Method of C-terminal peptide sequencing

A method of C-terminal peptide sequencing. The peptide is reacted with a mixed anhydride of isothiocyanic acid and a carboxylic, carbonic, or sulfonic acid, under basic conditions, to produce a C-terminal peptidyl thiohydantoin. The C-terminal amino acid can be identified by cleaving the thiohydantoin from the peptide and identifying the free amino acid thiohydantoin.

FIELD OF THE INVENTION 
The present invention relates to a method for determining the C-terminal 
amino acid of a peptide, and for sequencing a peptide from its C-terminal 
peptide end, and to a reagent useful in such methods. 
REFERENCES 
Cromwell, L. D., et al., Biochemistry 8:4735 (1969). 
Edman, P., Acta Chem Scand, 4:277 (1950). 
Hawke, D. H., et al, Anal Biochem, 166:298 (1987). 
Kenner, G. W., et al., J Chem Soc, 673 (1953). 
Meuth, J. L., et al., Biochemistry, 16:3750 (1982). 
Miller, M. J., et al, J Org Chem, 42:1750 (1977). 
Miller, C. G., et al, in "Techniques in Protein Chemistry (Hugh, T. E., 
ed), Academic Press, pp. 67-78 (1989). 
Miller, C. G., et al., Abstract T188 from the Third Symposium of the 
Protein Society, Seattle Wash. (July 29-Aug. 2, 1989). 
Parham, M. E., et al, Biochem Biophys Res Commun, 80:7 (1978). 
Rangarajan, M, in "Protein/Peptide Sequence Analysis: Current 
Methodologies" (Bhown, A. S., ed), CRC Press, pp 136-144 (1988). 
Schlesinger, D. H., et al, Anal Biochem, 95:494 (1979). 
Shively, J. E., et al, TIBS 14:246 (1989). 
Stark, G. R., in Methods in Enzymology (Hirs, C. H. W., et al, eds.), Vol 
25, p 369 Academic Press (1972). 
Tarr, G. E., in "Methods in Protein Sequence Analysis", (Whittmann-Liebold, 
B., ed) Springer Verlag, pp. 129-151 (1988). 
BACKGROUND OF THE INVENTION 
Determining the amino acid sequence, i.e., primary structure, of a peptide 
is central to understanding the structure of the peptide, as well as to 
manipulating the peptide to achieve desired properties in a modified or 
altered form. In addition, the amino acid sequence of a peptide is useful 
in a variety of recombinant DNA procedures for identifying the gene coding 
sequence of the peptide, for producing the peptide recombinantly, and/or 
for producing site-specific modifications of the peptide. 
Early attempts to determine the amino acid sequence of peptides relied on 
acid hydrolysis of the peptide or enzymatic degradation to separate the 
peptide into its component amino acids. Both of these methods were slow 
and produced complicated mixtures of amino acids which then had to be 
separated for analysis. 
The development of reagents to sequence peptides by more systematic means 
greatly facilitated the determination of amino acid sequences. The most 
widely used method involves reacting the N-terminus of the peptide with 
phenyl isothiocyanate (PITC), a process known as Edman degradation 
(Edman). Reaction of PITC with the free terminal amino group adds a 
phenylthiourea group, which cyclizes to form a free 
anilinothiothiazolinone (ATZ) of the N-terminal amino acid, and a 
shortened peptide. The ATZ-derivative of the N-terminal amino acid is 
extracted, converted to the corresponding phenylthiothiohydantoin (PTH) 
which is then analyzed by HPLC. The amino-acid-PTH derivatives produced in 
the Edman reaction are racemized in the course of the Edman reaction, and 
thus the reaction cannot be used to distinguish L- and D-form amino acids. 
N-terminal sequencing is carried out by successively converting the next-in 
N-terminal amino acid to the free amino acid PTH, and identifying each 
successively released amino acid. The method is generally reliable for 
N-terminal sequences up to about 20-40 or more amino acid residues. 
Despite the relative ease and reliability of N-terminal sequencing methods, 
it is often desired to obtain C-terminal amino acid sequence information 
which may be inaccessible or only obtained with difficulty by this method. 
Information about the carboxy terminal sequence may be useful for certain 
types of recombinant DNA procedures, particularly since the C-terminal end 
of the coding region of a protein corresponds to the end closest to a poly 
A tail, which is likely to be present in cDNA clones. 
Three general approaches have been proposed for C-terminals peptide 
sequencing: enzymetic, physical, and chemical. The enzymatic strategy, 
which involves analyzing the products resulting from treatment of the 
peptide with carboxypeptidase over time, is limited by the difficulty of 
controlling the extent of carboxypeptidase cleavage. Typically, the 
identification of the next-in amino acid becomes difficult after 
3-5-residues have been cleaved. 
