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Methods of Peptide Synthesis.

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Methods of Peptide Synthesis [*I
BY PROF. DR. T. WIELAND AND DR. H. DETERMANN
INSTITUT F O R ORGANISCHE CHEMIE DER UNIVERSITAT FRANKFURT/MAIN (GERMANY)
Dedicated to Dr. W. Foerst on fhc. occasion of his 60th birthday
I n the series of accounts of progress in the field ofpeptide synthesis, this review rurwy\
the literature from the beginning of1959 to the middle of‘1962.
Introduction
Protecting groups
Protecting group on the nitrogen
Protecting group on t h e carboxyl group
Methods of coupling
Use of chlorides
Use of hydrazides a n d azides
1I
IJse o f unsaturated compounds
Use of activated esters
Use of phosphorus derivatives
Use of azoles
Sundry methods
Synthesis of cyclic peptides
Racemization during the formation of peptidcs
Introduction
At the time of a previous review of peptide syntheses [I],
the chemistry of polypeptide hormones was gradually
being disclosed. In order to be able to build longer
chains from polyfunctional amino acids, attempts have
been made in recent years to find new protecting groups
which can be removed in a specific manner, to raise the
yields of the coupling reactions, and to avoid racemkation of the natural amino acids. These efforts have
led, for example, to the synthesis of corticotrophic peptides with the sequence of the first twenty-three [2] and
twenty-four [3] amino acids of natural corticotrophin.
Further accounts of the problems involved in peptide
syntheses [4-61 have since been added to those cited [I].
On saponification of dipeptide esters with alkali, the
carbobenzoxy group
which is probably still used
most - leads more or less easily to the formation of
urea derivatives [S]. MucLaren [9] has recently investigated this troublesome reaction thoroughly. He
found that carbobenzoxy-dipeptide esters in which the
ester group was attached to glycine rearranged in the
cold if excess alkali was used. For example, the ethyl
ester of carbobenzoxyglycylleucine can be hydrolysed
with 2 moles of alkali to give the carbobenzoxy-dipeptide without any trouble, whereas the ethyl ester of
carbobenzoxyleucylglycine gives a difficultly soluble
hydantoin derivative ( I ) under these conditions.
Protecting Groups
Protecting Group on the Nitrogen
The groups described below may be used for protecting
not only nitrogen, but also hydroxyl and mercapto
groups,mostly by methods that are similar in principle[7].
[*I Communication No. 28 in the series “Uber Peptidsynthesen”
(On peptide syntheses); Communication No. 27: T. Wieland, H .
Determann, and W . Kahle, Angew. Chem. internat. Edit. 2, 154
(1963). Peptide Syntheses IV: T . Wieland, Angew. Chem. 71,417
(1959).
[I] T . Wieland, Angew. Chem. 63, 7 (1951);ibid. 66, 507 (1954);
T . Wieland and B. Hrinke, ibid. 69, 362 (1957); T . Wieland, ibid.
71,417 (1959).
[2] K . Hofmann, H . Yajika, N . Yanaihara, T. Y. Liu, and S .
Lande, J. Amer. chem. Soc. 83, 487 (1961).
[3] H . Kappeler andR.Schwyzer,Helv.chim.Acta44,1136(1961).
[4] J. P . Greenstein and M . Winitz: Chemistry of the Amino
Acids. Wiley, New York 1961.
[5] E. Bricas, Bull. SOC.chim. France 1961,2001.
[6] N. F. Albertson, Org. Reactions 12, 157 (1962).
[7] See H . C. Beyerman and J . S. Bontekoe, Proc. chem. SOC.
(London) 1961, 249.
358
Birkojkr and coworkers [lo] were able to remove the
carbobenzoxy residue by hydrogenation with triethylsilane [ l l ] i n the presence Df catalytic quantities of PdC12
and triethylamine. They refluxed the carbobenzoxy
derivative in this solution for several hours and then
decomposed the resulting silyl derivative with methanol
to give the free amino acid or peptide. These conditions
also allow hydrogenolysis of beiizyl esters; benzyl linked
to sulfur is unaffected.
Substituted carbobenzoxy groups enjoy increasing
popularity because of the gradation of reactivity which
[8] F. Wessely and E . Konrm, Hoppe-Seylers Z. physiol. Chem.
174,306 (1928); F. Wessely, K . Schlogel, and G. Korger, Nature
(London) 169, 708 (1952);F. Wessely, K. Schlogel, and E . Wawersich, Mh. Chem. 83, 1426 (1952).
[9] J . A . MacLarew, Austral. J . Chem. 11, 360 (1958).
[to]L. Eirkhqfer, E. Eierwirth, and A . Ritter, Chem. Ber. 94,821
(1961).
[ I l l J . W . Jenkins and H . W . Post, J . org. Chemistry 15, 556
(1 950).
Angew. Cltern. internat. Edit. I Vol. 2 (1963) I No. 7
may be achieved. The nitro group in the p-nitrobenzyloxycarbonyl (PNZ) group reduces the electron density
at the benzyl carbon atom and hence makes re-elimination of the group with HBr in glacial acetic acid difficult. On the other hand, the PNZ group is removed
by catalytically activated hydrogen much more readily
than the carbobenzoxy group [121. These differences
have made it possible to prepare prolyl-N(E)-PNZ-lysine
from the carbobenzoxy compound [13]. The eflect of a
methoxyl group IS in the opposite sense; it considerably
facilitates proton-catalysed removal of the p-methoxybenzyloxycarbonyl (PMZ) group [14]. Introduction of
the PMZ group has been accomplished by Weygand and
Hunger [15] using the crystalline azide (2); anhydrous
trifluoroacetic acid at 0 ° C is sufficient to split it off.
The carbobenzoxy residue is not removed under these
conditions; it requires boiling for 30 minutes with trifluoroacetic acid [16]. A phenol must be added in order
to capture the benzyl cation and prevent side reactions.
For the use of the p-chlorobenzyloxycarbonyl group,
see [17].
Increased importance has been attained by the t-butyloxycarbonyl (BOC) group, which at the time of the
previous review [I] could only be introduced by reaction
Gf the isocyanate compounds with t-butanol. Several
more elegant ways have since been devised. First, the
preparation of t-butyl-p-nitrophenylcarbonate(3) was
reported; this reacts with amino acids within 20 minutes
in the presence of alkali at 100°C [18]. Acylation can
be carried out under milder conditions with t-butoxycarbonylazide (4) [20], which was made for the first
time by Carpino [19]. The imidazolide ( 5 ) [21] and the
cyanide (6) [22] may also be used for simple syntheses of
H,C
0
I
II
H,C-C-0-C-R
I
ti&
" .
.
14): R = -N,
(5):
F==7
R=-N'
\ I
[I21 C . Berse, R . Boucher, and L. PichC, J. org. Chemistry 22,805
( 1957).
[I31 J. E. Shields and F. H . Carpenter, J . Amer. chem. SOC.83,
3066 (1961).
1141 F. C. MeKay and N . F. Alhertson, J. Amer. chem. SOC.79,
4686 (1957).
[I51 F. Weygand and K. Hunger, Chem. Ber. 95, 1 (1962).
[I61 F. Weygand and W. Steglich, 2. Naturforsch. 146, 472
(1959).
[I71 L. Kisfaludy and S . Dualszky, Acta. chim. Acad. Sci.
Hungary 24, 301 (1960); ibid. 24, 309 (1960); Chem. Abstr. 55,
9295 (1961).
[18] G. W . Anderson and A . C . McGregor, J. Amer. chem. SOC.
79, 6180 (1957).
[19] L.A . Carpino, J. Amer. chem. SOC.79,98,4427 (1957);L. A.
Carpino, C. A . Giza, and B. A . Carpino, J. Amer. chem. SOC.81,
955 (1959); see also [20].
1201 R . Schwyzer, P. Sieber, and H . Kappeler, Helv. chim. Acta
42, 2622 (1959).
[21] W. KIee and M. Brenner, Helv. chim. Acta 44, 2151 (1961).
[22] L.A. Carpino, I. Amer. chem. SOC.82,2725 (1960).
Angew. Chem. internat. Edit. / Vol. 2 (1963) / No. 7
BOC derivatives. The BOC group can be split off at
room temperature by the procedure of Anderson and McCregor [18] using HBr in diethyl phosphite or glacial
acetic acid, or
separately from the carbobenzoxy
group - by the method of Schwyzrr [23] using anhydrous trifluoroacetic acid. The BOC group is not
removed by acetic acid, so that it is possible to split it
off separately if a trityl group I S also present [24].
The phthalyl group is being used less and less on
account of its sensitivity to alkali, which has recently
been measured quantitatively [25]. Moreover, the
commonly used cleavage with excess hydrazine hydrate
in alcohol cannot be applied t o base-sensitive peptides.
In this respect, Schwyzcr and coworkers [26] have made
the valuable observation that hydrazine is effective even
in weakly acid solution. We have observed that the
phthalyl group can be removed very easily by heating
with imidazole and a little water. Its introduction is now
performed at room temperature within 15 minutes
using N-ethoxycarbonylphthalimide(7) according to
Nefkens [27]. The triacylamrde (7) undergoes ring
opening to give a diacylamide (8), which splits off
urethane and gives the phthalyldmino acid (9). Phthalyl-
ation of amino acids is also possible by heating with
diphenyl phthalate [28]. The trifluoroacetyl (TFA)
group can be introduced in a similar way [29].
N-Formylamino acids are particularly prone to racemization during peptide syntheses [30]. Despite this,
attention should be paid to a method for specific
removal of the formyl group by oxidation with 15 %
aqueous hydrogen peroxide at 60°C for 2 hours [31].
Oxidation with peroxybenzoic acid is a mild way of
splitting off the ethylthioformyl group, C~HS-S-CO-.
A large number of amino acid and peptide derivatives
[23] R . Schwyzer, W . Rittel, H . Kuppeler, and B . Iselin, Angew.
Chem. 72, 915 (1960).
1241 R . Schwyzer and W. Riitel, H c l v . chim. Acta 44, 159 (1961).
[25] J. Rudinger, J. Krupitka, M . Zaoral, and W . Cernick,
Collect. czechoslov. chern. Cornmiin. 25, 3338 (1960).
[26] R . Schwyzer, A . Costopanagiotis, and P . Sieber, Chimia 16,
295 (1962).
1271 G. H . L. Nefkens, Nature (London) 185, 309 (1960); G . H.
L. Nefkens, G . J. Tesser, and R . J . Nivard, Recueil Trav. chim.
Pays-Bas 79, 688 (1961).
[28] F. Weygand and J . Kaclicke, Chcm. Ber. 95, 1031 (1962).
[29] F. Weygand and A. RCjpsch, Chem. Bcr. 02, 2095 (1959).
[30] J . C. Sheehan and D. H . Y m g . J. Amer. chem. SOC.80, 1154
( 1958).
[31] G.Losse and W . Z&nchen, Angew. Chem. 72, 385 (1960);
Liebigs Ann, Chem. 636, 140 (1960).
359
have been made recently in this way [32]. Dibenzylphosphoryl chlorides substituted in the p-position,
particularly thep-nitro compound, allow introduction of
phosphoric ester groupings, which may be re-eliminated
from the nitrogen by catalytic hydrogenation. During
their removal, N-phosphoryl derivatives that are
extremely labile to acids are formed as intermediates
[33]. The behavior of benzyl derivatives has been
amino acids (type [Oh), peptides were prepared in good
yields using dicyclohexylcarbodiimide, the protective
groups being split off with dilute mineral acid. The
reaction products of amino acids with 1,3-dicarbonyl
compounds ( e . g . acetoacetic ester, benzoylacetone, 2,4dioxopentane) are also hydrogen-bonded stable Schiffbases. They can be used in peptide syntheses, the
protecting group being split off by dilute acids after
coupling [41a].
A substituent which has been closely examined recently
is the trimethylsilyl group. N-Trimethylsilylamino esters
are obtained from trimethylchlorosilane and amino
esters 1421. The chloride reacts with salts of amino acids
to give trimethylsilyl N-trimethylsilylamino esters ( / I )
investigated further [34]. All benzyl residues may be
CII,
o
c1i3
I
II
removed from benzyl esters of dibenzylamino acids or
1 L,L-S1-NH-CIlK'-C-~1-~~l3
of peptides by treatment for 7 days with glacial acetic
c11~
c1-1~
acid saturated with HBr. The P-cyanoethyl group,
(11)
NC-CHz-CH2-, formed by addition of amino acids
onto acrylonitrile [35], is transferred over to aniline or
[43], which are also Formed from the sodium salts
[44] or hydrochlorides [45] with trimethylsilyldiethylother amines on heating [36]. Turba and coworkers [37]
recently worked out an interesting method for hydroamine, diethylamine being eliminated. These esters
genolytic release of amines and peptides from their 2,4are also formed with particular ease from free amino
dinitrophenyl derivatives. Large amounts of a platinum
acids by warming t Iicm in hexamethylsilazane,
catalyst are used; rn-phenylenediamine is formed from
(H3C)3Si-NHPSi(CH3)~, ammonia being evolved [46].
the dinitrophenyl residue.
The silyl ester of the trimethylsilylamino acid may be
The o-nitrophenylmercapto group, o-O~N-C~H~-S-, acylated on the nitrogen with carbobenzoxyamino acids
by the phosphorus oxychloride method [47] or by
which is easily removed from the nitrogen with weak
carbonyldiimidazole [48], the silyl group having no
acids, may be introduced by treating the sodium salts
adverse effects. On working up, the carbobenzoxyor esters of amino acids with o-nitrophenylsulfenyl
peptide is obtained directly owing to the extreme senchloride [38].
sitivity to water of the trimethylsilyl group [49]. Thus,
As the benzylidene group was used only in preliminary
the
trimethylsilyl group has the function here of prowork as a protecting group for peptide synthesis [39],
tecting
the carboxyl group and not the nitrogen.
and since its extreme lability to acid did not
give rise to any great hopes, the more stable hydrogen
bonded o-hydroxyarylidene compounds (1Oa) and (lob),
Protecting Groups on the Carboxyl Group
synthetized by Mclntire [40], have been used of late
[41]. Starting from the more stable naphthylideneThe carboxyl groups of N-acylamino acids may also be
substituted with the trimethylsilyl group if the salts of
the acids are treated with trimethylchlorosilane [50] or
hexamethylsilazane. The trimethylsilyl ester is probably
the most easily hydrolysable ester.
Tertiary butyl esters have become easily accessible in
ilea)
(Job)
the last few years and are particularly useful. The t-butyl
group,first used by Sheehan in the total synthesis of peni[32] A . Hajds, Chern. Ber. 94, 2350 (1961).
[33] A . Cosmntos, J. Photaki, and L. Zervas, Chem. Ber. 94,
2644 (1961).
[34] K . T . Poroshin and V. I . Maksimov, Izvest. Akad. Nauk
S.S.S.R., Otdel. khim. Nauk 1960, 1702; Chem. Abstr. 55, 9292
(1961);V. I . