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Enamine salts of retinals with secondary amines
Vision Res.
Vol. 8, pp. 1471-1482.
ENAMINE
Pamon
Press 1968. Printed in
SALTS OF SECONDARY JEANTOTI@
GreatBritain.
RETINALS AMINES’
WITH
and BARNETTROSENBERG
Department of Biophysics,Michipn State University,East Lamin& Michigan48823 (Received 2 July 1968)
INTRODUCTION THE VISUAL pigments consist of proteins of the opsin family combined with the 11-cis isomer of vitamin A aldehyde. The aldehyde, the chromophoric moiety, exists in nature in two forms, vitamin Ar aldehyde (retinal) and vitamin A2 aldehyde (3dehydroretinal). The linkage between the chromophore and the protein moiety is not known with certainty: it has been postulated to be either a protonated or unprotonated S&X’s base. Naturally occurring visual pigments based on retinal show absorption maxima over the range 440-620 nm. The postulate of a conjugate acid form of a S&X’s base can satisfactorily account for roughly only half the required spectral shift on union of retinal (Lax =385 nm in water solution) with an opsin (PITT, 1964). Available evidence indicates that the carbon-nitrogen linkage determined for N-retinylideneopsin-a possible photodegradation product of rhodopsin, the visual pigment of the rods-is present in rhodopsin itself (PITT, 1964). Considering the chemical properties of rhodopsin and its photodegradation products, Morton and Pitt proposed that in rhodopsin there are at least three links joining retinal to the opsin (MORTONand PITT, 1957). One of these is split in the conversion of rhodopsin to prelumirhodopsin (the photochemical step); another is broken when metarhodopsin converts to N-retinylideneopsin (or metarhodopsin I converts to metarhodopsin II in the case of cattle rhodopsin); and finally the carbon-to-nitrogen link is hydrolyzed to give retinaldehyde and opsin. N-retinylideneopsin has been shown to be a Schiff’s base (PITT, COLLJNS,MORTONand STOK, 1955). Morton and Pitt suggest that the linkage broken to form this compound is a retinylidene ammonium structure (Fig. l), the R and R'possibly representing different side groups of amino acid residues of the protein moiety. Protonation of solutions of retinal with imines or secondary amines produces compounds with structures comparable to the retinylidene ammonium structure. These compounds are enamine salts. A marked bathochromic shift is evident in their visible spectra, subsequent to protonation (resulting J!_‘s ranging from 450 to 650 nm). Working predominantly with indole (the precursor of the amino acid tryptophan), the nature of the reaction and the chemical properties of the resulting system were investigated. Its stability is extremely temperature dependent; it possesses indicator-like properties; and it 1 This Research was supported by Grants from National Institutes of Health -145 and GM-1422. 2 This research Represents a Partial Fulfillment of the Requirememts for the M. Sci. Degree. 1471
1472
JEXNTcmi AND BARNES
is readily hydrolyzed. These factors and the marked relevance to the chemistry of visual pigments.
ROSENBERG
bathochromic
shift indicate
its possible
FIG. 1. Proposed structure of retinylidene ammonium salt (after MORTONand Prrr, 1957). METHODS Spectra-grade carbon tetraehlcxide was the solvent employed in all studies, Other organic solvents (e.g. ethaxml, acetone, dioxane, chloroform, methanol) proved unsuitable, as proton&d solutions of retinal alone in these solven@ showed an initial bathochromic shift in the visible spectrum. (All protunation was accomplished by bubbling anhydrous HCl through the solution.) This was not seen when carbon tetrachloride was used as the solvent thus avoiding any ambiguity of resuIts for the proton&& system. ANruns retinal was obtained from DistiIlation Products and used without further purification. An initial spectral survey of the reactian of retinal with vari01.1~ imine~ and secondary amines was undertaken, for both the unprotors#d and pmtonated spccia. All spectra were measured in 1 cm path-length, stoppered quartz cuvettes, usiug either the Beckman D&B.S~pha~~i~ or the Bausch and Lomb spectronic 505. Further studies were undertaken with lndole.3 Solutions of indole and retinal were prepared and kept in the dark for indole auto-oxidiX$ in the presenca of oxygen and light, while retinal readily photoisomer&, A continuous variation study (Job’s plot) ~89 undertaken to ascertain the proportions of ants involved in the resulting compound Visible spectra were measured on 2-5 x 10-S M solutions of the compound. The course of the reaction in time, subsequent to protouation, was followed spectrally at 0” C and 38” C, (A 5 x 10-S M soIution w required for the 38” C murements to provide convenient optical densities in the long wavelength band of the protonated solution), Kinetic studies were also carried out for the protonated reaction at 0’ C and room temperature by monitoring the long wavelength band of the protouted system. The Beckman D.B. was employed for all temperature studies, as it was equipped with a w~~t-~~~t~e control. Using both a Perkin-Elmer Infrared Spectrophotometer and a Unicam SP,mO, measurements were made on solutions of all-trans retinai, indole and an unprotonated solution of retinal and indole, using carbon tetrachloride as the solvent in all cases. Since, at the concentrations required for i&a-red measurements, the protonated complex precipitates out of solution, the i.r. spectrum of a KBr pelIet of a precipitated and dried sample of the protonated com@nmd was measured. A sample of the precipitated compound was sent to Spaag Microanalytical Laboratory for analysis. EffEcts of pH on the visible spectra of the system were investigated. Anhydrous ammonia was bubbled through solutions of the protonated complex prepared in a I : 1.carbcm ~et~c~o~~~~h~~l so&e& This mix& solvent was used to avoid the complications of a precipitate of ammonium chloride, resulting from an excess of HCl in the system. Ammonium chloride is fairly solubIe in methanol and the proton&cl complex is stable for some time in the mixed solvent, when maintained at ice bath temperature. The phot~iti~~ of the systen$ both unprotonated and protonated, was also investigated spaczrophotometrically. RESULTS
All-tram retinal in carbon tetrachloride shows a &,, at 380 nm. Table 1 summarizes the spectral measurements of unprotonated and protonated solutions of retinal with various secondary amines. (The structural formulae af the reactants are shown in Fig. 2). The spectrum of the protonared earbazole system was measured at 0” C, as its stability is 3 The reaction of indole and retinal on protonation has been independently discovered by ABRAHAMSON and WV @rsonal communication).
Enamine Salts of Retinals with Secondary Amines
extremely temperature dependent. to hydrolysis.
1473
All the above systems proved to be extremely susceptible
TABLE 1. SPECTRAL MEASUREWWB OF REACilON OF RETINAL WITH SECONDARY AMINES
h,~unprotoIlated solution (nm)*
Amax-protonated solution (mn)
Piperidine
342
440
Pyrrolidine Piperazine Indoline
357 350 376 508 (indoline peak) 330
z 510 shoulder cu. 570 475-d peak 424,398,378subsidiary peaks 530 655 610 620
Secondary amine
3-Pyrroline Diphenylamine Pyrrole Indole Carbazole
380 380 380 380 360
* U.V. peaks typical of the secondary amim are not included.