The most common physical tools used for C-terminal sequencing are fast atom 
bombardment mass spectrometry (FAB/MS), and nuclear magnetic resonance 
(NMR) spectroscopy. FAB/MS analysis is applicable to 1-10 nmole amounts of 
peptide, but requires expensive mass spectrometry equipment. NMR analysis 
requires relatively large amounts of peptides, typically in the .mu.molar 
range, and also involves relatively expensive equipment. 
In view of the limitations of enzymatic and physical approaches to 
C-terminal sequencing, considerable effort has been invested in developing 
chemical methods for determining C-terminal amino acids residues, and for 
C-terminal sequencing. An inherent difficulty in C-terminal sequencing is 
the relatively poor reactivity of the carboxyl group, in contrast to the 
relative ease of addition at the N-terminal amino group. Of the reaction 
methods which have been proposed for C-terminal sequencing, three have 
received special attention. 
The first activation method involves generating a carboxyamido derivative 
at the C-terminal end of the peptide, followed by reaction with 
bis(I,I-trifluoroacetoxy)iodobenzen to form a derivative which rearranges 
and hydrolyses to a shortened carboxyamidopeptide and the aldehyde 
derivative of the C-terminal amino acid (Parham). The method has been 
successfully carried out only to 3-6 cycles before the reaction halts. In 
a second, related approach, the carboxy terminus is reacted with 
pivaloylhydroxamide to effect a Lossen rearrangement. One limitation of 
the method is that the chemistry does not degrade aspartic and glutamic 
acid residues (Miller, 1977). 
The most widely studied of the C-terminal chemistries is the thiohydantoin 
(TH) reaction. In one general method for carrying out the TH method, the 
carboxyl group is activated with an anhydride, such as acetic anhydride, 
in the presence of an ITC salt or acid, to form a C-terminal peptidyl-TH 
via a C-terminal ITC intermediate (Stark, 1972). The peptidyl-TH can be 
cleaved to produce a shortened peptide and a C-terminal amino acid TH, 
which can be identified, e.g., by high pressure liquid chromatography 
(HPLC). The coupling conditions in this method typically require about 90 
minutes at 60.degree.-70.degree. C. (Meuth), and often lead to degradation 
of some of the amino acid side chains in the peptide. Further, the 
anhydride reagent is relatively unstable, and therefore presents storage 
problems. 
A C-terminal TH sequencing method which can be carried out under milder 
conditions has been described by the inventor and co-workers (Hawke). 
Using trimethylsilyl ITC (TMS-ITC) as the reagent, TH formation was 
achieved by activation of the peptide with acetic anhydride for 15 min at 
50.degree. C., followed by reaction with TMS-ITC for an additional 30 min 
at 50.degree. C. The method suffers from the disadvantage, noted above, of 
peptide exposure to a highly reactive anhydride activating agent. In 
addition, and like the related TH-generating methods described above, the 
TH-amino acid reaction products are racemized, and thus the method cannot 
be used to distinguish D- and L-form amino acids. 
The C-terminal sequencing methods involving TH formation just described 
have commonly lead to racemized products. A modification of the C-terminal 
reaction employing phosphoryl isothiocyanatidate reagent has been proposed 
(Kenner). Although TH was produced, the reaction was too slow to be very 
useful. Miller et al have proposed a related method, but using a 
mercaptobenzothiazole derivative. The rationale for using this compound is 
that cyclization could occur with concommitant opening of the thiazole 
ring. 
SUMMARY OF THE INVENTION 
It is one general object of the invention to provide an improved method of 
determining the C-terminal amino acid residue of a peptide. 
It is a more specific object of the invention to provide such a method 
which is relatively rapid, and can be carried out under relatively mild 
reaction conditions, and in particular, under conditions which do not 
involve the use of anhydride activation reagents. 
The invention includes, in one aspect, a method for determining the 
C-terminal amino acid residue of a peptide. The peptide is reacted with a 
mixed anhydride of isothiocyanic acid and a carboxylic, carbonic or 
sulfonic acid, under basic conditions, to produce a C-terminal peptidyl 
thiohydantoin (TH). C-terminal hydrolysis of the peptidyl-TH bond releases 
the amino acid TH, which can then be identified, e.g., by HPLC, as an 
amino acid TH adduct. 
In one preferred embodiment of the invention, the mixed anhydride reagent 
used in the method has the form: 
##STR1## 
where R is an alkyl or aryl group. 
Where the method is used for C-terminal sequencing, the peptide is 
preferably attached to a solid support through an internal or N-terminal 
amino group. The peptide is subjected to successive rounds of C-terminal 
TH formation and amino acid TH release. 
According to one aspect of the invention, the formation of the TH-peptidyl 
complex, and subsequent cleavage under acidic conditions preserves the 
sterochemistry of the C-terminal amino acids. This is evidenced by the 
presence of only a single TH-amino acid (isoleucine) peak on HPLC, under 
chromatographic conditions in which diasteriomeric forms of Ile-TH are 
resolvable as a doublet. Thus the method can be practiced under conditions 
which permit identification of sterioisomeric forms of C-terminal amino 
acids. 
