W. FREDERICK HUBER,RUTHDAVISAND E. S. LUTTON
604
VOl. 59
THE POLYMORPHISM OF MONO- U-AMINOACYL TRIGLYCERIDES BY W. FREDERICK HUBER,RUTHDAVISAND E. S. LUTTOX Contribution f r o m the Research and Development Department oj The Procter & Gamble Company, Cincinnati, Ohio Received J a n u a r y 16, 1966
The polymorphism of a series of symmetrical 2-a-aminoacyl triglycerides and their acetate salts and of two X-acylated 2-a-aminoncyl glycerides is reported. The substitution of an amino group for a hydrogen atom on the a-carbon of the 2-acyl chain causes little change in the type of phase behavior of n triglyceride. The 2-a-aminoacyl triglycerides exhibit m.p. levels and long spacings similar to those of the corresponding 2-acyl triglycerides where the a-aminoacyl and 2-acyl groups contain the same number of carbon atoms. The acetates and N-acylated 2-a-atninoacvl glycerides have no counterpart in conventional glyceride molecules for direct comparisons, but sho\v polytnoi.phic behavior reminiscent of more familiar glycerides.
Previous papers from this Laboratory have reported the polymorphic behavior of various fatty acid glycerides. These studies have been estended to the mono-a-aminoacyl triglycerides, All the compounds discussed here are symmetrical diacid triglycerides of the XYX type in which Y is an a-aminoacyl and X is a saturated Cis, C16 or CISacyl group. The compounds for whirh Xray diffraction and melting characteristics are discussed are 2-alany1-ll3-dilaurin (LAL) , 2-alanyl1,3-dipalmitin (PAP), itc; acetate (PAPAc), 2alanyl-1,3-distearin (SAS), its acet8ate (SA4SAc), 2-a-aminobutyryl-l,3-distearin (SBS), 2-valyl-ll3distearin (SVS), its acetat,e (SVS=lc), 2-a-aminolauroyl - 1,3 distearin (SLNS), its acetate (SLNSAc), and also 2-N-acet..ylalanyl-113-dipalmitin and 2-N-palmitoylal,zuy1-1,3-dipalmi tin. The free-base glycerides exhibit alpha and either a beta or beta prime form, with one compound, SAS, showing sub-alpha in addition to alpha and beta forms. The acetate salts show typical alpha forms with SAS.4c1like its free base, also exhibiting a subalpha state. The higher melting forms of PAPAc and SASAc are beta-prime while those of SVSAc and SLNSAC, corresponding to no classical glyceride types, are designated as Forms I and 11. 2-N-Acetylalanyl-l13-dipalmitin and 2-N-palmitoylalanyl-l,3-dipalmitin both show alpha forms and two higher-melting forms. The latter do not fit classical types closely except for an intermediate beta-3 form for the first compound,
-
Experimental Purification of the Compounds.-The preparation of the mono-a-aminoacyl and acylnted mono-a-aminoacyl glycerides has been reported.] All compounds were recrystallized just prior to study. The amino acid glyceride acetates were recrystallized by addition of 10 to 20 volumes of petroleum ether to a Bolution of the coin ound in 2 t o 5 volumes of chloroform containing :bout 1 k of acetic acid. The free bnses were prepared as required by washing the acetates in chloroform solution with water at' 0'; this was followed by recrystallization fi,om 10 to 20 volumes of ether or petroleum ether a t 0" to -20". All compounds were 05 to 100% pure as indicated by pet,chloric acid titration of the amino group. Both 2-N-acetylalanyl-l,3-dipalmitin and 2-N-palmitoylalanyl-l,3-dipalmit~inwere recrystallized from petroleum ether. Kjeldahl nitrogen analysis indicat,ed the compounds to be 95 and 97% pure, respectively. Titration with perchloric acid indicated both to be 97% pure, based on the presence of no more than 3y0 unacylated amino groups. Thermal and X-Ray Diffraction Technique.-Established techniquesz were used for obtaining m.p. and diffraction (1) W. B. Huher. J. A m . Chem. Soc., 7 7 , 112 (lQ.55). (2) E. S. Lutton. F. L. Jackson and 0. T. Quimby, ibid., 70, 2441 (1948).
data. "Rapid complete m.p.'s" and "regular complete m.p.'s" were determined for each compound, the former for metastable phases, especially alpha, the latter usually for the stable form only. Compounds in melt,-filled glass capillaries tended to decompose on repeated thermal treatment; this caused a progressive clouding which greatly hampered observation of melting behavior. By filling the capillaries with powdered sample and sealing before heating, the useFul life of the capillaries was prolonged nnd more precise data could be obtained. All m.p. determinations reported here are on such powder-filled capillaries with the exception of tliose on 2-alanyldilaurin. Capillaries for the latter compound, due to its low m.p., were liquid-filled and then sealed. The possible existence OF intermediate melt,ing forms was explored as follow. Melted samples in capillaries wcre held a t 1 f 0.2' below the alpha m.p. for 10 minutw during which time they solidified and then a t 1 & 0.2" nbove the alpha m.p. where part,ial melting and resolidification occurred. The process took 10 to 20 minutes for the acetates and 2-N-acetylalanyl- and 2-N-palmitoylalanyl-1 ,3-tlipalmitin. The free bases required a period of several houra. Actually only one form other than alpha (or sub-alpha) was obtained from melt for any of these compounds with the exception of SLxSAc which showed two. Flat film X-ray diffraction patterns were mnde on all polymorphic forms with a General Electric XRD-I unit employing Cu K a nickel-filtered radiation and a 0.025 in. pinhole system. Sample-to-film distance I V ~ Sueually 10 em., with a few patterns being taken at 5 em. Lowmelting phases were kept below their m.p.s. in a small coldblock during exposure. Thermal and X-ray diffraction data are listed i n t,he Following order: compo,und, polymorphic form: m.p. C.; long spacing, L.S. (in A.); short spacings, S.S. (in A,). Utiless indicated otherwise, ni.p.'s of metastable forms are mpid c.m.p.s.; m.p.>. of stable forms nre c.m.p.s. Relative intensities of diffraction lines are indicated by ( V S ) = very strong, (S) = strong, ( A I ) = medium and ($1') = WPR . . ...k ... Mono-a-aminoacyl Glycerides: LAL. a-1 : 10" (approx.)3; L.S. 22.3; S.S. 4.10 (PI).p-3 (like): 26.504; L.S. 39.1; S.S. 4.70 ( M ) , 3.87 ( M ) . PAP. a - I : 29.9 rt 0.2'; L.S. 25.6; S.S. 4.09 (\I). p-3: 48.2"; L.S. 43.8; S.S. 5.38 is-). 5.04 IM-). 4.57 (1%). 4.01 (S).3.83 (S),3.70 (M-), 3.5i (M), 3.38 pi:). SAS. '~UIM-I: no m.p.6; L.S. 20.2; S.S.4.15 (S), 3.75 (W). a - I : 40.9 i 0.1'; L.S. 20.5; S.S.4.13 (VS). p-3: 56.6"; L.S. 48.1; S.S.5.31 (M-), 4.56 (S), 3.98 (RZ), 3.81 (14). SBS. a-1: 39.6 f 0.3'; L.S. 30.5; S.S. 4.14 ( 8 ) . p'-3: 56.3"; L.S. 55.2; S.S. 4.G9 (lV-), 4.20 (VS), 3.78 (S). SVS. a-1: 37.7 3~ 0.2"; L.S. 30.G; 8.8. 4.13 (SI+). p'-3: 56.5"; L.S. 57.i; s.S.5.1:~(n4-),4.74(~1),4.21(vS),3.8o(S). SL~S. a-2: 40.3 =I=O S o 6 ; L.S. 47.5; S.S. 4.08 (S). 8'4 (like): GG.8"; L.S. 66.4; S.S.5.16 (M-), 4.63 (S), 4.10 (VS),3.91 ,
~
I .
~
I ,
(SI.
Mono-a-aminoacyl Glyceride Acetates: PAPAc. a- 1: 32.2 f 0.4'; L.S. 31.5; S.S.4.10 (M). 6'-3 (like): 85.1'; L.S. 51.4; S.S.5.11 (W), 4.30 ( M - ) , 4.17 (VS), 4.07 (WI), (3) Starts to melt a n d rwlouds. (4) Obtained from melt only. ( 5 ) The sub-a-1 form, present a t O o , transfornis reversibly to tlie a-1 form below room temperature. (6) Softening point.
July, 1956
POLYMORPHISM O F
MONO-a-AMINOACYL TRIGLYCERIDES
3.80 ( M - ) , 3.60 (11). SASAc. sub-a-1: 110 m.p.7; L.S. 34.2. S.S. 4.16 (S), 3.79 ( M ) . a-1: 41.8 It 0.3'. L.S. 22.3; 8.8. 4.08 f S ) . 13'-3 (like): 86.7': L.S.55.2': S.S. ~:CSi'(M+),-4.60'(i~r),'4.30'(~j, 4.15 (SI, 4.03 3.76 (W+), 3.62 ( W + ) . SVSAC.a-1: 41.7 =t 0.2"; L.S. 32.5; S.S. 4.10(5). FormI-38: 75.2 =t 0.3"; L.S. 54.4; S.S. 4.73 (S), 4.25 ( W ) , 3.81 (S). Form 11-3: 79.6"; L.S. 55.2; S.S 4.88 (S), 4;17 (S), 3.85 (S), 3.77 (S-). SLNSAC.a-4: 57.4 & 0.2 : L.S. 93.1: S.S. 4.08 (VS). Form 1-2 (4P: 74.5 f 0.5';' L.S. 44.4 (88.8); S.S. 4.65 (14). Form 11-2 (4): 75.9". L.S.44.3 (88.6); S.S. 5.93 (W), 4.90 (AI-), 4.45 ( M + j , 4.19 (M-), 3.96(VS), 3.56 ( M - ) . Acylated Aminoacyl G1yce:ides. 2-N-Acetylalanyl-l,3dipa1mitin.-a-1: 32.G f 0.2 ; L.S. 28.1; S.S. 4.07 (S). p-3 (like)8: 62.8 i: 0.3"; L.S. 53.2; S.S. 5.06 ( M ) , 4.55 (S), 4.16 (W), 3.77 (VS), 3.49 (W).Foiin 1-3: G7.7'; L.S.56.2; S.S. 4.73 (S), 4.17 (VS), 3.95 (AI), 3.61 (RI-2. 2-N-Palmitoylalanyl-1,3-dipalmitin.a-3: 52.8 =t 0.3 ; T, S 74 1 . 8_ 8 .4.14 _ _ _ fVS). Form 1 - 3 : 72.0 f 0.5": L.S. 67.1: S.S.5.05 (iI),lh4.15 (T'S), 3.78 (RI). Form 11-2 (4): 75.2"; L.S. 44.3 (88.6); S.S.4.63 ( h I - ) , 4.15 (S), 3.92 ( A l ) , 3.71 (RI), 3.5G (\V).
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Discussion D,L-a-Aminoacyl Glycerides.-The lowest inelting forms of all the mono-a-aminoacyl glycerides studied are a-1, Le., single-chain-length a-forms with the exception of SLNS which is a-2. In addition, SAS shows a sub-a-form. Previous experience" has shown that symmetrical shortchain 2-acyl 1,3-diglycerides (an XYX type) such as 2-acetyl-, 2-propionyl-, 2-butyryl- and 2-caproyl1,3-distearin, also exist. in a-1 and possibly also sub-a-1 forms. Since the long spacings of the aforms of the 2-a-aminoacyl glycerides are essentially the same as those of the analogous 2-acyl glycerides, the long chains of the former like those of the latter must be perpendicular to the planes of the end groups. The amino group has little or no effect on the longitudinal dimension of the unit cell. This is illustrated in Fig. 1 which shows a plot of long spacings, with varying chain length of the 2-acyl and 2-a-aminoacyl groups in a series of 1,3-distearoyl triglycerides. Xote that the a-long spacings of both series follow practically the same path. The correspondence between the a-form m.p.'s of these 2-a-aminoacyl glycerides and those of t,he conventional X Y X type triglycerides is not as apparent; however, the general trend of the m.p. curves for the tu.0 series is the same. This is shown in Fig. 2, the nmiiioacyl glycerides usually melting 5 to 10" higher. Comparisons of the long spacings and m.p.'s of the stable forms of the 2-a-aminoacyl-l,3-distearins and the analogous 2-acyl glycerides are also show11 in Figs. 1 and 2, respectively. LAL, PAP and StlS all of which contain a 2-alanyl group ha1.e /3-3 (or p-like) stsable forms. SBS, (7) The sub-a-1 f o r m p t ' e w n t a t U o , traiiafot.inn reversibly t u the a-1 f o r m below 20".
