or by spraying or dipping with a 2% solution of ninhydrin in acetone. T.1.c. solvents used were as follows: n-butyl alcoho1:acetic acid:water, 12:3:5 (A); methanol: chloroform, 3: 1 (B); ethanol:water, 3: 1 (C); ethyl acetate:methanol, 3: 1 (D); acetone:acetic acid, 100:l (E); chloroform:acetone, 2: 1 (F); s-butyl alcohol :3 % ammonia, 25 :11 ( G ) ; n-propyl alcohol :water, 3 :1 (H), n-propyl alcohol: methanol:water, 7:2:3 (I); n-propyl alcohol: 1 N ammonia, 7:3 (J); pyridine: methyl ethyl ketone:acetic acid:water, 15:70: 3: 15 (K); n-butyl a1cohol:acetic acid:water, 4: 1 : 5 (L); methanol:chloroform, 1 :3 (M); chloroform: t-amyl alcohol :acetic acid 70: 30:0.5 (N). Amino acid analysis was carried out with the resin and by the procedure described earlier (45). The full four-buffer system was used as noted earlier (43, but for some runs a single buffer at pH 3.3 was used, corresponding to Pic0 Buffer A (Durrum Pic0 Buffer System Instructional Manual, Durrum Chemical Corp., 1971, Palo Alto, Calif.). Samples were hydrolyzed for 18-24 h in constant boiling HCI; the HCI was removed by repeated evaporation at 60°C under a Nz stream and the sample was applied in the starting buffer. Melting points (reported uncorrected) were 504

made on a Fisher-Johns hot stage; the stem correction, if made, is estimated as 2-3". Optical rotations were carried out in a Bendix NPL polarimeter, Type 143A, in a 5-cm cell. Samples for elementary analysis were generally prepared for analysis in a drying pistol in vacuo, at 100" or 110", over P,05. RESULTS AND DISCUSSION

N-Acetyl-O-tosyl-4-hydroxy-~-proline amide. NAc4-Hyp-NHZ(I) was made by mixed-anhydride amidation of N-Ac-4-Hyp after failure to ammonolyze N-Ac-4-HypOMe; the synthesis of N-acetyl-4-hydroxy-~-proline methylamide from the ester has been reported (7). Treatment of I with tosyl chloride, as earlier described for tosylation of N-Ac-4-HypOMe (46), led to a crystalline product (11) in low yield, which differed in m.p. and t.1.c. behavior from the ammonolysis product (111) of N-Ac-4Hyp(OTos)OCH, (IV). Infrared, n.m.r., and analytic data showed that compound I1 was actually N-Ac-4-Hyp(OTos) nitrile. The desired N-acetyl tosyl amide (111) was obtained in good yield by direct ammonolysis of IV. A point of interest in the characterization of I1 was the absence of a nitrile absorption band at 4.4-4.5 p. The occasional marked diminution or absence of this band in certain oxygen-rich compounds has been noted previously (47). Subsequent attempts were unsuccessful in displacing the tosyl group of 111 by treatment with thioacetate (as with serine conversion to cysteine (48,49)), or with thiobenzoate (as reported for the thiobenzoylation of N-O-ditosyl-4-HypOCH3 (50)). Gly-4-Hyp-X peptides. Only a few tripeptides with this sequence have been reported: Gly-4Hyp-Ro, Gly-CHyp4-Hyp (25), Gly-4-HypDL-Ser and Gly-4-Hyp-Glu (26). Gly-4-Hyp-Ala and Gly-4-Hyp-Ser were made commercially by undisclosed methods (43). To approach these syntheses, the N-hydroxysuccinimide ester of Z-Gly-4-Hyp-OH (V) was made. V itself was conveniently prepared via Z-Gly-ONSu in 5@-60"/, yield; other methods, such as differing mixed anhydride procedures (17,51), offered no advantage over the route via Z-Gly-ONSu. V was earlier reported as a product of the acid chloride method (1).

PEPTIDES OF

3- AND

Z-Gly-Hyp-ONSu (VI) was coupled with AlaO-CH3 and with free Ala. Crystalline products were obtained in each case; Z-Gly-4-Hyp-AlaOH (VIII)was also obtained by saponifying the corresponding ester (VII). Z-Gly-4-Hyp-Ser-OH (X) was readily made by direct coupling of serine with VI, but two products, distinguishable by t.l.c., resulted from comparable attempts to couple VI with L-leucine. The desired product Z-Gly-CHyp-Leu-OH (XII) was obtained, although in low yield, by fractional crystallization. The synthesis of Z-Gly-4-Hyp-4-Hyp-OH did not succeed, either by coupling VI to free 4-Hyp or to CHyp-OMe . HCl, or by coupling V with 4-Hyp-OMe HCl using DCC. Since Z-Gly-4Hyp-4-Hyp-OBz (XIV)has been made by a mixed anhydride procedure (25), XIV was made in the present work by coupling Z-Gly-4-Hyp and 4Hyp-OBz with DCC. Derivatives of 3-hydroxy-~-proline. Z-Gly-3DL-HYP-OH (XVI)was earlier made via coupling of Z-Gly-ONSu with 3 - ~ ~ - H (42); y p the product was not characterized. Present attempts to make XVI (using 3-Hyp rather than 3 - ~ ~ - H y pfailed ) to yield a crystalline product; in contrast with the synthesis of V, it was necessary to purify the product by extraction into dilute bicarbonate, and then, after acidification, into ethyl acetate. The product, while not crystalline, was analytically correct and yielded essentially pure Gly-3-Hyp (XVII)on catalytic hydrogenation. The tripeptides Gly-3-Hyp-CHyp (XX) and Gly4-Hyp-3-Hyp (XXII) were made via the respective carbobenzoxy benzyl esters. Z-Gly-3Hyp-CHyp-OBz (XIX) was obtained as a crystalline product; Z-Gly-4-Hyp-3-Hyp-OBz (XXI)failed to crystallize but seemed homogeneous by t.1.c. The free tripeptide (XX)obtained from XIX appeared essentially pure by amino acid analysis both of the peptide and of its acid hydrolysate. The free tripeptide (XXII)obtained from XXI, however, required purification by preparative chromatography on an amino acid analyzer column. Attempts to make Gly-Glu-CHyp were moti, vated by the difficulty in chromatographically separating this presumptive peptide from Gly-3Hyp-4-Hyp (45). Glu4Hyp (XXV)was prepared from Z-Glu(yOBz)-4-Hyp-OBz(XXIV).The syn-

