2-Alkoxy-S(4H)-oxazolonesfrom N-alkoxycarbonylamino acids and their implication in

carbodiimide-mediated reactions in peptide synthesis

N. LEOBENOITONA N D FRANCISM. F. CHEN
Department of Biochemistry, Uniuersity of Ottawa, Ottawa, Ont., Canada K I N 9A9
Received June 27, 1980
This paper is dedicated to Prof. Raymond U. Lemieux on the occasion of his 60th birthday

N. LEOBENOITON and FRANCISM. F. CHEN.Can. J. Chem. 59,384(1981).

Reaction of N-ethyl,N1-(y-dimethylaminopropy1)-carbodiimide HC1 with one equiv. of N-tert-butoxycarbonyl-L-valine (3a) in
Can. J. Chem. Downloaded from cdnsciencepub.com by 139.18.241.77 on 12/04/23

dichloromethane at 23'C gives, besides the symmetrical anhydride (5a), the optically pure 2-tert-butoxy-4-isopropyl-5(4N)-
oxazolone (40) which can be obtained in 50% yield under selected conditions. The 2-benzyloxycarbonyl-4-isopropyl-5(4N)-
oxazolone (46) is similarly obtainable from N-benzyloxycarbonyl-L-valine (36). Anhydrous acid converts 4a to the oxazolidine-
dione. Simple preparations of the N-carboxyanhydrides of valine and isoleucine have been devised from these reactions. Compound
4 reacts with 3 to give 5. Compound 4 reacts with an amino acid ester to give the optically pure peptide even in the presence of salts,
but partial racemization occurs for reactions in the presence of a tertiary amine. Evidence for the implication of 2-alkoxy-5(4N)-
oxazolones in the couplings of N-alkoxycarbonylamino acids is presented. Compound 4a has been isolated in 6-11% yield from
carbodiimide-mediated reactions of 3a with itself or amino acid methyl esters which have been terminated before completion.

N. LEOBENOITONet FRANCISM. F. CHEN.Can. J. Chem. 59,384(1981).

La reaction du chlorohydrate de N-Cthyl,N1(y-dimethylaminopropyl)carbodiimideavec un equivalent de N-tert-
butoxycarbonyl-L-valine (3a) dans le dichloromethane a 23°C donne en plus de I'anhydride symCtrique (5a), le compost tert-
butoxy-2 isopropyl-4 (4H)oxazolone-5 (4a) que I'on peut obtenir dans des conditions choisies avec un rendement de 50%. D'une
f a ~ o nanalogue, on obtient le benzyloxycarbonyl-2 isopropyl-4 (4H)oxazolone-5 (46) a partir de la N-benzyloxycarbonyl-L-valine
(36). L'acide anhydre transforme le compose 4a en oxazolidinedione. A partir de ces reactions, on a mis au point des preparations
simples de N-carboxyanhydrides de la valine et de I'isoleucine. Le compose 4 reagit avec 3 e t donne le compose 5. Leproduit 4 reagit
avec un ester d'acide amine pour conduire au peptide optimiquement pur mtme en presence de sels, mais il s e produit une
racemisation partielle au cours des reactions conduites en presence d'une amine tertiaire. On presente la preuve de I'implication des
alkoxy-2 (4H)oxazolones-5 dans les couplages des acides amines N-alkoxycarbonyles. On a isole le compose 4a partir des
reactions d'autocondensation du compose 3a ou de sa condentation sur les esters methyliques d'acides amines en presence de
carbodiimide qui s'arrttent avant d'avoir totalement reagi.
[Traduit par le journal]

