ISRAEL JOURNAL OF CHEMISTRY. Vol. 10, 1972, pp. 867-879

POLYDEPSJPEPTIDES I. SYNTHESIS AND CHARACTERIZATION OF COPOLYMERS OF a-AMINO AND a-HYDROXY ACIDS

Murray Goodman and Chaim Gilon* Department of Chemistry.

University of California, San Diego, La Jolla, Calgornia

Gerald S . Kirshenbaum** Polytechnic Institute of Brooklyn,

Brooklyn, N. Y. and

Yehuda Knobler Department of Orgunic Chemistry, The Hebrew University of Jerusalem

ABSTRACT R O R O

Copolymersofthe type [-m-z;c-) I D ;I. - [(-o-f;t )Y]

m were synthesized and characterized. The syntheses of these copolymers were achieved by copolymerization of an u-amino acid N-carboxy anhydride and an uhydroxy acid anhydrosulfite in the presence of a catalyst. The copolymers were characterized by spectroscopic methods including infrared, circular dichroism and nuclear magnetic re- sonance. Specific cleavage of the ester bonds by hydroxylamine in the cold confumed the A-B block structure where the A represents the poly(amino acid) segment and the B indicates the polyester segment. Measurements by nmr and viscosity indicate that the blocks consist of short chain lengths.

Poly-ar-amino acids and polypeptides with regularly repeating sequences of amino acid residues have been employed in recent years as interesting model compounds to investi- gate structural features present in proteins [ 141. In addition, they were also used to study the effect of specific amino acid residues on the stability of conformations adopt- ed by many proteins and synthetic polypeptides [2,4,5].

Depsipep tides have attracted considerable attention because of the unusual biological activity exhibited by various naturally occurring cyclic materials. The discovery that several naturally occurring cyclodepsipeptides may be efficient selective ion transporters

* Postdoctoral Fellow, 1971 - present, at the University of California, San Diego. On leave from the Hebrew University of Jerusalem.

** Submitted in partial fulfillment of the requirements for the degree of Doctor of Philosophy at the Polytechnic Institute of Brooklyn.

Received July 14,1972

f l

867

868 M. COODMAN ET AL. .Is. J. Chem.,

of alkali metal cations in biological systems [ 6 ] and that the antimicrobial activity ex- hibited by these depsipeptides is related to their complexing ability [7] led to an exten- sive effort in synthesis and conformational analysis of these cyclodepsipeptides and many of their synthetic analogs [8]. The synthesis of linear alternating polydepsipeptides has not been reported. However, the preparation of cyclodepsipeptides and their linear analogs has been extensively investigated by Shemyakin, Ovchinnikov and their asso- ciates [6,9-111. Stewart [12-141 succeeded in forming several sequential polydesi- peptides using active ester techniques.

This series of investigations involves the preparation, characterization and conforma- tional analysis of sequential polydepsipeptides in which ester bonds, derived from a or bhydroxy residues, [ 151 are incorporated into an otherwise peptidic backbone. Elimi- nation of part of the hydrogen bonding capacity of the chain can have a profound ef- fect on the conformational arrangements and stabilities of these copolymer chains.

RESULTS AND DISCUSSION

Synthesis of copolymers* A series of copolymers of the form poly(L-alaninex)poly(lactic acid’) (a) as well as

copolymers of the form poly(L-alaninex)poly(a-hydroxyisobutyric acidY) (b) and poly (a-aminoisobutyric acidx)poly(lactic acidY) (c) were synthesized by refluxing the appro- priate a-amino acid NCA(1) and a-hydroxy acid anhydrosulfite (11) in benzene for several days with a catalyst, triethylamine. The resulting copolymers were purified in order to re- move any homopolymer that might have been formed during the polymerization.