~HYDROXYPROLINE

thesis of XXV has been reported (52) without characterization. Satisfactory preparations of both XXIV and XXV are described below; however, the desired tripeptide Z-Gly-a-Glu4HypOH was not obtained, in attempts to couple Z-Gly-ONSu with XXV. As models for subsequent synthesis of dipeptides such as 3-Hyp-CHyp, a number of CHyp dipeptides were prepared. These included 2-4Hyp-CHyp-OBz (XXVI)which failed to yield the free dipeptide CHyp-CHyp (XXIX) on catalytic hydrogenation or treatment with HBr in acetic acid. XXIX was obtained instead via the reduction of Z-CHyp-4-Hyp-OH (XXVIII), itself a saponification product of Z-4-Hyp-4Hyp-OMe (XXVII). The amide, Z-4-Hyp-4Hyp-NHt (XXX),was obtained by ammonolysis of XXVII, and catalytic reduction of XXX yielded crystalline cyclo(4-Hyp-4-Hyp) (XXXI). Tripeptides as precursors of (-Gly-Pro+Hyp-),. An interest in obtaining polytripeptides with the internal sequence shown led to trials of various peptide combinations. Z-Pro-CHyp-OH (XXXII) was synthesized conveniently from Z-Pro-ONSu; earlier preparations of XXXII include that via Z-Pro-CI (14) or by saponification of Z-Pro-4Hyp-OMe (31). CHyp-Gly (XXXIV) was obtained, as previously reported (l), by catalytic hydrogenation of Z-4-Hyp-Gly-OBz (XXXIII);the latter compound was prepared in the present instance by DCC coupling instead of via the azide as earlier reported (1). As a route to Z-Pro-4Hyp-Gly-OH, attempts were made to couple Z-Pro-ONSu with XXXIV, in analogy with the preparation of XXXII by a comparable route, and with the coupling of Z-Pro-ONSu and Pro (unpublished). No product was obtained by this route, under varied conditions of temperature and relative concentration of components. Efforts to prepare Z-Pro-CHyp-Gly-ONP by coupling Z-Pro-4-Hyp-OH with HBr . Gly-ONP (DCC method) similarly failed. The desired sequence was therefore prepared in two other ways. In one, Z-4-Hyp-Gly (XXXVI) prepared by tlie saponification of Z4Hyp-GlyOEt (XXXV),was coupled with HBr . Pro-ONP (DCC, in acetonitrile). By another route, Z-ProCHyp was coupled with Gly-OEt.HC1 to form amorphous Z-Pro4Hyp-Gly-OEt (XXX505

N-AC-~-H~P(OTOS)-NHZ CI&I~'JZO~S

Z-Gly-4Hyp-0NS~ CIpH1I N308

Z-Gly-4-Hyp-Ala-OMe C19H25N307

.Z-Gly-4-Hyp-Ala-OH CigHz3N307

Gly-4-Hyp-Ala CIOHI~N~O~HZO

Z-Gly-4-Hyp-Ser-OH

Gly-4-Hyp-Ser CioHi,N306.4 Hz0

I11

VI

VII

VIII

IX

X

XI

N-Ac4HypNHz GHitNz03

N-Ac-~-H~~(OTOS)nitrile CI~HI~NZO~S

.

I1

I

Compound

42.3

52.8

43.3

55.0

16.0

54.4

(S,9.8)

51.5

6.3

5.7

6.9

5.9

6.2

5.1

5.6

54.5 5.2 (S, 10.4)

7.0

H

N

14.8

10.3

15.2

10.7

10.3

10.0

8.6

9.1

16.0

% Calculated

48.0

C 7.0

H

% Found

42.4

53.0

43.3

54.7

56.1

54.3

(S,9.7)

52.0

6.1

5.6

6.6

6.0

6.2

5.3

5.6

54.5 5.4 ( S , 10.0)

48.1

C

14.8

10.4

14.7

10.6

10.3

9.8

8.5

8.9

16.4

N

107-110

f

157-159

182-186

71-75

152-154

129-131

152-156

Melting point "C

TABLE 1 Analytical and other data on new compounds' T.I.c.~

0.80 (D), 0.15 (F)

0.71 (A), 0.80 (B), 0.70 (D) 0.99 (E), 0.65 (F)

0.46 (B), 0.18 (G)

0.31 (C), 0.22 (J)

- 74 (HzO, c = 0.62)

0.42 (C), 0.23 (G), 0.18 (H)

- 38 (MeOH, C= 1.1)

-118 (HIO, c = 0.73)

- 71 0.35 ( G ) (MeOH, c = 0.21)

- 85 0.86 (B), 0.27 (D), 0.81 ( G ) (MeOH, c = 0.64)

- 82 0.72 (D) (MeOH, c = 0.29)

- 20 (MeOH, c = 1.4)

d

-69 0.25 (A), 0.57 (B), 0.72 (C) (MeOH, c = 0.20)

[ z ] ~ degrees ~ ~ "

v)

P

0

3

x

b

P t:

