4596
ROLFPAULAND GEORGEIT.ANDERSON
VOl. 82
3000 and 2960 (aliphatic CH); 1620 ( N H ) ; 1600, 1560 and the residue heated t o boiling in 2 N hydrochloric acid
and 1545 (C=C, C=K); 1400 (unassigned).
(40 rnl.). T h e acidic solution was treated with Norit and
Anal. Calcd. for C5HsN5: C, 43.15; H, 6.52; N, 50.33. the filtrate neutralized t o pH 6 with concentrated ammonium hydroxide. T h e solid that deposited was collected by
Found: C,42.97; H, 7.01; N, 50.08.
Hydrogenolysis of
5-Amino-4-( l-benzylhydrazino)-6- filtration, dissolved in acetone (10 ml.), a small amount of
chloropyrimidine. A . Raney Nickel.-Raney
nickel (2.0 insoluble material removed by filtration, and the filtrate
evaporated to dryness in vacuo. T h e solid was dried in
g . wet, washed twice with pH 7 buffer solution) was added
to a mixture of 5-amino-4-( l-benzylhydrazino)-6-chloro- vacuo over PZOSa t 56" for 3 hours; yield 150 mg. (48.5%),
in mp: fiH 1,
pyrimidine (200 mg.) in pH 7 buffer solution (25 ml.) and m.p. 182-183' dec.; spectral data: , , ,A
334 (3.98); pH 7, 226 (11.2); -244 (9.33), 346 (2.78);
the resultant mixture refluxed for 2 hours. The mixture was
3265 ( N H ) ;
p H 13, 248 (8.46),280 (4.97); v in
filtered hot, the filtrate evaporated to dryness in z~ucuo,and
2940 (aliphatic C H ) ; 1655 (C=X); 1600, 1575, 1550 and
the residue extracted with chloroform ( 2 X 10 ml.). Evapo1480 (C=C, C=K); 740 and 700 (monosubstituted phenyl).
ration of the combined extracts and sublimation of the
L4naZ. Calcd. for ClaHjoCISj: C, 55.50; H , 33.85;
residue a t 124: (0.1 m m . ) gave a white solid; yield 75 mg.,
N , 27.00; C1, 13.67. Found: C, 55.06; H, 4.02; S ,
111.p. 130-132 . Recrystallization of this material from
27.01; C1, 13.87.
benzene gave a white solid, m.p. 136-137'.
This material
mas identified as 5-amino-4-benzylaminopyrimidineh y ele1,2-Dihydro-l-methylpyrirnido[
5,4-e]-as-triazine (XXIII,
mental analysis and by comparison of its infrared and
K = CH3) .--.4 solution of 5-amino-4-( 1-methy1hydrazino)ultraviolet spectra with those of 5-aniino-4-etliylaminopyrimidine (480 mg.) in formic acid (50 ml.) was refluxed
pyrimidinelo; spectral data: Amax in mp (E X
pH 1, for 0.5 hour and evaporated t o dryness under reduced
290 ( 1 2 . 7 ) ~PH 7, 255 (9.1); 290 (8.3); pH 13, 255 (9.61, pressure. The reddish-brown residue was then heated on a
290 (7.7); v in cm.-l: 3380 and 3200 ( N H ) ; 3060 (aromatic mater-bath under high vacuum until the color of the residue
CH); 2920 and 2880 (aliphatic C H ) ; 1680 (ru"); 1600, had changed t o yellow; yield 580 mg. This material
1590, 1570 and 1510 ( C = C , C=N); 1460 (aliphatic C H ) ; decomposes rapidly without melting above 175" and leaves
750 and 700 (monosubstituted phenyl).
a residue on combustion.
A portion (200 mg.) of this niaterial was extracted with
Anal. Calcd. for CjlH12K4: C, 65.98; H , 6.04; N,
ether (3 X 100 ml.); the extracts were combined and
37.98. Found: C, 66.09; H , 6.04;. hT,
. 28.20.
evaporated to dryness in a stream of nitrogen to give a
B. Palladium.--A
solution of 5-aniino-4-( l-benzylslightly brown solid; yield 100 m g . , m.p. 202' dec. (when
hydrazino)-6-~hloropyrimidine(1 .OO 9 , ) in 1 : 1 ethanoltaken rapidly from 175'); spectral data: , , ,A
in mp_(e X
water (100 ml.) containing magnesium oxide (1.00 g.) was
pH 1, 239 (shoulder, 5.1), 331 (4.55); pH l , 239
hydrogenated over a 5y0 palladium-on-charcoal catalyst.
335
(unstable);
pH
13,
unstable;
i j in cm.-':
(shoulder),
After the steady uptake of one equivalent of hydrogen,
3430.and 3220 (NH); 3040 (=CH); 2910 and 2810 (alithere vias a further, but much slower, absorption of hydrogen. T h e catalyst was removed by filtration and the phatic C H ) ; 1660, 1600 and 150G (C=C, C=X).
. 4 n d Calcd. for C6H7Nj: C, 48.31; H, 4.73; 5, 46.96.
residue was washed with ethanol (25 d.).