When an acylamino acid or a protected peptide
(1)is coupled with a nucleophile (amine or phenol),
racemization frequently occurs. The change in
stereochemistry is the result of the formation of the
2-alkyl-5(4H)-oxazolone (2) by cyclization of the
activated N-substituted amino acid moiety. Com-
pound 2 racemizes faster than it couples with the
nucleophile. However, no racemization occurs
when an N-alkoxycarbonylamino acid (3) is
coupled, as is done successively during the
build-up of a peptide chain starting from the C-
terminus. The reason accepted to explain the pre-
servation of stereochemistry has been that the cor-
responding 2-alkoxy-5(4H)oxazolones (4) are not
formed because of the unique nature of the
urethane carbonyl group (1). We describe here re-
sults which are incompatible with this basic tenet of
peptide chemistry. As a follow-up on our work on
the synthesis of symmetrical anhydrides (5) of
N-alkoxycarbonylamino acids (2), we describe the
preparation and characterization of the 2-alkoxy-
5(4H)-oxazolones 4a and 46 derived from Boc-Val
0008-40421811020384-06$01.00/0
0 1 9 8 1 National Research Council of CanadaIConseil national de recherches du Canada
(3a) and Z-Val (36) respectively,' as well as their
isolation from reaction mixtures similar to those
used in peptide ~ y n t h e s i s . ~
Reaction of the soluble carbodiimide EDC (6)
with two equiv. of 3 in dichloromethane followed
by washing with aqueous citric acid and sodium
hydrogen carbonate gave 70-80% yields of sym-
metrical anhydride (5) for several Boc- and Z-
amino acids but lower yields for Boc-Val and Boc-
Ile (2). Attempts to increase the yield of 5 by using
more EDC (1 equiv.) produced a second stable
neutral product which could be obtained pure
(yield, 2% by weight) after removal of solvent by
precipitating out the anhydride with light petro-
'Abbreviations: Boc, tert-butoxycarbonyl; Bzl, benzyl; Z,
benzyloxycarbonyl; DCC, N,N'-dicyclohexylcarbodiimide;
EDC, N-ethyl,N'-(y-dimethylaminopropyl)-carbodiimide~HCl;
EDU, N-ethyl,N1-(y-dimethylaminopropy1)-urea;NMM, N-
methylmorpholine; Val, valine. Amino acid symbols represent
the L-isomer.
ZPartof this work was presented at the Sixth American Pep-
tide Symposium, Washington, DC, June 1979 (3).
 BENOITON AND CHEN
leum. IH nuclear magnetic resonance spectroscopy
indicated it to be a derivative of valine with a tert-
butyl group displaced 0.13 ppm downfield from that
of a Boc or tert-butyl ester group, and which had
lost its -N-H proton, as evidenced by the sharp
a-proton doublet (4.13 ppm). Reaction with
anhydrous methylamine gave a quantitative yield
of Boc-Val-NHCH, thus establishing it to be an
activated form of Boc-Val. Elemental analysis cor-
responded with Boc-Val which had lost one mole of
water. Infrared absorption at 1845 and 1700 cm-I
Can. J. Chem. Downloaded from cdnsciencepub.com by 139.18.241.77 on 12/04/23
combined with the absence of absorption at 3400
(-N-H) and 1770 (urethane carbonyl) cm-I indi-
cated the 5(4H)-oxazolone structure (4, 5). The
compound is therefore 2-tert-butoxy-4-isopropyl-
5(4H)-oxazolone (4a). The aziridinone structure is
excluded on the basis of the mass spectrum and the
absence of absorption for a urethane carbonyl
group (4) in the ir spectrum. The high specific rota-
tion (-49.6" in CHCl,) indicated that the compound
was not appreciably racemized. A test for optical
purity (see below) established it to be optically pure
(<0.1% D-isomer).
Compound 4a could be detected and quantitated
in the presence of other derivatives of Boc-Val by
integration of the tert-butyl peaks of nmr spectra.
No 4a could be detected after reaction of EDC with
two equiv. of Boc-Val, or after a reaction carried out
in dimethylformamide. More 4a was formed at 23°C
(35%) than at 0°C (30%); the yield of 5a was greater
at 0°C (55%) than 23°C (40%). Other factors in-
creased the yield of 4a. Maximum yield (55%; iso-
lated) was achieved by adding the Boc-Val drop-
wise to adilute solution containing excess EDC and
some triethylamine- HC1. Compound 4a was stable
in deuterochloroform at -5°C for 5 days, in
dimethylformamide at 23°C for 2 h , and in
dichloromethane-ethanol(20: 1) at 23°C for 20 h. It
slowly decomposes in solutions containing water
giving rise to tert-butanol, and is converted to val-
ine N-carboxyanhydride (7a) on standing in tert-
butanol, anhydrous acid, or in uacuo. Since 5a is
also converted to 7a by dry HCl, the mixture of 4a
and 5a obtained from the reaction of Boc-Val and
EDC serves as a convenient source for the prep-
aration of 7a., The valine hydrochloride liberated
from 5a can be eliminated by simple filtration of the
solution of 7a in dichloromethane.
Evidence for the formation of the 2-tert-butoxy-
5(4H)-oxazolones (4) from the following Boc-
amino acids, using one equiv. of EDC, was also
)The generality of this approach for the preparation of N-
carboxyanhydrides is under investigation.
385
obtained (% yield): alanine, 15; leucine, 5;
isoleucine, 45; phenylalanine, 15. But in no case
could the product be isolated pure because the
symmetrical anhydride could not be precipitated
out completely. The best product from Boc-Jle
contained 5% of anhydride. The mixture of 4c and
5c obtained from Boc-Ile and EDC could be used to
prepare 7c in good yield.
When EDC was reacted with Z-Val (3b) as de-
scribed for reactions with Boc-Val, the analogous
2-benzyloxy-5(4H)-oxazolone (4b), [ol], -55.go
(CHCl,), was obtained in 20% yield using one
equiv. of EDC, and in 55% yield using the condi-
tions giving maximum yield. Elemental analysis,
nmr, ir, and mass spectra were in accord with this
structure. The compound is readily detected and
quantitated in a mixture on the basis of the
methyleneoxy protons of the nmr spectrum which
are 0.20 ppm downfield from those of a Z-group.
Compound 46 is more stable than 4a, not decom-
posing in uacuo, or on standing in tert-butanol or
wet dichloromethane for 24 h. After 36 h in ethanol,
it had been completely converted to the ethyl ester.
It reacted with p-nitrophenol to give the ester, and
with Z-Val to give the symmetrical anhydride (Sb),
but did not react with p-nitroaniline. Catalytic hy-
drogenation of 4b gave 7a.
Compound 4a reacted with an equiv. of Z-Val
and 46 with an equiv. of Boc-Val to give quantita-
tive yields of identical products whose nmr and ir
curves were consistent with the anhydride struc-
ture BOCNH~(CH,)~CH)CH-CO-O-CO-
CH(CH(CH3)2&NHZ.4 Unequivocal proof of the
absence of the anhydrides 5a or 56 in the product
cannot be provided.
Other 2-benzyloxy-5(4H)-oxazolones isolated
from reactions using conditions giving maximum
yields were those from Z-Phe (4d; 25%), Z-Gly
(5%), and Z-Cys(Bz1) (3%). All were pure as evi-
denced by nmr spectroscopy, but only 4d was com-
pletely characterized.
When EDC was reacted with two equiv. of 3a for
30 min as for the preparation of 5a, after work-up
the sole product was 5a. However, when such a
synthesis was terminated at 3 min by the addition of
aqueous sodium bicarbonate, and the products
were examined after removal of the water-soluble
side-products, a 37% yield of an equimolar mixture
of 4a and 5a was found. This corresponds to a 12%
yield of 4a. An additional experiment established
that 4a is present in the reaction mixture before
4The mppreviously reported for this compound whichis an oil
is an error (3).
 386 CAN. J. CHEM. VOL. 59, 1981