6.9

58.8

Z-3-Hyp-OH CioHisNOs

Z - G I U ( ~ - O B Z ) ~ H ~ ~ - O B66.9 Z C3zH34Nz08

a-Glu4Hyp C10H16Nz06 * CHoOH

=I11

XXIV

XXV

45.2

6.0

42.6

Gly4Hyp-3-Hyp CizH19N~O6* 2H20

XXII

5.7

6.2

6.6

44.3 Gly-3-Hyp4-H~~ CizH17N306 . CzH*Oz * HzO

Z-Gly-3-HyprlHyp-OBz C27H3iN308

XIX

6.6

xx

3-Hyp0B~HCl CIZHI~NOPCI

XVIII

42.6

5.6

6.0

Cily-3-H~~ C7HizNz04 4 Hz0

XVII

55.9

61.7

Z-Gly-3-Hyp-OH CI#I & 0 6

XVI

8.1

50.3

6.3

Gly4HypL.e~ C r i H z ~ N ~ *o4s Hz0

XI11

6.8

56.8

55.9

Z-Gly4Hyp-Leu-OH CziHz9N307 * 4 HzO

XII

9.6

4.9

5.3

12.7

11.1

8.0

5.4

14.2

8.7

13.5

9.5

44.9

66.7

58.3

42.7

44.8

61.2

55.2

41.9

56.1

50.1

57.1

6.9

6.2

5.7

6.9

6.8

5.7

6.5

6.2

9.6

4.9

5.3

12.5

11.6

8.2

5.6

14.2

8.2

13.0

7.6

5.9

9.4

6.6

0.3 ( G )

0.62 (C), 0.49 (J)

- 67 (MeOH, c = 0.6)

- 102 (HzO, C = 0.50)

(MeOH, c = 0.44)

- 15

d

d

(MeOH, c = 1.1)

- 69

(HZO, c = 0.28)

-3

-45 (HZO, c = 0.49)

E

(H20, c = 0.34)

-65

not -40.5 crystalline (MeOH, c = 0.4)

123-124

C

E

134-136

161-164

f

0.30 (A), 0.10 (G), 0.40 (J) minor ); spot 0.31 (I (ninhydrin-negative) 0.61 (I)

0.78 (B), 0.78 (D), 0.73 (G)

0.63 (B), 0.40 (D)

0.21 (J)

0.05 (A), 0.28 (J)

0.74 (B), 0.75 (D), 0.77 (G)

0.15 (A), 0.68 (G),0.67 (K)

0.29 (I), 0.31 (J)

not - 39 0.52 (B), 0.07 (D), 0.25 (GI crystalline (MeOH, c = 0.52)

c

134-138

i n

!i

3

e

? i U

W

'

P

3

x

59.1

57.3

Z-4-Hyp-4-Hyp-OH Ci~HzzNzO7

Z-4-Hyp-4-Hyp-NHZ CisHz3N306

Z-CHyp-Gly-OEt Ci7HzzNz06

2-4-Hyp-Gly-OH ci ,Hi sN206

Z-Pro-4-Hyp-Gly-OEt CzzHzsN309

XXVLII

XXX

XXXV

XXXVI

XXXVII

XXXVIII 2-Pro-4-Hyp-Gly-OH CzoHz5N307

6.0

6.5

5.6

6.3

6.1

5.9

6.2

6.0

10.0

9.4

8.7

8.0

11.1

7.4

7.1

6.0

N

57.1

58.9

56.2

58.6

56.6

56.9

58.2

64.1

C

6.2

6.8

5.9

6.6

6.1

6.0

6.3

5.9

H

% Found

10.0

9.3

8.6

8.3

11.0

7.0

7.0

6.0

N

0.55 (B), 0.72 (D), 0.31 (G)

0.80 (B), 0.20 (D), 0.64 (G)

- 77.9 (MeOH, c = 0.5)

- 55.6 (MeOH, c = 0.4)

- 56 0.56 (B), 0.06 (D) (MeOH, c = 0.54)

- 54 0.79 (D), 0.34 (F) (MeOH, c = 0.42)

0.74 (B), 0.63 (D), 0.74 (G)

0.67 (B), 0.46 (D), 0.50 (N)

145-148

T.I.c.~

-84.1 (MeOH, c = 1.0)

-83.1 (MeOH, c = 0.7)

degrees

-111 0.43 (B), 0.17 (M) (MeOH, c = 0.80)

not - 95 0.05 (D) crystalline (MeOH, c = 0.57)

195-198

148-152

144-145

190-195

135-140

144-147

Melting point "C

See text for other data such as ion-exchange chromatographic behavior and amino acid composition of free peptides. Solvents (in parentheses) are listed under Materials and Methods. Compounds listed gave single spots except where noted. All spots were visible by charring and by U.V. absorption (2-derivatives, tosyl compounds) or by ninhydrin (free peptides). Not listed for free peptides. ,I Quantity of pure material insufficient.

55.9

58.3

57.2

57.1

58.2

Z-4-Hyp-CHypOMe Ci9HuNz07

XXVII

64.1

H

% Calculated

Z4Hyp4-Hyp-OB~ CZSHZ~NZO~

C

XXVI

Compound

6

>

P 1 e

PEPTIDESOF

3- AND ~HYDROXYPROLINE

VII). Saponification of the latter yielded the crystalline product, Z-Pro-4-Hyp-Gly (XXXVII). DESCRIPTION OF COMPOUNDS

