388
METHODS FOR THE STUDY OF ANTIBIOTICS
[19]
p h y 15,19,2° w i t h s e n s i t i v i t y a t t h e 1 p p m level. D P P y i e l d s a w e l l - s h a p e d curve for I p p m c e p h a l o g l y c i n ( p r e v i o u s l y u n r e p o r t e d ) in 1 M H2S04 (Fig. 11) w i t h a l i n e a r p e a k c u r r e n t c o n c e n t r a t i o n r e l a t i o n (Fig. 12).
Acknowledgments The assistance of Lonel Brown, Jose Chang, and Marie Lefevre in obtaining some of the experimental data is gratefully acknowledged. ~9I. F. Jones, J. E. Page, and C. T. Rhodes, J. Pharm. Pharmacol., 20, 455 (1968); Anal. Abstr., 18, 2729 (1970). 2oD. A. Hall, J. Pharm. Sci. 62, 980 (1973).
[19] Proton Magnetic Resonance Spectroscopy of Antibiotics By GEORGE SLOMP I. II. III. IV.
Introduction . . . . . . . . . . . . . . . . . . Applicability . . . . . . . . . . . . . . . . . . Instrumentation and Sample Requirements . . . . . . . . . Interpreting the Spectrum . . . . . . . . . . . . . . A. Area Measurement . . . . . . . . . . . . . . . ]3. Spectral Analysis . . . . . . . . . . . . . . . . C. Chemical Shift Measurement . . . . . . . . . . . . V. Proposing a Structure . . . . . . . . . . . . . . . . A. Interpreting the Shifts . . . . . . . . . . . . . . B. Interpreting the Coupling Constants . . . . . . . . . . C. Trial Structures . . . . . . . . . . . . . . . . VI. Further Studies . . . . . . . . . . . . . . . . . A. Changing the Spectrum . . . . . . . . . . . . . . B. Changing the Sample . . . . . . . . . . . . . . .
388 390 392 394 394 395 396 396 397 398 400 400 400 403
I. I n t r o d u c t i o n P r o t o n m a g n e t i c r e s o n a n c e s p e c t r o s c o p y ( P M R ) of a n t i b i o t i c s is a specific a p p l i c a t i o n of t h e general n u c l e a r m a g n e t i c r e s o n a n c e ( N M R ) technique. T h e g r e a t e s t u t i l i t y of t h i s r e l a t i v e l y new f o r m of s p e c t r o s c o p y is in t h e a r e a of m o l e c u l a r s t r u c t u r e d e t e r m i n a t i o n , where it c o m p l e m e n t s some of t h e m o r e t r a d i t i o n a l m e t h o d s b u t d i s p l a c e s some others. T h e p r i n ciple is simple, b u t t h e i n s t r u m e n t a t i o n is c o m p l i c a t e d a n d expensive. T h e t e c h n i q u e is still u n d e r g o i n g r a p i d d e v e l o p m e n t , m o s t l y to i n c r e a s e its s e n s i t i v i t y . I t s a p p l i c a b i l i t y v a r i e s w i t h t h e size a n d n a t u r e of t h e u n k n o w n a n t i b i o t i c . I f t h e r e s u l t i n g s p e c t r a a r e difficult to a n a l y z e t h e y
[19]
P M R SPECTROSCOPY OF ANTIBIOTICS
389
can usually be manipulated to make them simpler, and several additional procedures are available when the interpretation is stalled. P M R spectroscopy is frequently employed in the study of time-dependent phenomena, such as the motions and interactions of large molecules of biological importance. 1,2 It also has quantitative applications, but its most widespread use is in the determination of molecular structure. It gives such minute structural detail that it is best used with more coarse methods like infrared and ultraviolet spectroscopy for detecting functional groups and characterizing unsaturation, or with mass spectrometry for molecular formula and fragment analysis. Since the integration of these analytical tools into a structure team most of the old fashioned degradations and chemical tests have been eliminated from structure proofs. The P M R method uses hydrogens as a probe for the molecular structure. It gives information on the environment around the hydrogens (i.e., what functional groups they are in), it tells which hydrogens are nearby each other (how the groups are connected), and it gives angular information (stereochemistry) on hydrogens which are vicinal, vinylic, or allylic. From this the rest of the structure can often be filled in. P M R is frequently augmented with the N M R of other nuclei, notably 13C. The two complement each other. Because the energy differences that are measured in this form of spectroscopy are so tiny (only slightly larger than thermal energy) and the whole radiofrequency spectrum which results is so narrow (the resolution must be in parts per billion) the instruments are quite costly. Even so, most laboratories are equipped for N M R studies at either 60, 100, or 220 MHz. Some commercial laboratories measure N M R spectra as a service. Although inherently an insensitive technique, compared to others, its great utility has generated many improvements. Recent advances based on relaxation (nuclear Overhauser effect3), complexation (shift reagents*), and pulsing (Fourier transform technique .r) help where the method was inherently weak previously. I~This series, Vol. 26, Section VII. D. W. Urry and M. Ohnishi, in "Spectroscopic Approaches to Biomolecular Conformation" (D. W. Urry, ed.), Chap. VII. American Medical Association, Chicago, Illinois, 1970. 3j. H. Noggle and R. E. Schirmer, "The Nuclear Overhauser Effect." Academic Press, New York, 1971. 4R. von Ammon and R. D. Fischer, Angew. Chem., Int. Ed. Engl. 11, 675 (1972). T. C. Farrar and E. D. Becker, "Pulse and Fourier Transform NMR." Academic Press, New York, 1971.
390
METHODS FOR THE STUDY OF ANTIBIOTICS
[19]
The reader is referred to other books, 6-1° reviews, 1~-13 and an introductory article TM for the theory of N M R , chemical shift, spin-spin coupling, double resonance, relaxation, etc., and for directions on how to obtain the best spectra. I t is intended here to discuss when and how to use P M R for molecular structure determination of antibiotics (as well as other organic molecules), what determinations to undertake or ask for, what size sample to provide, and especially how to interpret the data. 0 n l y recent or key references are cited, leaving it to the investigator to trace b a c k to the original contributions if he so desires.
I I . Applicability Success of the method depends greatly on the size of the molecule and the nature of the sample. Both influence the ability to analyze and interpret the N M R spectrum. Even when the spectrum cannot be completely "factored" much useful structural information can still be obtained. The analysis gets more difficult with larger molecules, having m a n y hydrogens, because of crowding and overlapping of the N M R signals. The best results have been on molecules of moderate size, up to about twenty nonmethyl hydrogens. Figure 1 shows the P M R spectrum which, together with MS, IR, and UV data, allowed a structure to be assigned to geldanamycin (I) 15 having 19 nonmethyl hydrogens, only 4 of which could not be analyzed. Larger molecules should be cleaved and studied in parts, as was done with lincomycin, TM streptolydigin, ~7 spectinomycin, TM and streptovaricin, TM 6j. W. Emsley, J. Feeney, and L. H. Sutcliffe, "High Resolution Nuclear Magnetic Resonance." Vols. 1 and 2. Pergamon, Oxford, 1965. 7L. M. Jackman and S. Sternhell, "Applications of Nuclear Magnetic Resonance Spectroscopy in Organic Chemistry." 2rid ed. Pergamon, Oxford, 1969. 8 E. D. Becker, "High Resolution NMR. Theory and Chemical Applications." Academic Press, New York, 1969. ~F. A. Bovey, "Nuclear Magnetic Resonance Spectroscopy." Academic Press, New York, 1969. 10N. Muller, in "Techniques of Chemistry" (A. Weissberger, ed.,), Vol. 1, Part IIIA, Chap. VII. Wiley (Interscience), New York, 1972. 11R. C. Ferguson and W. D. Phillips, Science 157, 257 (1967). 12 p. L. Corio, S. L. Smith, and J. R. Wasson, Anal. Chem. 44, 407R (,1972). la W. Mc.Farlane, Chem. Brit. 5, 142 (1969). 1~F. A. Bovey, Chem. Eng. News 43, 98 (1965). 15K. Sasaki, K. L. Rinehart, Jr., G. Slomp, M. F. Grostic, and E. C. Olson, J. Amer. Chem. Soc. 92, 7591 (1970). 1~G. Slomp and F. A. MacKellar, J. Amer. Chem. Soc. 89, 2454 (1967). 1TK. L. Rinehart, Jr., J. R. Beck, D. B. Borders, W. W. Epstein, T. H. Kinstle, L. D. Spicer, D. Krauss, and A. C. Button, Antimicrob. Ag. Chemother. 1963, 346 (1963).