These and other objects and features of the invention will become more 
fully apparent when the following detailed description of the invention is 
read in conjunction with the accompanying drawings.

DETAILED DESCRIPTION OF THE INVENTION 
In practicing the method of the invention, a mixed anhydride of 
isothiocyanic acid and a carboxylic, carbonic or sulfonic acid (Section A) 
is reacted with the C-terminal residue of a peptide, to form a peptidyl 
thiohydantoin (Section B). The peptidyl-TH is treated under conditions 
which cleave the C-terminal amino acid-TH from the peptide and the 
released compound is identified, for example by HPLC, to determine a 
C-terminal amino acid (Section C) or for C-terminal peptide sequencing 
(Section D). Related mixed anhydride compounds which may be used for 
C-terminal sequencing are also disclosed (Section E). 
A. Mixed anhydride Reagent 
The reagent employed in the method of the present invention is a mixed 
anhydride of isothiocyanic acid (ITC) and carboxylic, carbonic, or 
sulfonic acid. Several exemplary mixed anhydride reagents are illustrated 
in FIG. 1. These include: 
(a) an alkyl or aryl acyl-ITC compound, as exemplified by acetyl ITC (I), 
and benzoyl ITC (II). The alkyl group may be selected from the group of 
alkyl and cycloalkyl compounds, such as methyl, ethyl propyl, t-butyl, and 
related carbon-containing compounds linked to the acyl carbon through a 
carbon-carbon linkage. The aryl group may be a benzene, substituted 
benzene, or related compound linked to the acyl carbon through an aryl 
ring carbon atom. These two classes of compounds are also referred to 
herein as mixed anhydrides of ITC and carboxylic acids, since hydrolysis 
at the CO--N bond yields a carboxylic acid and HNCS. 
(b) an alkoxy or aryloxy carbonyl-ITC compound, as exemplified by ethoxy 
acyl-ITC (III), or benzoxy carbonyl-ITC (IV). The ether linked alkyl or 
aryl groups in the compounds are as described in (a). These two classes of 
compounds are also referred to herein as mixed anhydrides of isothiocyanic 
acid and carbonic acids, since hydrolytic cleavage of the compounds 
produce a carbonic acid ester and HNCS. 
(c) an alkyl or aryl sufonyl-ITC compound, as exemplified by 
benzylsulfonyl-ITC (V). The alkyl or aryl groups are as described in (a). 
These two classes of compounds are also referred to herein as mixed 
anhydrides of sulfonic acid and ITC, since hydrolytic cleavage of the 
compounds produce a sulfonic acid and HNCS. 
In one preferred embodiment of the invention, the compound is a mixed 
anhydride of a carboxylic acid and isothiocyanate, as described in (a), 
and having the general formula: 
##STR2## 
where R may be any carbon-containing group, such as an alkyl or aryl 
group, which allows reagent solubility in the solvent used in the 
TH-forming reaction, and reactivity toward a peptide acyl group, in the 
isothiocyanation reaction to be described. 
Some compounds in this class may be obtained commercially, or prepared 
according to known procedures. One method of preparing acyl-substituted 
ITCs involves dissolving an isothiocyanate in a dry, inert solvent 
containing a base, and slowly adding the desired acyl chloride. For 
example, a synthesis of benzoyl ITC employs benzoyl chloride in 
acetonitrile or dichlormethane or toluene as an inert solvent, and 
diisopropyl ethyl amine as the base. 
Mixed anhydrides of isothiocyanic acid and carbonic acid (ROC(O)NCS) may be 
formed by analogous methods, using a selected alkyl or aryl ester chloride 
in reaction with ITC salt. Similarly, mixed anhydrides of isothiocyanic 
acid and sulfonic acid are formed by reacting the desired benzensulfonyl 
chloride with an ITC, as outlined in Example 7. 
B. Reaction Method to form Peptidyl TH 
In practicing the method of the invention, the peptide whose C-terminal 
amino acid is to identified is reacted with the mixed anhydride reagent 
reagent under basic conditions in which the C-terminal acid group of the 
amino acid is deprotonated. The C-terminal amino acid is converted to a 
C-terminal amino acyl TH which is linked to the next-in C-terminal amino 
acid in the peptide through a ring-nitrogen amide bond. 
As used above, the term "peptide" is intended to include both peptides and 
proteins, these generally being distinguished by less than or more than 
100 amino acids, respectively. 
Where the method is used to identify only the C-terminal amino acid residue 
of the peptide, the peptide may be reacted in free form, i.e., unattached 
to a solid support, where the amino terminus is protected. Typically, 
where successive rounds of C-terminal amino acid identification are 
desired, for purposes of C-terminal sequencing, the peptide is first 
attached at its N-terminus or internal side chain to a solid support. 