Table 1 lists all new compounds which were characterized, including elementary analysis, melting point, specific rotation, and t.1.c. data. N-Acetyl-4-hydroxy-~-prolineamide ( I ) . N-Ac-4Hyp (5334) (17.4 g, 0.1 mol) was dissolved. in 100 ml of DMF, treated with 13.8 ml of TEA, chilled to -5" in an ice-salt bath, and, with stirring, treated with 10 ml of ethyl chloroformate, added dropwise over 25 min. After 10 min, triethylammonium chloride was removed by filtration and washed with cold DMF. The combined filtrate and wash was treated with 200 ml of DMF containing 15 ml of aqueous NH40H (14.8 M), solution at 0".After 24 h at 25", D M F was removed by evaporation. The resulting syrup crystallized on rubbing in isopropanol. First crop, 10 g (58%); a small second crop of crystals (1 g) was obtained from the mother liquor after treatment with ether. Total yield: 63%. The product was recrystallized from isopropanol. N-Acetyl-O-tosyl-4-hydroxy-~-prolinenitrile (ZI). Attempts to make N-Ac-CHyp(0Ts)-NH2 by direct tosylation of (I) led to a crystalline product that was identified as 11. A sample of I (2 g, 11.6 mmol) was suspended in 20 ml of pyridine and kept at - 15". p-Toluenesulfonyl chloride (2.3 g, 0.12 mmol) in 10 ml of pyridine at - 10" was added dropwise to the suspension of I in pyridine and the mixture was stirred at 4". A clear yellow solution resulted within 24 h. T.1.c. indicated a mixture of compounds including unreacted I. Crystals of I1 (100 mg) were obtained from the reaction mixture following ether extraction of the pyridine solution after addition of 2 vols of 1 M HCI. The nature of the product was initially obscure. Its U.V. spectrum was consistent with the tosyl group as was elementary analysis (Table 1) and the n.m.r. spectrum (see below). However, no i.r. band attributable to the nitrile (4.4-4.5 f i ) was seen on repeated spectra. The compound was finally judged to be the nitrile by its synthesis from the amide (permitting the possible dehydration of the amide by the tosyl reagent), by its elementary analysis and by its distinction from 111 by a number of criteria (see 111, below). N.m.r. (CDCI,, TMS), 6 2.0 (5.3, Me), 2.2 (s, l),

2.45 (m,3,CH3), 3.7-3.9 (d, each 1, C-5), 4 . 5 4 9 (m,l), 50-5.3 (m,l), 7.2-7.9 (m,4, aromatic H). Peaks remained unchanged on shaking with D20. N-Acet~vl-O-tosyl-4-hydroxy-~-proline amide ( I l l ) N-Ac-CHyp(0Ts)-OMe (IV) was prepared as described earlier (38); m.p., 69"-72" (lit. 78" (38), 60" (46)). A sample of IV (12.5 g, 0.037 mol) was dissolved in 400 ml of methanol, chilled to O", saturated with NH3 and kept at 25" for 3 days. The reaction mixture was concentrated to a thick yellow oil, taken up in acetone and treated with n-heptane. The resulting oil crystallized on scratching. Yield, 11.1 (93 %). The compound was recrystallized from acetonitrile. 1.r. resembled that of I1 except for an additional band (5.9 f i ) attributed to the primary amide group. N.m.r. (CDCls) showed peaks almost identical with those of I1 except for the presence of two peaks at 6 5.7 and 6.8-7.0 (n, 1 H each) which were obliterated on shaking with D20;these were attributed to the -C-NHz hydrogens and reI1 0 presented decisive evidence (together with elementary analysis) for assigning the nitrile designation to I1 and the amide designation to 111. Carbobenzoxy-glycyl-4-hydroxy-~-proline( V). This was made by coupling Z-Gly-ONSu ( 5 5 ) with CHyp essentially as for a previous synthesis of Z-Gly-Pro (55). The oil obtained by extracting the acidified reaction mixture with ethyl acetate crystallized on rubbing with ethyl acetate. In several preparations, the yield of crystals was 50-60% of theory. The first crop of crystals was recrystallized from ethyl acetate (usually 20 vol/g):m.p. 122"-124"; in one preparation, 126"-129" (lit., 124" (1)).

Carbobenzoxyglycyl-4-hydroxy-~-proline-N-hydroxysuccinimide ester (VI). V (13 g, 0.041 mol) and

N-hydroxysuccinimide (4.7 g, 0.041 mol) were dissolved in 130 ml of dioxane; the solution was chilled in an ice bath, and treated with DCC (8.5 g, 0.041 mol). After several hours at 5", the reaction mixture was kept overnight at 25", then filtered to remove dicyclohexylurea and concentrated to a yellowish oil. The oil crystallized after dissolving in hot ethanol and standing at room temperature. The first crop weighed 10.2 g 509

ELIJAHADAM (59%). Recrystallization from hot ethanol (10 vol.) yielded an analytically correct product in 88% yield.