(8) Solvent cryfitallizcd. (9) Obtained pure from inelt. T h e rod pellet prepared froin solvent crystals appears t o be i~ mixture of forms I and 11, with the former predominating. (10) Diffuse diffrac1,ion line. (11) (a) F. L. Jackson, R . L. \Ville and E. S. Lutton, J. Am. Chem. Soc., 73, 4280 (1981). (b) Heretofore unreported in.p.'s and long spacings of several XYX glynerjdrs a r e : tlie lowest melting form of 2-propionyldilaurin 9.8', 19.2 A . (a-1); 2-propionyldiyalinitin 24.4", 25.7 A. (a-1); 2-proi)ionyldistearin 3G.9', 27.6 A. ( a - l ) ,28.6 A. (sub a-1) and for t h e solvent-crystallized forms of 2-proi,ion),ldilaurbn 24.1°, 37.1 .i. (Form 1-3); 2-iiropionyldipalmitin 46.8', 44.0 A . ( p - 3 ) ; 2-propionyldistearin 5 5 . 6 " , 48.4 1.(P-3).
?.-Acyl
_--
a-
/---
,_
( S t a b l e Forms)
I,3-didcarlns
i D . L - ~ -W A m i n o a a y l
-
GO5
1,3-distearins
,is)
~ , ~ - Z - V u l y 1.3-distearin i
IN-1 Forms)
30 20
! - U L L i i
G 8 10 S o . of carbon atoms in tlie 2-acyl group. 2
4
12
Fig. 1.-Comparison of long spacings of symmetrical distearoj.1 triglycerides: a and stable forms. (The data for 2-lnuro~~ldistearin are those of T. Malkin and M. L. hlearn, J. Chem. Xoc., 1141 (1939).)
: c
2-Acyl 1,3-dislsorins 0
~ , ~ ~ 2 - 6 - A m i n o o c I,l-disteorino yl 1,3-dirtearin
a o.L-2-Volyi
i
l
1
-
L
l
l
-L ---L
2 4 ti 8 10 I2 S o . of carbon atonis i l l the 2-acyl group. Fig. 2.-Coinpnrison of melting points of s j m m e t r i c d distearo),l triglJ,cerides: O( 2nd stable fornie.
SVS and SLNS which have, in the %position, aaminoacyl groups containing 4 or more carbon atoms, exist in p'-3 (or p'-like) stable forms. The value for the p-stable form of SAS falls on the curve for the p-forms of the 2-acyl-1,3-distenrins. Stable forms of the remaining a-aminoacyldistearins, being p-prime, have values which follow a parallel but slightly higher curve. M.p. agreement is good, differences between corresponding members being only 1 t o 3". Figure 3 shows the long spacings and Fig. 4 the m.p.'s for the a- and stable forms of the 2alanyl and corresponding 2-propionyl symmetrical triglycerides in which the fatty acyl group varies from 12 to 18 carbons. It is readily apparent that introduction of the amino group into the 2-acyl chain causes lit,tle change in m.p. or type of phase behavior. The a-forms of the "free bases" transform rather slowly to the stable fornis. In the case of the somewhat similarly behaving palmitoyl- and stearoyl-diacetins, mixtures have shown ext'reme alpha stability, but a 50 : 50 mixture of tmhepresent PAP and SAS was little if any more a-stable than the individual components and after 24 hours was completely transformed to beta. These results shorn that the substitution of an amino group on the a-carbon of one acyl group of a (12) B. J. Baur, J. A m . Oil Chem. Soc., 31, 19G (1954).