Methods for attaching peptides to solid supports, such as activated glass 
beads, carboxylated or aminated resin beads, and the like are well known. 
In one preferred approach, employed in the method described in Example 1, 
the dipeptide Leu-Val was immobilized to aminated resin by a benzyl 
diisothiocyanate. 
The peptide, either in free or immobilized form, is dissolved or suspended 
in a suitable solvent under basic, preferably non-aqueous conditions 
effective to deprotonate the C-terminal carboxylic acid group. The solvent 
preferably also includes pyridine, which may serve an important catalytic 
role in thiohydantoin (TA) ring formation, as discussed below. One 
preferred solvent is anhydrous acetonitrile containing 10% anhydrous 
pyridine. A typically reaction volume is about 100 .mu.L reaction solvent 
per 1-3 mg peptide. 
The mixed-anhydride reagent from above is now added to the peptide 
suspension, preferably in molar excess of the peptide carboxyl group 
available for reaction in the reaction suspension. Typically, about 10 
.mu.L of the reagent are added per 100 .mu.L reaction solvent. 
The TH-forming reaction is preferably carried out at 50.degree.-70.degree. 
C., for about 10-60 minutes, preferably at about 50.degree. C. for 15-30 
minutes, to minimize reaction of the ITC reagent with non-carboxyl groups 
in the peptide. Following this, the reaction mixture is cooled to room 
temperature, and the peptidyl TH is recovered. Where the peptide is 
attached to a solid support, the support recovery is accomplished readily 
by washing the support, for example, with several volumes of acetonitrile, 
and drying the washed support, for example, by vacuum centrifugation. 
Where the peptide is reacted in free form, the peptidyl TH may be 
recovered by chromatographic separation or the like in a solvent system 
which does not produce end-terminal cleavage of the amino acid TH from the 
peptide. 
FIGS. 2 and 3 show the proposed mechanism of the reaction for peptidyl TH 
formation in accordance with the method of the invention. In the reaction 
mechanism shown at the top in FIG. 2, the mixed anhydride reagent VII 
reacts through an unstable ITC intermediate to form the peptidyl mixed 
anhydride shown at VI in the figure. The anhydride then reacts with a free 
thiocyanate ion produced in the anhydride reaction, to form the peptidyl 
ITC compound shown at VIII in the figure via a tetrahedral intermediate 
IX. In this proposed mechanism, the mixed anhydride reagent functions both 
as an activating reagent, to form a reactive peptidyl anhydride, and as a 
source of thiocyanate ions for reaction with the anhydride. 
An alternative reaction mechanism is shown in the second line in FIG. 2. 
Here the thiocyanate group in the tetrahedral intermediate IX in the 
figure migrates to the peptide carbonyl carbon, forming the tetrahedral 
intermediate IX, which rapidly collapses to form the peptide ITC compound 
shown at VIII. 
With continued reference to FIG. 2, there are two possible electrophilic 
sites of reaction of the peptidyl deprotonated (nucleophilic) oxygen atom. 
The first site, and the one shown in the FIG. 2 reaction scheme, is the 
carbonyl carbon. The second site is the the thiocarbonyl carbon, which 
would lead to the wrong reaction products. Experiments conducted in 
support of the present invention indicate that more electrophilic 
substitution at the carbonyl carbon favors reaction at the thiocarbonyl 
carbon, based on the percentage of the amino acid-TH compound formed. 
Thus, for example, alkyl and aryl carbonyl groups (the mixed anhydrides of 
isothiocyanic acid and carboxylic acids) give the highest percentage of 
the desired TH product, and the more electronegative ester reagents (mixed 
anhydrides of isothiocyanic acid and carbonic acid), a lower prercentage 
of the desired product. Under present conditions, the sulfonic acid mixed 
anhydride reagent gives the lowest percentage of the desired TH product. 
The reaction of the peptidyl ITC compound XI to form the desired peptidyl 
TH is shown in FIG. 3, and presumably involves an electrophilic attack by 
the thiocyanate carbon on the amide nitrogen in the peptide ITC compound, 
with rapid cyclization to form the peptidyl TH shown at XI in the figure. 
The cyclization reaction may be catalyzed by pyridine, suggesting that 
pyridine may be reacting with the thiocyanate moiety to enhance the 
reactivity of the reactive carbon center, as has been proposed (Miller, 
1988). 
Specific reaction conditions for forming an amino acid or peptidyl TH, in 
accordance with the present invention are given in Examples 1A, 2, 3, and 
6 for benzoyl-ITC; in Example 4, for trimethylacetyl-ITC; in Example 5, 
for ethoxycarbonyl; and in Example 7B, for benzensulfonyl-ITC. 
C. Formation and Identification of Amino Acid TH 
This section describes the final steps of the method of the invention, 
which includes (a) treating the peptidyl TH under cleavage conditions 
effective to cleave the amide linkage joining the TH to the peptide, (b) 
isolating the amino acid TH released by the cleavage reaction, and (c) 
identifying the isolated amino acid TH. 