[19]
P M R SPECTROSCOPY OF ANTIBIOTICS
391 s,
I H~O
u II
1
I
'
i
'
I
'
1
~'
t
M ]
(~
'
,o,
0 [
NO v~
i
r
II
B
/
H3
i r
R3.D
,
G
C
oJ~ I:
a=C
s3
'~3
F~
/'
![
CHF
v~*3
I1
~ J
~i ~!i~ Ti!'
-f,,
•
i
I
!"
[i
I : .... I0
I 9
,
[
, 8
I
7
.
I
6
..... I ......... I . . . . . . . . I . . . . . . . . . . . . . 5
6 , ppm
4
3
I............. I
from T M S
FIG. 1. Proton magnetic resonance spectrum obtained from a solution of geldanamycin acetate in deuterochloroform at 100 MHz.
for example. Cleavage or derivatization may also be necessary when large numbers of similar hydrogens are present, e.g., polysaccharides like neomycin, s° polyamino acids, ~ polyenes, :1 etc., since overlapping in one area would obscure the individual signals. Very large molecules like crythromycin, gramicidin, pimaricin, and novobiocin do not tumble well iH solution and therefore display broad signals that are poorly resolved and of little use in structure determination. On the other hand, molecules with relatively few hydrogens are difficult to analyze by P M R if they leave large gaps in the molecule in which there are no hydrogen probes. These are better studied by ~'~C N M R Y v-':~ P M R is most useful on molecules with continuous strings of neighboring hydrogens where their couplings can be used to identify the structure, working down a chain or around a ring. It also helps to know some history of the sample or other characteristics of the sample which give clues to what type of antibiotic to expect. 1~G. Slomp and F. A. MacKellar, Tetrahedron Lett. 521 (1962). ~' K. L. Rinehart, Jr., M. L. Maheshwari, F. J. Antosz, H. H. Mathur, K. Sasaki, and R. J. Schacht, J. Amer. Chem. Soc. 93, 6273 (1971). 2o M. Hichens and K. L. Rinehart, Jr., J. Amer. Chem. Soc. 85, 1547 (1963). 51R. C. Pandey, V. F. German, Y. Nishikawa, and K. L. Rinehart, Jr., J. Amer. Chem. Soc. 93, 3738 (1971). := J. B. Stothers, "Carbon-13 N M R Spectroscopy." Academic Press, N e w York, 1972. "~G. C. Levy and G. L. Nelson, "Carbon-13 Nuclear Magnetic Resonance for Organic Chemists." Wiley (Interscience), N e w York, 1972.
392
METHODS FOR T H E STUDY OF ANTIBIOTICS
[19]
TABLE I CONVENIENT SAMPLE SIZE FOR
PMR SPECTROSCOPY
Instrument type
Amount
Amount/400 mol. wt.
56-60 MHz 90-100 MHz 100 MHz, CW 100 MHz, FT 220-300 MHz, FT
12 ~moles 525 ~moles 1.25 ~moles 125 nmoles 50 nmoles
50 mg 10 mg 500 ~g 50 ~g 20 ~g
Unknown impurities should not be present in amounts greater t h a n about 5% lest an unrecognized signal from an impurity cause an error in the structure assignment. Signals from known impurities can be subtracted out of the spectrum. Binary mixtures are about four times more difficult to analyze t h a n are either of the components separately. Enantiomers are not distinguished except under special conditions using chiral solvents 24 or chiral complexing agents. 25 Presence of radicals, paramagnetic metal atoms or iron filings in the sample ruins the resolution and aborts the analysis.
III. Instrumentation and Sample Requirements M a n y kinds of N M R spectrometers are available commercially. 26 Most laboratories use a 90-100 M H z instrument for research. Some use 220-300 M H z superconducting spectrometers because sensitivity and resolution both improve as the frequency and magnetic field strength increase. Computerized spectrometers which add either spectra (CW) or pulsed interference patterns 5 (FT) to overcome noise increase the sensitivity by about 10- to 100-fold. Some instruments 2~ use a larger cell for samples of limited solubility. Sample requirements depend somewhat on the sensitivity of the particular m a k e of instrument and a lot on the L a r m o r frequency. A listing of convenient sample sizes is shown in T a b l e I. Often smaller amounts can be studied with extra care. Micro cells, which restrict the sample to the most closely coupled space, can be used, but they cause some loss of resolution and therefore are not recommended. W. H. Pirkle, R. L. Muntz, and I. C. Paul, J. Amer. Chem. Soc. 93, 2817 (1971). 25R. R. Fraser, J. B. Stothers, and C. T. Tan, J. Mag. Res. 10, 95 (1973). 26Anal. Chem. (Laboratory Guide issue) 45, 214 LG (1973). 27A. Allerhand, R. F. Childers, R. A. Goodman, E. Oldfield, and X. Ysern, Amer. Lab. 4 (11), 19 (Nov. 1972).
[19]
P M R SPECTROSCOPY OF ANTIBIOTICS
393
For P M R spectroscopy the sample should be a neat liquid or a concentrated solution of the recommended amount in enough solvent to make about 0.3 to 0.4 ml of solution. This makes the solvent requirement a rather stringent one. If proton-containing solvents are used they are usually deuterated to remove interference from the abundant solvent protons. Solvents with less than 0.5% of residual protons can be purchased and are acceptable. Each coupled deuterium (spin = 1) splits the residual proton signal into three lines of equal intensity. Two deuteriums give five lines with an intensity ratio of 1 : 2 : 3 : 2 : 1 owing to three overlaps in this triplet of triplets. The spectrum may v a r y slightly with solvent--especially the polar hydrogens which tend to associate. Often several solvents are used taking advantage of these shifts to uncover obscured lines. The most common solvent--the one to which most tabulations of shifts refer--is deuterochloroform. To give a suitable concentration in this solvent the samples must be of moderate molecular weight and must not be highly polar (not more than two or three hydroxyl groups). Polar hydrogens such as SH, NH, and especially OH exchange locations and therefore their collected signals often appear in the spectrum as a single [)road line at an averaged chemical shift. Sometimes it is so broad that it is undetected. The d-chloroform is easily evaporated, and the sample may be rerun in d6-benzene or d~-pyridine (having cone-shaped anisotropy effects) to scramble the lines. 28 Often this will uncover signals that were superimposed. A different scramble can then be obtained with d3-acetonitrile (rod-shaped anisotropy effects). For this reason the shift data in these solvents may not be reliable. For polar samples (most antibiotics d~,-dimethyl sulfoxide (DMSO) or dT-dimethyl formamide ( D M F ) are used. The latter, being less viscous, often gives sharper lines but has more residual proton absorptions to deal with. DMSO has a nice advantage; it binds water so tightly that exchange with OH or N H protons in the sample is prevented, and these can therefore be detected in the spectrum, coupled to any protons that were attached to the alpha carbon. :9 M a n y antibiotics will dissolve in deuterium oxide. These spectra should be calibrated with sodium 4,4-dimethyl-4-silapentane-l-sulfonate (DSS) ~° or sodium 3-trimethylsilyltetradeuteriopropionate (TSP) 31 as internal reference since tetramethyl silane (TMS) is not soluble in water. ~J. Ronayne and D. H. Williams, Annu. Rev. N M R Spectrosc. 2, 83 (1969). -~O. L. Chapman and R. W. King, J. Amer. Chem. Soc. 86, 1256 (1964). 30B. R. Donaldson and J. C. P. Schwarz, J. Chem. Soc. B 395 (1968). 31L. Pohl and M. Eckle, Angew. Chem. Int. Ed. Engl. 8, 381 (1969).
394
METHODS FOR THE STUDY OF ANTIBIOTICS
[19]
Exchange now produces a signal from the resulting HOD which could be large enough to obscure other absorptions. The difficulty can be minimized by several cycles of evaporation followed by addition of fresh D=,O. The final residual HOD can then be moved aside (upfield relative to the other signals) by observing the spectrum at a higher temperature revealing any signals that were hidden underneath it. Some samples can be made more soluble in D20 by the inclusion of some DC1 or NaOD to convert them to their salts. Carbamine hydrogens are shifted downfield by this treatment? 2 For some samples which are only moderately polar, d6-acetone or d~-methanol can be used in place of DMSO or DMF. When all else fails the sample can usually be dissolved in trifluoroacetic acid but the shift data are unreliable. Measuring the spectrum at elevated temperature also improves the solubility, but it lowers the sensitivity somewhat.