\V.FREDERICK HUBER.RI-TH DAVISAND E. 8. LUTTON
RO(i 55
T'ol. 59
1 0
0 -
2-Propionyl 1,3-diglycerides
0 o,~-2-Alanyl (3-diglycerides
( Siabie Forms
5
45
4 0
2 - P r o p i o n y l (3-diglycerides
0 ~ , ~ - 2 - A l a n y l1,3-diglycerides
25
20
$ 4
4 23
1
2
C '
1
I
I I I 14 I ti 1% S o . of carhoii atoms in the 1,3-di:tcyl group. Fig. :3.--CompnYison of long sptcings of xj~mmet,i,icnit 1.iglycerides.
12 14 10 18 S o . of cathoii :itoms in the lJ-di:tc,j.l gror~l)s Fig. 4.-Co1nparison of melt,ing points of symiiietrirnl t rigl>,ceiides.
t,riglyceride has little effect oil melting properties nnd t8hemajor feat'ures of crystal structure. D. L-a-Aminoacyl Glyceride Acetates.-The 2-alanyl glyreridc acet'iit.es, PAPAc and SASAc, she\\a-1 and ,3-prime-3-likest8iwtui*es. In adtlil ion, like its free l)tlic. SASAc exIiihit#sa. sill)-a-1 form. It, appeal-s, surprisingly, t,hat8its the 2-0-aminonc3'1 group lengthelis. t'he similarity of t,he diffrac3tioii pat,t,eriis to those of conrwitmioiial t>riglyceride formh diminishes. This is shown for SJ'S.4c aiicl f i L ~ s . 1n-hich ~ have a-1 nucl (apparently) a-4 forms, respecti\sely. They also sho\v iiitermet1i:tte (from solveiit8) aiid sfnhle forms, the diffrnc.tion patt.erns of u-hich d o not resemble classical hetla or 6-prime forms. SJ'SIc has t,riple-clia,iii-Ieiigtli intei-mediate (from sol\,eiit) and stable fovms which are designatled as Form 1-3 and Foim 11-3. Of these compounds only 8 L ~ S ; l cslio\\-s t\\m liighei, melting forms from melt, ivhich, being either double-or quadi*uple-chain-length,have 1)ecn tlesignated as Form 1-2 and Form 11-2 or 4. Form I, held near it's m.p. until partial melting and resolidificatioii has occurred, yields Form 11.
Acylated Aminoacyl Glycerides.-The acylizt e t l 2-al:inyldipalniitiii~, P-N-acetylalanyl-l,3-dipalmitin and 2-N-palniitoylalanyl-l,3-dipalmitin,e.wh sho\v t1ii.t.e polymorpliic forms. 2-S-.\cetylalanyl-l,3-dipalmitin has R typical a-1 form, m.p. 32.0", an intermediate 6-3-like structure (from solvelit), m.p. G2.8",and a highestmeltiiig triple-chaiii-laiigth form designated as Form 1-3, m.p. 67.7". The diffraction data on the intermediate 6-3-like structure \\-ere obtained on a rod pellet of the crystals from solvent,. *I11 attempt,s to prepare t8his form from melt ga7.e pi,oducts melt'iiig het'ween G3 and 67.5", and the X-ray patterns showed eiridence of part,ial traiisformation of this intermediate form t,o Form 1-3 or t'ransformnt'ion accompanied by decomposition. 2-N-Palmitoylal~nyl-l,3-dipalmit~iii has a typical a-3 form, m.p. 52.8", an intermediate Form 1-3 (from solvent), m.p. 72.0", and a highest melting form designated Form 11-2 (or 4), m.p. 75.2". Acknowledgment.-The authors n.isli to express their apprecist,ion to R. H. Lind:Lhl for making the X-ray diffraction patterns used in this paper.
I
12
