LITERATURE CITED
Tab€e111. The Results of Factor Analysis of Seven Cyclohexane/Hexane Mixtures Mixture
W ,a 1
(a)
Cyclohexane Hexane
(from FA)
(reported)
1.00 0.88 0.81
1.00
6
0.34 0.17
0.00 0.10 0.19 0.53 0.76 0.87
0.16
0.92 0.83 0.55" 0.23 0.12
7
0.00
1.00
0.00
0.00
1 2 3 4 5
a
1.00 0.79 0.84
0.66
(1)J. J. Kankare, Anal. Chem., 42, 1322 (1970). (2)2. 2. Hugus, Jr., and A. A. El-Awady, J. Pbys. Chem., 75,2954(1971). (3)J. T. Bulmer and H. F. Shurvell, J. Phys. Chem., 77,256,2085(1973);Can. J. Chem., 53, 1251 (1975).
Mole fraction cyclohexane
0.55" 0.30
(4)
D. Macnaughtan, Jr., L. B. Rogers, and G. Wernimont, Anal. Chem., 44,
1421 (1972). (5)J. E. Davis, A. Shepard, N. Stanford, and L. B. Rogers, Anal. Chem., 46, 821 (1974). (6)W. H. Lawton and E. A. Sylvestre, Technometrics, 13,617 (1971). (7)E. A. Sylvestre, W. H. Lawton, and M. S.Maggio, Technometrics, 16,353 (1974). (8)G.L. Ritter, S. R. Lowry, T. L. Isenhour, and C. L. Wilklns, Anal. Chem., 48, 591 (1976). (9) P. H. Weiner, E. R. Malinowski, and A. R. Levinstone, J. Phys. Chem., 74, 4537 (1970). (IO)E. R. Malinowski, D.G. Howery, P. H. Weiner, J. M. Soroka, P. T. Funke,
Used to represent the standard solution.
R. B. Selzer, and A. Levinstone, "FACTANAL-Target Transformation Factor Analysis", Program 320,Quantum Chemistry Program Exchange, Indiana University, Bloomington, Ind., 1976.
crudely since the original purpose was to determine the number of components by FA and not the compositions. ACKNOWLEDGMENT
RECEIVEDfor review August 25, 1976. Accepted November 12, 1976. M. McCue was supported by a Robert Crooks Stanley Fellowship.
The authors extend their thanks to Harry Rozyn for his help in carrying out the computations.
Gas Chromatography-Mass Spectrometry Study of Acetylacetonyl Dipeptide Methyl Esters Hartmut Frank,'
K. D. Haegele,2 and D. M. Desiderio"
Institute for Lipid Research and Marrs McLean Depatiment of Biochemistry, Baylor College of Medicine, Houston, Texas, 77025
Reaction conditions for derlvatizationof dipeptides with acetylacetone are optimized. Compatible procedures for derivatization of trifunctional amino acids are developed. GC/MS properties of acetylacetonyl dipeptide methyl esters are investigated and found to be suitable for identificationof components In a complex mlxture of derlvattzed dipeptides. Unamblguous identificationof individual components is achieved for unresolved GC peaks.
Most amino acid sequences of natural proteins and peptides have been elucidated by Edman degradation ( I ) . In spite of these indisputable successes, some difficulties have been encountered in sequence determination of peptides isolated in amounts of less than 50 nmol(2). Each successive step has a yield of about 98% with the sequenator, and the amount of detectable amino acid derivative is gradually lowered while the background increases. Other problems may arise during extraction. Slight solubility of polypeptides in the organic solvent used in this step complicates analysis of amounts of less than 25 nmol. Solubility of oligopeptides in organic solvents may also cause loss of C-terminal peptide (2). Another elegant technique for protein sequence determination employs one of the dipeptidyl aminopeptidases (DAP) (3-5).DAP hydrolyzes a polypeptide into a set of dipeptides starting from the amino terminus to the carboxyl terminus or Present address, Chemisches Institut, Universitat Tubingen, Tubingen, West Germany. Present address, Clinical Pharmacology Program, University of Texas, Health Science Center, Departments of Pharmacology and Pathology, San Antonio, Texas 78284.
to any Pro, Lys, or Arg residues. If the latter are present, they are removed with one step of the Edman procedure. The Nterminal amino acid is removed by a chemical method and the resulting polypeptide is again subjected to enzymic digestion with DAP yielding a second, overlapping set of dipeptides. DAP I has been most widely employed. The method is suitable for sequence elucidation in combination with trypsin which selectively cleaves on the carboxyl side of Arg and Lys. These bonds are not hydrolyzed by DAP I. One potential drawback is the requirement for very efficient methods to separate the mixture into components and identify each dipeptide. In Edman degradation one deals with only 20 different molecular species and determination of Rf values distinguishes the 20 PTH-amino acids. Identification of all dipeptides in a mixture resulting from DAP digestion is a much more complicated task. The number of molecular species potentially present is high, and properties governing separation (molecular weight, hydrophilicity, ionic charge, conformation) are often similar for different dipeptides and complete separation can only be expected when the mixture contains a small number of dipeptides with different characteristics. Therefore Rf values or retention times are insufficient for identification of dipeptides resulting from DAP hydrolysis of a polypeptide. One of the least tedious, and at the same time most informative methods, is the combination of gas chromatography and mass spectrometry (GC-MS). Several parameters can be determined in one analysis: retention time, molecular weight, sequence-related fragmentation, and the amino acid sequence. Amino and carboxyl groups of dipeptides must be derivatized to make them suitable for GC. Perfluoroacyl derivatives
ANALYTICAL CHEMISTRY, VOL. 49, NO. 2, FEBRUARY 1977
287
Table I. Derivatization of AlaLeu with Acetylacetone and (except for TMAOH) Diazomethane Base
Yield=
N - Methylmorpholine Triethylamine Trimethylanilinium hydroxide Pyridine
100 95 66 5
24OC
65°C
A
0
1 p o l H-Ala-Leu-OH + MU1 0.1 N QNICH3bOH
t
1pl CH$OCH.$OCH)
I pmol WAla-Leu-OH + MpI 0.1 N QNlCH3)30H
+
311 CHJCOCH~COCHJ A
r
loo
lWC 0 0
0
Largest GC peak area set to 100.
of amino functions (5-7) and methyl ester formation of carboxyl groups (8) are most commonly used. A recent approach is pertrimethylsilylation of dipeptides (9). A third approach is reduction of N -acyl dipeptides to corresponding polyamino alcohols (10-12). This methodology has the advantage of reducing molecular weight while improving volatility. The method has been used successfully by one group (20-12). No detailed investigations concerning yield or side products of the reduction have been reported. Complete separation of all possible dipeptides is unlikely with any derivative. Therefore in an unresolved GC peak the mass spectrum of the employed derivative must permit positive identification of each constituent. Electron impact (EI) mass spectra of N-acetylacetonyl ( N -Aca) dipeptide esters (13, 14) have a base peak, representing in most cases the Nterminal amino acid, and a molecular ion of relatively high abundance (25),and fulfill this requirement satisfactorily. We are reporting on combined GC-MS analysis of N-Aca dipeptide esters. We investigated the course of derivatization procedure under different conditions, derivatization methods for dipeptides containing trifunctional amino acids, GC-MS properties of these derivatives, and their applicability in the analysis of dipeptide mixtures. EXPERIMENTAL
Gas Chromatography was performed on a Barber-ColmanGC equipped with a FID, gas flow 150 ml/min with 6-, 9-, or 12-footUshaped columns of 1%SE-30,1%OV-1, 1%OV-17, or 1%PZ-176, on Gas Chrom Q (100-120 mesh) at a temperature of 180 "C and upward. Gas Chromatography-Mass Spectrometry was performed on an LKB 9000 instrument (ionizing voltage 20 eV, ionizing current 60 FA, accelerating voltage = -3,5 kV, source temperature 290 "C) equipped with coiled columns with the phases mentioned before. Data reported will be those obtained on I% SE-30 columns because the peptides had the shortest retention times on this phase. For unresolved GC peaks, MS scans were taken every 20 s. Evaluation of Reaction Rate of Acetylacetone with Dipeptides. One pmol AlaLeu (404 pg) is dissolved in 20 pl 0.1 N trimethylanilinium hydroxide (TMAOH)(or the base in Table I) and 2 p1 of a 1 M solution of methyl stearate in methanol as internal standard are added. One or three fi1 of acetylacetone are added and the mixture is heated at given temperatures (Figure 1)over molecular sieve. A t given times an aliquot of about 10 nmol is injected on a 6-ft 1%SE-30 GC column at 180 O C . Derivatization of Dipeptides. The synthetic mixture of dipeptides is dissolved in 0.1 M N-methylmorpholine in dimethylformamide/methanol (1:l).Sufficient