Optical rotatory dispersion and circular dichroism studies of C19-steroid N-acetylglucosaminides

649

OPTICAL ROTATORY DISPERSION AND CIRCULAR DICHROISM STUDIES OF C 19-STEROID N-ACETYLGLUCOSAMINIDES

David K. Fukushima and Michio Matsui* From the Institute for Steroid Research, Montefiore Hospital and Medical Center, New York, N . Y . 10467

Received September 20, 1969

ABSTRACT The optical rotatory dispersion spectra of the anomeric N-acetylD-glucosaminides of androsterone, etiocholanolone, 17/3-hydroxy-5~androstan-3-one and 5/3-androstane-3~,17/3-diol and of the parent steroids have been determined. The molar rotatory dispersion differences ([M] of steroid conjugate minus [M] of steroid) demonstrate that the intensities of the e-anomers are considerably more positive than those of the corresponding/3-anomers; at 230 mM these differences are greater than 5500. The circular dichroism spectra of the anomeric N-acetylD-glucosaminides of 17/3-hydroxy-5~-androstan-3-one, 3~-hydroxy5/3-androstan-17-one and 5/3-androstane-3~,17/3-diol and of the parent steroids have been determined. The molar ellipticity differences ([g] of steroid conjugate minus [g] of steroid) for the anomeric glucosaminides are distinctly different. A solvent effect on the circular dichroism spectra of the anomers of cyclohexyl and methyl N-acetylglucosaminides in water and 2,2,2-~rlfluorethanol is reported. Optical rotatory dispersion measurement was employed as the principai means for assigning the o~-glycoside configuration to the naturally occurring 3/3-hydroxy-A5-pregnen-20~-yl 2'-acetamido2'-deoxy-D-glucopyranoside by Arcos and Lieberrnan (1) based on studies of the anomers of methyl N-acetylglucosaminide (2). It was subsequently demonstrated by synthesis that the steroid conjugate had the/~-configuration (3). It is known that bulkier alcohol groups at

*Visiting Scientist from Tokyo Biochemical Research Institute, Japan 1967-1969.

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Dec. 1969

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Fig 3. A, Molar optical rotatory dispersion [M] and B, molar optical rotatory dispersion difference AIM] for androsterone 17-keto-5~-androstan-3~-yl 2'-acetamido-2'-deoxy-/3-D-glucopyranoside . . . . and its o-anomer . . . . . . .

C-1 of the sugar enhance the optical rotatory properties of alkyl glycosides and these effects were observed with C19-steroid and cyclohexyl N-acetylglucosaminides at the sodium D line (4). In the present paper the optical rotatory dispersion (ORD) and circular dichroism (CD) spectra of anomeric C19-steroid and cyclohexyl 2'-acetamido-2'-deoxy-D-glucopyranosides have been investigated. METHODS AND MATERIALS The synthesis of the C19-steroid and cyclohexyl N-acetyI-Dglucosaminides employed in this study have been reported previously (4). The anomeric methyl N-acetyI-D-glucosaminides were prepared by the method of Zilliken and coworkers (5).

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Dec. 1969

ST E R O I DS

Optical rotatory dispersion (ORD) spectra were determined in methanol at 27° with a Cary Model 60 spectropolarlmeter using a Xenon arc. XBO-450 w/4 lamp and are reported in molar rotaHon [M] X values. The molar rotation difference, A[M]x, was obtained by subtracting the [M];~ of the steroid from the [M]), of the corresponding steroid glucosaminide. Cylindrical quartz cell with 1.0 cm path length was used. The slit-width of the polarlmeter was programmed for half band-widths of less than 1.5 mM through the entire range. Circular dichrolsm (CD) spectra were measured in freshly distilled 2,2,2-trifluorethanol with a 6001 circular dlchroism attachment on the Cary Model 60 spectropolarimeter and are reported in molar ellipticlty [g] values. The CD of the simple N-acetylglucosamlnides were also determined in water. The molar elllpticity difference, A[g] A was obtained by subtracting the [g]~, of the steroid from [g]~ of the corresponding steroid glucosamlnide.