A variety of cleavage reaction conditions are known. Hydrolytic cleavage 
with 12 N HCl, dilute alkali (Kenner), or saturated aqueous triethylamine 
have been reported. These cleavage reactions are reported to yield up to 
70% percent cleavage, but the extreme pH conditions can lead to ring 
opening and/or damage to internal peptide side chains. More recently, 
cleavage by treatment with acetohydroxamate in pyridine at pH 8.0 was 
reported (Meuth). The method affords recovery yields of up to 60-80% of 
the C-terminal TH (Miller, 1988). A related method involves treatment with 
primary or secondary amines in acetonitrile. 
The cleavage reactions are typically carried out at room temperature or 
higher, for 15-60 minutes, depending on the cleavage agent used. The 
course of the reaction can be readily followed, for purposes of optimizing 
TH release, and minimizing ring opening and damage to the shortened 
peptide, by examining the integrity of the released amino acyl TH and/or 
shortened peptide, according to analytical methods described below, during 
the course of the reaction. Suitable solutions for carrying out the 
cleavage reaction include a 5-10% solution of trialkyl amine in 
acetonitrile and a 5-15% solution of tripropylamine in acetonitrile. 
In one cleavage method, described in Examples 1, 2, 3, and 4, the peptidyl 
TH is treated with 10% propylamine in acetonitrile at room temperature for 
15 minutes. In a related method, described in Example 5, the peptidyl-TH 
was cleaved with tetra-N-butyl ammonium hydroxide in water containing 1 
mg/ml dithiothreitol (DTT). 
According to one aspect of the invention, it has been discovered that the 
TH-forming method, when carried out under acidic conditions, preserves the 
stereoisomeric form of C-terminal amino acid. This is illustrated in 
Example 6, where t-BOC protected Ile is deprotected with trifluoroacetic 
acid (TFA), in 25% H.sub.2 O, to yield a single L-form Ile-TH compound, as 
seen in FIG. 7B. By contrast, reaction formation of deprotected Ile-TH by 
reaction with trimethylsilyl isothiocyanate (TMS-ITC), and deprotection 
(cleavage) with 12N HCl, in accordance with prior-art C-terminal analysis 
methods (Hawke), produced the two diastereoisomeric forms seen as a 
doublet in FIG. 7A. 
The results of the cleavage reaction are illustrated at the bottom in FIG. 
3. In the FIG. 3 reaction, the cleavage produces a peptide shortened by 
one amino acid (compound XIII) and an amino acid TH XIV whose R.sub.1 
group is, of course, the amino acid side chain of the original C-terminal 
amino acid. 
The amino acyl TH released from the peptide or the shortened peptide, or 
both, are analyzed to identify the C-terminal amino acid. Where the 
peptide is coupled to a solid support, the released TH can be easily 
separated by removing the cleavage-reaction solvent, e.g., by vacuum 
centrifugation, and extracting the released TH in a suitable solvent such 
as acetonitrile, as detailed in Example 1. Where the peptide is free in 
the cleavage reaction mixture, the mixture may be separated, e.g., by 
HPLC, as part of the method for identifying the compound. 
The released amino acid TH compound may be identified by known 
chromatographic methods, such as high pressure liquid chromatography 
(HPLC), according to standard procedures. Compound identification can be 
made conveniently by comparing the retention times in the columns with the 
retention times of known reference amino acid THs, prepared according to 
standard methods. The HPLC methods detailed in Example 1 are suitable. 
FIGS. 5A and 5B, for example, show HPLC profiles of leucine-TH (L-TH) and 
methionine-TH (M-TH), and FIGS. 6A and 6B, of L-TH and phenylalanine 
(F-TH). 
Alternatively, the released and isolated amino acid TH can be identified by 
other available tools, such as mass spectrometry or NMR. 
D. C-Terminal Amino Acid Sequencing 
The method described in Section C above is designed for determining the 
C-terminal amino acid residue of a peptide. It will be appreciated that 
repeated application of the method can be used for C-terminal amino acid 
sequencing of the peptide. 
FIG. 8 illustrates the sequencing method. The peptide to be sequenced is 
coupled to a solid support S, as indicated. The peptide is then carried 
through a first round of steps to (a) produce the C-terminal peptidyl TH, 
(b) cleave the terminal TH, yielding the C-terminal amino acyl TH 
(AA.sub.n) and a shortened peptide whose C-terminal residue is now 
AA.sub.n-1, and (c) identify the released amino acyl TH. 
After washing the peptide support, the shortened peptide is treated with a 
second round of the above steps to release the next-in amino acid as an 
amino acid TH (AA.sub.n-1) and yield a peptide shortened by two C-terminal 
residues. This procedure is repeated until the peptide has been sequenced 
or until loss of resolution, due to incomplete TH formation and release, 
makes further sequencing impossible. The sequencing method is illustrated 
in Example 5, for the determination of the two C-terminal amino acid 
residues of leucine enkephalin (YGGFL). The HPLC chromatogram from the 
first round of analysis, shown at 6A shows the C-terminal L-TH peak. The 
chromatogram from the second round shows the expected F-TH. 