ethanol addition. Yield 100 mg (77%). Amino acid analysis of the unhydrolyzed peptide yielded a single peak at 3 h (single buffer method); analysis of a hydrolyzate showed only Gly, Ala, Carbobenzoxyglycyl-4-hydroxy-~-proline-~-alanine and Hyp in ratios 1.00:1.01:0.93.T.1.c and ionmethyl ester ( VII). 1.05g (0.0025 mol) of VI wasdis- exchange chromatography agreed with a comsolved in 20 ml of dimethoxyethane and treated mercial preparation of this peptide (43). whose with 0.35 g (0.0025 mol) of Ala-OMe.HC1 identity was established both by composition and and 0.35 ml (0.0025 mol) of TEA. The reaction sequence analysis (43). mixture was stirred for 60min at 25" then treated with 300 ml of cold H 2 0 and kept overnight at 5". Carbobenzoxyglycyl-4-hydroxy-~-prolyl-~-serine Crystals which formed were collected and the (X).This was made essentially as described for mother liquor concentrated about two-fold, VIII B, from 2.1 g (0.005 mol) of VI and equimolar with appearance of further crystals. Both crops Ser. The concentrated reaction mixture crystaltogether weighed 0.65 g (64%).The compound lized on acidification. Yield 1.6 g (78%). was recrystallized from ethyl acetate in 80% yield. Glycyl-4-hydroxy-~-prolyl-~-serine (XI).This was made from 250 mg (0.61 mol) of X essentially Carbobenzoxyglycyl-4-hydroxy-~-prolyl -L-alanine as described for the preparation of IX. Yield, (VIII). This was made both by saponification of 146 mg, 86%. Amino acid analysis (single buffer) VII (A) and by direct coupling of VI with Ala (B). gave a single peak at 130 min. Analysis of a hydroA. 0.3 g (0.74 mmol) of VII was dissolved in 6 lyzate gave approximately equimolar Hyp and ml of acetone + 3 ml of methanol and treated with Ser and a major Gly peak that was not integrable 0.8 ml of 1 N NaOH. After 1 h at 25", the reaction for technical reasons. mixture was made acid with 1 N HCI and evaporated to a salt-oil residue. 3 ml of H 2 0 were added and the solution was extracted twice Carbobenzoxyglycyl-4-hydroxy-~-prolyl-z-e with 10 ml of ethyl acetate. The ethyl acetate ( X I I ) . This was made essentially as described for solution was evaporated to yield an oil which VII B, from 2.1 g (0.005 mol) of VI and equimolar Leu. The concentrated, acidified reaction solidified on scratching with heptane. B. 2.1 g (0.005 mol) of VI was suspended in mixture was extracted with ethyl acetate. Treat40 ml of ethanol, and treated with 0.45 g (0.005 ment of the ethyl acetate solution with ether mol) of Ala and 0.84 g (0.01 mol) of NaHC03 yielded a crumbly precipitate (1.2 g) with a in 20 ml of H 2 0 . After 24 h at 25", the reaction broad m.p. (90-120"). T.1.c. (solvent G ) showed mixture was acidified with 6 N HCI, evaporated two equally intense spots made visible by charto an oil-salt residue brought up in 12 ml of ring, at Rr 0.1 and Rr0.3 respectively. After several H 2 0 and saturated with NaCI. The aqueous weeks at 5", a second crop of crystals had appearphase was extracted twice with 50 ml of ethyl ed in the mother liquor (ethyl acetate-ether). The acetate. The extracts were combined, washed with second crop of crystals weighed 230 mg (1 1 %), 15 ml and evaporated to an oil. The oil was gave only the spot of higher Rr and appeared to dissolved in 100 ml of refluxing ethyl acetate be the desired product (Table 1 and XIII). and allowed to cool, with the formation of crystals. Yield 1.1 g (56%); m.p. and t.1.c. agreed Glycyl-4-hydroxy-~-prolyl-~-leucine (XIII). 100 with the product of method A (Table 1). mg (0.22 mmol) of XI1 was hydrogenated exactly as in the preparation of XI. Crystals obtained Glycyl-4-hydroxy-~-prolyl-~-alanine ( I X ). V I I I by treating the methanol filtrate with ethanol (200 mg, 0.51 mmol) in 50 ml of methanol weighed 50 mg (71%). Amino acid analysis of containing 2 ml of acetic acid and 100 mg of the peptide gave a single major peak at 345 min Pd catalyst, was hydrogenated for 30 min. After by the single buffer method. Analysis of a hydroremoval of the catalyst, the methanol solution lyzate gave only Gly, Hyp and Leu in the ratio, was concentrated to an oil and crystallized on Gly 1 .00,Hyp 0.95, Leu 1.08. 510

PEPTIDESOF

3- AND CHYDROXYPROLINE

Carbobenzoxyglycy1-4-hydroxy+prolyl-4-hydroxy-L-proline benzyl eater (XZV).This compound,

which has previously been synthesized via a mixed anhydride procedure (25), was made by DCC coupling from 1.61 g (0.005 mol) of V and 1.27 g (0.005 mol) of CHyp-OBz HCI (1). After removal of dicyclohexylurea, the filtrate was concentrated and taken up in ethyl acetate to remove a further crop of urea. The filtrate was taken to dryness and crystallized on dissolving in hot methanol, treatment with ether, and storage at 5". After drying, the product weighed 1.6 g (61%), m.p. 152"-156" (lit. (25), 155"-156). T.1.c.: (G) single spot, visible by U.V. absorption and charring, Rr 0.84.

-

100 mg of Pd catalyst. After removal of the catalyst, the methanol solution was concentrated to small volume. Addition of ether resulted in a somewhat sticky precipitate. Trituration with ether, filtration and drying, yielded a white powder: 130 mg (87%). Amino acid analysis (standard 4-buffer system) showed a single major peak at 112 min, eluting just before glycine. The sample contained a small glycine contaminant estimated as less than 5 % on a molar basis. Analysis of a hydrolyzate showed only equimolar Gly and 3-Hyp (Gly 1.00; 3-Hyp 1.00). 3-Hydroxy-~-proline benzyl ester hydrochloride

(XVZII). This was made by a procedureessentially as that for 4-Hyp-OBz . HCI (l), from 500 mg(3.8 Glycyl-4-hydroxy-~-prolyl-4-hydroxy-~-proline mmol) of 3-Hyp. Addition of 2 vol. of ether to ( X V ) . 400 mg (0.76 mmol) of XIV was hydro- the benzyl alcohol solution resulted in a pregenated in a solution of 50 ml of methanol con- cipitate which crystallized on standing at 5". taining 2 ml of acetic acid and 200 mg of Pd The product was washed with ether, dried to catalyst. After 2 h the catalyst was removed by yield 830 mg (87%), and recrystallized from filtration and the methanol filtrate evaporated benzyl alcohollether in 73% yield. to an oil; the oil, after trituration with ether, solidified at 5". Yield, 200 mg (89%). Chromato- Carbobenzoxyglycyl-3-hydroxy-~-prolyl-4graphy on the amino acid analyzer indicated hydroxy-L-proline benzyl ester ( X I X ) . 420 mg a major peak at 125 min (single buffer). The (1.3 mmol) of XVI was dissolved in 10 ml of principal visible contaminant was a peak re- dioxane. 4-Hyp-OBz.HC1 (1) (336 mg, 1.3 presenting 9% of free 4-Hyp on a molar basis. mmol) was added, together with 0.2 ml of TEA. Carbobe~~zoxyglycyl-3-hydroxy-~-proline ( X VI). This was made essentially as described for V, except for an additional purification step, extraction of the product into dilute KHCOs. To 0.76 g (0.0025) of Z-Gly-ONSu dissolved in 15 ml of dimethoxyethane, was added 0.33 g (2.5) mmol) of 3-Hyp and 0.21 g (2.5 mmol) of NaHCOn in 3 ml of water. After 18 h at 25", the reaction mixture was acidified and extracted with three 10-ml vol. of ethyl acetate, The ethyl acetate solution (evaporated to 7 ml) was extracted with two 15-ml portions of 1 M KHC03, the KHCOl solution was acidified and extracted with two 10-ml portions of ethyl acetate. The product appeared pure by t.1.c. but failed to crystallize on a number of trials with various solvents in several different preparations. Drying in uacuo yielded an easily pulverized but hygroscopic glass:0.54 g (67%). Clycyl-3-hydroxy-L-proline (XVII). 260 mg (0.8 mmol) of XVI was hydrogenated for 2 h in 30 ml of methanol containing 1 ml of acetic acid and

Water (2 ml) was added to dissolve the suspension. The solution was chilled on ice, treated with 294 mg (1.4 mmol) of DCC and kept overnight at 5". Precipitated dicyclohexylurea was filtered offand the filtrate was concentrated and brought up in ethyl acetate. After concentration to small volume a further crystalline precipitate (presumptive triethylammonium chloride) was removed and the filtrate was again concentrated to an oil and treated with heptane. The oil solidified at 5" and was filtered and dried (400 mg, 58%). Recrystallization from hot ethyl acetate yielded a small crop of crystals (120 mg). A second crop (40mg) had a low m.p, and was discarded.

Glycyl-3-hydroxy-~-prolyl-4-hydroxy~-prolitte ( X X ) . X I X (113 mg, 0.22 mmol), dissolved in 30 ml of methanol containing 1 ml of acetic acid and 100 mg of PD-charcoal, was hydrogenated for 1 h. The catalyst was filtered off and the methanol solution was evaporated to dryness. Scraping the residue in ethanol yielded a fluffy solid. After filtration, washing with ether 51 1

ELIJAHADAUS and drying, the product weighed 45 mg (54%). The compound was eluted from the amino acid analyzer column as a single peak (4 buffer system) at 133 min. Hydrolyzates of this material showed approximately equimolar Gly, 3-Hyp, and 4-Hyp (Gly 1.00, 3-Hyp 0.90, CHyp 1.00). An earlier preparation, made with jH-CHyp, gave the expected sequence by the Edman procedure (45).

Carbobenzoxy-a-L-glutamyl-(y-benzylester-4-hydroxy-L-proline benzyl ester (XXIV).This was made by DCC coupling in CHC13 from 0.544 g (1.47 mmol) of Z-Glu-7-OBz and equimolar CHyp-OBz .HCI. The product was a viscous oil (800 mg, 95%).

a-~-Glutatnyl-4-hydroxy-~-proline (XXV). XXIV (760 mg, 1.32 mmol) was hydrogenated for 4 h in 50 ml of MeOH containing 2 ml of acetic acid CarbobenzoxygIycyI-4-hydroxy-~-prolyl-3-hydroand 110 mg of Pd-charcoal. The catalyst was xy-L-proline benzyl ester ( X X I ) . This was made removed by filtration, the solvent evaporated essentially as described for XIX, from 0.81 g off and the oily residue treated with ether, (2.5 mmol) of V and equimolar XVIII. After yielding a white solid of 265 mg (78%). The removal of dicylohexylurea and triethylammonproduct (nominally 5 pmol) was eluted from the ium chloride, the ethyl acetate filtrate was conamino acid analyzer (4-buffer system) as a single centrated to a yellow oil which failed to crystallize peak at 150 min: a small peak (0.03 pmol) on triturating with ether or heptane. T.1.c. in the position of Glu was present. Analysis of a showed a single spot visible by U.V. absorption hydrolyzed sample of the product gave only and charring, and which migrated faster than equimolar Glu and CHyp (Glu, 1.00; Hyp, 0.98). both of the starting compounds. Rr 0.67 (B), T.1.c. showed a single major spot visible both by Rr 0.44 (D). The dried oil weighed 1.3 g (100%). ninhydrin and charring and a faint second spot, Further characterization was not attempted and ninhydrin negative, consistent with asmall amount the product was subjected to hydrogenation. of diketopiperazine (Table 1). An attempt to synthesize Z-Gly-Glu-4-Hyp Glycyl-4-hydroxy-~-proIyI-3-hydroxy-~-proIine (XXIZ). Hydrogenation of 1.3 g (2.5 mmol) of by coupling Z-Gly-ONSu with XXV in dimethXXI under the conditions for XX yielded a oxyethane did not yield a homogeneous product. white granular precipitate of 700 mg (87%) on evaporating the solvent. An amino acid analyzer Curbobenzoxy-4-hydroxy-~-prolyI-4-hydroxy-~run (4-buffer system) on a nominal 5 pmoles of the proline benzyl ester ( X X V I ) . Z-4-Hyp was made product showed a major peak at 103 min and as noted for Z-3-Hyp (XXIII). 2.65 g (0.01 mol) several small peaks in the position of 3-Hyp, of Z 4 H y p dissolved in 20 nil of dioxane was Gly-4-Hyp, and Gly. Because of the presence of added to a suspension of 2.58 g (0.01 mol) of small quantities of contaminants, the major 4-Hyp-OBz . HCI and 1.4 ml of TEA. Addition peak was isolated by chromatographing 50 mg of 4 ml of water and warming brought undisof the impure product on a 0.9 cm amino acid solved material into solution. The solution was analyzer column, using the first two buffers chilled in ice, treated with 2.26 g (0.011 rnol) of of the standard 4-buffer system. Fractions DCC and kept at 5" overnight. Dicyclohexylurea corresponding to the major peak were pooled and was removed and the dioxane solution concendesalted with a Dowex-50-H+ column, eluting trated to small volume and taken up in ethyl with 1 M NH,OH. The NHIOH eluate was acetate; further precipitated urea and triethyllyophilized yielding 17 mg of a yellowish powder. ammonium chloride were removed by filtration. Amino acid analysis of a hydrolyzed sample The filtrate was concentrated, the residue was dissolved in 25 ml of CHCI, and washed with N gave Gly 1.00, 4-Hyp. 1.00, 3-Hyp 0.94. HCI, 1 N NaHC0, and HzO, dried over Na2S04 Carbobenzoxy-Ehydroxy-~-proline( X X I I I ) . This and concentrated to an oil which crystallized was made by a general procedure for carbo- from ethyl acetate. Yield 2.8 g (60%). benzoxy amino acids (56), using excess NaHCO,, Hydrogenation of XXVI was carried out under from 0.262 g (0.5 mmol) of 3-Hyp. The product conditions described by Davis & Smith (15) crystallized from ethyl acetate. Yield, 0.31 g for Z-Pro-Pro and Z-Pro-Hyp which yielded the dipeptides rather than the diketopiperazines. The (57%). 512

PEPTIDES OF

3- AND CHYDROXYPROLINE

product of hydrogenating XXVI was a gummy solid which by t.l.c., and from the small fraction retained on Dowex 5O-H+, appeared to contain the diketopiperazine and free dipeptide in an approximate 4: 1 mixture. Attempts to remove the benzyl ester group selectively by saponification or by HBr in acetic acid (57) were unsuccessful.

Carbobenzoxy-4-hydroxy-~-prolyl-4-hydroxy-~-

proline amide (XXX). XXVII (700 mg, 1.8 mmol) was dissolved in 20 ml of methanol, gassed with dry NH3 at room temperature for 1 h, then transferred to a pressure bottle and kept at 25" for 3 days. The solution was evaporated to an oil and triturated with ether, yielding a granular solid. Yield, 550 mg of an impure product giving two Carbobenzoxy-4-hydroxy-~-prolyl4-hydroxy-~- spots on t.l.c., oneconsistent with residual XXVII. proline methyl ester (XXVZI). Z-4-Hyp (2.65 g, The product (400mg) was purified by crystalliza0.01 mol) was coupled with CHyp-OMe * HCl tion from ethyl acetate-methanol (70: 10) -ether. (made as earlier described (l)), exactly as des- Yield 300 mg (61 %). cribed for the synthesis of XXVI. After the removal of residual dicyclohexylurea and triCyclo-(4-hydroxyl-prolyl-4-hydroxy-~-prolyl) ethyl ammonium chloride from the ethyl acetate (XXXI). 250 mg (0.66 mmol) of XXX was solution, the filtrate was concentrated to a dense hydrogenated for 1 h in 50 ml of methanol with oil and crystallized from ethyl acetate. The first 100 mg Pd-charcoal. The catalyst was removed crop weighed 1.8 g (46%). A small second crop by filtration and the solvent evaporated to yield (0.5 g, 13%) had a low m.p. and was discarded. a glassy deposit that could be scraped up in ether as white floccules. After redissolving the Carbobenzoxy4-hydroxy-~-prolyl-4-hydroxy-~product in a small volume of ethanol and storage proline (XXVZII). This was made by saponiat 5", successive crops of crystals were obtained. fication (1 equivalent of NaOH, 1 h, 25") of 786 Total yield, 80 mg (54%), m.p. 250"-252". T.1.c. mg (2 mmol) of XXVII,dissolved in dioxan :water showed a single ninhydrin-negative spot visible (8 ml: 1 ml). After acidification (1 N HCI) and by charring:Rr 0.60 (B). This compound was concentration to a residue of oil and salt crystals, first described as a product of 4-Hyp-OEt (58) the residue was extracted with two portions and gave m.p. 245"-246" (58); a more recent (20 ml each) of ethyl acetate, the ethyl acetate preparation from the ethyl ester gave m.p. solution dried over Na2S04 and concentrated 248"-249" (32). to a dense oil which crystallized on scraping in a small volume of ethyl acetate. Yield 430 mg Carbobenzoxy-~-prolyI-4-hydroxy-~-proline (57%). (XXXII). This compound, which had been pre4- Hydroxy-~-prolyl-4-hydroxy-~-proline (XXZX). pared earlier from Z-Pro-C1 (14), was made by XXVIII (315 mg, 0.8 mmol) was hydrogenated the N-hydroxysuccinimide method, essentially under the conditions used for XXVI (15). The as for the synthesis of Z-Pro-Gly ( 5 9 , from solvent was evaporated off and the residue was 10.4 g (0.03 mol) of Z-Pro-ONSu (55) and equiscraped up in ether, yielding a white flocculent molar CHyp. The concentrated acidified reaction precipitate. Yield after drying, 45 mg (75%). mixture crystallized on rubbing in ethyl acetate T.1.c. (Solvent J) indicated a major spot, R, = (9.7 g (90%)) and was recrystallized from hot 0.21, made visible by charring and ninhydrin, ethanol, map. 220"-221" (lit. (14), 217"-218"). and a faint spot, Rf = 0.58, visible only by charbenzyl ring and judged to be the diketopiperazine. The Carbobenzoxy-4-hydroxy-~-prolylg~cine dipeptide was purified by absorption to and ester (XXXIZI). This compound, reported earlier subsequent elution from Dowex 50-H+ with 1 M (1) as a product of the azide method, was made NH40H. The dipeptide was eluted from the by a DCC coupling procedure from 6.6 g (0.025 amino acid analyzer column (4-buffer system) mol) Z-CHyp and equimolar GIy-Obz Ts. and 132 min, as a peak with predominant 440 After removal of dicyclohexylurea, the CHCI, nm absorbance. An acid hydrolysate of the solution was washed successively with 1 N product showed only 4-Hyp by amino acid HCI and 1 N NaHC03, and dried over Na2S04. Concentrating the solution led to a heavy crop analysis.

513

ELIJAH ADAMS

of crystals which was recrystallized from ethyl acetate. Yield, 6 g (46%), m.p. 148"-152" (lit. 153" (1)). 4-Hydroxy-~-proIylglycine(XXXI V). This compound was prepared by hydrogenating 4.0 g of XXXIII, essentially as described earlier (1). Yield 1.6 g (89%). T.1.c. : (Solvent J) single spot, R, = 0.5, visible both by ninhydrin and charring.

Carbobenroxy-4-hydroxy-~-proIyIgIycineethyI ester (XXXV).This was made essentially as described for XXXIII from 6.6 g (0.025 mol) of 2-4-Hyp and equimolar Gly-OEt . HCI. The product crystallized on concentrating the washed CHCI, solution. Yield, 7.3 g (83%). Recrystallization from ethyl acetate yielded 6.1 g of crystals with unchanged m.p.

3.6 ml of 2 N NaOH, and kept at room teniperature for 2 h. A small precipitate was removed by filtration, and the reaction mixture was extracted with 25 ml of ethyl acetate. The aqueous phase was acidified and extracted with ethyl acetate continuously for 4 h at room temperature. The ethyl acetate extract was concentrated to small volume and kept at 5". Two crops of crystals weighed 1.33 g (50%). ACKNOWLEDGMENTS

The synthetic work described was begun in the Department of Biophysics, Weizmann Institute of Science, Rehovot, Israel, and the synthesis and identification of compounds 1-111 were carried out there with the advice of Drs. M. Wilchek, A. Patchornik, N. Lotan and A. Berger. I thank Dr. E. Gehra, Department of Chemistry, Weizmann Institute, for help with the spectral data distinguishing Compounds 11 Carbobenzoxy-4-hydroxy-~-prolyIgIycine and 111. Although the remaining compounds des(XXXVZ). XXXV (6 g, 0.017 mol) was dissolved cribed were subsequently prepared in Baltimore, this in 90 ml of methanol and treated with 10 ml of work too owes much to the initial guidance in current 2 N NaOH. After 75 min at 25" the solution was peptide synthetic methods that I obtained from the acidified and the product crystallized on con- Biophysics Department group (Weizmann Institute). centration. Yield 4.1 g (75%). Recrystallization I thank the Guggenheim Foundation for financial of 100 mg from 2 ml of hot water yielded 40 mg support and Dr. E. Katchalsky-Katzir, then Head of the Department of Biophysics, Weizmann Institute, of crystals. The unrecrystallized product gave for his hospitality and encouragement during my an acceptable elementary analysis (Table 1). visit to Rehovot. Dr. Franz Kasler, Department of Chemistry, University of Maryland at College Park, Carbobenzoxy-~-prolyl-4-hydroxy-~-prolyIgIycinecarried out most of the elementary analyses cited, Mrs. ethyl ester (XXXVIZ). 3.6 g (0.01 mol) of XXXII M. Lamon of our laboratory did the amino acid was suspended in 40 ml of CHCI,; to this was analyses and Mr. D. Blass made many of the optical added 1.3 g (0.01 mol) of Gly-OEt . HCI and rotation measurements cited. Mrs. S. Ramaswamy 1.4 ml of TEA. The suspension was chilled in assisted with a number of the preparations, particularly ice and treated with 2.17 g (0.0105 mol) of DCC. XXIII, XXXVII and XXXVIII. This work has been The suspension was stirred at 25"for an additional supported by PHS Grant GM-11105. 24 h, at which time t.1.c. on a small filtered sample REFERENCES showed essentially complete disappearance of XXXII. The dicyclohexylurea was removed by 1 . SMITH, E.L. & BERGMANN, M. (1944) J. Biol. Chem. 153,627451. filtration, the CHCl, filtrate was washed succes2. MAUGER, A. B. & WirKoP, B. (1966) Chem. sively with 1 N HCI, 1 N KHCO,, water, and Rev. 66,47-86. dried over Na2S04. On concentration and 3. CARTER, H. E. & LOO,Y.H. (1948) J. Biol. Chem. bringing up in ethyl acetate, a small, further 174,723-726. crop of urea crystals formed and was removed. 4. SHIBNEV, V. A., DEBABOV, V. G. & BAULINA, G. The ethyl acetate was concentrated to a thick A. (1964) Bull. Acad. Sci. USSR, Div. Chem. Sci. oil and dried in uucuo to an amorphous powder. No. 6, 975-978. Yield, 3.9 g (87%). 5. SCHNABEL, E. (1967) Liebig's Ann. Chem. 702, 188-196. Carbobenzoxy-~-proIyI-4-hydroxy-~-proIyIgIycine 6. SHIBNEV, V. A., CHUVAEVA, T. P. & POROSHIN, K. T. (1968) Bull. Acad. Sci. USSR, Div. Chem. (XXXVZZZ). 2.9 g (6.5 mmol) of XXXVII was Sci. No. 8, 1725-1728. dissolved in 8 ml of methanol, treated with

514

PEPTIDES OF

3-

AND 4-HYDROXYPROLINE

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Address : Dr. Ehjah Adams Department of Biological Chemistry University of Maryland School of Medicine Baltimore, Maryland 21201 USA.

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