A 5y0solution
Found: C, 47.84; H, 4.90; K, 47.13.
of sodium carbonate (50 ml.) was added t o the combined
wash and filtrate, and the whole evaporated to dryness.
l-Benzyl-l,2-dihydropyrimido[5,4-e]-as-triazine (XXIII,
Extraction of the residue with chloroform ( 2 X 50 ml.) K = CHzCeH5).-A solution of 5-amino-4-( 1-benzylhydraand evaporation of the combined extracts gave 550 mg. of
zino)-pyrimidine (460 mg.) in 98-10070 formic acid (25 rnl.)
material, m.p. 101' with softening from 95". Sublimation was refluxed for 30 minutes, evaporated to d r y n e s , and the
of this sample a t 105' (0.1 mm.) gave pure 5-amino-4-(1residue dissolved in 0.5 A' h3-drochloric acid (15 d . 1 .
benzy1hydrazino)-pyrimidine(XXI, R = CHyC6Hs);yield The solution was neutralized with 1 N sodium hydroxide, and
510 mg. (59%), m.p. 100-lO1°; spectral data: A,,
in mp
the oil that deposited extracted with ether (3 X 50 inl.).
(E X
pH 1, 293 (shoulder) ( 8 . 3 ) ,314 (9.3); pH 7- Evaporation of the ether and trituration of the residue with
265 (7.6), 307 (8.4); pH 13, unstable; ij in cm.?: 3410
a small amount of methanol gave a light yellow solid;
and 3300 (XH); 3040 (aromatic C H ) ; 1650 (NH); 1600, yield 220 mg. (46%).
1575, 1550 and 1490 ( C = C , C=N); 1460 (aliphatic C H ) ;
-2small sample of this material was recrystallized from
730 and 700 (monosubstituted phenyl).
benzene by the addition of Skellysolve C ; spectral data:
in xnp ( E X 10-3): p H 1, 335 (4.3); pH 7 , 341 (3.9);
Anal. Calcd. for CIlHl~N5:C, 61.37; H , 6.09; N, , , ,X
pH 13, unstable; 7 in cm.-l: 3200 ( K H ) ; 2950 (aliphatic
32.54. Found: C,60.93; H , 6.08; N,32.29.
l-Benzyl-5-chloro-l,2-dihydropyrimido[5,4-e]-as-triazine C H ) ; 1660 ( C = S ) ; 1600, 1590, 1570 and 1490 (c=c,
(XXII).-A
solution of 5-amino-4-( l-benzylhydrazino)-6- C=N); 775 and 700 (monosuhstituted phenyl).
Anal. Calcd. for C11H1,N5: C, ti:3.98; 11, .1.!!3; N ,
chloropyrimidine (320 mg.) in formic acid (10 nil.) mas
refluxed for 4 hours, evaporated t o a small volume in Z'UCZLO, 31.09. Found: C,68.91; 11,4.91; K,30.96.
N,N'-Carbonyldiimidazole, a New Peptide Forming Reagent'
BY ROLFPAULAND GEORGE\V..\NDCRSON
RECEIVED
FEBRUARY
9, 1960
S,"-Carbotiyldiililiddzole
was shown t o be a useful peptide forming reagent Conditions mere worked out for dc\.uidiW
racemization in the formation of ethyl carbobenzoxyglycyl-~-phenylalanylglycinate,
a sensitive case
I t has been demonstrated by \\Yeland and Schneider2 that peptide derivatives can be synthesized
through acylation of the imidazole ring of methyl
N-benzoyl-L-histidinate followed by reaction with
the appropriate amine. Their method, however,
(1) Preliminary communication G W. Anderson a u d K ,Paul, Tms
80, 4423 (19%).
( 2 ) T . Wieland a n d C, Schneider. Ann.. 580, 1.59 ( 1 9 3 ) ; see also
31 Lierginaiin and L. Zervas. %. p l i r s i u l . C h e m . . 175, 142 (lU28).
JOURNAL,
was not suitable for general use because of low
yields. It occurred to us that a more direct agent
for making acyl-imidazoles might be N,N'-carbonyldiimidazole. This would be a convenient
reagent since the by-products, carbon dioxide and
imidazole, are innocuous. The carbon dioxide
evolution would provide a driving force for the reaction.
Sept. 5, 1960
N,N’-CARBONYLDIIMIDAZOLE
Models show t h a t the amide nitrogen of the imidazole can be brought into contact with the carboxylate suggesting a simultaneous bond formation and
carbon dioxide elimination. An alternate path is,
I
+
of course, a simple displacement by a second mole/=N + co,
cule
of imidazole.
HE 1
k L
It is possible to obtain peptides from acylimidaStaab‘ has shown that S,N’-carbonyldiimidazole zoles by reaction with an amino acid ester hydro(I) is reactive toward amines and alcohols forming chloride instead of the free base and even by using
ureas and carbonates. Extending this, we found an aqueous sslution of an amino acid salt. I n the
that acids react to give acyl-imidazoles, 2nd tlie re- latter case, the yields are lower. Carbobenzoxyaction can be used in peptide synthesis. Ethyl glycine, for example, was treated with the reagent
carbobenzoxyglycyl-L-tyrosinate(II), for instance, in T H F iollowed by a solution of sodium DL-phenylalaninate in water to give a 53% yield of carbowas made in 95% yield by this method.
Difficulties were encountered in following the benzoxyglycyl-DL-phenylalanine(IV) . X limitaliterature3 preparation of the reagent. These were tion to the last method is the low solubilities of
solved by preparing N,K’-carbonyldiimidazole some amino acid salts in water. Likewise a limitafrom imidazole and phosgene in rigorously dried tion on the use of amino acid ester hydrochlorides is
benzene. It was important to use a slight excess their insolubilities in organic solvents.
The importance of using precise amounts of asof imidazole to prevent contamination of the product by half-reacted phosgene. Likewise it was sayed N,S’-carbonyldiimidszole was overlooked
found important to remove all the imidazole hy- a t first since the examples studied gave reasonablydrochloride from the reagent. Contaminated N,- good yields and little trouble was encountered in
purifying them. It was assumed t h a t the desired
?; I-carbonyldiimidazole gave poor yields in peptide
synthesis and was deliquescent. The crude rea- reactions went a t a much faster rate than side reacgent was assayed for carbon dioxide on hydrolysis; tions. As more reactions were studied it became
the purity ranged from 91 to 100yoand the melting evident, however, that while acylimidazole formation was relatively fast and complete, the reaction of
point of the better material was 113-115’.
X typical peptide reaction was run by treating a the acylimidazole with an amine to form a peptide
solution of 0.010 mole of an acylamino acid in 10 was about as fast as the reaction of amine with unml. of tetrahydrofuran (THF) with 0.010 mole of reacted N,N’-carbonyldiimidazole to form ureas.
reagent, adjusted for that quantity from the assay. Ureas being difficult to separate, it was best to avoid
The evolution of carbon dioxide was immediately their formation through the use of precise amounts
observed. After an hour the desired amino acid or of assayed reagent. On using an excess of reagent
peptide ester was added in 0.010 molar quantity. in a preparation of ethyl carbobenzoxyglycyl-LX small amount of heat was evolved. The reaction tyrosinate, the crude product had a poor melting
has been worked up after 15 min., but longer stand- point and was difficult to purify. By the time a
ing is probably beneficial. The product was isolated reasonable melting point was achieved, the yield
by removing the solvent under vacuum followed by had dropped to 64% as compared to 95y0obtained
washing the residue with
acid, saturated bicar- previously.
bonate and finally water.
I n another experiment to determine tlie best solIt is critical t o maintain absolute dryness during vent for the reaction little difference was noted
the reaction of the acid with the r\’,N’-carbonyldi- among six tried. The Oilly one out of line was
imidazole since the reagent decomposes almost in- methylene chloride which gave a higher yield. This
stantly on contact with water. The intermediate difference could be accounted for by the fact t h a t
acylimidazole is stable toward hydrolysis for short the solvent was boiled off rather than removed a t
periods of time.
room temperature and the heating made the reacT o get a better picture of the course of the reac- tion go farther. T o test this, t-butyl trifluoroacetioil, cdrbobenzoxyglycine mas treated with the tylglycyl-L-prolinate (IT)
was obtained in 53%
reagent and the carbobenzoxyglycylimidazole (111) crude yield when prepared in the usual fashion.
was isolated and analyzed. It was treated further In another run, after the addition of the t-butyl Lwith ethvl L-tyrosinate to give an 83% yield of prolinate, the reaction mixture was heated oti a
ethyl carbobenzoxyglycyl-i,-tyrosinate.A possible steam-bath for 1 hour, giving a higher crude yield,
mechanism for the acyl-in-idazole formation would 61yo. However, heating is not recomniended
be an attack on the carbonyl carbon of the reagent as a general procedure since it increascs side reacby the oxygen of the acid or a carboxylate ion fol- tions.
lowed by elimination of a n imidazole molecule to
In several other experiments (see Table I) the
give
best conditions for the formation of one particular
dipeptide were worked out. Mixing the acid and
amine followed by the reagent gave no isolatable
product. Removal of the reaction solvent at room
temperature before work-up gave a slight (2%) improvement, The best yield was obtained in a run
where the reaction of reagent with acid was perL,
mitted t o go for at least 30 minutes at room temperature before the amine was added.
ROLFPAULAND GE(IRGE W. ANDERSON
4598
Vol. 82
TABLE
I
N,N'-Carbonyldiimidazole3(I).--A11 equipment used in
this reaction was dried iii an oven a t 115'. A 2.2-1. portion
VARIATION
OF REACTION
CONDITIOKS
IN THE FORMATIOX
OF
of benzene was placed on 5-10 g . of calcium hydride and 2.0
Z.gly-tyr .OEt(L) (11)
1. was distilled into a N i t e r , 3-necked flask containing 112
Reg . (1.65 moles) of imidazole and equipped with a stirrer aiid
crystd.
drying tube. When the benzene had been distilled over,
yield,
the condenser was replaced b y a gas inlet tube and the misVariation
Solvent
70 A f . p . , O C .
ture heated and stirred t o obtain a clear solution. A 38.6Used excess I m C O
1°F
63
125-128
g . (0.40 mole) quantity of phosgene was collected as a liquid
Heated before a d d n . of H , t y r . O E t
?'ina Dry Ice condenser arid dibtilled, by warnling by hand,
(L)
THP
59
127-129.5
into the reaction mixture. -% precipitate formed at this
Heated a f t e r addn. of H . t y r . O E t
point.
After all the phosgene was distilled, the system w a i
(L)
THF
70
127-128
flushed with dry nitrogen for a few minutes, cooled t o 30"
>-one
THF
71
137-128
and let stand until the liquid phase was clear (from 1 t o 24
Sone
(CHaOCH2)? 67
127-128
lir.), then warmed t o 50" anti filtered. Most of the betiBoiled solvent oR
CHzCIz
78
126-127
zene was removed under reduced pressure a t 60". T h e
None
Pyridine
i4
128-127
rcsulting slush was cooled and quickly filtered. Overnight
None
DMF
titi
126 A-128
drJ-ing under reduced pressure gave 50.4 g . of N,X'-carNone
(Et0)zPOH
73
136.5-128
bonyldiiinitlazole, a 77y0yield. T h e material, on a capillary
Used H B r . H . t y r . O E t ( L ) ; boiled
melting point, shrank a t I l l o , started t o liquify a t 1 1 3 O ,
off solv.
CHzClz
80
126.5-127.5
was a cloudy liquid a t 115' and all clear a t 117" (lit.3
Calcd. arnt. of ImzCO used here
1n.p. 115.5-116°).
T h e reagent was assayed for Carl,
a n d below
CN2Cl?
YO
12-1-1?5.5
dioxide on hydrolj-sis. A calculated C o n value of 27.2
Removed sol!.. before workup; 2
compared with the 26.7 i 0.5yo COa found b y aualy
recrystn.
THF
80
121-126.5
showed the final product t o be 98 =k 276 pure.
silver
K e p t solvent; 2 recrystn,
THF
84 125-127
nitrate test 011 a n acidified sample in water solution was
R a n a t -13"; 4 recrystn.
I1 hl F
79
127-127
n?gative. On several runs yields of 75-8270 and purities
Combined Z.gly.OH
H.tyr.OEt TIIF
Son-crystalfrom 91 t o 99.57, were obtained.
(L) then added lrn?CO
liaable oil
Ethyl Carbobenzoxyglycyl-L-tyrosinate(11).-To a solu30 min. for 1 9 t step, 12 hr. Znd,
tion of 2.09 g. (0.010 mole) of carhobenzozyglycine' in 10
vac. off solv., 1 recrystn.
THF
95
127-128
ml. of dry T H F was added 1.62 g . (0.010
of s,s'30 min. f o r 1st s t e p , 12 hr. 2 n d ,
cnrhoriyldiimidazole. One-half hour later, 2.09 g . (0.010
vac. off solv., 1 recrystn.
CH2Clz
89
12.;-127
e ~ added. After standing
mole) of ethyl ~ - t > - r o s i n a t was
overnight, t h e THF was removed by an air stream and 50
Racemization was investigated in the reaction ml. of 1 '1' hl-drochloric acid was added. Cooling of the
of carbobenzoxyglycyl-L-phenylalanineand ethyl solution gave a solid. This was washed with water, triglycinate, a very sensitive case.4 I t was necessary turated with 20 ml. of 570 sodium bicarbonate solution,
filtered and again washed with water. T h e product was
to determine the purity of the phenylalanine deriva- dried
in a steam cabinet and yielded 3.93 g . (98%), havirig
tive since commercial L-phenylalanine may contain :I melting point of 125.5-127'. Recrystallization from 50D/G
a s x u c h as 5.&!
DL-material. The sample used for ethanol gave 3.79 g. (95%) of material with a melting poi112
1.0
the racemization study was carefully purified by of 127-128". T h e optical rotation, [ c Y ] * ~ D-k 18.2
( r ,5, abs. ethanol), and the melting point compare favorably
fractional crystallization. It was checked for race- with
Lit. vxiues'oof I . 1 2 4 ~
19.3 i 0.1', 171.p. 125-126.5",
mic content and shown to be pure L-isomer. In a n d yield 680jc.
running the test on N,N'-carbonyldiimidazolea t
I ~ Jnaiting
15 tniii. before t h e addition of thc ctll? 1 Lroom teinperature in T H F , 5% racemic material t i rosinate an 83$7, x-ield of r e u s - s t a l k e d materid, 111 I).
was found in the tripeptide thus produced. How- 12C,-12io, !vas obtained.
+
*
+
N-( Carbobenzoxyglycy1)-imidazole
ever, ii the reaction was run iii DMF (diinethyl- ~Itljimidaznle (1.91 g . , 0.01 1 mole,
forrliaiiiitlc) t i t - 1 O 3 less tli:~ii O.,??;) racemic nia- g. fO.010 mole) of carbnbenznxygl
i i i 10 ml. of d r y 'TIIF.
After the cffe
tcrial was detectctl.
solution was diluted with 30 nil. nf
The possibility ol using r)tlicr r e a p i t s siiiiilar i o erystallizd
and w a s cnllecterl b y filt~itioii. I t was tv:isl:(.d
N,S'-carl~r~rivldii~iiidatzole
was investigated. X,- w i t h etlier and drird i n a desiccator t o gi\-e I.U2 g.. 111.1'.
N ' - C a r t ~ o r i ~ l d i b c i i ~ i r i i i ~ 1W;LS
~ ~ zmade
~ l e s and foulid 1 1 .?--I 18'. 'rlie niother liquors yic.lrletl a11 nt1tliti~ili:d
to be less reactii-e t.han the irnidaznle c ~ o n i ~ ~ o u ~ i(c)l,.I 6g . , m . p . 117--118", giving R total !.ieItI o f XO%. 0 1 1
from TIIF-etiier, I .57 g . (60?0), 111.1).
Scveral attelripts to make r\.,Tu"-sullotiyldiiniida- rccryst:illization
1 l~l-120°,wasobtairied.
zole failed. 'Table I1 gives yields for other pepfor C131H13K:jOI:C , ( i O . Z ; 11, 5.0-5; s ,
tides niadc with N.K'-carbonyldiimidazole aiid
C, 60.41; 11, j.31; N,16.04.
coinpares them to literature preparations. An
partially decomposed o n standitlg in t l ~ c
attempt has been made to quote the higher and opcn :veriiight as determined b y a drop in nielting point t o
80-88 . A vialed sample decomposed slightly over t h e
oftinies the highest literature yields.
of a month t o 113-118'.
Perhaps this was due t o
The use of K,:,"-carbonyldiimidazole in the spn- tperiod
h r inoisture in the air of t h e vial.
thesis of angiotensin is currently under investigaX 0.259-g. (0.001 mole) sample of Z.gly.Im was treated
tion.
wit!] 0.209 g. (0.001 mole) of ethyl L-tyrosinate in 2 ml., Of
Acknowledgments.--We thank R4r. L. Brancone THF. After standing overnight water was added giving
olution and a n oil. The oil was crashed wit11
and staff for analyses and Mr. W. Fulmor and
acid and mater. Drying in an oven gave 0.362
staff for optical rotations.
of material, m . p . 121-126'.
Recrystallization
1 g.
yield), 111.p. 1?8-186', of ethyl carboExperimental
benzosyglycyl-L-tyrosinate.
Melting Points.-All
melting points were run on a calibrated Fisher- Johns block unless otherwise indicated.
Solvents-All solvents used were dried; T H F u-as distilled
from calcium hydride; DMF was dried b y azeotroping with
benzene and storing over anhydrous magnesium sulfate.6
(4) G. W. Anderson and 17. 51. Callahan, THISJ O U R K A L , 80, 2002
(1958).
( 5 ) H. A. S t a a b and G. Seel, A n n . , 612, 187 (1968).
( 0 ) A. B. T h o m a s a n d E. G. Rochow, THIS JOURNAL, 7 9 , 1843
(1957).
( 7 ) hI. Bergmann and L. Zervas, B o . , 66, 1192 (1932).
( 8 ) I n some of t h e earlier experiments t h e necessity of having each
b a t c h of X.N,-carbonyldiimidazole assayed was n o t recognized.
(0) E. Fischer, Bey., 34, 433 (1901).
(10) J . R . Vaughan, Jr., a n d R . L. Osato, T m s J O U R N A L . 74, li7C
(1952).
N,N'-CARBONYLDIIMIDAZOLE
Sept. 5 , 1960
4599
TABLE
I1
PEPTIDES
DERIVATIVES
PREPARED
USINGN,K'-CARBOSYLDIIMIDAZOLE~
Pure
yield,
Product
NO
1
2
3
4
J
6
7
8
9
10
11
12
Z.gly-tyr.OEt(L)"
B .phe-gly.OEt(L)"
Z .gly-phe.OEt(DL)'
Z .gly-phe.OEt(DL)'
Z .gly-phe.OH(DL)
Z.gly-phe.OH(L)
Phth.phe-gly-gly.OEt(m)'
Z .ala-gly.OEt(L)
Z .gly-leu .OH( L ) ~
Z.gly-gly.OEt"'
TFA.gly-pro .OtBu( L)'
Z.phe-tyr.OEt(L,~)*
L-HZ
%
95
78
80
82
55
40
56
65
68
60
51
57
I
h4.p..
oc.
127-128
8849.5
90-91
91-92
162.5-163.5
126.5-127.5
163-164.5
98-99
103-101
82.5-83
90-91
158-160
13 2 .asp-gly . O C H Z C ~ H ~ ( L ) ' 35 177-1 79
27 184
14 Zaileu-his OMe(L,L)
87 119.9-120.3
1 5 Z .gly-phe-glyOEt(L)w
Lit.
yield,
Recrystn.
solvent
[U]r'D
50% ethanol
Pet. ether
Benzene-pet. ether
Benzene-pet. ether
50% ethanol
Water
Benzene-pet . ether
EtOAc-pet. ether
EtOAc-pet. ether
50% ethanol
Methylcyclohexane
50% ethanol
Water
Methanol-water
Abs. ethanol
(6)
-I-18.2 f 1 . 0 " (abs. E t O H )
- 4 . 2 =k 1 . 2 ( E t O H )
4-40.7 & 1 . 7 (abs. E t O H )
-21.7 & 0 . 5 (abs. E t O H )
-18.2 i 0 . 5 (1 N XaOH)
-83 ( E t O H )
- 9 . 2 & 0 . 5 (EtOH)
+1 . 8 ( D M F )
-12.2 & 1 . 2 5 ( E t O H )
c/o
Lit.
F.P.,
C.
68h 125-126.8
81d 86-87
86'
9 1-92
86'
91-92
€ 1 3 ~ 160-182
12i.j"
162-163
77' 100
62' 104
83
86-87"
69'
89-90
46b 189-160'
50'
50"
96"
181-183
186-189
120-120.5
Z = Carbobenzoxy. * Ref. 10; mixed anhydride method.
B = t-butyloxycarbonyl,
G. W. Anderson and A. C.
McGregor, THISJOURNAL,79, 6183 (1957). Recrystallized t o 35% yield, m.p. 89.5-90", from ethyl acetate-petr. ether;
pyrophosphite method. e Free H.phe.OEt(DL) used. f Ref. 12; mixed anhydride method. 0 Using HBr.H.phe.OEt( D L ) ' directly.
~
Ref. 4. a P h t h = phthaloyl; from Phth.phe.OH and H,gly-gly.OEt. j M. Bergmann, L. Zervas, J. S.
Fruton, F. Schneider and H. Schleich, J . Biol. Chem., 109, 325 (1935); acid chloride method.
The ethyl ester is an oil
and was saponified; the yield is the over-all yield.
H. S. Goldschmidt and H. Lautenschlager, Ann., 580,68 (19.53); phosphorus trichloride method, followed by saponification of the ethyl ester thus formed.
Using HC1,H.gly.OEt directly.
Ref. 14a using the pyrophosphite method. 0. Siis, Ann., 572, 104 (1951), reports m.p. 82.5-83" and a 25y0 yield using
the phosphorus trichloride method.
T F A = trifluoroacetyl. p Ref. 13; pyrophosphite method, crude yield. * Amorphous. I. J. S. Fruton and M. Bergmann, J . Biol. Chenz., 145,262 (1942), using 2.phe.Cl obtain 4670,m.p: 162'. * Amine
used as benzenesulfonate; product was recrystallized three times. ' H. K. ,Miller and H. Waelsch, Arch. Bzochem. Biophys.,
35, 176 (1952); diethyl chlorophosphite method. * H. Schwarz, F. M. Bumpus and I. H. Page, THISJOURNAL, 79, 5697
(1957); mixed anhydride method.
Ref. 4; pyrophosphite method.
The general procedure used for the synthesis of
Z.gly-tyr,OEt(L) was used in each case.
Ethyl Carbobenzoxyglycyl-DL-phenylaiadnate.-Ethyl DLphenylalaninate was prepared from 2.75 g. (0.010 mole)
of ethyl DL-phenylalaninate hydrobromide" on treatment
n i t h a n excess of triethylamine in T H F followed by filtration and concentration. A solution of 2.09 g. (0.010 mole)
of carbobenzoxyglycine in 10 ml. of d r y T H F was treated
with 1.62 g. (0.U10 mole) of K,N'-carbonyldiimidazole.
When the effervescence ceased, the previously prepared
ethyl DL-phenylalaninate was added. After 15-30 minutes,
the mixture was air-dried and 50 ml. of 1 N hydrochloric
acid added. On cooling, a solid formed. This was collected, triturated first with water, then with a 5% sodium
bicarbonate solution, and again with water. Drying
gave 3.20 g. (83y0 yield) of ethyl carbobenzoxyglycyl-DLphenylalaninate with a melting point of 90-91". The material was recrystallized from benzine-petroleum ether, resulting in 3 . 1 0 ~ (80%
.
yield).
product had a melting
point of 90-91 , lit. value12 91-92 . This experiment was
repeated except t h a t the ethyl DL-phenylalaninate hydrobromide was added directly t o the reaction mixture without
first removing the H B r with triethylamine. An 82% yield
of a product having a melting point of 91-92' was obtained
by this method.
Carbobenzoxyglycyl-DL-phenylalanine (IV).-X
solution
of 2.09 g. (0.010 mole) of carbobenzoxyglycine in 10 ml. of
tlricd T H F was treated with 1.62 g. (0.010 mole) of S , S ' carbon>-ldiiinidazole. IVhen the effervescence stopped, a
solution of 1.64 g. (0.010 mole) of DL-phenylalanine in 10
nil. ( J f 1 S sodium hydroxide was added. After 15-30
minutes, 30 ml. of 1 A hydrochloric acid was added. The
oily liquid thus formed crystallized in a few minutes. The
product, carbobenzoxyglycyl-DL-phenylalanine, was collected, washed with water and dried t o give 2.51 g. (707,
yield) of material having a melting point of 158-160'.
TF
(11) Made f r o m Dr.-phenylalanine, ethanol a n d d r y hydrogen bromide; m.p. 131-132'.
A f d . Calcd. for CIIHlaKO?Br: C, 48.19;
H , 5.88; N, 5.11. F o u n d : C . 48.32; H, 0.01; N,5.39.
(12) J. R. Vaughan, Jr., a n d R . L. Osatn, THISJ O U R N A L , 73, 5553
(1951).
The product was recrystallized from 35 nil. of 507; ethanul
giving 1.97 g. (557, yield) of the dipeptide with a melting
point of 162.6-163.5", lit. valuelo 160-162".