Dr. Irving kistowsky of the Albert Einstein College

of Medicine made the spectropolarimeter available to us and we gratefully acknowledge his aid and helpful discussion. RESULTS AND DISCUSSION The ORD spectra of the anomeric N-acetyI-D-glucosaminides of androsterone, gtiocholanolone, 17/3-hydroxy-5~-androstan-3-one and 5/~-.androstane-3o~,17/~-diol and the parent steroids are reported in Figures (1A to 4A). The spectra of the ketonic compounds are dominated by the Cotton effect of the ketonic carbonyl group. The AIM] spectra (Fig 1B to 4B, [M];k of the steroid N-acetylglucosamlnide minus [M];k of the steroid) are those of the anomeric amino sugar moiety. Thus the A[M] curves for the/3-anomers are similar to each other and to the ORD spectra

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of methyl and cyclohexyl N-acetyI-D-glucosaminides. They all have a negative Cotton effect for the amide group with a trough at about 230 ml~ in agreement with earlier studies on methyl N-acetyI-D-glucosaminide in aqueous solution (2, 6). The AIM] curves for the o~-anomer are also similar to each other and to the ORD spectra of methyl and cyclohexyl glucosaminides. The Cotton effect near 230 ml~ is present but may be barely discernable as in the curve derived from the etiocholanolone conjugate, Fig 1B. The intensities of the c~-anomers of steroid and cyclohexyl glucosaminides are considerably more positive than those of the corresponding/3-anomers; at 230 ml~ these differences are greater than + 5500. The circular dichroism spectra, Fig 6, of the anomeric N-acetylD-glucosaminides of 17/3-hydroxy-5e-androstan-3-one and of 3~-hydroxy5/3-androstan-17-one in 2,2,2-trifluorethanol demonstrate the Cotton effects of both carbonyl groups. The molar ellipticity differences, A[g] = [g] of steroid conjugate minus [g] of steroid, cancel out the effects of the absorption of the ketonic groups and thus show only the molar ellipticity of the amino sugar moiety due to the amide carbonyl absorption, Fig 6B and 6D. The non-ketonic steroid, 5/3-androstane-3~,17/3-diol, does not give a circular dichroism spectrum and thus the curves of the N-acetyl glucosaminides of this compound are those of the amino sugar moiety Fig 7A. In all three compounds the spectra for the anorneric sugars are distinctly different; the /3-.glycosides have a small negative band centered near 210 mls whereas the a-glycosides show the beginning of a strong positive band below 200 ml~. The differences are undoubtedly due to a solvent

Dec. 1969

ST E R O I DS

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effect.

The spectra for the anomers of cyclohexyl 2'-acetamido-2'-deoxy-

D-glucopyranoside in 2,2,2-trifluorethanol, Fig 7B, are similar to those derived from the steroid glucosaminldes, a positive Cotton effect for the c~-anomer and a negative band for the /3. However the spectra of the anomeric cyclohexyl glucosaminides in water are almost identical,

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large negative Cotton effect centered around 210 mlJ Fig 7E. Such curves have been obtained previously for methyl N-acetylglucosaminides in water by other investigators (7). The spectra of these glycosides in water obtained in these laboratories are shown in Fig 7D and compared with those derived in trifluorethanol, Fig 7C. The latter exhibit a positive Cotton effect for the rv-anomer and a negative ellipticity for the/3. The effect of the solvent on the ellipticity of the anomers may be the result

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Dec. 1969

ST E R O I DS

ACKNOWLEDGEMENT The authors appreciate the interest and support of Dr. T. F. Gallagher throughout the investigation. We gratefully acknowledge the invaluable assistance of Julia S. Liang and Roland Bloch. This work has been supported by grants from the American Cancer Society and from the National Cancer Institute, N[H, grant CA 07304.

REFERENCES ,

M. Arcos and S. Lieberman, BIOCHEMISTRY 6, 2032 (1967).

2.

S. Beychok and E. A. Kabat, BIOCHEMISTRY 4, 2565 (1965).

3.

M. Matsui and D. K. Fukushima, BIOCHEMISTRY 8, 2997 (1969).

4.

G. Sauer, M. Matsui, R. Bloch, J. S. Liang and D. K. Fukushima, J. ORG. CHEM., in press.

.

F. Zilliken, C. S. Rose, G. A. Braun and P. Gyorgy, ARCH. BIOCHEM. BIOPHYS. 54, 392 (1955).

.

I. Listowsky, G. Av:.gad and S. Englard, CARBOHYDRATE RES. 8, 205 (1968).

.

E. A. Kabat, K. O. Lloyd and S. Beychok, BIOCHEMISTRY 8, 747 (1969).

657