R R’ I I I

I C = 0 + C H 3 4 -C = 0 ----+ C H 3 4 -

I 1 m 0 N H O

‘C’ II II 0 0 I I1

a R = R’ = H, poly(L-Ala’) - poly(Lac y,

b R = H R‘ = CH3, poly(L-AlaX)-poly(a-HIBAY) c R = CH3 R’ = H, poly(a-AIBAX)poly(LacY)

A series of three copolymers of the type (a) were synthesized, (L-Ala?’ )n- (Lac”), , (L-Alas0)n(Lac50), and (L-Alas0)n-(Lac20),,, . The first two polymers were not syn- thesized independently but were obtained by a polymerization carried out at an anhydride-

* Copolymers were named according to Abbreviated nomenclature of synthetic polypeptides, Bb- polymers, 11: 321 (1950).

Vol. 10, 1972 POLY DEPSIPEPTIDES. I 869

to-initiator ratio of 500. The two polymers were separated on the basis of their difference in solubility in chloroform. The third copolymer was prepared independently from a polymerization reaction in which the anhydride-to-initiator ratio was 50. Since an increase in the catalyst concentration increases the rate of the NCA polymerization, the third co- polymer contained a higher alanine content than the first two copolymers.

The copolymer (L-Ala75),-(a-HIBA2S)m was synthesized by copolymerization of Lalanine NCA and a-hydroxyisobutryic acid anhydrosulfite (a-HIBA) in the presence of triethylamine (A/I = 50). Because of the steric effect of the extra methyl group in the 4 position, a higher alanine content was found in this copolymer than in the lactic acid co- polymers described above.

zation of a-aminoisobutyric acid NCA (a-AIBA) and S-lactic acid anhydrosulfite in the presence of triethylanine (A/I = 50). The composition of this copolymer is reasonable since a-aminoisobutyric acid NCA polymerizes more slowly than alanine NCA.

Several attempts were made to copolymerize the two optically inactive monomers, a-aminoisobutyric acid NCA and a-hydroxyisobutyric acid anhydrosulfite. When the poly- merization was carried out thermally, without the presence of a catalyst, after 1 week only the a-hydroxyisobutyric acid homopolymer could be isolated. This indicated that the NCA is too stable to polymerize at these temperatures without a catalyst. When the polymerization was repeated with a catalyst (triethylamine or sodium methoxide at an anhydride-to-initiator ratio of 50) a polymer formed within 3 days. This turned out to be the homopolymer of the a-aminoisobutyric acid. It appears that the catalyst increased the rate of the polymerization of the NCA but had no noticeable effect on the polymeri- zation of the anhydrosulfite.

A copolymer of the structure (a-AIBA25),-(Lac75)m was synthesized by copolymeri-

Infrared studies The (L-Ala" ),-(Lac7' )m and (L-Ala50),-(LacSo)m copolymers exhibit a sharp ester

peak at about 1450 cm-' while (L-Ala20),-(Lac20)m exhibits a weak one. All three co- polymers show a broad amide I1 peak at about 1535-1525 cm-'. The copolymer (L-Ala2'), -(Lac7') shows a split amide I absorption with peaks at 1650 and 1625 cm-' while (LAlaSo~-(LacSo),,, and (L-Alas0),-(LacZo), exhibit a strong band at 1650 cm-' and a definite shoulder at 1625 cm-'. The splitting of the amide I band into the 1650 cm-' and 1625 cm-' bands is typical of polypeptides which exhibit a &structure [2,16,17]. The application of the split amide I band to the conformation of these copolymers will be discussed elsewhere.

The copolymer (L-Ala75),-(a-HIBA25)m exhibits a weak ester peak at 1750 cm-' , an amide I1 peak at 1525 cm-' and a strong amide I peak at 1650 cm-' with a shoulder at 1625 cm-' .

a weak amide I1 peak at 1525 cm-' , and a medium amide I peak at 1655 cm-' . The amide I peak was not split.

The copolymer (a-AIBA25),-(Lac75)m shows a very strong ester peak at 1755 cm-' ,

CD studies Figure 1 shows the circular dichroism spectra of the copolymers (L-AlaX),-(LacY),.