taining a nitrogen nucleophile. A normal coupling

of Boc-Val with Val-OMe- HCl (NMM) mediated
by EDC (3 mmol of each) was terminated after
3 min by the addition of aqueous citric acid.
Work-up as usual followed by precipitation of the
protected dipeptide with light petroleum and re-
moval of solvent by evaporation gave 45 mg (7.5%
yield) of pure 4a. A similar experiment using DCC
gave essentially the same results except that the
product was contaminated with an equal amount of
non-peptide Boc-derivative (N-acylurea). Since 4a
Can. J. Chem. Downloaded from cdnsciencepub.com by 139.18.241.77 on 12/04/23

is known to react with amines to give amides, and

since there is no 4a in the above reaction mixtures if
they are left for 30 min, we conclude that 4a is an
intermediate in the carbodiimide-mediated coup-
ling of Boc-Val with Val-OMe. As in the previous
case, some 0-acylisourea must cyclize to form 4a
which then reacts with Val-OMe to give the pro-
tected peptide 9 (Scheme 1). An additional possi-
bility is that some of the 4a reacts with 3a to form 5a
which then reacts with Val-OMe to give the pep-
tide. Evidence pointing to this comes from the fol-
contact with water. Compound 4a does not decom- lowing competitive experiment. When 4a was
pose upon standing in dichloromethane at 23"C, added to a mixture of Z-Val and Val-OMe (1 equiv.
and reacts within minutes with 3a to give 5a. Com- of each), the product consisted of Z-Val-Val-OMe
pound 5a did not give rise to any 4a when it was left (15%) in addition to Boc-Val-Val-OMe. Formation
several hours in dichloromethane, even in the pres- of the anhydride (or 46) must be invoked to account
ence of triethylamine. HCl. We therefore conclude for the appearance of Z-Val-Val-OMe. Implication
that 4a is an intermediate in the reaction of EDC with of the anhydride in these couplings, however,
3a to give 5a. When 3a is not present in amount would run contrary to the contention that the 0 -
sufficient to react with all of the activated 3a, 4a acylisourea and not the symmetrical anhydride is
accumulates, to the extent of >25%. The initial the activated intermediate for synthesis in solution
product of the reaction of EDC with 3a is the 0- (6). Other competitive experiments were carried
acylisourea (8);5 8 then reacts with 3a to give 5a. out, but these could not be rationalized because of
But some of 8 must cyclize to form 4a which then the complexity of the system examined.
reacts with 3a to give 5a (Scheme 1). The question which immediately arises with
Having succeeded in isolating 4a from a reaction respect to the implication of 2-alkoxy-5(4H)-oxa-
mixture containing an oxygen nucleophile, an at- zolones in couplings is whether it is a phenomenon
tempt was made to isolate it from a mixture con- general to carbodiimide-mediated couplings. The
results of an attempt to answer this question are
given in Table 1. The 2-alkoxy-5(4H)-oxazolone
was detectable in most of the reaction mixtures
examined, the amount being independent of the
nature of the carbodiimide or the side-chain of the
amino acid ester nucleophile involved. Three fac-
tors had an important bearing on the amounts iso-
lated: temperature, N-protecting group, and resi-
due activated. The amount was 6-1 1% for Boc-Val
and Boc-lle at 23°C. It dropped to 0.3-1% at OOC, or
for Boc-Leu and Z-Val at 23°C: No 5(4H)-oxazo-
lone was detectable for Z-Val or Boc-Leu at 0°C.
The amount detected represents the balance be-
tween the rate of formation of the 5(4H)-oxazolone
and its rate of reaction with the ester. Failure to
detect the 5(4H)-oxazolone in the latter cases can-
 BENOITON AND CHEN 387