base is added to render the pH of the solution to about 9. Molecular sieve and 5 p1 acetylacetone per 100 p1 of solution (equivalentto 1-2 pmol dipeptide) are added and the mixture is heated to 100 "C for 2 h. The solution is cooled to room temperature, the base neutralized with an appropriate volume of 1 M formic acid in methylene chloride,and the solution blown to dryness with nitrogen. The residue is dissolved in 200 pl methanol and an excess of a solution of diazomethane in ether is added. After 30 min at room temperature, the solvent is evaporated, the residue dissolved in 30 pl pyridine and 15 pl hexamethyldisilazane and the mixture heated to 60 "C for 30 min. Aliquots of the mixture are analyzed by GC-MS. Simultaneous Methylation of Carboxyl and Hydroxyl Groups. Two pmol N-Aca dipeptide are dissolved in 200 pl methylene chloride, cooled to -10 O C and 10 mol % BFa-etherate in ether are added. Di288
------0
zw
1M R E A C T I O N TIME l m l n l
Flgure 1. Reaction rate of
2
1
"
, 300
AlaLeu with acetylacetone
, 350 INJECTOR TEMPERATURE T I OCI
, 400
Figure 2. Relative detector response as a function of injector temper-
ature azomethane in methylene chloride is added slowly until gas evolution ceases and the solution is kept at -10 'C for 10 min. RESULTS AND DISCUSSION One prerequisite for a derivatization method is completeness of the reaction (Figure 1).The influence of different parameters on the yield of the reaction between an a-amino group and acetylacetone was investigated. TMAOH was the base used. After 3 h at room temperature, a yield of only 20% is observed, while a t 100 "C reaction is virtually completed within 2 h. Data in Figure 1indicate that a three-fold increase of the acetylacetone concentration accelerates the reaction at room temperature, has no effect a t 60 "C, but has an adverse effect a t 100 "C. This phenomenon may be due to self-condensation of acetylacetone (16). Large differences in yield are noticed when different bases are used for N-derivatization (Table I). N- Methylmorpholine is superior compared to TMAOH. At elevated temperatures the reaction mixture with N-methylmorpholine remains colorless, whereas with TMAOH, decomposition products color the reaction mixture yellow. Esterification of the carboxyl group is effected with diazomethane except when TMAOH was used for pyrolytic esterification. Whether the lower yield in this case (66%, Table I) is due to incomplete Nor C-derivatization, or t o side reactions has not been investigated. Pyrolytic esterification is dependent on several factors. Increasing amounts of TMAOH result in lower yield. Detector response also depends on injector temperature (Figure 2), optimizing a t 330 "C. The use of TMAOH has similar shortcomings for probe mass spectrometry. Pyrolytic esterification requires 100-120 "C, a temperature too high for microdistillation of more volatile dipeptide esters. The large amount of N,N- dimethylaniline simultaneously released into the ion source reduces sensitivity. Hydroxyl groups are generally converted to trimethylsilyl ethers. Under the conditions employed in this reaction, free carboxyl groups are esterified. TMS esters of N-Aca dipep-
ANALYTICAL CHEMISTRY, VOL. 49, NO. 2, FEBRUARY 1977
a
TMS-ESTERS
b
M E T H Y L - ESTERS
2P
,
1
19
.. I 10
0
C I S O T H ---180°
I 0
MIN
190"
200"
210°
Figure 4. Gas chromatogram of a mixture of K A c a dipeptide methyl esters on a 12-ft column of 1 % SE-30 on Gas Chrom Q (100-120
I O MIN
Flgure 3. Gas chromatogram of a mixture of a) K A c a dipeptide TMS ester, b) K A c a dipeptide methyl ester of (1) Alalle, (2) Glylle, (3) Leulle
mesh)
(1)LeuAla, (2) Alalle, (3)LeuGly, (4) Glylle
tides have undesirable GC properties (Figure 3a) as compared to the methyl esters (Figure 3b). Therefore carboxyl groups must be esterified with diazomethane before hydroxyl groups are converted to TMS ethers. Carboxyl and hydroxyl groups are derivatized simultaneously with diazomethane in an aprotic solvent with a Lewis acid as catalyst ( I 7). Boron trifluoroetherate and tetrafluoroboric acid proved to be the most practical catalysts. Less practical are aluminum chloride, ferric chloride, and phenylboric acid; they are poorly soluble in organic solvents and/or their removal is tedious. This one-step procedure yields only one derivative for Tyr-containing dipeptides, while the two-step procedure always produces two peaks, representing the corresponding methyl ether and TMS ether. His-residues are methylated in the side chain, whereas this reaction is not complete in the two-step procedure. Arg residues are converted to Ns-2-(4,6-dimethyl)pyrimidylornithine residues (16)under the described derivatization conditiqns. Dipeptides with N-terminal Asp readily undergo an intramolecular ester condensation to the corresponding dehydropiperidine derivative. (See Scheme 1).
B-
P
-CH30H
SCHEME I
Therefore, with an unknown peptide the N -Aca derivative is formed, followed by esterification of the C-terminus. Trimethylsilylation derivatizes side chains. Arg is converted to
+
Ser(TMS)Gly, (5) Asp(0Me)Ala. (6) Leulle, (7) Ser(TMS)Leu,(8) MetGly, (9) ValGlu(OMe),(IO)ProVal, (1 1) LeuMet, (12) ProLeu Metlle, (13)GlyPhe, (14) Thr(TMS)Met, (15)PheAsp(OMe), (16) MetGlu(OMe), (17) Glu(OMe)Glu(OMe), (18)MetMet. (19) Tyr(TMS)Gly, (20) LeuTyr(TMS)
+
a pyrimidyl-0rn derivative during the first step. After these three derivatization steps, the mixture is subjected to GC analysis. It may seem disadvantageous to employ three steps, but this ensures that all residues are amenable to GC. For example, in the one-step TMS-derivatization procedure ( 9 ) , some dipeptides containing Asn, Gln, and His and all those with Arg do not elute. A typical GC separation of a mixture of N-Aca dipeptides is shown in Figure 4. An aliquot (1111) of a synthetic mixture of 22 dipeptides (10-20 nmol each) is injected on a 12-ft column of 1%SE-30. Complete resolution is not achieved because of the usual broad peak shape of dipeptide derivatives and the large number of components in the mixture. Some dipeptides have undesirable GC properties. Those containing His and Trp are considerably less volatile and produce broad, tailing peaks. Replacement of a methyl group by a trifluoromethyl group was assumed to reduce retention time and GC properties of trifluoroacetylacetonyl dipeptide methyl esters were investigated. Retention of these derivatives is indeed lowered 20-50 units on the Kovacs scale, but broad, tailing peaks are produced. Hexafluoroacetylacetonyl derivatives are strongly absorbed on the column. Dipeptides in an unresolved peak are identified by MS because of their simple, fragmentation pattern (Figure 5 ) . The most abundant fragment ion (usually 30-80% of the total ion current, 8)arises from cleavage between C" of the N-terminal amino acid and the carboxamide group and is denoted A+. This process may be favored by formation of a six-membered ring (pathway a). Further evidence for the fragmentation-
1TTE-NH..] M+'
r
* - - b- - CH3
R'/
AS
1 a
,
cH3qyR
CH3
+
230" T I T I
220'
1
CH3
R' +
'OC-NH
...
Figure 5. Fragmentation pattern of N-Aca dipeptide methyl esters ANALYTICAL CHEMISTRY, VOL. 49, NO. 2, FEBRUARY 1977
289
w
I
>
c
5
25-
E W
250
406
I.
I
I ,
M+
462 I
,
m ;
s
rn/e
TMS I-GLY-OME
flCR-TYR( b
100-
29.3
179
2-
c
2; z
75-
W
c
z
50-
>
14
307
227
W
A+
c
346
290
25-
M+
W
w
;
'
#
'
I
3
,
) I 1 # ' ' !
3
I
,/
I
,
,
,
I I
5
directing nature of the conjugated a-electron system is the fragment ion of low abundance arising from cleavage of the side chain of the N-terminal amino acid (pathway b). Other fragments of low abundance are m/e 43 (CH3CO+) and the frequently present ion at m/e 110 (CH3COCH=C(CH3)N=CH)+, Ions typical for individual amino acids are found in addition to those ions due to pathways a and b: m/e 70 (Pro); (M - 15)+, (M - 44)-+,and (M - go)-+in the case of TMS ethers of Ser, Thr and Tyr, respectively; nonetherified Ser and Thr do not change the mass spectra completely, but give rise to a set of ions at (M - 18).+and either (M - 30)-+ or (M - 44).+, respectively, for every fragment species containing the hydroxyl function. When Tyr, Trp, Met, His, or Arg are present, fragmentation is dependent upon whether they are in the N-terminal position (position 1)or the C-terminal position (position 2). In the mass spectrum of the LeuTyr(TMS) derivative (Figure 6a), A+ (m/e 168) comprises 70% 2 and is the base peak. When Tyr(TMS) is in position 1 (Figure 6b), the base peak (m/e 179) represents the side chain, while A+ ( m / e 290) has only 10-20% relative intensity (RI). Other intense ions are (M - 99)-+,( m / e 307, loss of CH&OCH=C(CH3)NH2) and (M - 179)+ ( m / e 227). Similarly, dipeptides with Met in position 2 yield mass spectra with A+ as the base peak (50-60% 8).When Met is in position 1,A+ or (A-CH3SH)+ may be the base peak (15-20% 8)but additional ions [(A - 18)+,(M - 30)-+, (M - 74).+ (McLafferty rearrangement)] complicate the spectrum. N L mmethyl-His and Trp-containing derivatives produce relatively intense molecular ions (20-5Wh RI). When Nim-methyl-Hisis in position 1, A+ is the base peak, while when in position 2, A+ and the C-terminal fragment (OC-His(Me) -OCH3)+ have similar relative intensities. Mass spectra of dipeptides containing Trp have a base peak at mle 130 (side chain), but when Trp is in position 1,ions at (M - 90)+ and (M - 129)+may be of similar abundance. Mass spectra of dipeptides with Asp(0Me) in position 1 contain a molecular ion 32 amu lower than expected. In most cases this ion is the base peak. No peak corresponding to A+ 290
406
374 1 1 ' s
,
I
I
,I
/ / / I
,
I
I
I
I
'
111t
I
I,
I
/I I
I
!
1
m
w" S
1
occurs, but an intense (M - 43)+ (up to 90% RI) is observed indicating that the molecular ion corresponds to the cyclic structure shown in Scheme 1. Dipeptides with Glu(0Me) in a - or y-linkage are readily distinguished. a-Glu dipeptides reveal the usual mass spectra with A+ (m/e 198,60% 2 ) as the base peak while the base peak of y-Glu dipeptides (m/e 184,6-10% 2 ) is 14 amu lower. In summary three different fragmentation patterns are observed 1)dipeptides containing Ala, Gly, Glu, Ile, Leu, Phe, Ser, Thr, or Val in position 1produce simple mass spectra with A+ as base peak and M.+ as second most abundant ion; 2) dipeptides containing Arg, Asn, Gln, His, Met, or Pro in position l produce mass spectra with A+ as base peak and one or more ions of high abundance originating from the side chain; 3) dipeptides containing Asp or Tyr in position 1,His in position 2, or Trp in either position produce mass spectra governed by ions characteristic for each particular amino acid. Because of these observations, mass spectra of dipeptides can be predicted qualitative!y. An unknown dipeptide is identified readily by 1)the base peak (Table 11)determining the N-terminal amino acid, 2) the molecular ion, 3) the difference in mass between M.+ and the base peak determining the C-terminal amino acid. The determination of the components of the unresolved GC peak 11/12 in Figure 4 is described as an example. When the peaks emerge, mass spectra are taken every 20 s and two spectra are shown in Figure 7. The most abundant peaks between m / e 107 and 228 (the mass range for all base peaks except for Asp(0Me) in position 1)are at m/e 138,152,168, and 186. Consequently dipeptides containing N-terminal Pro (152), Leu or Ile (168), and Met (138, 186) are present. The most abundant peaks in the range from m/e 228 (M.+ of GlyGly) to m/e 584 [Ma+ of Tyr(TMS)Tyr (TMS)] are at m/e 281, 284, 324, and 358. These peaks may represent the molecular ions of the following dipeptides: 281: AsnAla, AlaAsn, GlyGln, GlnGly 284: ValAla, AlaVal, GlyLeu(Ile), Leu(I1e)Gly
ANALYTICAL CHEMISTRY, VOL. 49, NO. 2, FEBRUARY 1977
-
168
Table 11. Assignment of Base Peaks
Base peak, amu 107
N-terminal amino acid
126 130 138 144
Tyr(Me) GlY Ala Trpa Metb Trp(Me)
170 179 184 186 192 198 202 206 214 228 M.+
Thr(Me) Tyr(TMS) r-Glu(OMe) Metb His a-Glu(0Me) Phe His(Me) Ser(TMS) Thr(TMS) Asp(OMe)d
112
186 152
-
152
168
186
138
a Also when Trp is C-terminal. Either ion may represent the base peak. Dehydration to the corresponding nitrile. Due to cyclization to the corresponding 2-methyl-3-acetyl-4-oxo-2-
324: ProLeu(Ile), Leu(I1e)Pro 358: ProPhe, PhePro, Thr(TMS)Ala, AlaThr(TMS), MetLeu(Ile), Leu(I1e)Met Since the amino acids in position 1 are Pro, Leu(Ile), and Met, six dipeptides remain (eleven if one counts the Ile-containing sequences): 284. Leu(I1e)Gly 324: ProLeu(Ile), Leu(I1e)Pro 358: ProPhe, MetLeu(Ile), Leu(I1e)Met Furthermore, two dipeptides can be deleted from this list because of their retention times which are different from that of peak 11/12: Leu Gly, (peak 3 in Figure 4) and ProPhe. The latter must have a longer retention time, indicated by the higher sum of the corresponding retention index increments (18). The list is now reduced to the dipeptides ProLeu(Ile), Leu(Ile)Pro, MetLeu(Ile), and Leu(1le)Met. ProLeu(I1e) and MetLeu(I1e) must be components of the mixture since they are the only remaining dipeptides which can contribute the respective A-fragments a t m/e 152 and 186. The last question is whether both Leu(I1e)Pro and Leu(I1e)Met are present or only one of them. Because the intensity of m/e 168 (A+ of Leu) decreases while the intensity of mle 324 (Ma+ of LeuPro) increases, these two ions cannot arise from the same compound. Therefore LeuPro is not present. Peak 11/12 consists of ProLeu(Ile), MetLeu(Ile), and Leu(I1e)Met. The peaks a t m/e 281 and 284 are fragment ions, the former, (324 - 43)' arising from loss of the acetyl moiety from N-
-lm 1D
,40 ,5D
lBD
170
180
~~
terminal Pro, and the latter, (358 - 74).+, from loss of vinyl thioether from N-terminal Met* LITERATURE CITED (1) P. Edman and G. Begg, Eur. J. Biochem., 1, 80 (1967). (2) H. D. Niall, "Peptides: Chemistry, Structure, Biology", R. Walter and J. Meienhofer, Ed., Ann Arbor Science Publishers, Inc., Ann Arbor, Mich., 1975, pp 975-984. (3) J. K. McDonald, P. X. Callahan, and S. Ellis, Methodshzymoi., 25,272-298 (1972). (4) Y. A. Ovchininkov and A. A. Kiryushkin, FEBSLett., 21, 300 (1972). (5) R. M. Caprioli, W. E. Seifert, and D. E. Sutherland, Biochem. Biophys. Res. Commun., 55, 67 (1973). (6) F. Weygand, Fresenius' 2. Anal. Chem., 205, 406 (1964). (7) B. A. Anderson, Acta Chem. Scand., 21, 2906 (1967). (8) A. L. Chibnall, J. L. Manzan, and M. W. Rees, Biochem. J., 68, 114 (1950). (9) H. C. Koutzsch and J. J. Pisano, Ref. 2, pp 985-990. (10) H. Nau, Biocbem. Biophys. Res. Commun., 59, 1088 (1974). (11) G. M. Hass, H. Nau, K. Biemann, D. T. Grahn, L. H. Ericsson, and H. Neurath, Biochemistry, 14, 1334 (1975). (12) G. Hudson and K. Biemann, Biochem. Biophys. Res. Commun., 71, 212 (1976). (13) G. M. Schier, B. Halpern, and G. W. A. Milne, Biomed. Mass Spectrom., 1, 21 (1974). (14) G. M. Schier, P. D. Bolton, and B. Halpern, Biomed. Mass Spectrom., 3, 32 (1976). (15) R. A. Day, H. Falter, J. P. Lehmann, and R. E. Hamilton, J. Org. Chem., 38, 782 (1973). (16) H. Vetter-Diechti, W. \letter, W. Richter, and K. Biemann, Experientia, 24, 340 (1968). (17) E. Muller, M. Bauer, and W. Rundei, TetrahedronLett., 1961, 136. (18) H. Nau, H. T. Foster, J. A. Kelley, and K. Biemann, Biomed. Mass Spectrom., 2, 326 (1975).
RECEIVEDfor review June 24, 1976. Accepted November 1, 1976. This work has been supported by Grant GM-13901 of the National Institutes of Health.
ANALYTICAL CHEMISTRY, VOL. 49, NO. 2, FEBRUARY 1977
291