IV. Interpreting the Spectrum Three major types of information must be extracted from the spectrum before a molecular structure can be proposed for the sample. First, the relative area of each multiplet should be noted on the spectrum. The area tells how many hydrogens are represented by the multiplet. Next, the multiplets should be analyzed. These are the "pitchforks" on the spectrum in Fig. 1. The types and sizes (as measured by the coupling constant, J) of the multiplets tell how the hydrogens are arranged in the molecule. Last, the original absorption frequency (the so-called chemical shift) which results from the factoring should be obtained from the spectrum. The shift identifies the hydrogen type.
A. Area M e a s u r e m e n t
Areas can be measured with a planimeter, by cutting and weighing, or more easily from the step integral curve produced by the spectrometer. Relative areas can be determined by setting the total area equal to the number of hydrogens in the molecular formula, from mass spectrometry (MS) or elemental analysis, or by assigning a recognizable signal the appropriate number of hydrogens for calibration. If this is not satisfactory, a simple proton-containing derivative (acetate, methyl ester, tetramethylammonium salt, etc.) can be prepared, and the calibration can be taken from the area of the added proton signal. If adiacent multiplets come out as fractions, it usually means that partial overlapping is occurring. 3: G. Slomp and J. G. Lindberg, Anal. Chem. 39, 60 (1967).
[191
P M R SPECTROSCOPY OF ANTIBIOTICS
395
B. Spectral Analysis Factoring the multiplets is probably the most difficult part of the interpretation. It requires an understanding of spin-spin coupling and a foreknowledge of what the common multiplets look like in their various perturbations. The investigator is referred elsewhere 33-36 for a more comprehensive discussion of spin-spin coupling. The multiplets change as the strength of the coupling increases. They can be divided into two groups. First-order multiplets result from weakly coupled nuclei that have small couplings compared to the difference in their chemical shifts, J/Av ~ 0.15. These first-order multiples can be factored by inspection because they have approximately symmetrical patterns of N ~ 1 lines (where N is the number of coupled nuclei), and they are insensitive to the signs of the coupling constants. With practice considerable deviation from first-order appearance can be tolerated (J/±v up to 0.4) and the multiplets can still be recognized and factored. Second-order multiplets result from more strongly coupled nuclei, those which have large couplings compared to the difference in their chemical shifts, J/Av ~ 0.15. They give patterns which have more than N ~ 1 lines. These second-order multiplets can usually be recognized by their complexity and asymmetry. As J/Av increases the outside lines of the partners split, separate and move outward, diminishing in intensity. They are usually identified by the number and arrangement of the lines with reference to systematic plots of calculated spectra2 ,9 Like first-order multiplets, some of the line separations match up in the partners but these line separations, S, are rarely a measure of the coupling constant, J. For first approximations the line separations can be used in place of the coupling constants if an accuracy of ± 2 - 3 Hz can be tolerated. To measure the actual J values the multiplets must be analyzed mathematically per instructions in the literature 6 or occasionally they can be determined conveniently by construction methods27 The analysis can be checked by computing the spectrum 3s and if desired the accuracy of the J and ±v parameters can be improved by iteration, as 33E. W. Garbisch, J. Chem. Educ. 45, 311, 402, 480 (1968). 34R. J. Abraham, "Analysis of High Resolution Spectra." Elsevier, Amsterdam, 1971. ~J. N. Murrell, The theory of nuclear spin-spin coupling in high resolution NMR spectroscopy, in "Progress in NMR Spectroscopy" (J. W. Emsley, J. Feeney, and L. H. Sutcliffe, eds.), Vol. 6. Pergamon, Oxford, 1971. 3~p. Diehl, R. K. Harris, and R. G. Jones, in "Progress in NMR Spectroscopy" (J. W. Emsley, J. Feeney, and L. H. Sutcliffe, eds.), Vol. 3, Chap. 1. Pergamon Press, New York, 1967. 3, G. Slomp, Appl. Spectrosc. Rev. 2, 263 (1969). 38p. DieM, H. Kellerhals, and E. Lustig, Computer assistance in the analysis of high resolution NMR spectra, in "NMR" (P. Diehl, E. Fluck, and R. Kosfield, eds.), Vol. 6. Springer-Verlag, Berlin and New York, 1972.
396
METHODS FOR THE STUDY OF ANTIBIOTICS
[19]
First determine which multiplets are coupled partners by finding those with matching line separations using dividers. Check the assignment by noting the severity and direction of the intensity slant. In first-order multiplets the number of splittings must also match the partners' relative area. If there is any uncertainty in pairing the multiplets, it can be solved by decoupling 1~ them to see which ones are interacting and what pattern they decouple to. An I N D O R sweep is a convenient way to do this since it also locates buried partners. If the multiplet does not decouple to a singlet, additional partners should be sought. Next, factor the first-order multiplets by drawing in and labeling the pitchforks. Record the coupling constants, J, measured from the line separations then analyze the secondorder multiplets where possible. In the analysis be on the alert for deceptively simple spectra, virtual coupling, long-range coupling, and asymmetric nonequivalence. If there is difficulty in analyzing second-order multiplets they can usually be simplified by observing the spectrum at a higher Larmor frequency, which increases ±v relative to J. Switching solvents or adding a shift reagent may accomplish the same result. C. Chemical Shift Measurement
The frequency scale along the bottom of the spectrum is usually calibrated in Hz relative to internal TMS, a9 or a suitable substitute. ~°'31 For first-order multiplets the resonance frequency of the center of the multiplet is read from this scale. For second-order multiplets the resonance frequency is obtained from appropriate line frequencies by a mathematical, construction or computer analysis in accordance with instructions in the literature. If such analysis is not done, the resonance frequency may be approximated from the center of gravity of the multiplet. Since spectrometers operate at various Larmor frequencies~ the chemical shift is usually expressed in dimensionless units 8, which are common to all: ~ppm. = (robs. -- Vref.) X 106/Larmor frequency
where a positive value indicates the line was on the downfield side of TMS. V. Proposing a Structure
The results of the spectrum analysis are conveniently summarized in a diagram representing each hydrogen by a letter of the alphabet. The diagram for geldanamycin is shown in Fig. 2. 3~p. Laszlo, A. Speert, R. Ottinger, and J. Reissi, J. Chem. Phys. 48, 1732 (1968).
[19]
PMR
2.02
Ps
7.36
I ~1 A
6.54
B
SPECTROSCOPY OF ANTIBIOTICS 0.94
W3
2.47
6.5 3.0 5.12
11.5
397
N~ , ~ 5 0 ~ 0 ~
2.25
L
D
1.90 ~ Q - - ~ //~ 6.0 I I 9.5 ~3.6 I /z 1.11 V 3 I / I 4.38 G 3.51 / / " t0.0 I ~1 1.50 U ~ I 5.07 E 17 ~ / T ~ 1o9 FIG. 2. Analysis of the nuclear magnetic resonance spectrum of geldanamycin. Signals are labeled with a chemical shift, $, beside their symbol. Coupling constants, J, are shown beside the attaching lines. Couplings shown by the dashed lines were inferred. 5.79
C
1.76
1.0 S3
4.87 F 7.0 I
A. I n t e r p r e t i n g t h e Shifts T h e chemical shifts i d e n t i f y the h y d r o g e n type. A s s i g n m e n t is m a d e from e m p i r i c a l correlations u s i n g charts, 4°-44 tables, 45-49 a d d i t i v i t y rules, 5°-53 a n d model compounds. M a k e a list of p a r t i a l s t r u c t u r e s to fit each h y d r o g e n type, T a b l e II. "° K. Nukada, 0. Yamamoto, T. Suzuki, M. Takeuchi, and M. Ohnishi, Anal. Chem. 35, 1892 (1963). 41N. F. Chamberlain, "The Practice of NMR Spectroscopy with Spectra Structure Correlations for Hydrogen-l." Plenum, New York, 1974. 4~R. M. Silverstein and G. C. Bassler, "Spectrometric Identification of Organic Compounds," 2nd ed., p. 110. Wiley, New York, 1967. 4~O. Yamamoto, T. Suzuki, M. Yanagisawa, and K. Hayamizu, Anal. Chem. 40, 568 (1968). 4, N. F. Chamberlain, "Nuclear Magnetic Resonance Chemical Shifts of Oxygenated Unsaturated Aliphatics," Anal. Chem. 40, 1317 (1968). 4~N. S. Bhacca, D. P. Hollis, L. F. Johnson, E. A. Pier, and J. N. Shoolery, "NMR Spectra Catalog" Vols. 1 and 2. Varian Associates, Palo Alto, California, 1963. 46F. A. Bovey, "NMR Data Tables for Organic Compounds." Wiley, New York, 1967. *' W. Briigel, "Nuclear Magnetic Resonance Spectra and Chemical Structure," Vol. 1. Academic Press, New York, 1967. ,8 H. A. Szymanski and R. E. Yelin, "NMR Band Handbook." Plenum, New York, 1967. *9"Sadtler NMR Reference Spectra." Sadtler Research Laboratories, Philadelphia. Pennsylvania. 5oj. S. Martin and B. P. Dailey, J. Chem. Phys. 39, 1723 (1963). 51R. F. Zurcher, Helv. Chim. Acta 46, 2054 (1963). ~2A. I. Cohen and S. Rock, Steroids 3, 243 (1964). 5~U. E. Matter, C. Pascual, E. Pretsch, A. Pross, W. Simon, and S. Sternhell, Tetrahedron 25, 691, 2023 (1969).