Compounds containing two nitrogen atoms in a conjugated ring structure (imidazole, pyrazole, indazole, purine) failed to produce any marked spectral shifts when mixed with retinal in solution. The spectra of the protonated species are essentially the combined
7 7 7 r /N-hCH“C//C\C/N\C/c\CH I2 I II II I ,“;,-,i2 W CHZ HZC
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A
k
iI
Piperazine --
Corbazple
Diphenylamine
2
Pyrrolidine
3-Pyrrolina -
Piperidine -
HC-CH
Pyrr0k FIG.
lndole
lndoline
2. Structural formulae of retinal and secondary amines forming enamine complexes.
1414
JEAN TOTH AND BARNETTROSENBERG
spectra of the pure constituents, with a slight broadening of the absorption band characteristic of retinal. The retinal-indole system Since the visible spectrum of the unprotonated solution of retinal and indole is not evidently different from the combined spectra of the pure constituents, difference spectra were measured for a continuous variation study. The results show the reaction is definitely one-to-one (Fig. 3).
0.15
2
010
0.05 .I_n 7~3 6:4 FIG. 3. Continuous
5~5 4:6 3:i’ RETINAL: INDOLE
variation study (Job’s plot) of the reaction of indole and retinal. peak at a mole ratio of one indicates a 1 : 1 complex.
The
The stability of the protonated solution proved to be temperature dependent. Kinetic studies, monitoring the absorption band of the protonated species, clearly show this dependence (Fig. 4). If the system is prepared and maintained at 0’ C and the reaction,
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Room
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40
60
80
100
120
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Fro. 4. Kinetics of the protonation reaction of a 1 : 1 molar ratio of indole with retinal in CC14 saturated with anhydrous HCl at two different temperatures, 0” and 24“ C. The o.d. was followed in time at a wavelength of 610 nm.
1475
Enamine Salts of Retinals with Secondary Amines
subsequent to protonation, is followed throughout the visible spectrum, an isosbestic point is clearly evident at approximately 463 nm (Fig. 5). The band characteristic of the unprotonated species red-shifts upon protonation (from 380 to 387 nm) and its o.d. decreases
FICL5. Time dependence of the absorption spectrum of a 1 : 1 molar ratio of indole and all-trans retinal in CC14 at 0” C, sulxequent to protonation with anhydrous HCl. The appearance of the isosbestic point at 463 nm indicates the formation of a single product.
progressively with time, while a long wavelength band, characteristic of the protonated species, emerges. This long wavelength band is initially extremely broad and its &,_ lies at approximately 610 nm. The final spectrum shows a h, at 580 nm, with U.V. peaks at 276 and 324 nm (Fig. 6). When prepared at higher concentrations, this protonated species precipitates out of solution, A filtered, washed and dried sample remains stable, even at room temperature.
SocltDn (lmXld3Ml ---- PrUcnntd Wtbn (25X IO%
-khpmhltd
0.6
i
/
i
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1
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FIG. 6. Absorption spectra of the initial and fbal product of the reaction of indole with all-rruns retinal, in CClh, upon protonation with anhydrous HCI at 0” C.
If the system is prepared and maintained at room temperature, the resulting reaction is very different. The band characteristic of the unprotonated species fails to red-shift on
1476
JEANTOTHAND BARNETTROSENBERG
protonation, although its o.d. does decrease progressively with time; the o.d. of the long wavelength band of the protonated compound initially increases with time but soon reaches a maximum and begins to decrease, as the A,, shifts toward the blue region of the spectrum (Fig. 7). The nature of the compound this system eventually produces has
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---
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FIG. 7. Time dependence of the absorption spectrum of a 1 : 1 molar ratio of indole and ail-rwrs retinal in CC14 at 38” C, subsequent to protonation with anhydrous HCI. No isosbestic point appears in this reaction. The left-hand side of the figure, at lower concentration, indicatea the monotonic disappearance of the 380 nm band. The right-hand side of the figure shows an initial incmase in a.d. at about 600 nm, with a subsequent decrease.
not been investigated. Its visible spectrum is markedly different from that of the compound prepared at 0’ C (Fig. 8). It is also possible to produce this compound by allowing a solution, prepared and protonated at 0” C, to warm up. Successive periods of cooling and warming of this solution indicate the initial reversible formation of an intermediate compound. Eventually, however, the system is no longer reversible and the resulting compound has the same visible spectrum as one prepared at room temperature.
FIG, 8. Absorption spectrum of the final product of the reaction of a 1 : 1 molar ratio of indole and all-trans retinal in CC14 at 38” C, subsequent to protonation with anhydrous HCl.
Enamine Salts of Retinais with Secondary Amines
1477
Condensation of retinal and indole gives rise to an OH group, as evidenced by the emergence of a characteristic OH band in the i.r. spectrum of a one-to-one unprotonated solution. In general, however, the spectrum is a mere superposition of bands characteristic of the pure ~nstituen~, indicating that the equi~b~um of the unprotonat~ solution favors the reactants. The i.r. spectrum of a KBr pellet of the protonated species is totally lacking the bands characteristic of the NH group of indole (3420 and 3480 cm -1) and CH=O group of retinal (1650 cm -1). A C=N type structure is indicated by the presence of several weak-to-moderate bands between 2490 and 2700 cm-t. However, it is impossible to ascertain whether this linkage is charged or uncharged. The results of the microanalysis agree with the theoretical percentages for a one-to-one reaction-with two molecules of HCI reacting with each molecule of the retinal-indole compound. (Required: C 73.66 per cent, H 7.72 per cent, N 3.06 per cent, Cl 15.53 per cent; found: C 73.57 per cent, H 7.69 per cent, N 3.19 per cent, Cl 1546 per cent). One molecule of HCI reacts at the linkage between the two constituents, producing the enamine salt. Since the 3-position of the indole nucleus is known to be extremely reactive once the nitrogen is involved in chemical binding (SUMPTJIR and MKLLER,1954), it was postulated that the second molecule of HCl reacts at this position, adding across the double bond. To check this hypothesis, the enamine salt of all-trans retinal and indole-3-propionic acid was prepared and sent to Spang Microanalytical Laboratory. The results of this analysis indicated that only one molecule of HCI reacted with each molecule of unprotonated compound, thus subst~~at~g the hypothesis. The protonated retinal-indole system has definite indicator-like properties, as evidenced by the marked alteration in its visible spectrum when anhydrous ammonia has been bubbled through the solution. Figure 9 shows the spectral measurements after several
FIG. 9. Absorption speotra of a 1 : 1 molar solution of indole and all-rranr retina3 in a CC~J-CH~OH solvent, indicating spectral reversibility between acidic form (long wavelength peak *y 600 nm) and basic form (shorter wavelength peak - 480 nm). For further explanation see text.