TABLEI . 2-Alkoxy-5(4Ej)-oxazolonesisolated from coupling chiral stabilities of the two types of 5(4H)-
mixturesa oxazolones is therefore one of degree.
In view of the above, we submit that the tenet
Temperature 4
3 Esterb Reagent "C %yield
that 3 does not racemize when it is coupled because
it does not form the 5(4H)-oxazolone must now be-
Boc-Val EDC 23 7.5' rejected. The 5(4H)-oxazolone can and does form
Boc-Val EDC 23 6.4'
Boc-Val DCC 23 6"
in some cases. The reason no racemization obtains
Boc-Ile EDC 23 8" is that even if the 2-alkoxy-5(4H)-oxazolone does
Boc-Ile DCC 23 11" form, it is chirally stable under normal conditions of
Boc-Leu EDC 23 0.3' coupling. Caution with technique should be exer-
Boc-Ile EDC 0 0.9'
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Boc-Ile DCC 0 > I'

Z-Val EDC 23 0.8'
Z-Val EDC 23 0.3'
Z-Val EDC 0 0
Boc-Leu EDC 0 trace'
"Couplmgs (3 mmol) rn d~chloromethanewere termrnated after 3 mm Y~elds
represent neutral roduct remalnlng In mother lrquor aRer normal work-up followed
by preclpltatron ofpeptrde and N-acylurea wlth llght petroleum
as hydrochlonde, neutralized wlth N-methylmorphollne
' Isolated pure (40-45 mg)
dMlxed wrth an equal amount of Inert unestenfied Boc denvatlve (N acylurea)
Mlxed wrth 115 of ~ t welght
s of peptlde
'Not pure Add~tlonof methylamrne ellmrnated the a hydrogen doublet In the nmr
spectrum producrng the CH,NH doublet of the methylam~de
not be taken as proof that it was not formed. Kinetic
data are required before the results can be com-
pletely rationalized. We nevertheless conclude that
the data in Table 1 provide compelling evidence in
favor of the tenet that the 2-alkoxy-5(4H)-oxazo-
lone is implicated generally in the carbodiimide-
mediated couplings of 3.
Both 4a and 4b were shown to be optically pure
when assessed by a procedure used routinely in our
laboratory. The method consists in coupling the
Boc-amino acid, in this case the 5(4H)-oxazolone,
with L-Lys(Z)-OBzl followed by deprotection and
analysis for the diastereomeric peptides with an
amino acid analyzer (7). The ester is used as the
hydrochloride salt, which is neutralized by adding
NMM. Besides demonstrating the optical integrity
of the 2-alkoxy-5(4H)-oxazolones, the test also
demonstrated that they couple without racemiza-
tion, even in the presence of salt. These results
were mandatory if the 2-alkoxy-5(4H)-oxazolones
are to be implicated in coupling reactions which are
known to proceed without racemization. So there is
no inconsistency between the results and the pro-
posals. The chiral stability of the 2-alkoxy-5(4H)-
oxazolones (4) contrasts with that of the 2-alkyl-
5(4H)-oxazolones (2) which cannot be coupled
without extensive racemization (l,8). When 4a and
4b were coupled in the presence of a tertiary amine
base, however, some racemization did occur, 6 and
15%, respectively, in the presence of 0.2 equiv. of
triethylamine. As much as 50% of 4b was
racemized when it was coupled in the presence of
1.0 equiv. of triethylamine. The difference in the
cised, however, because 4 can racemize in the
presence of excess base.
When 4a was first encountered by us, the only
reference in the literature to the existence of a
2-alkoxy-5(4H)-oxazolone was a communication
by Jones and Witty (9). They reported that the
cyclic compound derived from Z-Phe and phosgene
or thionyl chloride followed by triethylamine,
which had been described as the N-benzyloxy-
carbonyl-3-benzylaziridin-2-one (lo), was in fact
the 2-benzyloxy-5(4H)-oxazolone 4d. The so-
called N-acylaziridinones had been suggested for
use in peptide synthesis since they reacted with
amino acid esters to give unracemized products
(10). The full paper by Jones and Witty (1 1) became
available to us shortly after our presentation at the
Washington Symposium. They have shown with
kinetic data that 4d reacts with amine nucleophiles
(anilines) more quickly, and racemizes in the pres-
ence of a tertiary amine base more slowly, than the