398
METHODS FOR THE STUDY OF ANTIBIOTICS
[19]
TABLE II ABSORPTIONS IN THE SPECTRUM OF GELDANAMYCIN ACETATE
Shift, ~ Label 8.67 7.36 7.24 7.17 6.54 5.79 5.12 5.07 5.00 4.87 4.38 4.04 ~3.6 3.36 3.33 -~3.0 2.47 2.25 2.02 ~-~1.9 ~1.9 1.82 1.76 1.50 1.11 0.94 0.59
X A
Multiplet
Area Suggested structure
Singlet Doublet, S = 11.5 Singlet Singlet Doublet doublet, S = 11.5, 11.5 Doublet doublet, S = 9.5, 11.5 Broad doublet, S = 9.0, -~1 Broad singlet, S = ~ 1 Singlet Doublet doublet, S = 7.0, 4.0 Broad doublet, S = 9.5, ~ 1 Singlet Complex multiplet Singlet Singlet Complex multiplet Doublet doublet, J = -12.5, 7.0 Doublet doublet, J = -12.5, 5.8 Broad singlet, S = =o Z I"-o
R 1 = --C(CHs) s or C(CDs) 3 R~ = --C(CHs) s o r C(CDs) s or - - C F 2 - - C F 2 - C F 3 Rs = - - H o r - - D M = Eu, P r , Yb, o r other r a r e e a r t h
(m)
402
METHODS FOR THE STUDY OF ANTIBIOTICS
[19[
sample it complexes with electron-rich functions on the molecule (CO--NH > - - N H > --OH > C-~-O > - - 0 - - > --CN), and by a pseudocontact mechanism it changes the shielding (AS) of nearby protons in an amount depending on their distance (R) and angle of deviation from the coordination axis (8): A S = K [ ( 3 cos 2 0 - - 1 ) / R 3 ] , where K is a scalar constant for the particular experiment including the binding ability of the reagent, its concentration, etc. The shifts in the spectrum can be paramagnetic (downfield) or diamagnetic (upfield) depending on 0 and the metal of the reagent. The desired effect is to increase the separation of the signals in order to make them less overlapped and more firstorder. The method works best in nonpolar solvents and with substrates that have a dominant binding site and therefore does not always work
R = -- C ( C H s ) s o r -- C F 2 - C F z - - C F 3
(iv) on antibiotics. If a chiral shift reagent (IV) is used enantiomers shift differently and can therefore be distinguished because the complex is diasterotopic. ~5 Computer programs are available 6~ which test the molecular geometry of proposed structures by computing the agreement factor between the observed and calculated shifts. This procedure is very useful when choosing from more than one proposed structure. A subtle change in the spectrum brought on by the nuclear Overhauser effects can sometimes be used to confirm the close proximity of protons across space. The test depends on internuclear relaxation, not on spin coupling. A weak radiation is applied to a given proton at its resonance frequency which partially saturates that proton's resonance signal changing its Boltzmann distribution. If a second proton is nearby across space and therefore depends on the perturbed proton for its relaxation, it too will experience a disruption of its Boltzmann distribution with the result that its resonance intensity will grow as much as 50%. The effect falls off rapidly with distance (1/R 6) and is a specific test for hydrogens in an eclipsed 5- or 6-position. R. E. Davis, M. R. Willcott, III, R. E. Lenkinski, W. yon E. Doering, and L. Birladeanu, J. Amer. Chem. Soc. 95, 6846 (1973).
[19]
P M R SPECTROSCOPY OF ANTIBIOTICS
403
TABLE III SHIFTS RESULTING FROM ACETYLATION OF GELDAN:kMYCIN
Proton
Shift, A~
Proton
Shift, A~
A X M B C D E Y2 F G Ha I Ja
+0.38 +0.09 -0.14 -0.04 -0.06 --0.67 --0.13 +0.19 +1.31 +0.04 --0.07 ? 0.01
K3 L N O
+0.05 -0.16 +0.02 -0.11
P3
--0.02
Q
T Ra $3 U Va Wa
?
--0.14 Absent -0.01 -0.26 -0.19 --0.01
The use of higher Larmor frequency has already been noted, and the N M R of other nuclei, such as 13C or 15N, may provide the needed information to solve the P M R spectrum. In the case of geldanamycin acetate (II) the first spectra were observed at 60 MHz. Comparison of the 60 and 100 MHz spectra helped identify the multiplets.
B. Changing the Sample For antibiotics a very useful reaction is acetylation or carbamation of the sample. The alcohols and amines can be characterized from the resulting shifts noting that secondary carbino166 or carbamine 32 hydrogens shift downfield much more than primary ones. The acetyl methyls can usually be counted, and the polarity may be diminished enough to allow additional observations in a less polar solvent. The carbamation reaction can be carried out in the N M R cell by the addition of trichloroacetyl isocyanate reagentY In the geldanamycin example, the acetate (II) was prepared with acetic anhydride and pyridine and the shifts recorded in Table I I I helped identify the molecular structure. Other reactions such as ketone derivatization, hydrogenation, or borohydride reduction have been used to help interpret spectra. In the geldanamycin case the methanolysis product (V) was prepared from (I) with C. R. Narayanan and M. R. Sarma, Tetrahedron Lett. 1553 (1968). 8, V. W. Goodlett, Anal. Chem. 37, 431 (1965).
404
METHODS FOR THE STUDY OF ANTIBIOTICS
[20]
CHsO~H o
~/ HaC/~ C
"VO
~'~NH~ CHaO"
O CH3 CHa C ~
H
3
0
OH
~ OCONH2
(V) p o t a s s i u m c a r b o n a t e in refluxing m e t h a n o l - c h l o r o f o r m ( 1 : 1 ) . T h e P M R s p e c t r u m of (V) h a d the I, Q, a n d T m u l t i p l e t s u n c o v e r e d , a n d a large upfield shift of the a r o m a t i c p r o t o n signal (M) showed t h a t the a m i d e m u s t h a v e been a t t a c h e d ortho to it.
[20] The Use of 13C Labeling in the Study of Antibiotic Biosynthesis By NORBERT NEUSS I. II. III. IV.
Introduction . . . . . . . . . . . . . . . Instrumental Requirements . . . . . . . . . . . Satellite Method . . . . . . . . . . . . . . Experimental Conditions . . . . . . . . . . . . A. Preliminary 14C Experiment . . . . . . . . . . B. Natural Abundance CMR Spectrum . . . . . . . C. Selection of an Appropriate 13C-Enriched Precursor . . D. Consideration of Nuclear Overhauser Effect (NOE) . . E. Conditions of Labeling . . . . . . . . . . . V. CMR in Biosynthetic Studies of Antibiotics . . . . . . VI. Biosynthesis of fl-Lactam Antibiotics . . . . . . . . A. Synthesis of Model Compounds and 1'C Precursors . . B. Fermentation, Labeling, and Isolation . . . . . . . C. Recording of the Spectra . . . . . . . . . . . D. Assignments of Chemical Shifts in the CMR Spectra . . E. Determination of Incorporation Levels of 13C Precursors F. Discussion and Conclusions . . . . . . . . . .
. . . . . .
. . . . . .
. . . . . . . . . . . . . . .
. . . . . . . . . . . . . . . . . . . . . . . . . . . . .
404 405 406 406 407 407 407 407 407 408 410 411 411 414 415 417 418
I. I n t r o d u c t i o n T h e i n t r o d u c t i o n of a n t i b i o t i c s into the t h e r a p y of b a c t e r i a l i n f e c t i o n s p r o m p t e d chemical i n v e s t i g a t i o n s in m a n y l a b o r a t o r i e s t h r o u g h o u t the
METHODS FOR THE STUDY OF ANTIBIOTICS
[19]
p h y 15,19,2° w i t h s e n s i t i v i t y a t t h e 1 p p m level. D P P y i e l d s a w e l l - s h a p e d curve for I p p m c e p h a l o g l y c i n ( p r e v i o u s l y u n r e p o r t e d ) in 1 M H2S04 (Fig. 11) w i t h a l i n e a r p e a k c u r r e n t c o n c e n t r a t i o n r e l a t i o n (Fig. 12).
Acknowledgments The assistance of Lonel Brown, Jose Chang, and Marie Lefevre in obtaining some of the experimental data is gratefully acknowledged. ~9I. F. Jones, J. E. Page, and C. T. Rhodes, J. Pharm. Pharmacol., 20, 455 (1968); Anal. Abstr., 18, 2729 (1970). 2oD. A. Hall, J. Pharm. Sci. 62, 980 (1973).
[19] Proton Magnetic Resonance Spectroscopy of Antibiotics By GEORGE SLOMP I. II. III. IV.
Introduction . . . . . . . . . . . . . . . . . . Applicability . . . . . . . . . . . . . . . . . . Instrumentation and Sample Requirements . . . . . . . . . Interpreting the Spectrum . . . . . . . . . . . . . . A. Area Measurement . . . . . . . . . . . . . . . ]3. Spectral Analysis . . . . . . . . . . . . . . . . C. Chemical Shift Measurement . . . . . . . . . . . . V. Proposing a Structure . . . . . . . . . . . . . . . . A. Interpreting the Shifts . . . . . . . . . . . . . . B. Interpreting the Coupling Constants . . . . . . . . . . C. Trial Structures . . . . . . . . . . . . . . . . VI. Further Studies . . . . . . . . . . . . . . . . . A. Changing the Spectrum . . . . . . . . . . . . . . B. Changing the Sample . . . . . . . . . . . . . . .
388 390 392 394 394 395 396 396 397 398 400 400 400 403
I. I n t r o d u c t i o n P r o t o n m a g n e t i c r e s o n a n c e s p e c t r o s c o p y ( P M R ) of a n t i b i o t i c s is a specific a p p l i c a t i o n of t h e general n u c l e a r m a g n e t i c r e s o n a n c e ( N M R ) technique. T h e g r e a t e s t u t i l i t y of t h i s r e l a t i v e l y new f o r m of s p e c t r o s c o p y is in t h e a r e a of m o l e c u l a r s t r u c t u r e d e t e r m i n a t i o n , where it c o m p l e m e n t s some of t h e m o r e t r a d i t i o n a l m e t h o d s b u t d i s p l a c e s some others. T h e p r i n ciple is simple, b u t t h e i n s t r u m e n t a t i o n is c o m p l i c a t e d a n d expensive. T h e t e c h n i q u e is still u n d e r g o i n g r a p i d d e v e l o p m e n t , m o s t l y to i n c r e a s e its s e n s i t i v i t y . I t s a p p l i c a b i l i t y v a r i e s w i t h t h e size a n d n a t u r e of t h e u n k n o w n a n t i b i o t i c . I f t h e r e s u l t i n g s p e c t r a a r e difficult to a n a l y z e t h e y
[19]
P M R SPECTROSCOPY OF ANTIBIOTICS
389
can usually be manipulated to make them simpler, and several additional procedures are available when the interpretation is stalled. P M R spectroscopy is frequently employed in the study of time-dependent phenomena, such as the motions and interactions of large molecules of biological importance. 1,2 It also has quantitative applications, but its most widespread use is in the determination of molecular structure. It gives such minute structural detail that it is best used with more coarse methods like infrared and ultraviolet spectroscopy for detecting functional groups and characterizing unsaturation, or with mass spectrometry for molecular formula and fragment analysis. Since the integration of these analytical tools into a structure team most of the old fashioned degradations and chemical tests have been eliminated from structure proofs. The P M R method uses hydrogens as a probe for the molecular structure. It gives information on the environment around the hydrogens (i.e., what functional groups they are in), it tells which hydrogens are nearby each other (how the groups are connected), and it gives angular information (stereochemistry) on hydrogens which are vicinal, vinylic, or allylic. From this the rest of the structure can often be filled in. P M R is frequently augmented with the N M R of other nuclei, notably 13C. The two complement each other. Because the energy differences that are measured in this form of spectroscopy are so tiny (only slightly larger than thermal energy) and the whole radiofrequency spectrum which results is so narrow (the resolution must be in parts per billion) the instruments are quite costly. Even so, most laboratories are equipped for N M R studies at either 60, 100, or 220 MHz. Some commercial laboratories measure N M R spectra as a service. Although inherently an insensitive technique, compared to others, its great utility has generated many improvements. Recent advances based on relaxation (nuclear Overhauser effect3), complexation (shift reagents*), and pulsing (Fourier transform technique .r) help where the method was inherently weak previously. I~This series, Vol. 26, Section VII. D. W. Urry and M. Ohnishi, in "Spectroscopic Approaches to Biomolecular Conformation" (D. W. Urry, ed.), Chap. VII. American Medical Association, Chicago, Illinois, 1970. 3j. H. Noggle and R. E. Schirmer, "The Nuclear Overhauser Effect." Academic Press, New York, 1971. 4R. von Ammon and R. D. Fischer, Angew. Chem., Int. Ed. Engl. 11, 675 (1972). T. C. Farrar and E. D. Becker, "Pulse and Fourier Transform NMR." Academic Press, New York, 1971.
390
METHODS FOR THE STUDY OF ANTIBIOTICS
[19]
The reader is referred to other books, 6-1° reviews, 1~-13 and an introductory article TM for the theory of N M R , chemical shift, spin-spin coupling, double resonance, relaxation, etc., and for directions on how to obtain the best spectra. I t is intended here to discuss when and how to use P M R for molecular structure determination of antibiotics (as well as other organic molecules), what determinations to undertake or ask for, what size sample to provide, and especially how to interpret the data. 0 n l y recent or key references are cited, leaving it to the investigator to trace b a c k to the original contributions if he so desires.
I I . Applicability Success of the method depends greatly on the size of the molecule and the nature of the sample. Both influence the ability to analyze and interpret the N M R spectrum. Even when the spectrum cannot be completely "factored" much useful structural information can still be obtained. The analysis gets more difficult with larger molecules, having m a n y hydrogens, because of crowding and overlapping of the N M R signals. The best results have been on molecules of moderate size, up to about twenty nonmethyl hydrogens. Figure 1 shows the P M R spectrum which, together with MS, IR, and UV data, allowed a structure to be assigned to geldanamycin (I) 15 having 19 nonmethyl hydrogens, only 4 of which could not be analyzed. Larger molecules should be cleaved and studied in parts, as was done with lincomycin, TM streptolydigin, ~7 spectinomycin, TM and streptovaricin, TM 6j. W. Emsley, J. Feeney, and L. H. Sutcliffe, "High Resolution Nuclear Magnetic Resonance." Vols. 1 and 2. Pergamon, Oxford, 1965. 7L. M. Jackman and S. Sternhell, "Applications of Nuclear Magnetic Resonance Spectroscopy in Organic Chemistry." 2rid ed. Pergamon, Oxford, 1969. 8 E. D. Becker, "High Resolution NMR. Theory and Chemical Applications." Academic Press, New York, 1969. ~F. A. Bovey, "Nuclear Magnetic Resonance Spectroscopy." Academic Press, New York, 1969. 10N. Muller, in "Techniques of Chemistry" (A. Weissberger, ed.,), Vol. 1, Part IIIA, Chap. VII. Wiley (Interscience), New York, 1972. 11R. C. Ferguson and W. D. Phillips, Science 157, 257 (1967). 12 p. L. Corio, S. L. Smith, and J. R. Wasson, Anal. Chem. 44, 407R (,1972). la W. Mc.Farlane, Chem. Brit. 5, 142 (1969). 1~F. A. Bovey, Chem. Eng. News 43, 98 (1965). 15K. Sasaki, K. L. Rinehart, Jr., G. Slomp, M. F. Grostic, and E. C. Olson, J. Amer. Chem. Soc. 92, 7591 (1970). 1~G. Slomp and F. A. MacKellar, J. Amer. Chem. Soc. 89, 2454 (1967). 1TK. L. Rinehart, Jr., J. R. Beck, D. B. Borders, W. W. Epstein, T. H. Kinstle, L. D. Spicer, D. Krauss, and A. C. Button, Antimicrob. Ag. Chemother. 1963, 346 (1963).
[19]
P M R SPECTROSCOPY OF ANTIBIOTICS
391 s,
I H~O
u II
1
I
'
i
'
I
'
1
~'
t
M ]
(~
'
,o,
0 [
NO v~
i
r
II
B
/
H3
i r
R3.D
,
G
C
oJ~ I:
a=C
s3
'~3
F~
/'
![
CHF
v~*3
I1
~ J
~i ~!i~ Ti!'
-f,,
•
i
I
!"
[i
I : .... I0
I 9
,
[
, 8
I
7
.
I
6
..... I ......... I . . . . . . . . I . . . . . . . . . . . . . 5
6 , ppm
4
3
I............. I
from T M S
FIG. 1. Proton magnetic resonance spectrum obtained from a solution of geldanamycin acetate in deuterochloroform at 100 MHz.
for example. Cleavage or derivatization may also be necessary when large numbers of similar hydrogens are present, e.g., polysaccharides like neomycin, s° polyamino acids, ~ polyenes, :1 etc., since overlapping in one area would obscure the individual signals. Very large molecules like crythromycin, gramicidin, pimaricin, and novobiocin do not tumble well iH solution and therefore display broad signals that are poorly resolved and of little use in structure determination. On the other hand, molecules with relatively few hydrogens are difficult to analyze by P M R if they leave large gaps in the molecule in which there are no hydrogen probes. These are better studied by ~'~C N M R Y v-':~ P M R is most useful on molecules with continuous strings of neighboring hydrogens where their couplings can be used to identify the structure, working down a chain or around a ring. It also helps to know some history of the sample or other characteristics of the sample which give clues to what type of antibiotic to expect. 1~G. Slomp and F. A. MacKellar, Tetrahedron Lett. 521 (1962). ~' K. L. Rinehart, Jr., M. L. Maheshwari, F. J. Antosz, H. H. Mathur, K. Sasaki, and R. J. Schacht, J. Amer. Chem. Soc. 93, 6273 (1971). 2o M. Hichens and K. L. Rinehart, Jr., J. Amer. Chem. Soc. 85, 1547 (1963). 51R. C. Pandey, V. F. German, Y. Nishikawa, and K. L. Rinehart, Jr., J. Amer. Chem. Soc. 93, 3738 (1971). := J. B. Stothers, "Carbon-13 N M R Spectroscopy." Academic Press, N e w York, 1972. "~G. C. Levy and G. L. Nelson, "Carbon-13 Nuclear Magnetic Resonance for Organic Chemists." Wiley (Interscience), N e w York, 1972.
392
METHODS FOR T H E STUDY OF ANTIBIOTICS
[19]
TABLE I CONVENIENT SAMPLE SIZE FOR
PMR SPECTROSCOPY
Instrument type
Amount
Amount/400 mol. wt.
56-60 MHz 90-100 MHz 100 MHz, CW 100 MHz, FT 220-300 MHz, FT
12 ~moles 525 ~moles 1.25 ~moles 125 nmoles 50 nmoles
50 mg 10 mg 500 ~g 50 ~g 20 ~g
Unknown impurities should not be present in amounts greater t h a n about 5% lest an unrecognized signal from an impurity cause an error in the structure assignment. Signals from known impurities can be subtracted out of the spectrum. Binary mixtures are about four times more difficult to analyze t h a n are either of the components separately. Enantiomers are not distinguished except under special conditions using chiral solvents 24 or chiral complexing agents. 25 Presence of radicals, paramagnetic metal atoms or iron filings in the sample ruins the resolution and aborts the analysis.
III. Instrumentation and Sample Requirements M a n y kinds of N M R spectrometers are available commercially. 26 Most laboratories use a 90-100 M H z instrument for research. Some use 220-300 M H z superconducting spectrometers because sensitivity and resolution both improve as the frequency and magnetic field strength increase. Computerized spectrometers which add either spectra (CW) or pulsed interference patterns 5 (FT) to overcome noise increase the sensitivity by about 10- to 100-fold. Some instruments 2~ use a larger cell for samples of limited solubility. Sample requirements depend somewhat on the sensitivity of the particular m a k e of instrument and a lot on the L a r m o r frequency. A listing of convenient sample sizes is shown in T a b l e I. Often smaller amounts can be studied with extra care. Micro cells, which restrict the sample to the most closely coupled space, can be used, but they cause some loss of resolution and therefore are not recommended. W. H. Pirkle, R. L. Muntz, and I. C. Paul, J. Amer. Chem. Soc. 93, 2817 (1971). 25R. R. Fraser, J. B. Stothers, and C. T. Tan, J. Mag. Res. 10, 95 (1973). 26Anal. Chem. (Laboratory Guide issue) 45, 214 LG (1973). 27A. Allerhand, R. F. Childers, R. A. Goodman, E. Oldfield, and X. Ysern, Amer. Lab. 4 (11), 19 (Nov. 1972).
[19]
P M R SPECTROSCOPY OF ANTIBIOTICS
393
For P M R spectroscopy the sample should be a neat liquid or a concentrated solution of the recommended amount in enough solvent to make about 0.3 to 0.4 ml of solution. This makes the solvent requirement a rather stringent one. If proton-containing solvents are used they are usually deuterated to remove interference from the abundant solvent protons. Solvents with less than 0.5% of residual protons can be purchased and are acceptable. Each coupled deuterium (spin = 1) splits the residual proton signal into three lines of equal intensity. Two deuteriums give five lines with an intensity ratio of 1 : 2 : 3 : 2 : 1 owing to three overlaps in this triplet of triplets. The spectrum may v a r y slightly with solvent--especially the polar hydrogens which tend to associate. Often several solvents are used taking advantage of these shifts to uncover obscured lines. The most common solvent--the one to which most tabulations of shifts refer--is deuterochloroform. To give a suitable concentration in this solvent the samples must be of moderate molecular weight and must not be highly polar (not more than two or three hydroxyl groups). Polar hydrogens such as SH, NH, and especially OH exchange locations and therefore their collected signals often appear in the spectrum as a single [)road line at an averaged chemical shift. Sometimes it is so broad that it is undetected. The d-chloroform is easily evaporated, and the sample may be rerun in d6-benzene or d~-pyridine (having cone-shaped anisotropy effects) to scramble the lines. 28 Often this will uncover signals that were superimposed. A different scramble can then be obtained with d3-acetonitrile (rod-shaped anisotropy effects). For this reason the shift data in these solvents may not be reliable. For polar samples (most antibiotics d~,-dimethyl sulfoxide (DMSO) or dT-dimethyl formamide ( D M F ) are used. The latter, being less viscous, often gives sharper lines but has more residual proton absorptions to deal with. DMSO has a nice advantage; it binds water so tightly that exchange with OH or N H protons in the sample is prevented, and these can therefore be detected in the spectrum, coupled to any protons that were attached to the alpha carbon. :9 M a n y antibiotics will dissolve in deuterium oxide. These spectra should be calibrated with sodium 4,4-dimethyl-4-silapentane-l-sulfonate (DSS) ~° or sodium 3-trimethylsilyltetradeuteriopropionate (TSP) 31 as internal reference since tetramethyl silane (TMS) is not soluble in water. ~J. Ronayne and D. H. Williams, Annu. Rev. N M R Spectrosc. 2, 83 (1969). -~O. L. Chapman and R. W. King, J. Amer. Chem. Soc. 86, 1256 (1964). 30B. R. Donaldson and J. C. P. Schwarz, J. Chem. Soc. B 395 (1968). 31L. Pohl and M. Eckle, Angew. Chem. Int. Ed. Engl. 8, 381 (1969).
394
METHODS FOR THE STUDY OF ANTIBIOTICS
[19]
Exchange now produces a signal from the resulting HOD which could be large enough to obscure other absorptions. The difficulty can be minimized by several cycles of evaporation followed by addition of fresh D=,O. The final residual HOD can then be moved aside (upfield relative to the other signals) by observing the spectrum at a higher temperature revealing any signals that were hidden underneath it. Some samples can be made more soluble in D20 by the inclusion of some DC1 or NaOD to convert them to their salts. Carbamine hydrogens are shifted downfield by this treatment? 2 For some samples which are only moderately polar, d6-acetone or d~-methanol can be used in place of DMSO or DMF. When all else fails the sample can usually be dissolved in trifluoroacetic acid but the shift data are unreliable. Measuring the spectrum at elevated temperature also improves the solubility, but it lowers the sensitivity somewhat.