The above experiment was repeated using L-phenylalanine in place of DL-phenylalanine. X 407, yield of material
was obtained which had a melting point of 126.5-127.5'
and a n optical rotation [CY]*~D 40.7 f 1.7" (c 2.9, absolute
ethanol); the corresponding lit. values4 were m.p. 127.5'
and [ c Y ] ~ ~ D38.8 f 0.5" ( c 5, absolute ethanol).
&Butyl Trifluoroacetylglycyl-L-prolinate (V).-The
solution resulting from the reaction of 1.71 g. (0.01 mole) of
trifluoroacetylglycine's in 10 ml. of T H F with 1.74 g. (0.01
mole, 93y0 pure) of iX,K'-carbonyldiimidazole was permitted to stand for a n hour. Then 1.71 g. of t-butyl Lprolinate14 was added. After standing overnight, the solvent rvas removed under vacuum and the residual yellow
oil triturated with 35 ml. of N acid. The product crystallized readily. The colorless solid was collected, triturate?
with water and dried, giving 1.70 g. (53y0), m.p. 89-90 .
One recrystallization from methylcyclohexane gave 1.65
g . (517, yield) of material, m.p. 90-91".
I n a second run the reaction was warmed on a steam-bath
for 1 hr. after the addition of the amine instead of leaving
i t overnight a t room temperature. In this case, the crude
yield was 1.97 g. or 61%, m.p. 89-90', [ a I z 5 D - 83" (c 2 ,
ethanol); lit.14m.p. 89-90",
Racemization Studies with N,N'-Carbonyldiimidazo1e.The reaction of carbobenzoxyglycyl-L-phenylalaninewith
ethyl glycinate is very sensitive t o ra~emization.~.'6 I t
was ~ h o r n nthat
~ ~ ~using
~
tetraethylpyrophosphite as the
peptide-forming agent gave no racemization. T o test the
purity of the starting dipeptide acid the reaction was run
using tetraethylpyrophosphite and no racemic material
+
+
(13) F. Weygand a n d E. Leising, Be?., 87, 248 (1954).
(14) G. W. Anderson a n d F. M. Callahan, THISJ O U R N A L , 82, 3369
(19GO).
(15) (a) G. W. Anderson a n d R . W. Young, i b i d . , 74, 5307 (1952);
(b) G. W. Anderson, J. Blodinger a n d A. D. \Velcher, ibid., 74, 5309
(1952); ( c ) J. It. Vaughan, Jr., ibid.. 74, GI37 (1952).
4600
DEKEKG . S X Y T I I ,
XTSUO NL\G.CvL4TSU i\ND JOSEPH
s. F R U T O N
Vol. 82
was fourid. Procecdiiig from there, a solution of 3.56 g.
[having
(0.010 mole) of carbobenzoxyglycyl-L-phenylalanine
a melting point of 127.5-128.0", and a n optical rotation
[a]"D f 38.2" (c 5 , abs. ethanol) compared with values4
38.8 i.0.5" (c 5. abs. ethanol)]
of m.p. 127.5' and [ a ] " D
in 10 ml. of dried (calcium hydride) dimethj-liormamide
was cooled t o - 10' and 1.65 g. (0.010 mole based on 98y0
purity) of N,N'-carbonyldiimidazole was added. When
the slow effervescence stopped, 1.03 g. (0.010 mole) of freshlydistilled ethyl glycinate was added. The reaction solution
was allowed t o warm t o room temperature and permitted
t o stand 15-30 min. Then 50 ml. of 1 N hydrochloric
acid was added. JYlien t h e oily liquid thus formed solidified, i t was washed with 20 m1. of a 57, sodium bicarbonate
solution and with water. On drying, 4.22 g. (96% yield)
with a melting point of l15.~5-l17.0° was obtained. The
product was dissolved in 210 ml. of absolute ethanol t o
give a 27, solution. After cooling t o O", the solution was
seeded with a crystal of ethyl carbobcnzoxyglycyl-DLphenylalarij~lgl~~ciiiate.
Fractions were cut as follows:
133.5", the percentage of DL-tripeptide is estimated from
the melting points t o be much less than 0.5%.
In other reactions, run in the same way, varying oiic or
two factors, more racemization was found. Running the
reaction in T H F a t room temperature gave 57, DL-tripeptide. Using ethyl glycinate hydrochloride a t room tempcrature in T H F gave 8% DL-tripeptide. Running the rcaction in dimethylformamide a t 0-5' gave 1.3yc raceniization.
I n these studies, the melting point of the carbobenzoxyglycyl-L-phenylalanine used was very important since
commercial L-phenylalanine may contain several per cent .
of the DL-isomer.
Ethyl Carbobenzoxyglycyl-DL-phenylalaninateusing N,N'Carbonyldibenzimidazo1e.--A solution of 2.09 g . (0.010 mole)
of carbobenzosygl!.cine in 10 1111. of dry THE; was treated
with 2.62 g. (0.010 mole) of S,S'-carboiiyldibeiiziniicl~z(~le.b
When no effervescence was noted, and the reagent only
partially dissolved, another 10 ml. of T H F was added.