870

34

M

26

22

I8

14

-s 10

B s p 0

0 % 6 . 0 . 2

- Q, -2 u

-6

-10

- I4

-18

-22

-26

M. COODMAN ET AL. Isr. J. Chem.,

185 190 m 210 220 230 240 250 260

X,nn

FIG. I. CD spectra of copolymers (cAlaX),-(Lacy), in HFIP. a) PolyS-lactic acid b) Copolymer (GAlaa'),-(LacTs )m c) Copolymer &-Alas O),-(Lac'O), d) Copolymer (GAla'O),-(Lac'O)m e) Poly-LAla from hydroxylaminolysis of copolymer

(L-Ala' o),-(Laca "), f) Poly-L-Ala (DP = 37) All the spectra were recorded in HFIP with a concen- tration of 1 mg/ml. A cell whose path length was 0.1 mm was used. The mean residue molecular weight was used in calculating the molar ellipticities.

It also shows the spectra of poly-S-lactic acid, poly-Lalanine (% = 37) and poly-L- alanine from the hydroxylaminolysis of (L-Ala80)n-(LacZo),,, . All spectra were measured in hexafluoroisopropanol (HFIPL

We can see that poly-LAla (DP= 37) ( f ) exhibit a typical a-helical spectrum [ 18, 191

Vol. 10. 1972 POLYDEPSIPEPTIDES. I 871

with a negative n+n* band at about 21 1 nm ([dl tion. This n+r* transition consists of a negative band at about 202 nm ([el and a positive band at 187 nm ([el broad positive band from 185 to 235 nm with a maximum of [ e ] ~ = 5.9 x lo3 at about 207 nm. The spectra of the copolymers appear to be a combination of the spectral features of both homopolymers. The (L-Alas'), - (Lac"), (c) and (L-Ala*'),-(Lac2'), (d) copolymers exhibit typical a-helical spectra with a trough at 213 nm which arises from the lactic segments. The (LAla2S),-(Lac7s)m (b) exhibits a spectrum which appears to be a direct combination of the two homopoly- mers spectra: a weak positive peak at about 210 nm, a trough at 202 nm, and a positive peak at 188 nm. The CD spectra are consistent with a block-like structure of the alanine and lactic acid segments.

The CD spectrum of the (L-Ala7'),-(a-H1BA2' ),,, copolymer in H F P was a typical poly-L-alanine a-helical spectrum with a shoulder at 217 nm ([dl tive peak at 202 nm ([el This spectrum is slightly less than half the intensity of the poly-Lalanine (Fig. 1). Since the a-hydroxyisobutyric acid is optically inactive, we could expect a decrease in the absorption because of a dilution effect. The CD spectrum of (a-AIBA2S),-(Lac7s ),,, copolymer in TFE exhibits a typical poly-S-lactic acid spectrum: a broad absorption band between185 and 230 nm with a maximum at 210 nm of [el = 2 x lo3. Since the a-aminoisobutyric acid residues are optically inactive, we can expect a decrease in absorp tion because of a dilution effect. These findings also support block structure for the above-mentioned copolymers.

= -15 x I d ) and a split m* transi- = -25 x lo3

= 34 x lo3). Poly-S-lactic acid (a) exhibits a

= 4600), a nega- = -%00) and a positive peak at 189 nm ([el, = +7500).

NMR studies The chemical shifts of the various protons of all the copolymers (LAlaX),-(Lacy),

were essentially the same. The 220 MHz NMR data for copolymer (LAlaS0),-(Lacs0),,, are listed in Table I, for a 3% polymer concentration in TFA, and the spectrum is shown in Fig. 2a.