The above-detailed technique is easily automated using technology known for 
the automated sequencing of the N-terminal residues of peptides. One 
embodiment of a device to automatically sequence a peptide from the 
C-terminal end employs a solid support contained in a reaction vessel to 
which fresh solvent and reagents are added, and from which reaction 
mixtures and solvent washes are removed. The released amino acid TH's are 
isolated from the stored material for later identification by HPLC. 
E. Alternative Mixed Anhydride Reagents 
Although the present invention encompasses the mixed anhydrides of 
carboxylic, carbonic, and sulfonic acid described in Section A, additional 
mixed anhydrides as disclosed below may be suitable for forming C-terminal 
amino acid-TH compounds. 
One of the alternative reagents is a carbamate mixed anhydride of 
isothiocyanic acid, having the general formula: 
##STR3## 
where R may be any amine, and preferably a quarternary amine, such as 
trimethyl amine. This reagent would be expected to have about the same 
electronegativity as the carbonate mixed anhydrides, and thus should 
provide a reasonable yield of the desired peptidyl-TH product. 
Another alternative mixed anhydride is a benzoyl-mercaptobenzothiazole 
(MBT) XV, such as illustrated at the right in FIG. 9. This compound may be 
prepared according to the synthetic method described in Example 8. 
Briefly, MBT XVI is dissolved in a dry, inert solvent in the presence of a 
suitable base, such as diisopropyl ethyl amine. A selected acyl chloride, 
e.g., benzoyl chloride XVII, is then slowly added at a reduced temperature 
to form the desired product. Related acyl MBT compounds, such as acetyl 
MBT, can be prepared by a similar method, using the desired acyl chloride 
starting material. 
FIG. 10 shows the proposed reaction steps in the formation of a peptidyl 
phenyl TH, employing an MBT reagent. The reaction conditions may be 
substantially similar to those described in Section B, with suitable 
adjustment in reaction time, if necessary, to ensure completion of the 
TH-formation reaction. Typical reaction conditions are given in Example 8. 
With reference to FIG. 10, the deprotonated peptide (XVIII) is reacted with 
benzoyl-MBT (BzMBT) to form, in the proposed reaction pathway, the 
intermediate peptide benzothiazole compound indicated at XIX. The 
formation of the intermediate may proceed via an activated anhydride, with 
subsequent attack by a benzathiazole ion, or may involve a concerted 
rearrangement reaction, as discussed with reference to FIG. 2. 
The cyclization of the intermediate benzathiazole to form the peptidyl aryl 
TH likely proceeds along the same pathway as described in FIG. 3, with the 
electrophilic thio ring carbon reacting with the amide nitrogen, followed 
by cyclization to form the desired peptide phenyl TH compound XIV. 
Still another possible mixed anhydride is a mixed anhydride of isocyanic 
acid and carboxylic acid, such as benzoyl isocyanate. Such a reaction, 
which is described in Example 9, produces a reasonable yield of the 
desired amino acid-TH product, as reported in the example. 
From the foregoing, it can be appreciated how various objects and features 
of the invention are achieved. The method of the invention permits 
peptidyl TH formation under substantially milder conditions than prior-art 
methods relying on anhydride activation of the peptide's carboxyl group. 
At the same time, the TH-formation reaction can be carried out relatively 
rapidly, e.g., 15-30 minutes, thus allowing sequencing to be carried out 
quickly in an automatic system. 
In addition, when the cleavage reaction in the method is carried out under 
acidic conditions, the method preserves the stereochemistry of the 
C-terminal amino acid, and thus can be used to determine L- or D-form 
amino acids. 
The following examples are intended to illustrate the synthesis of various 
acyl compounds, and their use in determining C-terminal amino acid groups, 
and for C-terminal sequencing. The examples are in no way intended to 
limit the scope of the invention. 
Materials 
Pyridine was used as supplied from Aldrich, as was mercaptobenzothiazole, 
propylamine and triethylamine. Benzoyl isothiocyanate was from Aldrich, as 
supplied or vacuum distilled. Leucine enkephalin was from Sigma, 
dipeptides were from Bachem Biosciences. Polydimethyl acrylamide resin was 
prepared according to J. Sparrow, and had about 0.7 meq/g of amino groups. 
Conversion of those amino groups to isothiocyanates was performed using 
thiocarbonyl diimidazole and diisopropylethyl amine (DIPEA) in 
acetonitrile. Nuclear magnetic resonance spectra were collected on a Jeol 
FX90 spectrometer. Mass spectral analyses were performed by the Berkeley 
Mass Spectrometry Laboratory under the direction of Dr. J. Leary. 