JEANTcm
1478
AND
BARNETT ROSENBERG
consecutive bubblings of ammonia and HCI. The o.d. changes are mainly the result of dilution with methanol (to ensure complete solubilization of any ammonium chloride formed). Dilution with methanol rather than filtration was used to remove this precipitate since the protonated compound is readily adsorbed on its surface, as would be expected if it is truly an enamine salt with chloride as the anion. Both unprotonated and protonated soiutions of the retinal-indole system proved to be photosensitive. Indole itself is photosensitive, showing marked spectral changes subsequent to irradiation with U.V.light. However, no appreciable alterations of the absorption bands representative of the indole moiety in the spectrum of the complexed system were evident following irradiation. The principal effect evident for the unprotonated solution was the disappearance of the 380 mn band with the emergence of a doublet, peaking at 320 and 336 rrm. When the protonated species was irradiated, the o.d. of all absorption bands decreased, as the &ax of the long wavelength band gradually shifted blue-wards. Spectral measurements were also undertaken with the 9-cis isomer of retinal, the only isomer, other than the 11-c;is isomer, capable of reacting with the various opsins and forming what are known as the isopigments. The X,, of a solution of 9-cis retinal in carbon tetrachloride is 372 nm. As with all-trans retinal, the spectrum of the unprotonated retinal-indole system is basically a combination of the spectra of the pure constituents. Subsequent to proto~tion, at 0’ C, the 372 nm band shifts to 385 nm and its o.d. decreases progressively with time, as the long wavelength band of the protonated species emerges. An isosbestic point is clearly evident at approximately 462 nm. The final spectrum shows a xmoxat 585 nm with U.V.peaks at 272, 334 and 392 nm (Fig. 10). Irradiation of an unpro-
2.9
X 10.~
M
0.6.
E-
I 6
X IO4
l/mole-cm
1 260 300
400
A
500
600
700
(mu)-
FIG. IO. Absorption spectrum of a 1 : 1 molar ratio of indole and 9-cis retinal in CC14 ad protonated with anhydrous HCl, at 0’ C.
tonated solution of this system produces spectral changes identical to those for the alltrans. The changes in the spectrum of the protonated solution, following irradiation, are somewhat different. Optical densities in the range 370-520 nm progressively increase with
Enamine Saltsof RetinaIs with Secondary Amines
1479
each instalment of irradiation. Since the main absorption bands of the system decrease with irradiation (the long wavelength band simultaneously blue shifting), the resulting spectrum contains what approximates to two isosbestic points, at 520 and 370 mn. The protonated compound, precipitated and dried, has proved extremely unstable in all solvents tested. It is virtually insoluble in such non-polar solvents as carbon tetrachloride, cyclohexane and benzene, as would be expected of an organic salt. In such solvents as ethanol, methanol, acetone, dioxane, chloroform, formamide, sulfolane and hexylmethylphosphoramide, at both room temperature and fyDC, it has proved extremely unstable, completely ~ss~a~g or producing the compound that forms when the reaction proceeds at room temperature. In this respect it is strongly akin to the cone pigments. It has been postulated that the continued failure to extract cone pigments is probably the result of destruction or dissociation of the pigments by the commonly used extraction solvents and procedures (MORTON and Prrr, 1957). DISCUSSION
In recent years much attention has been given to the structure, preparation and reactions of enamines. The term “enamine” is synonymous with a+-unsaturated amine (Sz~uz~ov~cz, 1963). Typical enamine compounds can be formed by condensation of aldehydes and ketones with secondary amines in the presence of dehydrating agents (BLAHA and CERWNKA, 1966). In the case of derivatives of aromatic ~dehyd~, the formation of carbinolamines has been observed (KWYANOVSKII and BYSTROV, 1963). Investigating similar condensation reactions of imines and saturated heterocyclic amines with aldehydes, KOSTYANOVSKIIand PAN’SHIN (1963) prepared and isolated carbinol type compounds. The structure of the products was confirmed by proton magnetic resonance spectra. They are unstable at room temperature, both in the crystalline state and in solution, but stable at dry ice temperature. The low stability arises from their ease of ovation, or reversibility. Immonio-carbon compounds of the type (IW =CHz) X-, where X is a more electronegative anion than OH, can be obtained by reaction with H (Halides). These compounds are identical in structure to the products of protonation of simple enaminescompounds called enamine salts (BLAHAand CEWINU, 1966). The experimental results reported here indicate that the nature of the reaction of retinal with secondary amines is identical with the reactions identified by these Russian workers. The emergence of an OH band in the i.r. spectrum of the unprotonated compound is evidence for the formation of a carbinol type compound. Its low stability, or reversibility, is indicated by the fact that this spectrum is predominantly a superposition of the bands of the pure constituents. The existence of the @I+ =C) X- type linkage for the protonated system is not definitely confhmed by its i.r. spectrum, though bands indicative of a C=N type linkage are present. Formation of a positively charged nitrogen atom, however, can satisfactorily account for the marked bathochromic shift evident in its visible spectrum. Although the effects of introducing a positively charged atom into a polyene structum have not been rmfficiently investigated, the work of Prrr, et al. (1955) indicates that the introduction of such an atom results in a fairly extensive shift in the visible spectrum. The proposed reaction scheme is summarized in Fig. 11. From the structural formulae of Fig. 2 and the spectral measurements summarized in Table 1, it is evident that an increase in conjugation of the reacting compound results in an increase in the extent of bathochromic shift evident on protonation. This may be a combined effect of increased conjugation of the system as a whole and an increase of the
JEANTOTH AND BARNET~ROSENBERG
1480
+ I
!
HC 2 ,
,7-CH3 5
C%
A
k
;i
All-Tm-Retinal
HV’
\
/i Secondary
Amine
I
Ht
1
N-Aminocorbinol
FIG. 11. Proposed reaction scheme of retinal with secondary amines to form an intermediate N-amjnocarbinol which, upon protonation with HCl, forms an enamine salt.
positive charge on the nitrogen from inductive effects. The extent of shift expected from the addition of such conjugated compounds as studied here is difficult to estimate, as all were cyclic in nature. Although much work has been done on the spectral effects of increased conjugation in polyene systems (JAFFEand ORCHIN, 1962; MURRELL, 1963) linear conjugated chains of carbon atoms were primarily investigated. The hypothesis that a red shift in the A,, can be attained by increasing the positive charge on the nitrogen, by the inductive effect of substituents, was proposed by ROSENBERG and KRIGAS (1967) from their work with SchifF’s bases of retinal. The Amax’sof protonated S&S’s bases formed from retinal and various singly substituted meta- and para-anilines were compared. As the substituents become more “electron withdrawing” the h,,, of the resulting protonat~ compounds becomes increasingly red shifted. The gradual blue-shifting of the h,,, of the protonated retinal-indoie system with time is most probably a result of loss of conjugation in the system. The first effect of the addition of HCI is probabiy at the carbon-nitrogen linkage, splitting off a molecule of water and producing the enamine salt, with an extension of the conju~tion of the system through the indole moiety. A second molecule of HCI would then attack the 3-position of the indole nucleus, adding across the double bond and thus decreasing the conjugation of the system. This loss of conjugation could explain the 30 nm shift (from 610 to 580 nm) with time of the long wavelength band of the protonated compound. The failure of compounds containing two nitrogen atoms in a conjugated system to react with retinal cannot be readily explained. One such compound, imidazole, also fails to react with nitrous acid, despite the presence of the imino group. This has been taken to indicate that the imino group of this class of compounds has different reactive properties from the typical imino grouping (HOFMANN, 1953). This may possibly explain why reaction of such compounds with retinal was not evidenced. The extensive bathochromic shift, the ease of hydrolysis, the temperature dependence of stability, and the indicator-like properties of this system indicate its possible relevance
Enamine Salts of Retinals with Secondary Amines
1481
to the chemistry of visual pigments. However, enamine salts are easily reduced with the common reducing agents (BLAHA and CERVINKA, 1966). Thus, the enamine salt linkage does not appear to offer a direct explanation for the failure of rhodopsin to react with such strong reducing agents as borohydride until the metarhodopsin stage of its photodegradation. It also cannot explain the direction of the pH dependence of the interconversion of metarhodopsin I and II in the case of cattle rhodopsin (HUBBARD, BOWNDS and YOSHIZAWA, 1965). The exact nature of the opsin involved and its 3-dimensional configuration with respect to the chromophoric moiety may be of considerable importance in these cases. Of ultimate interest is the nature of the transduction process of vision. ROSENBERG (1966) has postulated a photoconductivity theory to explain this phenomenon. In connection with this theory, the photovoltaic effect and photoconduction properties of the protonated retinal-indole systems are presently being investigated. Preliminary results, using different isomers of retinal, indicate that the system is indeed photoconductive. A prominent band appears in all action spectra, corresponding to the main absorption band of the compound. REFERENCES BLAHA,K. and CERVINIU, 0. (1966). Cyclic enamines and iminea, pp. 147-227. In Adwmccsin Heterucyclic Chemistry, Volume 6 (edited by A. R. khTRlTZKY and A. J. MOULTON),Academic Press, New York. HOPMANN, K. (1953). Zmiaizzole ard Its Derivatives Part I. The Chemistry of Heterocyciic Compormds, Vol. 6, Interscience, New York. HUBBARD,R., BOU~NDS,D. and YOSIUZAWA,T. (1965). The Chemistry of visual photoreception, Co&i Spring Harb. Symp. quant. Biol. 30, 301-315. JAFFE,H. H. and OR-, M. (1962). Theory and Application of Ulfraviolef Spectroscopy, pp. 220-241,
John Wiley, New York.
KOSTYANOVSIUI, R. G. and BY~TROV,V. F. (1963). Aryi-N-Ethyleneiminocarbinols, Bull. Acad. Sci., USSR. Div. Chem. Sci. 1, 151-153. K~STUNO~U, R. G. and PAN’SHIN, 0. A. (1963). N-Piperidenccarbinol, Bull. Acad. Sci., USSR. Div. Chem. Sci. i, 164-167. MORTON, R. A. and Prrr, G. A. J. (1957). Visual Pigments, Fort&r. Chem. Org. Nafurstoffe 14, 244-316. M-I., J. N. (1963). The Theory of the Electronic Spectra of Organic Molecules, pp. 67-90, John Wiley, New York. prrr, G. A. J., COLLINS,F. D., MORTON, R. A. and STOK,P. (1955). Studies on rhodopsin. 8. Retinylidenemethylatnine an indicator yellow analogue. Biochem. J. 59, 122-128. m, G. A. J. (1964). A survey of the chemistry of visual pigments. ExprZ Eye Res. 3, 316-326. ROSENBERG B. (1966). A physical approach to the visual receptor process, pp. 193-241. In Advances in Radiation Biology, Volume 2 (edited by L. AUOENS~, R. MAXIN and M. ZELLE),Academic Press, New York. ROSENBERG, B. and KRIGAS,T. M. (1967). Spectral shifts in retinal Schiff base complexes. Phorochem. Photobiol. 6, 769-773. Sum, W. C. and MILLER, F. M. (1954). Heterocyclic Compounds with In&de and Carbazole Systems. me Chemistry of Heterocydic Compounds, Vol. 8, Interscience, New York. Swov~cz, J. (1963). Enaminca, pp. l-l 14. In Advances in Organic Chemistry. Methoak and Results (&it& by R. A. RAPHAEL,E. C. TAYLOR and H. WYNB~RG),Interscience, New York. Abstract-Retinal
condenses with secondary amines to produce a carbinol type compound.
Protonation of this system with anhydrous HCl results in the formation of an enamine salt, with an extensive bathocbromic shift in its visible spectrum (A,,, ranges from 450 to 650 nm). Extensive investigations of thii system were undertaken with indole. The stabiity of the protonated product is temperature dependent; the resulting solution is readily hydrolyzed and possesses indicator-like properties; and the precipitated protonated speciea is extremely unstable in all available solvents. Both unprotonated and protonated solutions are photosensitive, as evidenced by marked alterations in their visible spectra. The various chemical properties and the extensive bathochromic shift indicate the possible relevance of this system to the chemistry of visual pigments.
1482
JEAN TOTHAND
BARNET~ ROSENBERG
RCum&Le retinal se condense avec des amines secondaires pour produire un compose du type carbinol. La protonation de ce systeme avec HCI anhydre forme WI se1 d’&tamine, avec un large d&a&e bathochrornique dans le visible (A,,, varie entre 450 et 650 nm). On entreprend une etude approfondie de ce systeme par l’indole. La stabilite du produit proton6 d&end de la temperature; la solution resultante est aisCment hydrolysee et po&de des prop&t&s d’un indicateur; enfin le produit protoni precipite est extremement instable dam tous les solvants possiblea. Les solutions tant non proton&s que proton&a sont photosensibles, comme le prouvent les changements marques dans leurs spectres visiblea. Ces diverses propri&es chimiques et le decalage bathochromique important sugg&ent une application possible de ce systeme a la chimie des pigments visuels. Znaammem&ung-Retinal verbindet sich mit sekumhiren Ammen um eine karbinolartige Verbindung zu erg&en. Die Protonisierung dieses Systems mit wasserfreiem HCI resuhiert in der Bildung einea &am&&es mit einer ausgepr&ten bathochromen Verschiebung im sichtbaren Spektrmn (&,, erstreck; sich von 450 bis 650 nm). Au&h&he Untersuchungen dieaes Systems wurden mit Indol untemommen. Die Stabilitiit des protonierten Prod&s hlLagt von der Temperatur ab. Die entstehende Lijsung wird leicht hydrolysiert und zeigt indikatorffhnliche Eigenschaften. Die ausfallende, protonisierte Substanz ist iiugerst unstabil und zerf%llt in allen verftigbaren Lostmgsmitteln. SowohI nichtprotonisierte, als such protonisierte LiWngen sind lichtemptindlich, was sich in den ausgeprggten Ver&tderungen ihrer sichtbaren Spektren zeigt. Die verschiedenen chemischen Eigenschaften und die weitgehende bathochrome Verschiebung weisen auf die miigliche Bedeutung dieses Systems ftir die Chemie der Sehstoffe hin. Pe3mme - Perrinanb xorurencnpyercff co eropriYHbiMEl aMmmh4r.irf naer coenmfemie xap6mronM.ioro rrina. TIpoTotmponamie 3~ol CricreMbr c 6e3nonrrot HC!l rtprfno~~~ K o6pa3oBaEmo ~~~MxH~J~oBcorm, c 6onbmrrM 6aToxpo&mb&t CZJB~~~OM B ee BWII~MOM cneKqx(A,cMenraerca 0T 450 AO 650 HM). 3Ta
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Vol. 8, pp. 1471-1482.
ENAMINE
Pamon
Press 1968. Printed in
SALTS OF SECONDARY JEANTOTI@
GreatBritain.
RETINALS AMINES’
WITH
and BARNETTROSENBERG
Department of Biophysics,Michipn State University,East Lamin& Michigan48823 (Received 2 July 1968)
INTRODUCTION THE VISUAL pigments consist of proteins of the opsin family combined with the 11-cis isomer of vitamin A aldehyde. The aldehyde, the chromophoric moiety, exists in nature in two forms, vitamin Ar aldehyde (retinal) and vitamin A2 aldehyde (3dehydroretinal). The linkage between the chromophore and the protein moiety is not known with certainty: it has been postulated to be either a protonated or unprotonated S&X’s base. Naturally occurring visual pigments based on retinal show absorption maxima over the range 440-620 nm. The postulate of a conjugate acid form of a S&X’s base can satisfactorily account for roughly only half the required spectral shift on union of retinal (Lax =385 nm in water solution) with an opsin (PITT, 1964). Available evidence indicates that the carbon-nitrogen linkage determined for N-retinylideneopsin-a possible photodegradation product of rhodopsin, the visual pigment of the rods-is present in rhodopsin itself (PITT, 1964). Considering the chemical properties of rhodopsin and its photodegradation products, Morton and Pitt proposed that in rhodopsin there are at least three links joining retinal to the opsin (MORTONand PITT, 1957). One of these is split in the conversion of rhodopsin to prelumirhodopsin (the photochemical step); another is broken when metarhodopsin converts to N-retinylideneopsin (or metarhodopsin I converts to metarhodopsin II in the case of cattle rhodopsin); and finally the carbon-to-nitrogen link is hydrolyzed to give retinaldehyde and opsin. N-retinylideneopsin has been shown to be a Schiff’s base (PITT, COLLJNS,MORTONand STOK, 1955). Morton and Pitt suggest that the linkage broken to form this compound is a retinylidene ammonium structure (Fig. l), the R and R'possibly representing different side groups of amino acid residues of the protein moiety. Protonation of solutions of retinal with imines or secondary amines produces compounds with structures comparable to the retinylidene ammonium structure. These compounds are enamine salts. A marked bathochromic shift is evident in their visible spectra, subsequent to protonation (resulting J!_‘s ranging from 450 to 650 nm). Working predominantly with indole (the precursor of the amino acid tryptophan), the nature of the reaction and the chemical properties of the resulting system were investigated. Its stability is extremely temperature dependent; it possesses indicator-like properties; and it 1 This Research was supported by Grants from National Institutes of Health -145 and GM-1422. 2 This research Represents a Partial Fulfillment of the Requirememts for the M. Sci. Degree. 1471
1472
JEXNTcmi AND BARNES
is readily hydrolyzed. These factors and the marked relevance to the chemistry of visual pigments.
ROSENBERG
bathochromic
shift indicate
its possible
FIG. 1. Proposed structure of retinylidene ammonium salt (after MORTONand Prrr, 1957). METHODS Spectra-grade carbon tetraehlcxide was the solvent employed in all studies, Other organic solvents (e.g. ethaxml, acetone, dioxane, chloroform, methanol) proved unsuitable, as proton&d solutions of retinal alone in these solven@ showed an initial bathochromic shift in the visible spectrum. (All protunation was accomplished by bubbling anhydrous HCl through the solution.) This was not seen when carbon tetrachloride was used as the solvent thus avoiding any ambiguity of resuIts for the proton&& system. ANruns retinal was obtained from DistiIlation Products and used without further purification. An initial spectral survey of the reactian of retinal with vari01.1~ imine~ and secondary amines was undertaken, for both the unprotors#d and pmtonated spccia. All spectra were measured in 1 cm path-length, stoppered quartz cuvettes, usiug either the Beckman D&B.S~pha~~i~ or the Bausch and Lomb spectronic 505. Further studies were undertaken with lndole.3 Solutions of indole and retinal were prepared and kept in the dark for indole auto-oxidiX$ in the presenca of oxygen and light, while retinal readily photoisomer&, A continuous variation study (Job’s plot) ~89 undertaken to ascertain the proportions of ants involved in the resulting compound Visible spectra were measured on 2-5 x 10-S M solutions of the compound. The course of the reaction in time, subsequent to protouation, was followed spectrally at 0” C and 38” C, (A 5 x 10-S M soIution w required for the 38” C murements to provide convenient optical densities in the long wavelength band of the protonated solution), Kinetic studies were also carried out for the protonated reaction at 0’ C and room temperature by monitoring the long wavelength band of the protouted system. The Beckman D.B. was employed for all temperature studies, as it was equipped with a w~~t-~~~t~e control. Using both a Perkin-Elmer Infrared Spectrophotometer and a Unicam SP,mO, measurements were made on solutions of all-trans retinai, indole and an unprotonated solution of retinal and indole, using carbon tetrachloride as the solvent in all cases. Since, at the concentrations required for i&a-red measurements, the protonated complex precipitates out of solution, the i.r. spectrum of a KBr pelIet of a precipitated and dried sample of the protonated com@nmd was measured. A sample of the precipitated compound was sent to Spaag Microanalytical Laboratory for analysis. EffEcts of pH on the visible spectra of the system were investigated. Anhydrous ammonia was bubbled through solutions of the protonated complex prepared in a I : 1.carbcm ~et~c~o~~~~h~~l so&e& This mix& solvent was used to avoid the complications of a precipitate of ammonium chloride, resulting from an excess of HCl in the system. Ammonium chloride is fairly solubIe in methanol and the proton&cl complex is stable for some time in the mixed solvent, when maintained at ice bath temperature. The phot~iti~~ of the systen$ both unprotonated and protonated, was also investigated spaczrophotometrically. RESULTS
All-tram retinal in carbon tetrachloride shows a &,, at 380 nm. Table 1 summarizes the spectral measurements of unprotonated and protonated solutions of retinal with various secondary amines. (The structural formulae af the reactants are shown in Fig. 2). The spectrum of the protonared earbazole system was measured at 0” C, as its stability is 3 The reaction of indole and retinal on protonation has been independently discovered by ABRAHAMSON and WV @rsonal communication).
Enamine Salts of Retinals with Secondary Amines
extremely temperature dependent. to hydrolysis.
1473
All the above systems proved to be extremely susceptible
TABLE 1. SPECTRAL MEASUREWWB OF REACilON OF RETINAL WITH SECONDARY AMINES
h,~unprotoIlated solution (nm)*
Amax-protonated solution (mn)
Piperidine
342
440
Pyrrolidine Piperazine Indoline
357 350 376 508 (indoline peak) 330
z 510 shoulder cu. 570 475-d peak 424,398,378subsidiary peaks 530 655 610 620
Secondary amine
3-Pyrroline Diphenylamine Pyrrole Indole Carbazole
380 380 380 380 360
* U.V. peaks typical of the secondary amim are not included.
Compounds containing two nitrogen atoms in a conjugated ring structure (imidazole, pyrazole, indazole, purine) failed to produce any marked spectral shifts when mixed with retinal in solution. The spectra of the protonated species are essentially the combined
7 7 7 r /N-hCH“C//C\C/N\C/c\CH I2 I II II I ,“;,-,i2 W CHZ HZC
'N' A
“+
c-c\cp
2\N/
A
k
iI
Piperazine --
Corbazple
Diphenylamine
2
Pyrrolidine
3-Pyrrolina -
Piperidine -
HC-CH
Pyrr0k FIG.
lndole
lndoline
2. Structural formulae of retinal and secondary amines forming enamine complexes.
1414
JEAN TOTH AND BARNETTROSENBERG
spectra of the pure constituents, with a slight broadening of the absorption band characteristic of retinal. The retinal-indole system Since the visible spectrum of the unprotonated solution of retinal and indole is not evidently different from the combined spectra of the pure constituents, difference spectra were measured for a continuous variation study. The results show the reaction is definitely one-to-one (Fig. 3).
0.15
2
010
0.05 .I_n 7~3 6:4 FIG. 3. Continuous
5~5 4:6 3:i’ RETINAL: INDOLE
variation study (Job’s plot) of the reaction of indole and retinal. peak at a mole ratio of one indicates a 1 : 1 complex.
The
The stability of the protonated solution proved to be temperature dependent. Kinetic studies, monitoring the absorption band of the protonated species, clearly show this dependence (Fig. 4). If the system is prepared and maintained at 0’ C and the reaction,
‘OT------06’
1
5.0 X bM Temperature
Room
l...,..‘..“.J 0 20
40
60
80
100
120
i 0
i x)
40
60
w
Fro. 4. Kinetics of the protonation reaction of a 1 : 1 molar ratio of indole with retinal in CC14 saturated with anhydrous HCl at two different temperatures, 0” and 24“ C. The o.d. was followed in time at a wavelength of 610 nm.
1475
Enamine Salts of Retinals with Secondary Amines
subsequent to protonation, is followed throughout the visible spectrum, an isosbestic point is clearly evident at approximately 463 nm (Fig. 5). The band characteristic of the unprotonated species red-shifts upon protonation (from 380 to 387 nm) and its o.d. decreases
FICL5. Time dependence of the absorption spectrum of a 1 : 1 molar ratio of indole and all-trans retinal in CC14 at 0” C, sulxequent to protonation with anhydrous HCl. The appearance of the isosbestic point at 463 nm indicates the formation of a single product.
progressively with time, while a long wavelength band, characteristic of the protonated species, emerges. This long wavelength band is initially extremely broad and its &,_ lies at approximately 610 nm. The final spectrum shows a h, at 580 nm, with U.V. peaks at 276 and 324 nm (Fig. 6). When prepared at higher concentrations, this protonated species precipitates out of solution, A filtered, washed and dried sample remains stable, even at room temperature.
SocltDn (lmXld3Ml ---- PrUcnntd Wtbn (25X IO%
-khpmhltd
0.6
i
/
i
Of 0.
1
/ I
r-I.8
X
IO’
I/mole-cm
\
IO
FIG. 6. Absorption spectra of the initial and fbal product of the reaction of indole with all-rruns retinal, in CClh, upon protonation with anhydrous HCI at 0” C.
If the system is prepared and maintained at room temperature, the resulting reaction is very different. The band characteristic of the unprotonated species fails to red-shift on
1476
JEANTOTHAND BARNETTROSENBERG
protonation, although its o.d. does decrease progressively with time; the o.d. of the long wavelength band of the protonated compound initially increases with time but soon reaches a maximum and begins to decrease, as the A,, shifts toward the blue region of the spectrum (Fig. 7). The nature of the compound this system eventually produces has
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-
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0 D.
---
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00.
L---Y ‘~ ‘\\
_,--.. ,’
,’
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\
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FIG. 7. Time dependence of the absorption spectrum of a 1 : 1 molar ratio of indole and ail-rwrs retinal in CC14 at 38” C, subsequent to protonation with anhydrous HCI. No isosbestic point appears in this reaction. The left-hand side of the figure, at lower concentration, indicatea the monotonic disappearance of the 380 nm band. The right-hand side of the figure shows an initial incmase in a.d. at about 600 nm, with a subsequent decrease.
not been investigated. Its visible spectrum is markedly different from that of the compound prepared at 0’ C (Fig. 8). It is also possible to produce this compound by allowing a solution, prepared and protonated at 0” C, to warm up. Successive periods of cooling and warming of this solution indicate the initial reversible formation of an intermediate compound. Eventually, however, the system is no longer reversible and the resulting compound has the same visible spectrum as one prepared at room temperature.
FIG, 8. Absorption spectrum of the final product of the reaction of a 1 : 1 molar ratio of indole and all-trans retinal in CC14 at 38” C, subsequent to protonation with anhydrous HCl.
Enamine Salts of Retinais with Secondary Amines
1477
Condensation of retinal and indole gives rise to an OH group, as evidenced by the emergence of a characteristic OH band in the i.r. spectrum of a one-to-one unprotonated solution. In general, however, the spectrum is a mere superposition of bands characteristic of the pure ~nstituen~, indicating that the equi~b~um of the unprotonat~ solution favors the reactants. The i.r. spectrum of a KBr pellet of the protonated species is totally lacking the bands characteristic of the NH group of indole (3420 and 3480 cm -1) and CH=O group of retinal (1650 cm -1). A C=N type structure is indicated by the presence of several weak-to-moderate bands between 2490 and 2700 cm-t. However, it is impossible to ascertain whether this linkage is charged or uncharged. The results of the microanalysis agree with the theoretical percentages for a one-to-one reaction-with two molecules of HCI reacting with each molecule of the retinal-indole compound. (Required: C 73.66 per cent, H 7.72 per cent, N 3.06 per cent, Cl 15.53 per cent; found: C 73.57 per cent, H 7.69 per cent, N 3.19 per cent, Cl 1546 per cent). One molecule of HCI reacts at the linkage between the two constituents, producing the enamine salt. Since the 3-position of the indole nucleus is known to be extremely reactive once the nitrogen is involved in chemical binding (SUMPTJIR and MKLLER,1954), it was postulated that the second molecule of HCl reacts at this position, adding across the double bond. To check this hypothesis, the enamine salt of all-trans retinal and indole-3-propionic acid was prepared and sent to Spang Microanalytical Laboratory. The results of this analysis indicated that only one molecule of HCI reacted with each molecule of unprotonated compound, thus subst~~at~g the hypothesis. The protonated retinal-indole system has definite indicator-like properties, as evidenced by the marked alteration in its visible spectrum when anhydrous ammonia has been bubbled through the solution. Figure 9 shows the spectral measurements after several
FIG. 9. Absorption speotra of a 1 : 1 molar solution of indole and all-rranr retina3 in a CC~J-CH~OH solvent, indicating spectral reversibility between acidic form (long wavelength peak *y 600 nm) and basic form (shorter wavelength peak - 480 nm). For further explanation see text.