TABLE I. 220 MHz NMR data for (L-Alaso)n-(LacsO)m in TFA

chromophore ppm from TMS peak shape

NH 1.14 broad singlet -NH,' 1.3 broad singlet a C H , lactic 5.36 sharp quartet a C H , alanine 4.14 broad singlet -CH, , lactic 1.7 sharp doublet -CH, , alanine 1.55 broad singlet

The copolymer (Alaa0),-(Lac2'),,, in TFA has a similar spectrum except for the broadness of the peaks assigned to the lactic acid residue (at 5.36 and 1.7) ppm (Fig. 2b). The sharpness of the lactic acid peaks in copolymer (LAla5'),-(LacS0), tends to indicate that the lactic acid segment in this polymer is short.

872 M. GOODMAN ET AL. Isr. J. Chem.,

b

I I I 15 50 2 5

PPU

FIG. 2.220 MHZ NMR spectra of copolymers (L-Ala'),-

a) Copolymer (L-Alas O),-(LacSo Irn b) Copolymer (L-Ala80)n- (Lac"), c) Poly-L-Ala from hydroxylaminolysis of copolymer (L-A~~*O), , - (L~C'~ 1, AU the spectra were recorded in 100% TFA with a concen- tration of 3% TMS was used as the internal standard.

~ (Lac'), in TFA

The 60 MHz NMR spectrum of copolymer (Ala7'),-(a-HIBA2' ),,, contains a broad peak centered at 7.8 ppm, a broad aCH peak centered at 4.75 ppm and overlapping CH3 peaks between 1.8 and 1.5 ppm.

The 220 MHz NMR spectrum of the copolymer (a-AIBA25)n-(Lac75), shows a broad NH peak at 7.8 ppm, a broad aCH peak at 5.4 ppm and overlapping CH3 peaks between 1.8 and 1.5. Measurements of the ratio of the areas of the ester segment protons and the peptidic segment protons were used to calculate the copolymer composition. These calculations were in agreement with the findings of the hydroxylaminolysis.

Vol. 10, 1972 POLYDEPSIPEPTIDES. I 873

Detailed NMR studies as well as conformational analysis of these copolymers will be discussed elsewhere.

Hydroxylaminolysis A series of hydroxylaminolysis experiments were carried out on the copolymers which

confirm the block structure of these copolymers. Since the results of the hydroxylaminoly sis experiments were essentially the same, only the hydroxylaminolysis of the copolymer (L-Ala80)n-(Lac20),,, will be discussed.

In these experiments, the copolymer was treated with hydroxylamine in basic media in the cold. The undissolved residue did not show the 1750 cm-' ester band in the IR spectrum. The NMR spectra in TFA is shown in Fig. 2c. It did not show the a-CH and the -CH3 peaks of the lactic acid residues at 5.36 ppm and 1.7 ppm, respectively. This con- firms the A-B block structure of the copolymer where A represents the poly(a-amino acid) segment and B, the polyester segment.

Measurements of the ratio of.the areas of the peaks located at 7.3 ppm (-NH3+) and at 7.74 ppm and 7.5 ppm (peptide NH) yielded a number-average molecular weight of about 1600 (%= 22) [20] for the polyalanine remaining after hydroxylaminolysis. The CD spectra in HFIP are shown in Fig. lc. 'The intensity of the 202 and the 217 nm peaks is lower than that of poly-Lalanine (s = 37). This supports the shortness of the poly-Lalanine segment.

acid ferric complex corresponding to the lactic acid residue content of the copolymer. This absorbance was compared with the absorbance of the lactylhydroxamic acid ferric complexes derived from hydroxylaminolysis of poly-S-lactic acid and DL-lactic acid methyl ester under the same conditions. The (L-Ala)/(Lac) ratio calculated by this method was in agreement with the ratios calculated from NMR.

To ascertain that no peptide bonds undergo hydroxylaminolysis under these condi- tions, poly-Galanine was subjected to hydroxylamine under the above-mentioned condi- tions. No absorbance was observed at the visible range.