corresponding 2-phenyl-5(4H)-oxazolone.Our re-
sults are in complete harmony with those of Jones
and Witty as well as with the observations of
Miyoshi (lo).
Our conclusion on the chiral stability of 2-
alkoxy-5(4H)-oxazolones is the same as that of
Jones and Witty (1 1). As pointed out by them, it has
already been clearly demonstrated that the 4-
proton in the analogous 2-alkoxy- Y4H)-thiazolone
is much less acidic than that in the corresponding
2-alkyl-5(4H)-thiazolone (12), so the observations
are as expected.
Explanations invoked to account for the appar-
ent reluctance of 3 to form 4 have been the lower
acidity of the -N-H (11, 13) and the lower nu-
cleophilicity of the carbonyl (14) of the urethane
group. Cyclization of 3 is considered to require an
exceptionally good leaving group such as chloride
at the carboxyl group (1, 11) and, until the report of
Jones and Witty, was considered to be accom-
panied by expulsion of the alkyl group giving rise to
the N-carboxyanhydride 7 (1). Cyclization without
loss of the alkyl group obviously does occur, and it
occurs under conditions less forcing than previ-
ously thought. In fact, the conditions used to make
 388 CAN. J. CHEM. VOL. 59, 1981
2-alkyl-5(4H)-oxazolones (2) using EDC (15) are
identical with those used here to make the 2-
alkoxy-5(4H)-oxazolones (4) except that 0°C is
used in the former case, to avoid racemization. The
differences in nature between the amide and
urethane groups of the two substrates are therefore
of little consequence in the cyclization. A more
important difference resides in the fate of the prod-
ucts once formed. Compound 2 undergoes no ap-
parent reaction with the substrate, while 4 reacts to
Can. J. Chem. Downloaded from cdnsciencepub.com by 139.18.241.77 on 12/04/23
form 5. This can account in part for the much higher
yields of 2 than 4 from the reactions.
Our results on the racemization attending cou-
plings of 4 in the presence of base indicate that the
2-tert-butoxy group allowed less racemization than
the 2-benzyloxy group. This is consistent with the
report that the racemizing tendency of the tert-
butoxycarbonyl group is four times lower than that
of the benzyloxycarbonyl group (16). It suggests
also that the lability of the 4-proton in 4 is affected
by the electron donating property of the Zalkoxy
substituent. Inspection of molecular models indi-
cates that steric effects cannot be invoked to ex-
plain the different ionizing tendencies of the 4-
proton in 5(4H)-oxazolones. The electronic effects
of the 2-alkoxy substituent must also be responsible
for the unusually low acidity of the 4-proton in 4
relative to that in 2.
I Experimental
General
EDC (17) was purchased from Sigma Chemical Co., St. Louis.
Light petroleum refers to the 30-60°C fraction, from Baker
(Canlab). Magnesium sulfate was used as drying agent. Solvents
were removed at room temperature using a rotary evaporator.
Melting points were taken by the capillary method and are
uncorrected. Optical rotations were measured with a Perkin-
Elmer model 141 polarimeter using a 1-dm tube. The ir spectra
were taken on a Unicam SPllOO spectrometer. Proton and
carbon-13 nmr spectra were taken on Varian T60 and FT80
instruments, respectively. Chemical shifts are given in ppm
relative to tetramethylsilane as internal standard for solutions in
deuterochloroform. Elemental analyses were done by Mr. H.
Seguin, National Research Council, Ottawa. The stereochemical
purities of 4 and 7 were established by adding the products
(0.5 mmol) to a solution of L-L~S(Z)-OBZ~.HCI (NMM) in
dichloromethane. Peptide products were isolated, deprotected,
and analyzed for the diastereomers as previously described (7).
For assessing the racemization of 4a and 4b during couplings in
the presence of base, the base was added to the solution of the
ester.
(i) Using Reactants in Equimolar Amounts
A solution of Boc-Val (434 mg; 2 mmol) and EDC (384 mg;
2 mmol) in 20 mL of CH,CI, was stirred at OOC for 30 min. The
solution was washed with cold 10% aqueous citric acid (20 mL),
saturated aqueous NaHCO, (20 mL), and water (20 mL) and
dried. The solvent was removed and light petroleum was added
to the residue. After 18 h at -5"C, the crystalline symmetrical
anhydride (5a) (207 mg; 50%) mp 84-85°C was filtered off and
the solvent was removed to leave a clear oil (80 mg; 20%); [aJD2)
-49.6"(c 2.0, CHCI,); ir(liquid film): 1845,1700~m-~; 'Hnmr6:
0 . 9 4 ( d , 3 , J = 7 H z ) , 1 . 0 6 ( d , 3 , J = 7 H z ) , 1.60(s,9),2.18(m, l),
4.13 (d, 1, J = 4 Hz); 13Cnmr6: 175.7(s, C*), 155.9(s, C=N),
85.4 (s, C(CH,),), 71.0 (d, C-N), 31.1 (d, C(CH,),), 27.5 (q,
C(CH3),), 18.8 and 17.3 (q, C(CH,),); ms mle: 199 (M+ absent),
184 (2, M+ - CH,), 144 (2,7aH+), 116 (5,7aH+ - CO), 101 (20,
(CH,),COCO+), 57 (100, (CH,),C+), 41 (90, C3H,+). Anal.
calcd. for CloH17N03(Boc-Val - H20): C 60.28, H8.60, N 7.03;
found: C 60.04, H 8.47, N 7.02; optical purity >99.8%.
(ii) Conditions Giuing Maximum Yield
Boc-Val (2 mmol) in 20 mL of CH2C12was added dropwise
over 20 min to a stirred solution of EDC (3 mmol) and Et,N. HCI
(3 mmol) in 100 mL of CH,CI,. Work-up as in (i) gave a 20%
yield of 5a and a 50% yield (200 mg) of 4a.
(iii) From a Prematurely Terminated Synthesis of 5a
A solution of Boc-Val(2 mmol) and EDC (1 mmol) in 20 mL of
CH2C12was stirred at 23'C for 3 min. The mixture was washed
with acid and aqueous bicarbonate as in (i), and the solvent was
removed. Analysis showed the residue (160 mg; =37%) was an
equimolar mixture of 4a and 5a. Removal of 5a by precipitation
(-5°C) with light petroleum and elimination of solvent gave pure
4a (40 mg, 10%). When the reactants were stirred for 20 min
before work-up, no 4a could be detected in the product.
(iu) From a Prematurely Terminated Coupling
T o a stirred solution of Boc-Val, Val-OMe HCI, and NMM
(3 mmol of each) in 60 mL of CH2C12at 23°C was added EDC
(3 mmol). After3 min the solution was washed and dried as in(i),
the solvent was removed, and light petroleum was added. After
15 h a t -Sac, the Boc-Val-Val-OMe and any 5a were filtered off.
Elimination of the solvent gave 4a (45 mg; 7.5%).
Similar work-up after 3 min of a coupling mixture containing
DCC instead of EDC gave a 6% yield of 4a mixed with an equiv.
of inert Boc-derivative (see Table 1). The presence of 5a in the
mixture is excluded on the basis of reaction of the mixture with
CH,NH, in ether. Extraction of the solution with N hy-
drochloric acid, and analysis of the aqueous extract (nmr)
showed the presence of Val-NHCH, but no valine.
2-Benzyloxy-4-isopropyl-~-5(4H)-oxazolone (4b)
A synthesis starting with Z-Val (2 mol) and EDC (2 mmol)
carried out as described in (i) for the preparation of 4a gave
crystalline 5b (243 mg; 50%) mp 99-10l0C, followed by 4b a s an
oil (93 mg; 20%); [aIDZ3-55.8" (c 1.0, CHCI,); ir (liquid film):
1845, 1700cm-I; IH nmr6: 0.93 (d, 3, J = 8Hz), 1.07 (d, 3, J =
8 Hz), 4.20 (d, 1, J = 4 Hz), 5.40 (s, 2), 7.48 (s, 5); 13C nmr 6:
175.3 (s, C=O), 158.3 (s, C=N), 134.4-128.6 (aryl carbons),
71.6(t,PhC),71.O(d,C-N),31.0(d,C(CH3),), 18.6and 17.2(q,
C(CH,),); ms mle: 233 (3, M+), 205 (8, M+ - CO), 152 (17,
PhCH20CONH3+), 107 (20, PhCH20+), 91 (100, PhCH,').
Anal. calcd. for Cl,HISN03:C 66.93, H 6.48, N 6.01; found: C
67.10, H 6.65, N 6.15; optical purity >99.8%.
Under conditions giving maximum yield as in (ii) Z-Val
(2 mmol) gave 160 mg of 5b, followed by 253 mg(54%) of 4b.
2-Benzyloxy-4-benzyl-~-5(4H)-oxazolone (4d)
Using the conditions giving maximum yield as in (ii) Z-Phe
(2 mmol) gave 140 mg (25%) of 4d which could be crystallized
from ether - light petroleum. Melting point 65-66°C; [aID2,
-36.5" (c, 1.5, tetrahydrofuran) (lit. (1 I) mp 73-735°C; [aID
-36.0"); lH nmr 6: 3.14 (d, 1, J = 6 Hz), 3.16 (d, 1, J = 5 Hz),
4.60 (2d, 1, J = 5 and 6Hz), 5.33 (s, 2). 7.22 (s, 5), 7.40 (s, 5).
Anal. calcd. for C17Hl,N03:C 72.58, H 5.36, N 4.98; found: C
72.32, H 5.52, N 4.89.
 BENOITON AND CHEN 389

Reaction of 4a with Methylamine Acknowledgements

To 4a (153 mg) was added excess CH,NH, in anhydrous
ether, obtained by extraction from an aqueous solution satu-
rated with NaCI, and the solvent was removed after 10 min. The
residue was dissolved in ether, and the solution was washed with
5% aqueous citric acid and aqueous NaHCO,, dried, and the
solvent was removed. The residue (mp 98-102'C) was recrys-
tallized from CH,CI, - petroleum ether to give 165 mg (94%) of
Boc-Val-NHCH,, mp 113-114'C; 'H nmr 6: 0.95 (d, 6), 1.42 (s,
9),2.10(m,1),2.82(d,3,J=5Hz),3.90(dd,l,J=9and5Hz),
5.10 (br, l), 6.1 (br, 1). Anal. calcd. for C, ,H,2N,03: C 57.4, H
9.6, N 12.2;found: C57.2, H9.5, N 12.1.
Can. J. Chem. Downloaded from cdnsciencepub.com by 139.18.241.77 on 12/04/23
We thank the Medical Research Council of
Canada for financial support, Mr. H. Seguin for the
elemental analyses, and Dr. J. H. Jones for the free
exchange of information subsequent to our initial
presentati~n.~ N.L.B. is Career Investigator of the
M.R.C.C.
1. M. BODANSZKY, Y. S. KLAUSNER, and M. A . ONDETTI.
Peptide synthesis. 2nd ed. Wiley-Interscience, New York,

NY. 1976. p. 137.