IV. Interpreting the Spectrum Three major types of information must be extracted from the spectrum before a molecular structure can be proposed for the sample. First, the relative area of each multiplet should be noted on the spectrum. The area tells how many hydrogens are represented by the multiplet. Next, the multiplets should be analyzed. These are the "pitchforks" on the spectrum in Fig. 1. The types and sizes (as measured by the coupling constant, J) of the multiplets tell how the hydrogens are arranged in the molecule. Last, the original absorption frequency (the so-called chemical shift) which results from the factoring should be obtained from the spectrum. The shift identifies the hydrogen type.
A. Area M e a s u r e m e n t
Areas can be measured with a planimeter, by cutting and weighing, or more easily from the step integral curve produced by the spectrometer. Relative areas can be determined by setting the total area equal to the number of hydrogens in the molecular formula, from mass spectrometry (MS) or elemental analysis, or by assigning a recognizable signal the appropriate number of hydrogens for calibration. If this is not satisfactory, a simple proton-containing derivative (acetate, methyl ester, tetramethylammonium salt, etc.) can be prepared, and the calibration can be taken from the area of the added proton signal. If adiacent multiplets come out as fractions, it usually means that partial overlapping is occurring. 3: G. Slomp and J. G. Lindberg, Anal. Chem. 39, 60 (1967).
[191
P M R SPECTROSCOPY OF ANTIBIOTICS
395
B. Spectral Analysis Factoring the multiplets is probably the most difficult part of the interpretation. It requires an understanding of spin-spin coupling and a foreknowledge of what the common multiplets look like in their various perturbations. The investigator is referred elsewhere 33-36 for a more comprehensive discussion of spin-spin coupling. The multiplets change as the strength of the coupling increases. They can be divided into two groups. First-order multiplets result from weakly coupled nuclei that have small couplings compared to the difference in their chemical shifts, J/Av ~ 0.15. These first-order multiples can be factored by inspection because they have approximately symmetrical patterns of N ~ 1 lines (where N is the number of coupled nuclei), and they are insensitive to the signs of the coupling constants. With practice considerable deviation from first-order appearance can be tolerated (J/±v up to 0.4) and the multiplets can still be recognized and factored. Second-order multiplets result from more strongly coupled nuclei, those which have large couplings compared to the difference in their chemical shifts, J/Av ~ 0.15. They give patterns which have more than N ~ 1 lines. These second-order multiplets can usually be recognized by their complexity and asymmetry. As J/Av increases the outside lines of the partners split, separate and move outward, diminishing in intensity. They are usually identified by the number and arrangement of the lines with reference to systematic plots of calculated spectra2 ,9 Like first-order multiplets, some of the line separations match up in the partners but these line separations, S, are rarely a measure of the coupling constant, J. For first approximations the line separations can be used in place of the coupling constants if an accuracy of ± 2 - 3 Hz can be tolerated. To measure the actual J values the multiplets must be analyzed mathematically per instructions in the literature 6 or occasionally they can be determined conveniently by construction methods27 The analysis can be checked by computing the spectrum 3s and if desired the accuracy of the J and ±v parameters can be improved by iteration, as 33E. W. Garbisch, J. Chem. Educ. 45, 311, 402, 480 (1968). 34R. J. Abraham, "Analysis of High Resolution Spectra." Elsevier, Amsterdam, 1971. ~J. N. Murrell, The theory of nuclear spin-spin coupling in high resolution NMR spectroscopy, in "Progress in NMR Spectroscopy" (J. W. Emsley, J. Feeney, and L. H. Sutcliffe, eds.), Vol. 6. Pergamon, Oxford, 1971. 3~p. Diehl, R. K. Harris, and R. G. Jones, in "Progress in NMR Spectroscopy" (J. W. Emsley, J. Feeney, and L. H. Sutcliffe, eds.), Vol. 3, Chap. 1. Pergamon Press, New York, 1967. 3, G. Slomp, Appl. Spectrosc. Rev. 2, 263 (1969). 38p. DieM, H. Kellerhals, and E. Lustig, Computer assistance in the analysis of high resolution NMR spectra, in "NMR" (P. Diehl, E. Fluck, and R. Kosfield, eds.), Vol. 6. Springer-Verlag, Berlin and New York, 1972.
396
METHODS FOR THE STUDY OF ANTIBIOTICS
[19]
First determine which multiplets are coupled partners by finding those with matching line separations using dividers. Check the assignment by noting the severity and direction of the intensity slant. In first-order multiplets the number of splittings must also match the partners' relative area. If there is any uncertainty in pairing the multiplets, it can be solved by decoupling 1~ them to see which ones are interacting and what pattern they decouple to. An I N D O R sweep is a convenient way to do this since it also locates buried partners. If the multiplet does not decouple to a singlet, additional partners should be sought. Next, factor the first-order multiplets by drawing in and labeling the pitchforks. Record the coupling constants, J, measured from the line separations then analyze the secondorder multiplets where possible. In the analysis be on the alert for deceptively simple spectra, virtual coupling, long-range coupling, and asymmetric nonequivalence. If there is difficulty in analyzing second-order multiplets they can usually be simplified by observing the spectrum at a higher Larmor frequency, which increases ±v relative to J. Switching solvents or adding a shift reagent may accomplish the same result. C. Chemical Shift Measurement
The frequency scale along the bottom of the spectrum is usually calibrated in Hz relative to internal TMS, a9 or a suitable substitute. ~°'31 For first-order multiplets the resonance frequency of the center of the multiplet is read from this scale. For second-order multiplets the resonance frequency is obtained from appropriate line frequencies by a mathematical, construction or computer analysis in accordance with instructions in the literature. If such analysis is not done, the resonance frequency may be approximated from the center of gravity of the multiplet. Since spectrometers operate at various Larmor frequencies~ the chemical shift is usually expressed in dimensionless units 8, which are common to all: ~ppm. = (robs. -- Vref.) X 106/Larmor frequency
where a positive value indicates the line was on the downfield side of TMS. V. Proposing a Structure
The results of the spectrum analysis are conveniently summarized in a diagram representing each hydrogen by a letter of the alphabet. The diagram for geldanamycin is shown in Fig. 2. 3~p. Laszlo, A. Speert, R. Ottinger, and J. Reissi, J. Chem. Phys. 48, 1732 (1968).
[19]
PMR
2.02
Ps
7.36
I ~1 A
6.54
B
SPECTROSCOPY OF ANTIBIOTICS 0.94
W3
2.47
6.5 3.0 5.12
11.5
397
N~ , ~ 5 0 ~ 0 ~
2.25
L
D
1.90 ~ Q - - ~ //~ 6.0 I I 9.5 ~3.6 I /z 1.11 V 3 I / I 4.38 G 3.51 / / " t0.0 I ~1 1.50 U ~ I 5.07 E 17 ~ / T ~ 1o9 FIG. 2. Analysis of the nuclear magnetic resonance spectrum of geldanamycin. Signals are labeled with a chemical shift, $, beside their symbol. Coupling constants, J, are shown beside the attaching lines. Couplings shown by the dashed lines were inferred. 5.79
C
1.76
1.0 S3
4.87 F 7.0 I
A. I n t e r p r e t i n g t h e Shifts T h e chemical shifts i d e n t i f y the h y d r o g e n type. A s s i g n m e n t is m a d e from e m p i r i c a l correlations u s i n g charts, 4°-44 tables, 45-49 a d d i t i v i t y rules, 5°-53 a n d model compounds. M a k e a list of p a r t i a l s t r u c t u r e s to fit each h y d r o g e n type, T a b l e II. "° K. Nukada, 0. Yamamoto, T. Suzuki, M. Takeuchi, and M. Ohnishi, Anal. Chem. 35, 1892 (1963). 41N. F. Chamberlain, "The Practice of NMR Spectroscopy with Spectra Structure Correlations for Hydrogen-l." Plenum, New York, 1974. 4~R. M. Silverstein and G. C. Bassler, "Spectrometric Identification of Organic Compounds," 2nd ed., p. 110. Wiley, New York, 1967. 4~O. Yamamoto, T. Suzuki, M. Yanagisawa, and K. Hayamizu, Anal. Chem. 40, 568 (1968). 4, N. F. Chamberlain, "Nuclear Magnetic Resonance Chemical Shifts of Oxygenated Unsaturated Aliphatics," Anal. Chem. 40, 1317 (1968). 4~N. S. Bhacca, D. P. Hollis, L. F. Johnson, E. A. Pier, and J. N. Shoolery, "NMR Spectra Catalog" Vols. 1 and 2. Varian Associates, Palo Alto, California, 1963. 46F. A. Bovey, "NMR Data Tables for Organic Compounds." Wiley, New York, 1967. *' W. Briigel, "Nuclear Magnetic Resonance Spectra and Chemical Structure," Vol. 1. Academic Press, New York, 1967. ,8 H. A. Szymanski and R. E. Yelin, "NMR Band Handbook." Plenum, New York, 1967. *9"Sadtler NMR Reference Spectra." Sadtler Research Laboratories, Philadelphia. Pennsylvania. 5oj. S. Martin and B. P. Dailey, J. Chem. Phys. 39, 1723 (1963). 51R. F. Zurcher, Helv. Chim. Acta 46, 2054 (1963). ~2A. I. Cohen and S. Rock, Steroids 3, 243 (1964). 5~U. E. Matter, C. Pascual, E. Pretsch, A. Pross, W. Simon, and S. Sternhell, Tetrahedron 25, 691, 2023 (1969).