The remaining solution exhibits a typical absorbance at 492.5 nm of lactylhydroxamic

Conclusions The synthesis and characterization of copolymers of a-amino and a-hydroxy acids

is described. The spectroscopic data consistently support a block structure which was found by specific cleavage of the ester bond to be of an A-B type where A represents the poly(amino acid) segment and B, the polyester segment.

EXPEN MENTAL *

A) Commercial materials and solvents

Japan. Poly-L-alanine (DP = 37) was obtained from the Pilot Chemical Company (Lot

* Melting points are uncorrected. Analyses were carried out by the Calbraith Laboratories, Knox- ville. The results of these analyses are discussed in Section E.

Optically active L-alanJe was purchased from Ajinomoto Company, Inc., of Tokyo,

874 M. COODMAN ET AL. Is. J . Gem.,

No. AL52). S-Lactic acid was acquired from the Mann Research Laboratories, while a-hydroxyisobutyric acid was supplied by the Aldrich Chemical Company. a-Aminoiso- butyric acid was bought from Fisher Scientific Company. Triethylamine (from Metro Scientific) was refluxed with phenylisocyanate for 1 hr and then freshly distilled prior to use. Deuterochloroform was purchased from the Diaprep Corporation. Hexafluoro- isopropanol was supplied by Columbia Organic Chemicals. Trifluoroacetic acid, tetra- methylsilane, and trimethyl phosphate was acquired from Aldrich Chemical Company. Trifluoroethanol was bought from Halocarbon Products. Tetrahydrofurane from Metro Scientific was dried over molecular sieves while dioxane was refluxed over so- dium for l week, dried over lithium aluminum hydride for 24 hr, and then distilled prior to use. Phosgene was supplied by the Matheson Gas Company. Petroleum ether and thionyl chloride were purchased from Metro Scientific. Unless specifically stated, all of the chemicals and solvents were used without further purification.

'

B) Apparatus and measurements The circular dichroism spectra were obtained using Cary models 60 and 61 recording

spectropolarimeter with a circular dichroism accessory. An &ram Xenon lamp was used as the light source. Visible measurements were obtained using a Cary 14 spectrometer. The infrared spectra were recorded on a Perkin-Elmer model 52 1 grating infrared spectro- meter. The spectra were obtained in the solid state as potassium bromide pellets. The nuclear magnetic resonance measurements were carried out on a Varian Associates A 4 0 analytical NMR spectrometer and a Varian Associates HR-220 high resolution NMR spectrometer. Tetramethylsilane (TMS) was used as the internal standard. Concentra- tions of 10% were used for the A40 instrument, while concentrations of 3% were used with the HR-220 instrument. All of the spectra were recorded at room temperature. The melting points were obtained using capillary tubes and a Thomas-Hoover capillary melting point apparatus. The viscosities of the copolymers were determined in a thermo- stated, constant temperature water bath at 30°C using standard Cannon viscometers.

C) Reparation of the compounds 1) Synthesis of the monomers a ) L-Alanine Ncarboxyanhydtide (4-methyl-L-2,5-oxazolidinedione) [2 1,221 . G

Alanine (7.5 g, 0.084 mole) was suspended in 300 ml of dry tetrahydrofuran. The system was purged with nitrogen for 1 hr. The suspension was then treated with phosgene at 49.5-50' for 3 hr until the L-alanine had completely dissolved. The system was then purged with nitrogen for 2 hr. The solvent and remaining gases were removed under reduced pressure, and a white material was obtained which was washed with hexane. This material was recrystallized several times from ethyl acetate-hexane in a dry box. This reaction yielded 7.0 g (73%) of the Ncarboxyanhydride, mp 90-91' [lit. [21,22] mp 90'1.

b ) eAminoisobutyric acid N-carboxyanhydride (4,4-dimethyl-2,5 oxazolidinedione). This compound was prepared according to the procedure in Sorenson and Campbell [23] @. 355) on a 0.15 mole scale. This reaction furnished the desired product in 709% yield with a mp 95%" [lit. [23] mp 95-97'].

VoL 10,1972 POLYDEPSIPEPTIDES. I 875

c ) eHydroxyisobutyric acid mhydrosulfite [23-251. This compound was prepared according to the procedure in Sorenson and Campbell [23] (p. 359) on a 0.20 mole scale. This reaction furnished the desired product in 40% yield with a bp 55' at 10 mm Hg [lit. [23] bp et-55' at 16 mm Hg] . The compound was prepolymerized prior to use.

d ) S-Lactic acid unhydrosulfite. This compound was prepared using the procedure in Sorenson and Campbell [23] for the a-hydroxyisobutyric acid anhydrosulfite. Instead of purifying this monomer by prepolymerization, it was distilled three times under re- duced pressure. The reaction furnished the desired product in 40% yield with a bp 7 1-72' at 16 mm Hg [lit. [24] bp 72-74' at 19 mm Hg] .

2) Synthesis of the copolymers a) General procedure. Most of the copolymers were prepared according to a procedure

similar to that outlined in Sorenson and Campbell [23] (p. 359) for the polymerization of a-hydroxyisobutyric acid anhydrosulfite.

A round-bottom flask was dried in the oven for 3 hr. To the flask, which was cooled under nitrogen, was added 40 ml of benzene. In order to make sure that the system was completely dry, about 20 ml of the benzene was distilled from the reaction vessel. The flask was cooled under nitrogen in an iceaalt bath until the benzene had frozen, and the reactants were then added to the flask. The reaction mixture was refluxed for several days under nitrogen to give a cloudy, colorless gel. The gel was separated by fdtration to give a solid polymer. The polymer was washed as outlined and dried in vacuo at 100°C.

b) &polymers of L-alonine and S-lactic acid. Raaction I : The general procedure (a) was followed for this reaction. LAlanine Ncarboxyanhydride (1 .O g, 0.0087 mole), S- lactic acid anhydrosulfite (1 .O ml) and dry triethylamine (0.00243 ml, 1.74 x lo-' mole, anhydride-to-initiator ratio of 500) were added to the reaction flask. This was refluxed for 4 days under nitrogen. A white precipitate was obtained which was isolated by fd- tration. This powder was washed with hot benzene to eliminate any polylactic acid homopolymer that might have been formed during the polymerization The white pow- der was dissolved in hot chloroform. However, the product only partially dissolved, and therefore a soluble fraction (Sample 1) and an insoluble fraction (Sample 2) were iso- lated by filtration. The insoluble fraction was reprecipitated from trifluoroethanol- petroleum ether, while the soluble fraction was reprecipitated from chloroform-petroleum ether and then trimethyl phosphate petroleum ether. Integration of the NMR spectra and hydroxylaminolysis showed that Sample 1 consisted of 75% lactic acid residues for 25% alanine residue, while Sample 2 contained alanine residue for each lactic acid resi- due. Both samples decomposed between 250-280". The reaction furnished about 200 mg of each sample The infrared spectra of both compounds exhibited an ester peak at 1755 cm-', an amide I1 peak at 1540 cm", and a split amide I peak at 1655 and 1625 cm-'.

the integration of the NMR spectnun);C, 50.17; H, 5.94; N, 4.88. Anal. Sample 1 : Calcd. for C 12H1 7N07 (Monomer ratio as determined from

Found: C, 45.79; H, 5.87; N, 3.92. Sample 2: Calcd. for C6H9N03 (mohomer ratio as determined from inte-

gration of the NMR spectrum): C, 50.34; H, 6.33; N, 9.78. Found: C, 47.28; H, 5.82; N, 8.26.