Reactions of 4 with 3 to giue 5 2. F. M. F. CHEN,K. KURODA, and N. L. BENOITON. Synthe-
To 4a in CH,CI, was added an equiv. of 3a. After 15 min, the sis, 928 (1978).
solution was washed with aqueous NaHCO,, dried, and the 3. N. L . BENOITON and F. M. F. CHEN.In Peptides: structure
solvent was removed. Addition of light petroleum gave 5a and biological function. Edited by E. Gross and J.
(>90% yield). Compound 5b was obtained similarly from 4b and Meienhofer. Pierce Chemical Co., Rockford, IL. 1979. p.
3b. Reaction of 4a with 3b, or 4b with 3a, gave the same product 261.
(oil) whose nmr spectrum indicated one Boc-group and one 4. M. W. WILLIAMSand G. T. YOUNG.J. Chem. Soc. 3701
Z-group per two isopropyl side-chain groups of ~ a l i n e . ~ (1964).
L- Valine-N-carboxyanhydride (7a)
5. M. GOODMAN and W. J. MCGAHREN. J. Am. Chem. Soc.
87,3028 (1965).
(i) Synthesis from Boc-Val 6. J. REBEKand D. FEITLER.J. Am. Chem. Soc. 96, 1606
A solution of Boc-Val (2 mmol) was treated with EDC under (1914).
cond~tionsgiving a maximum yield of 4a. After work-up and 7. N. L. BENOITON, K. KURODA, S. T. CHEUNG, and F. M. F.
removal of solvent, the residue (0.36 g) containing 5a (34%) and CHEN.Can. J. Biochem. 57,776 (1979).
4a (50%) was left in 20 mL of CH,CI, saturated with dry HCI at 8. D. S. KEMP. In The peptides. Analysis, synthesis, biol-
0°C for 2 h. The solvent was removed by evaporation, more was ogy. Vol. 1. Major methods of peptide bond formation.
added, and evaporation was repeated. CH2CI, was added and Edited by E. Gross and J. Meienhofer. Academic Press.
the mixture was filtered. The solvent was removed, and the New York, NY. 1979. p. 317.
residue was recrystallized from CHCI, -light petroleum to give 9. J. H. JONESand M. J. WITTY.J. Chem. Soc. Chem. Com-
185 mg (65%) of N-carboxyanhydride 7a, mp 57-58°C; [aIDz4 mun. 281 (1977).
-45.0" (c 1.0, acetone) (lit. (18) mp 71°C; [a], -43"; lit. (19) mp 10. M. MIYOSHI.Bull. Chem. Soc. Jpn. 43,3221 (1970); 46,212
67-6YC, [a], -44.4"); ir (CHCI,): 3345,1860, and 1795cm-I; 'H (1973); 46, 1489 (1973).
nmr6: 1.06(d,3,J=6Hz), 1.10(d,3,J=6Hz),2.30(m,1),4.28 11. J. H. JONESand M. J. WITTY.J. Chem. Soc. Perkin Trans.
(d, 1, J = 4 Hz), and 7.15 (br, 1). Anal. calcd. for C6H,N0,: C I, 3203 (1979).
50.34, H 6.34, N 9.79; found: C 50.36, H 6.49, N 9.77; optical 12. J . H . DAVIES,R. H. DAVIS,and R. A. G. CARRINGTON. J.
purity >99.8%. The product is completely soluble in CHCI, Chem. Soc. Perkin Trans. I, 1983 (1972).
indicating the absence of p ~ l y m e r . ~ 13. 1. ANTANOVICS, A. L. HEARD,J. HUGO,M. W. WILLIAMS,
(ii) Reaction of 4a with Hydrogen Chloride and G. T. YOUNG.In Peptides. Proc. Sixth European Pep-
When 4a was left in CHZCI, containing dry HCI at O°C, a 90% tide Symposium, Athens, 1963. Edited by L. Zervas. Per-
yield of 7a was obtained. gamon Press, Oxford. 1966. p. 139; G. T . YOUNG.In Pep-
(iii) Catalytic Hydrogenation of 4b tides. Proc. Eighth European Peptide Symposium,
When 4b was hydrogenated in tert-butanol in the presence of Noordwijk, 1966. Edited by H. C. Beyerman, A. Van d e
5% palladium-on-charcoal catalyst, a quantitative yield of 7a Linde, and W. Massen Van den Brink. North-Holland,
was obtained. Amsterdam. 1967. p. 55.
14. H. DETERMANN, J. HEUER,P. PFAENDER,and M. L.
L-lsoleucine-N-carboxyanhydride (7c) REINARTZ.Ann. Chem. 694, 190(1966).
This compound was obtained from Boc-Ile as described for 15. F. M. F. CHEN,K. K U R O D A N. , ~ L.
~ ~BENOITON.Synthe-
the preparation of 7a. Yield: 65%; mp 65-66'C$ [aIDZ4-27.0" (c sis, 230 (1979).
2.0, acetone) (no data reported (19,20)); ir (CHCI,). 3360, 1860, 16. J. K o v ~ c sR., COVER,G. JHAM,Y. HSIEH,and T. KALAS.
and 1795 cm-I; lH nmr 6: 1.05 (m, 6), 1.45 (m, 2), 2.00 (br, l), I n Peptides: chemistry, structure and biology. Edited by R.
4.25 (d, 1, J = 4 Hz), and 7.10 (br, 1). Anal. calcd. for Walter and J. Meienhofer. Ann. Arbor Science Publishers,
C,HllN0,:C53.49,H7.06,N 9.91;found: C53.66,H7.18,N Ann Arbor, MI. 1975. p. 317.
8.89; optical purity >99.8%. 17. J. C. SHEEHAN,P. A. CRUICKSHANK, and G. L. BOSHART.
Isolation of 4from Prematurely Terminated Couplings (Table 1) J. Org. Chern. 26,2525 (1961).
Reactions and work-up were as described for the isolation of 18. M. OYA,R. KATAKAI,H. NAKI,and Y. IWAKURA. Chem.
4a according to (iu). The identity of the products was confirmed Lett. 1143(1973).
by adding anhydrous methylamine and not~ngthe change in the 19. R. HIRSCHMANN, H. SCHWAM,R. G. STRACHAN,E. F.
nmr spectrum. SCHOENEWALDT, H. BARKEMEYER, S. M. MILLER,J. B.
CONN,V. GARSKY,D. V. VEBER,and R. G. DENKEWAL-
6The mp gradually increases after storage of the N- TER. J. Am. Chem. Soc. 93,2746 (1971).
carboxyanhydride at -5°C (4" in four days). Removal of 20. P. PFAENDER,E . KUHNLE,B. KRAHL,A. BACKMAWSSON,
polymerized material by taking up the mixture in chloroform and G. GNAUCK, and H. BLECHER.Hoppe-Seyler's Z. Physiol.
filtering, gives product (80% recovery) with the original mp. Chem. 354,267 (1973).