Peptides were attached to resin in one of two ways: (1) by a urea linkage, 
or (2) by a thiourea linkage (using isothiocyanato-resin, above). 
(1) Urea link: The resin is neutralized with excess DIPEA and allowed to 
react with excess DSC in 10% pyridine/NMP for 1-2 hours at ambient 
temperature. After several washes, finally with ACN, a solution of the 
peptide in 10% pyridine/water is added to the moist resin and allowed to 
stand overnight. The resin is washed extensively and dried under vacuum. 
Typical loading is &gt;100 nanomoles/ml. Confirmation is by amino acid 
analysis, and one round of C-terminal analysis. 
(2) Thiourea link: A solution of the peptide is added to either ITC (above) 
or DITC resin, and incubated at 45.degree. C. overnight. After rinsing and 
drying, attachment is confirmed by TFA cleavage of the peptide (less the 
aminoterminal amino acid) from the resin followed by characterization on 
HPLC and by sequence analysis. 
EXAMPLE 1 
C-Terminal Analysis of a Leu-Val Dipeptide 
A. Coupling 
Peptidyl resin (about 1 mg, Leu-Val attached via a urea-linkage) was 
suspended in 100 .mu.l of 10% pyridine/acetonitrile (PA). Benzoyl 
isothiocyanate (BITC) (10 .mu.l) was added, mixed by agitation, and 
allowed to react for 30 minutes at 60.degree. C. The resin was extensively 
washed with several volumes of acetonitrile, and dried under vacuum. 
B. Hydrolysis 
The resin from above was wetted with 10 .mu.l of 10% propylamine in 
acetonitrile. After 15 minutes at room temperature volatiles were removed 
and the amino acid TH extracted with acetonitrile for analysis. 
C. HPLC 
The separation of the hydrophobic amino acid thiohydantoins was readily 
achieved on a narrow-bore system (Model 120A, Applied Biosystems) using a 
PTH-C18 column (2.1 mm.times.22 cm, ABI) and a TFA-water-acetonitrile 
gradient system. The column was first equilibrated in A solvent (0.1% TFA 
in water, v/v), held in 100% A, 0% solvent B for 5 minutes after 
injection, then a linear gradient was developed to 40% B solvent (0.85% 
TFA in 70% acetonitrile) over 30 minutes. The percentage of B was then 
increased to 90% over 5 minutes, and held there for 20 minutes. The flow 
rate was 200 .mu.l/ min at ambient temperature. Effluent was monitored at 
269 nm for TH, and 214 nm for peptides. 
The thiohydantoin is identified by comparison with the elution of authentic 
material (part D, below). The HPLC of the extract in part B is shown in 
FIG. 4B. The major peak is readily identified as TH-V. The assignment was 
confirmed by coinjection. FIG. 4A shows the TH-V product generated in a 
control experiment in which the resin was reacted with trimethylsilyl 
isothiocyanate (Hawke), in the an anhydride activating reagent. 
D. Preparation of Authentic Standards 
Amino acid thiohydantoins of the hydrophobic residues are preparable by 
classical methods (such as Cromwell). Briefly, the amino acid is treated 
with a thiocyanate salt in acetic acid/acetic anhydride at up to 
90.degree. C. for 30 minutes. After vacuum drying, the residue is taken up 
in 12 N HCl and allowed to stand at room temperature for up to 1 hour. The 
mixture is again vacuum dried, and the residue recrystallized, usually 
from boiling water. The structures were consistent by NMR and mass 
spectrometry. 
EXAMPLE 2 
C-Terminal Analysis of A-M Dipeptide 
Following the procedures described in Example 1, immobilized Ala-Met (urea 
linkage) was degraded for one cycle using BITC and PA. The assignment of 
methionine TH was confirmed by coinjection. 
EXAMPLE 3 
Leucine Enkephalin C-Terminal Analysis 
Leu-enkephalin (YGGFL) linked through a thiourea linkage to resin was 
treated with BITC and PA as in Example 1. The released L-TH was detected 
by HPLC. 
The resin was further rinsed with acetonitrile and dried, then treated with 
25% TFA in water at 60.degree. C. for 10 minutes to release the peptide 
components from the resin. HPLC analysis followed by isolation and both 
sequencing and FAB mass spectrometry showed a major peak corresponding to 
the desleucine peptide, confirming the loss of the C-terminal amino acid. 
EXAMPLE 4 
C-Terminal Analysis of N-Protected Phe Gly 
N-protected t-Boc Phe-Gly was obtained commercially. The peptide was 
suspended in 100 .mu.l of 10% pyridine/acetonitrile (PA). Trimethylacetyl 
ITC (10 .mu.l) was added, mixed by agitation, and allowed to react for 30 
minutes at 60.degree. C. The resin was extensively washed with several 
volumes of acetonitrile, and dried under vacuum. 