876 M. GOODMAN ET AL. Isr. J. Chem..

Reaction 2: This reaction was performed exactly as in Reaction 1 except in this case the anhydride-to-initiator ratio was 50. A white precipitate was isolated by fdtration. The powder was washed with the following hot solvents: benzene, THF and chloroform to eliminate any homopolymer that might have formed during the polymerization. This reaction furnished about 0.75 g of the copolymer. Integration of the NMR spectrum showed that this copolymer consists of 80% alanine residues for 20% lactic acid residue. The material decomposed between 240 and 260". The infrared spectrum exhibited an ester peak at 1745 cm-', an amide I1 peak at 1525 cm-', and an amide I peak at 1655 cm-' with a shoulder at 1626 cm-' . The intrinsic viscosity in trifluoroacetic acid at 30" was 0.1 9.

Anal. Calcd. for C1SH24N406 (monomer ratio as determined from the integration of the NMR spectrum): C, 50.56; H, 6.79; N, 15.09. Found: C,47.52;H,6.35;N, 13.77.

c ) Copolymer of L-alanine and ol-hydroxyisobutyric acid. The general procedure (a) was followed for this reaction. LAlanine Ncarboxyanhydride ( 1 .O g, 0.0087 mole), a-hydroxyisobutyric acid anhydrosulfite (1 .O ml) and dry triethylamine (0.0243 ml, 1.74 x This was refluxed for 4 days under nitrogen. A white precipitate was obtained which was isolated by filtration. The compound was washed with hot benzene and hot chloroform in order to eliminate any homopolymer that might have been formed during the reaction. The reaction furnished about 0.6 g of the copolymer. The material did not melt or de- compose below 275". Integration of the NMR spectrum showed that the copolymer con- sists of 75% of alanine and 25% a-hydroxyisobutyric acid residue. The intrinsic viscosity of the polymer in trifluoroacetic acid at 30" was 0.084. The infrared spectrum exhibited a medium ester peak at 1745 cm-' , an amide 11 peak at 1525 cm-' , and a strong amide 1 peak at 1650 cm-' with a shoulder at 1625 cm-' .

Anal. Calcd. for C13H21N305 (monomer ratio as determined from integration of

mole, anhydride-to-initiator ratio of 50) were added to the reaction flask.

the NMR spectrum): C, 52.16; H, 7.07; N, 14.04. Found: C, 49.09; H, 6.87; N, 13.44.

d ) Copolymer of S-lactic acid and a-aminoisobutyric acid. The general procedure (a) was followed for this reaction. a-Aminoisobutyric acid N-carboxyanhydride (0.2 g, 0.00155 mole), S-lactic acid anhydrosulfite (0.2 ml), and dry triethylamine (0.00432 ml, 3.1 x lo-' mole, anhydride-to-initiator ratio of 50) were added to the reaction flask. This was refluxed under nitrogen for 1 week but no precipitate had formed. A small amount of petroleum ether was added, and a white precipitate immediately formed. This material was isolated by filtration. Upon adding more petroleum ether and cooling in the refrigerator overnight, a second precipitate had formed. This second crop proved to be polylactic acid homopolymer. The copolymer was reprecipitated from benzene- petroleum ether and about 150 mg of the polymer was obtained. The copolymer a p peared to soften between 133-140" and underwent total decomposition by 230'. Inte- gration of the NMR spectrum showed that the copolymer consists of 75% lactic acid residues per 25% a-aminoisobutyric acid residue. The infrared spectrum exhibited a

Vol. 10, 1972 POLYDEPSIPEPTIDES. I 877

very strong ester peak at 1755 cm-' , and weak to medium amide peaks at 1655 and 1528 cm-'

D) Hydroxylaminolysis 1) Hyd-oxylaminolysis of poly-S-lactic acid PolyS-lactic acid (25 mg, 0.35 mmol) was mixed with hydroxylamine sulfate (lN,

1.75 ml) and sodium hydroxide ( lN, 5.25 ml) was added. The mixture was adjusted to 25 ml and stirred for 48 hr at room temperature at which time all the polymer had dis- solved. A 1 ml aliquat was taken and adjusted to 10 ml with ferric ammonium sulfate (2% solution in 1 N H2SO4 W/V). The solution showed an absorbance of 0.945 at the b,492.5 nm (ferric ammonium sulfate solution as a reference).

2) Hydroxylaminolysis of the copolymer (Ala80),,(Lac20), The copolymer (Ala80),(Laczo), (75 mg, 1.05 mmol) was treated with hydroxyl-

amine as in the above-mentioned procedure. After 48 hr, the undissolved material was collected. A 1 ml aliquot of the Mtrate was taken and adjusted to 10 ml with ferric ammonium sulfate (22% solution in 1N H2S04 W/V). The solution exhibited an absorb ance of 0.575 at the A,,,= 492.5 nm (ferric ammonium sulfate as a reference). The ratio between the alanine residues and the lactic acid residues in this copolymer as calculated from the absorbance at 492.5 nm was found to be 4: 1. This ratio is in a good agreement with the ratio calculated from the integration of the NMR spectra.

The undissolved material was washed with water, ethanol, water and acetone and dried over phosphorus pentoxide in vacuum. NMR showed a typical poly-L-alanine spectra (Fig. 2c). There were no peaks at 5.36 ppm (&H, lactic) and at 1.7 ppm (CH3, lactic). Inte- gration of the NH3+ peak at 7.3 ppm and the NH peaks at 7.5 and 7.74 ppm yielded a number average weight of 1600 (a = 22) [20]. The IR spectrum did not show the ester C = 0 peak at 1750 cm" . The CD spectra showed a typical poly-Lalanine spectra (Fig. le).

Anal. Calcd. for poly-L-alanine + 0.4 mol% H20: C, 46.04; H, 7.44; N, 17.89. Found: C, 46.64; H, 6.79; N, 17.

3) Hydroxylaminolysis of DL-methyl lactate DLmethyl lactate (36.6 mg, 0.35 mmol) was treated with hydroxylamine according

to the above-mentioned procedure. After 48 hr, a 1 ml aliquat was taken and adjusted to 10 ml with ferric ammonium sulfate (22% solution in 1N H2SO4 W/V). The solution showed an absorbance of 0.94 at the &= 492.5 nm (ferric ammonium sulfate solution as a reference).

4) ikztment of poly-L-lllanine with hydroxylamine Poly-Lalanine (25 mg, 0.35 mmol) was treated with hydroxylamine according to the

above-mentioned procedure. After 48 hr, the precipitate was filtered off and 1 ml ali- quat was adjusted to 10 ml with ferric ammonium sulfate (22% solution in 1N H2S04 W/V). The solution did not absorb at 492.5 nm.

878 M. GOODMAN ET AL. Isr. J. Chem.,

E) Elemental analysis The quantitative elemental analyses were carried out by the Galbraitb Laboratories,

Knoxville, Tennessee. In almost every case, the results obtained from Galbraith did not agree with the chemical compositions calculated from the integration of the NMR spec- tra and by the hydroxylaminolysis experiments. For many of the copolymers, the re- sults reported from Galbraith could not have corresponded to copolymers of any compo- sition of the initial monomers. There are several possible explanations for these deviations For these copolymers the elemental analyses were almost all too low for carbon, nitrogen, and hydrogen. These low analyses may be attributed to the fact that polypeptides do not vaporize completely on decomposition which leads to incomplete combustion. Reports of residual ash on combustion of higher oligomers have been cited in elemental analysis results for compounds submitted by other workers in this laboratory. In addition, the effect of the catalyst and end groups on the chemical analysis of low molecular weight polymers may also be a factor for deviations from theoretical values.

ACKNOWLEDGEMENT

We wish to thank the National Institutes of Health (Grant GM 08974 at the Polytechnic Institute of Brooklyn and Grant GM 18694' at the University of California, San Dtego) for the generous support of this research. We also wish to thank Drs. P. Temussi, R. Rupp, F. Naider and N. Ueyama for many helpful discussions and suggestions.

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