398
METHODS FOR THE STUDY OF ANTIBIOTICS
[19]
TABLE II ABSORPTIONS IN THE SPECTRUM OF GELDANAMYCIN ACETATE
Shift, ~ Label 8.67 7.36 7.24 7.17 6.54 5.79 5.12 5.07 5.00 4.87 4.38 4.04 ~3.6 3.36 3.33 -~3.0 2.47 2.25 2.02 ~-~1.9 ~1.9 1.82 1.76 1.50 1.11 0.94 0.59
X A
Multiplet
Area Suggested structure
Singlet Doublet, S = 11.5 Singlet Singlet Doublet doublet, S = 11.5, 11.5 Doublet doublet, S = 9.5, 11.5 Broad doublet, S = 9.0, -~1 Broad singlet, S = ~ 1 Singlet Doublet doublet, S = 7.0, 4.0 Broad doublet, S = 9.5, ~ 1 Singlet Complex multiplet Singlet Singlet Complex multiplet Doublet doublet, J = -12.5, 7.0 Doublet doublet, J = -12.5, 5.8 Broad singlet, S = =o Z I"-o
R 1 = --C(CHs) s or C(CDs) 3 R~ = --C(CHs) s o r C(CDs) s or - - C F 2 - - C F 2 - C F 3 Rs = - - H o r - - D M = Eu, P r , Yb, o r other r a r e e a r t h
(m)
402
METHODS FOR THE STUDY OF ANTIBIOTICS
[19[
sample it complexes with electron-rich functions on the molecule (CO--NH > - - N H > --OH > C-~-O > - - 0 - - > --CN), and by a pseudocontact mechanism it changes the shielding (AS) of nearby protons in an amount depending on their distance (R) and angle of deviation from the coordination axis (8): A S = K [ ( 3 cos 2 0 - - 1 ) / R 3 ] , where K is a scalar constant for the particular experiment including the binding ability of the reagent, its concentration, etc. The shifts in the spectrum can be paramagnetic (downfield) or diamagnetic (upfield) depending on 0 and the metal of the reagent. The desired effect is to increase the separation of the signals in order to make them less overlapped and more firstorder. The method works best in nonpolar solvents and with substrates that have a dominant binding site and therefore does not always work
R = -- C ( C H s ) s o r -- C F 2 - C F z - - C F 3
(iv) on antibiotics. If a chiral shift reagent (IV) is used enantiomers shift differently and can therefore be distinguished because the complex is diasterotopic. ~5 Computer programs are available 6~ which test the molecular geometry of proposed structures by computing the agreement factor between the observed and calculated shifts. This procedure is very useful when choosing from more than one proposed structure. A subtle change in the spectrum brought on by the nuclear Overhauser effects can sometimes be used to confirm the close proximity of protons across space. The test depends on internuclear relaxation, not on spin coupling. A weak radiation is applied to a given proton at its resonance frequency which partially saturates that proton's resonance signal changing its Boltzmann distribution. If a second proton is nearby across space and therefore depends on the perturbed proton for its relaxation, it too will experience a disruption of its Boltzmann distribution with the result that its resonance intensity will grow as much as 50%. The effect falls off rapidly with distance (1/R 6) and is a specific test for hydrogens in an eclipsed 5- or 6-position. R. E. Davis, M. R. Willcott, III, R. E. Lenkinski, W. yon E. Doering, and L. Birladeanu, J. Amer. Chem. Soc. 95, 6846 (1973).
[19]
P M R SPECTROSCOPY OF ANTIBIOTICS
403
TABLE III SHIFTS RESULTING FROM ACETYLATION OF GELDAN:kMYCIN
Proton
Shift, A~
Proton
Shift, A~
A X M B C D E Y2 F G Ha I Ja
+0.38 +0.09 -0.14 -0.04 -0.06 --0.67 --0.13 +0.19 +1.31 +0.04 --0.07 ? 0.01
K3 L N O
+0.05 -0.16 +0.02 -0.11
P3
--0.02
Q
T Ra $3 U Va Wa
?
--0.14 Absent -0.01 -0.26 -0.19 --0.01
The use of higher Larmor frequency has already been noted, and the N M R of other nuclei, such as 13C or 15N, may provide the needed information to solve the P M R spectrum. In the case of geldanamycin acetate (II) the first spectra were observed at 60 MHz. Comparison of the 60 and 100 MHz spectra helped identify the multiplets.
B. Changing the Sample For antibiotics a very useful reaction is acetylation or carbamation of the sample. The alcohols and amines can be characterized from the resulting shifts noting that secondary carbino166 or carbamine 32 hydrogens shift downfield much more than primary ones. The acetyl methyls can usually be counted, and the polarity may be diminished enough to allow additional observations in a less polar solvent. The carbamation reaction can be carried out in the N M R cell by the addition of trichloroacetyl isocyanate reagentY In the geldanamycin example, the acetate (II) was prepared with acetic anhydride and pyridine and the shifts recorded in Table I I I helped identify the molecular structure. Other reactions such as ketone derivatization, hydrogenation, or borohydride reduction have been used to help interpret spectra. In the geldanamycin case the methanolysis product (V) was prepared from (I) with C. R. Narayanan and M. R. Sarma, Tetrahedron Lett. 1553 (1968). 8, V. W. Goodlett, Anal. Chem. 37, 431 (1965).
404
METHODS FOR THE STUDY OF ANTIBIOTICS
[20]
CHsO~H o
~/ HaC/~ C
"VO
~'~NH~ CHaO"
O CH3 CHa C ~
H
3
0
OH
~ OCONH2
(V) p o t a s s i u m c a r b o n a t e in refluxing m e t h a n o l - c h l o r o f o r m ( 1 : 1 ) . T h e P M R s p e c t r u m of (V) h a d the I, Q, a n d T m u l t i p l e t s u n c o v e r e d , a n d a large upfield shift of the a r o m a t i c p r o t o n signal (M) showed t h a t the a m i d e m u s t h a v e been a t t a c h e d ortho to it.
[20] The Use of 13C Labeling in the Study of Antibiotic Biosynthesis By NORBERT NEUSS I. II. III. IV.
Introduction . . . . . . . . . . . . . . . Instrumental Requirements . . . . . . . . . . . Satellite Method . . . . . . . . . . . . . . Experimental Conditions . . . . . . . . . . . . A. Preliminary 14C Experiment . . . . . . . . . . B. Natural Abundance CMR Spectrum . . . . . . . C. Selection of an Appropriate 13C-Enriched Precursor . . D. Consideration of Nuclear Overhauser Effect (NOE) . . E. Conditions of Labeling . . . . . . . . . . . V. CMR in Biosynthetic Studies of Antibiotics . . . . . . VI. Biosynthesis of fl-Lactam Antibiotics . . . . . . . . A. Synthesis of Model Compounds and 1'C Precursors . . B. Fermentation, Labeling, and Isolation . . . . . . . C. Recording of the Spectra . . . . . . . . . . . D. Assignments of Chemical Shifts in the CMR Spectra . . E. Determination of Incorporation Levels of 13C Precursors F. Discussion and Conclusions . . . . . . . . . .
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404 405 406 406 407 407 407 407 407 408 410 411 411 414 415 417 418
I. I n t r o d u c t i o n T h e i n t r o d u c t i o n of a n t i b i o t i c s into the t h e r a p y of b a c t e r i a l i n f e c t i o n s p r o m p t e d chemical i n v e s t i g a t i o n s in m a n y l a b o r a t o r i e s t h r o u g h o u t the