Cleavage of both the t-Boc group and the Gly-TH was achieved by heating at 
60.degree. in 25% TFA/water. The reaction, as followed by HPLC, showed 
rapid loss of starting material (judged after 15 minutes of reaction), 
along with the appearance of G-TH and a minor component (probably the 
Boc-deprotected dipeptidyl-TH). After two hours reaction, only G-TH was 
detectable. 
EXAMPLE 5 
C-Terminal Analaysis of Leu-Enkephalin 
Immobilized leucine enkephalin (urea linkage) was degraded for two cycles 
of chemistry. The couplings were performed with ethoxycarbonyl 
isothiocyanate (instead of BITC) for 5 minutes at 60.degree. C. Cleavages 
were effected by adding 10 .mu.l of 10% tetra-N-butyl ammonium hydroxide 
in water containing 1 mg/ml DTT to the dry resin, and heating for 45 
minutes at 60.degree. C. Chromatograms are shown in FIGS. 6A and 6B. The 
cycles were assigned as leucine and phenylalan respectively. The 
assignments were confirmed by coinjection. 
EXAMPLE 6 
Preparation of Single Isomer of Isoleucine 
Approximately 10 .mu.moles of t-Boc-isoleucine was dissolved in 100 .mu.l 1 
10% pyridine/acetonitrile. This was heated with 10 .mu.l BITC at 
60.degree. for 20 minutes. HPLC analysis of the product showed a single 
major new peak, the t-Boc-Ile-thiohydantoin. A small portion of this 
mixture was cleaved in 25% TFA at 60.degree. C. for 10 minutes. The 
reaction mixture was diluted with acetonitrile and analysed by HPLC. The 
result was a single peak corresponding to Ile-TH. Running the peak 
isocratically (11% B) gave an improved resolution of isomers, shown in 
FIG. 7B, and allows estimation of the upper bound of about 2% 
epimerization. 
T-Boc-isoleucine (10 .mu.mol) was dissolved in 100 .mu.l 10% TMS-ITC in 
acetic anhydride. The reaction was heated at 60.degree. for 20 minutes. 
The material was cleaved in 25% TFA at 60.degree. C. for 10 minutes, and 
the reaction mixture was diluted with acetonitrile and analysed by HPLC., 
as above. The result was a doublet peak, corresponding to the 
diasteromeric forms of lle-TH, as seen in FIG. 7A. 
EXAMPLE 7 
Reaction of Sulfonyl ITC Reagent with N-Protected Amino Acid 
A. Preparation of benzenesulfonyl isothiocyanate (BzS-ITC) 
Benzenesulfonyl chloride was obtained from Aldrich Chemical Co. The 
compound (1 mmol) was dissolved in 10ml CH.sub.2 Cl.sub.2 with 2 mmol 
pyridine ethyl amine to a final volume. To this mixture was added 1 mmol 
of TMSITC, and the reaction mixture was stirred for 1 hour at 25.degree. 
C. The benzylsulfonyl isothiocyanate (BzSITC) was recovered by filtering 
the salrts which formed, and removing the solvent and volatile 
side-products by vacuum. 
B. Reaction to form amino acid TH 
One mmol of t-Boc-Leu was dissolved in dichloromethane containing pyridine, 
and 1 mmol of BxSITC prepared as in A. After reacting overnight, the 
solvent was removed by rotary evaporation and the t-Boc-Leu-TH which 
formed was purified by chromatography on silica gel. The identify of the 
TH compound was confirmed by NMR and HPLC. 
EXAMPLE 8 
Reaction of bZ-MBT with Peptidyl Resin 
A. Preparation of BzMBT 
Mercaptobenzothiazole (1 mmole) and diisopropyl ethylamine (1 mmole) were 
dissolved in 5 ml acetonitrile. Benzoyl chloride (1 mmole) was added 
slowly by syringe while stirring rapidly. After 2 hours at room 
temperature solvent was partially removed to permit precipitation of the 
amine salt. The supernatant was further concentrated, and chromathographed 
on silica get (9:1, heptane:ethyl acetate). 
B. Reaction 
Peptidyl resin (1 mg Lenk, thiourea linkage) was suspended in 100 .mu.l of 
10% pyridine in acetonitrile containing 1 mg Bz-MBT. The reaction was 
heated at 60.degree. C. for 30 minutes, then washed and dried as before. 
The arylthiohydantoin was cleaved with 10% propylamine at room temperature 
for 15 minutes. Treatment with TFA released the remaining peptide 
fragments from the resin, as confirmed by the loss of the C-terminal 
residue as assessed by HPLC. 
EXAMPLE 9 
Reaction with Benzoyl Isothiocyanate 
One mole of t-Boc-Leu was reacted with a slight excess of benzoyl 
isothiocyanate in a reaction mixture similar to that in Example 7. The 
t-Boc-Leu-TH was chromatographed, then deprotected to give a moderate 
yield of hydantoin, as confirmed by NMR.