Again nothing happened and the mixture was heated undcr
reflux for 10 min. A solution formed and 1.93 g. (0.010
No.
Time, hr.
LVt., 6 .
SI.p., o c ,
mole) of ethyl DL-phenylalaninate (freshly distillcd) was
1 3
0,0091
120.0-133.5
added. After heating for 15-30 niin. on a steam-bath,
2 6
,0097
119.0-128.5
most of the solvent boiled off. X 50-ml. quantity of 1 fi
hydrochloric acid was then added. Cooling and scratcli3 10
,0214
118.5-119.5
ing gave a solid product. This was washed first with wxter,
4 24
2.3201
119.8-120.1
then with 20 nil. of 5 7 , sodiurn bicarbonate solution and
Concd.
I . 4787
119.9-120.3
finally with water again. The crude product weighed 3.48
Residue
0.3271
g. (777, yield) and had a melting point range of 83.5-58.0".
Kecrystallization from 20 ml. of ethyl acetate and 40 ml. of
0.0188 0 .457,
DL-Isomer
petroleum ether resulted in 2.75 g . of compound with a
L-Isomer
3.8202 87%
[ a ] * j-12.2
~
& 1.25'
melting range of 85-88', This material was recrystallized
(c 2, E t O H )
again, this time from 20 ml. of benzene a n d 40 mi. of peRcsidue
0,3271
troleum ether, giving 2.63 g. (69y0 yield) of ethyl carbobenzoxyglJ-cyl-DL-phenvlalaninate with a melting point of
Material
89.5-91.0' as compared t o a previously cited melting point
balance 4.1661
of 90-91". Because of the poorer yield and the inure
Since the melting point of pure ethyl earhoberizox~-glycj-l- rigorous conditions nccessary for a reaction, N,S'-carbonyltlibenziiiiidazole is considered inferior t o S,N'-carbonyldiDL-phenylalan?-lglyCinatC was reported4 t o be 132-133'
and has been found in other reactions by us t o be 133.0- imidazole.
+
[CONTRIBUTIOX FROX THE
DEPARTMEXT
O F BIOCHEMISTRY,
YALE
UNIVERSITY]
Some Reactions of N-Ethylmaleimide
BY DEREKG. SMYTII,?
ATSUONAGAMATSU~
AKD
JOSEPH
S. FRUTON
RECEIVED
MARCH7, 1960
T h e reactions of S - e t h y l m a l e h i d e (NEM) with the amino group :if peptides, with imidazole and with cysteine have been
investigated. IVith the first two classes of compound, an S-acylation reaction appears t o occur, followed in t h e case of
imidazole by a catalytic polymerization of S E M . \\'it11 cysteine, reaction proceeds through addition of the thiol t o the
olefinic bond of XEM ; in alkaline sulution. the c p t r i n e adduct undergoes an intramolecular transamidation reaction to
form a thiazane derivative.
During the course of studies on the action of
cysteine-activated cathepsin C4on glycyl-L-histidinamide a t fiH 7.4, X-ethylmaleimide (KELT) was
used to facilitate chromatographic examination of
the composition of the incubation mixture. The
reaction of NEM with sulfhydryl corn pound^^^^
had been used by Hanes, et al.,; to stabilize glutathione and other sulfhydryl peptides in paper
(1) This investigation w a s supported by grants from t h e Kational
Science Foundation (G-7451) a n d the United States Public Health
Service (RG-6452).
(2) Alexander Brown Coxe Fellow of the Yale School of Medicine,
1059-1960.
(3) James Hudson Brown Fellow of t h e Yale School of hledicine,
1!158-1059.
On leave from t h e Depnrtment $ i f Biochemistry Kyushu
7.niversity. F u k u o k a , Japan.
(4) N . Izumiya and J . S. Frruton, .I. B i d Chewi., 218, 59 (1<#.56)
(.5) 13. Friedmann, D 11. l l a r r i a n and 1. Simon-Rruss, Rvi' J .
I'iini,in~: 01 , 4 , 105 ( 1 9 4 ! I i , Biorhiiii. I : i o p k y s : l r / , r , 9 , 161 ( 1 9 2 2 ) .
(6) D. 1%.tlarriazi, J . Chuni.Soc.. 1515 (1940).
( 7 ) C . S. Hanes, F. J . R . Hird and F. A . Ishrrw~oud.B i o L i i i i ~ :J . . 51,
2 3 (11132i.
chromatography. n'hen the NEM-treated incubation mixtures of cathepsin C and glycyl-Lhistidinamide were chromatographed, a number of
Pauly-reactive components of widely different
Rf values were observed.e Subsequent control
experiments demonstrated, however, t h a t the new
products (other than the expected hydrolytic
product, glycyl-L-histidine, or the unchanged dipeptide amide) arose in incubation mixtures
to which no enzyme had been added. Further
investigations showed t h a t one of the new Paulypositive products was noted only when NE31
had been used before paper chromatography-, and
led to the recognition that NEM is not specific
tnward sulfhydryl compounds, a s had previously
1)c.eii supposed. 111 thc present comiiiunicatioii
IW report some reactions of NE11 with ili1id:tLolc
and its derivatives, with the a-amino group of
( 8 ) J Barndbds unpublished experlments