JEANTcm
1478
AND
BARNETT ROSENBERG
consecutive bubblings of ammonia and HCI. The o.d. changes are mainly the result of dilution with methanol (to ensure complete solubilization of any ammonium chloride formed). Dilution with methanol rather than filtration was used to remove this precipitate since the protonated compound is readily adsorbed on its surface, as would be expected if it is truly an enamine salt with chloride as the anion. Both unprotonated and protonated soiutions of the retinal-indole system proved to be photosensitive. Indole itself is photosensitive, showing marked spectral changes subsequent to irradiation with U.V.light. However, no appreciable alterations of the absorption bands representative of the indole moiety in the spectrum of the complexed system were evident following irradiation. The principal effect evident for the unprotonated solution was the disappearance of the 380 mn band with the emergence of a doublet, peaking at 320 and 336 rrm. When the protonated species was irradiated, the o.d. of all absorption bands decreased, as the &ax of the long wavelength band gradually shifted blue-wards. Spectral measurements were also undertaken with the 9-cis isomer of retinal, the only isomer, other than the 11-c;is isomer, capable of reacting with the various opsins and forming what are known as the isopigments. The X,, of a solution of 9-cis retinal in carbon tetrachloride is 372 nm. As with all-trans retinal, the spectrum of the unprotonated retinal-indole system is basically a combination of the spectra of the pure constituents. Subsequent to proto~tion, at 0’ C, the 372 nm band shifts to 385 nm and its o.d. decreases progressively with time, as the long wavelength band of the protonated species emerges. An isosbestic point is clearly evident at approximately 462 nm. The final spectrum shows a xmoxat 585 nm with U.V.peaks at 272, 334 and 392 nm (Fig. 10). Irradiation of an unpro-
2.9
X 10.~
M
0.6.
E-
I 6
X IO4
l/mole-cm
1 260 300
400
A
500
600
700
(mu)-
FIG. IO. Absorption spectrum of a 1 : 1 molar ratio of indole and 9-cis retinal in CC14 ad protonated with anhydrous HCl, at 0’ C.
tonated solution of this system produces spectral changes identical to those for the alltrans. The changes in the spectrum of the protonated solution, following irradiation, are somewhat different. Optical densities in the range 370-520 nm progressively increase with
Enamine Saltsof RetinaIs with Secondary Amines
1479
each instalment of irradiation. Since the main absorption bands of the system decrease with irradiation (the long wavelength band simultaneously blue shifting), the resulting spectrum contains what approximates to two isosbestic points, at 520 and 370 mn. The protonated compound, precipitated and dried, has proved extremely unstable in all solvents tested. It is virtually insoluble in such non-polar solvents as carbon tetrachloride, cyclohexane and benzene, as would be expected of an organic salt. In such solvents as ethanol, methanol, acetone, dioxane, chloroform, formamide, sulfolane and hexylmethylphosphoramide, at both room temperature and fyDC, it has proved extremely unstable, completely ~ss~a~g or producing the compound that forms when the reaction proceeds at room temperature. In this respect it is strongly akin to the cone pigments. It has been postulated that the continued failure to extract cone pigments is probably the result of destruction or dissociation of the pigments by the commonly used extraction solvents and procedures (MORTON and Prrr, 1957). DISCUSSION
In recent years much attention has been given to the structure, preparation and reactions of enamines. The term “enamine” is synonymous with a+-unsaturated amine (Sz~uz~ov~cz, 1963). Typical enamine compounds can be formed by condensation of aldehydes and ketones with secondary amines in the presence of dehydrating agents (BLAHA and CERWNKA, 1966). In the case of derivatives of aromatic ~dehyd~, the formation of carbinolamines has been observed (KWYANOVSKII and BYSTROV, 1963). Investigating similar condensation reactions of imines and saturated heterocyclic amines with aldehydes, KOSTYANOVSKIIand PAN’SHIN (1963) prepared and isolated carbinol type compounds. The structure of the products was confirmed by proton magnetic resonance spectra. They are unstable at room temperature, both in the crystalline state and in solution, but stable at dry ice temperature. The low stability arises from their ease of ovation, or reversibility. Immonio-carbon compounds of the type (IW =CHz) X-, where X is a more electronegative anion than OH, can be obtained by reaction with H (Halides). These compounds are identical in structure to the products of protonation of simple enaminescompounds called enamine salts (BLAHAand CEWINU, 1966). The experimental results reported here indicate that the nature of the reaction of retinal with secondary amines is identical with the reactions identified by these Russian workers. The emergence of an OH band in the i.r. spectrum of the unprotonated compound is evidence for the formation of a carbinol type compound. Its low stability, or reversibility, is indicated by the fact that this spectrum is predominantly a superposition of the bands of the pure constituents. The existence of the @I+ =C) X- type linkage for the protonated system is not definitely confhmed by its i.r. spectrum, though bands indicative of a C=N type linkage are present. Formation of a positively charged nitrogen atom, however, can satisfactorily account for the marked bathochromic shift evident in its visible spectrum. Although the effects of introducing a positively charged atom into a polyene structum have not been rmfficiently investigated, the work of Prrr, et al. (1955) indicates that the introduction of such an atom results in a fairly extensive shift in the visible spectrum. The proposed reaction scheme is summarized in Fig. 11. From the structural formulae of Fig. 2 and the spectral measurements summarized in Table 1, it is evident that an increase in conjugation of the reacting compound results in an increase in the extent of bathochromic shift evident on protonation. This may be a combined effect of increased conjugation of the system as a whole and an increase of the
JEANTOTH AND BARNET~ROSENBERG
1480
+ I
!
HC 2 ,
,7-CH3 5
C%
A
k
;i
All-Tm-Retinal
HV’
\
/i Secondary
Amine
I
Ht
1
N-Aminocorbinol
FIG. 11. Proposed reaction scheme of retinal with secondary amines to form an intermediate N-amjnocarbinol which, upon protonation with HCl, forms an enamine salt.
positive charge on the nitrogen from inductive effects. The extent of shift expected from the addition of such conjugated compounds as studied here is difficult to estimate, as all were cyclic in nature. Although much work has been done on the spectral effects of increased conjugation in polyene systems (JAFFEand ORCHIN, 1962; MURRELL, 1963) linear conjugated chains of carbon atoms were primarily investigated. The hypothesis that a red shift in the A,, can be attained by increasing the positive charge on the nitrogen, by the inductive effect of substituents, was proposed by ROSENBERG and KRIGAS (1967) from their work with SchifF’s bases of retinal. The Amax’sof protonated S&S’s bases formed from retinal and various singly substituted meta- and para-anilines were compared. As the substituents become more “electron withdrawing” the h,,, of the resulting protonat~ compounds becomes increasingly red shifted. The gradual blue-shifting of the h,,, of the protonated retinal-indoie system with time is most probably a result of loss of conjugation in the system. The first effect of the addition of HCI is probabiy at the carbon-nitrogen linkage, splitting off a molecule of water and producing the enamine salt, with an extension of the conju~tion of the system through the indole moiety. A second molecule of HCI would then attack the 3-position of the indole nucleus, adding across the double bond and thus decreasing the conjugation of the system. This loss of conjugation could explain the 30 nm shift (from 610 to 580 nm) with time of the long wavelength band of the protonated compound. The failure of compounds containing two nitrogen atoms in a conjugated system to react with retinal cannot be readily explained. One such compound, imidazole, also fails to react with nitrous acid, despite the presence of the imino group. This has been taken to indicate that the imino group of this class of compounds has different reactive properties from the typical imino grouping (HOFMANN, 1953). This may possibly explain why reaction of such compounds with retinal was not evidenced. The extensive bathochromic shift, the ease of hydrolysis, the temperature dependence of stability, and the indicator-like properties of this system indicate its possible relevance
Enamine Salts of Retinals with Secondary Amines
1481
to the chemistry of visual pigments. However, enamine salts are easily reduced with the common reducing agents (BLAHA and CERVINKA, 1966). Thus, the enamine salt linkage does not appear to offer a direct explanation for the failure of rhodopsin to react with such strong reducing agents as borohydride until the metarhodopsin stage of its photodegradation. It also cannot explain the direction of the pH dependence of the interconversion of metarhodopsin I and II in the case of cattle rhodopsin (HUBBARD, BOWNDS and YOSHIZAWA, 1965). The exact nature of the opsin involved and its 3-dimensional configuration with respect to the chromophoric moiety may be of considerable importance in these cases. Of ultimate interest is the nature of the transduction process of vision. ROSENBERG (1966) has postulated a photoconductivity theory to explain this phenomenon. In connection with this theory, the photovoltaic effect and photoconduction properties of the protonated retinal-indole systems are presently being investigated. Preliminary results, using different isomers of retinal, indicate that the system is indeed photoconductive. A prominent band appears in all action spectra, corresponding to the main absorption band of the compound. REFERENCES BLAHA,K. and CERVINIU, 0. (1966). Cyclic enamines and iminea, pp. 147-227. In Adwmccsin Heterucyclic Chemistry, Volume 6 (edited by A. R. khTRlTZKY and A. J. MOULTON),Academic Press, New York. HOPMANN, K. (1953). Zmiaizzole ard Its Derivatives Part I. The Chemistry of Heterocyciic Compormds, Vol. 6, Interscience, New York. HUBBARD,R., BOU~NDS,D. and YOSIUZAWA,T. (1965). The Chemistry of visual photoreception, Co&i Spring Harb. Symp. quant. Biol. 30, 301-315. JAFFE,H. H. and OR-, M. (1962). Theory and Application of Ulfraviolef Spectroscopy, pp. 220-241,
John Wiley, New York.
KOSTYANOVSIUI, R. G. and BY~TROV,V. F. (1963). Aryi-N-Ethyleneiminocarbinols, Bull. Acad. Sci., USSR. Div. Chem. Sci. 1, 151-153. K~STUNO~U, R. G. and PAN’SHIN, 0. A. (1963). N-Piperidenccarbinol, Bull. Acad. Sci., USSR. Div. Chem. Sci. i, 164-167. MORTON, R. A. and Prrr, G. A. J. (1957). Visual Pigments, Fort&r. Chem. Org. Nafurstoffe 14, 244-316. M-I., J. N. (1963). The Theory of the Electronic Spectra of Organic Molecules, pp. 67-90, John Wiley, New York. prrr, G. A. J., COLLINS,F. D., MORTON, R. A. and STOK,P. (1955). Studies on rhodopsin. 8. Retinylidenemethylatnine an indicator yellow analogue. Biochem. J. 59, 122-128. m, G. A. J. (1964). A survey of the chemistry of visual pigments. ExprZ Eye Res. 3, 316-326. ROSENBERG B. (1966). A physical approach to the visual receptor process, pp. 193-241. In Advances in Radiation Biology, Volume 2 (edited by L. AUOENS~, R. MAXIN and M. ZELLE),Academic Press, New York. ROSENBERG, B. and KRIGAS,T. M. (1967). Spectral shifts in retinal Schiff base complexes. Phorochem. Photobiol. 6, 769-773. Sum, W. C. and MILLER, F. M. (1954). Heterocyclic Compounds with In&de and Carbazole Systems. me Chemistry of Heterocydic Compounds, Vol. 8, Interscience, New York. Swov~cz, J. (1963). Enaminca, pp. l-l 14. In Advances in Organic Chemistry. Methoak and Results (&it& by R. A. RAPHAEL,E. C. TAYLOR and H. WYNB~RG),Interscience, New York. Abstract-Retinal
condenses with secondary amines to produce a carbinol type compound.
Protonation of this system with anhydrous HCl results in the formation of an enamine salt, with an extensive bathocbromic shift in its visible spectrum (A,,, ranges from 450 to 650 nm). Extensive investigations of thii system were undertaken with indole. The stabiity of the protonated product is temperature dependent; the resulting solution is readily hydrolyzed and possesses indicator-like properties; and the precipitated protonated speciea is extremely unstable in all available solvents. Both unprotonated and protonated solutions are photosensitive, as evidenced by marked alterations in their visible spectra. The various chemical properties and the extensive bathochromic shift indicate the possible relevance of this system to the chemistry of visual pigments.
1482
JEAN TOTHAND
BARNET~ ROSENBERG
RCum&Le retinal se condense avec des amines secondaires pour produire un compose du type carbinol. La protonation de ce systeme avec HCI anhydre forme WI se1 d’&tamine, avec un large d&a&e bathochrornique dans le visible (A,,, varie entre 450 et 650 nm). On entreprend une etude approfondie de ce systeme par l’indole. La stabilite du produit proton6 d&end de la temperature; la solution resultante est aisCment hydrolysee et po&de des prop&t&s d’un indicateur; enfin le produit protoni precipite est extremement instable dam tous les solvants possiblea. Les solutions tant non proton&s que proton&a sont photosensibles, comme le prouvent les changements marques dans leurs spectres visiblea. Ces diverses propri&es chimiques et le decalage bathochromique important sugg&ent une application possible de ce systeme a la chimie des pigments visuels. Znaammem&ung-Retinal verbindet sich mit sekumhiren Ammen um eine karbinolartige Verbindung zu erg&en. Die Protonisierung dieses Systems mit wasserfreiem HCI resuhiert in der Bildung einea &am&&es mit einer ausgepr&ten bathochromen Verschiebung im sichtbaren Spektrmn (&,, erstreck; sich von 450 bis 650 nm). Au&h&he Untersuchungen dieaes Systems wurden mit Indol untemommen. Die Stabilitiit des protonierten Prod&s hlLagt von der Temperatur ab. Die entstehende Lijsung wird leicht hydrolysiert und zeigt indikatorffhnliche Eigenschaften. Die ausfallende, protonisierte Substanz ist iiugerst unstabil und zerf%llt in allen verftigbaren Lostmgsmitteln. SowohI nichtprotonisierte, als such protonisierte LiWngen sind lichtemptindlich, was sich in den ausgeprggten Ver&tderungen ihrer sichtbaren Spektren zeigt. Die verschiedenen chemischen Eigenschaften und die weitgehende bathochrome Verschiebung weisen auf die miigliche Bedeutung dieses Systems ftir die Chemie der Sehstoffe hin. Pe3mme - Perrinanb xorurencnpyercff co eropriYHbiMEl aMmmh4r.irf naer coenmfemie xap6mronM.ioro rrina. TIpoTotmponamie 3~ol CricreMbr c 6e3nonrrot HC!l rtprfno~~~ K o6pa3oBaEmo ~~~MxH~J~oBcorm, c 6onbmrrM 6aToxpo&mb&t CZJB~~~OM B ee BWII~MOM cneKqx(A,cMenraerca 0T 450 AO 650 HM). 3Ta
CECTeMa
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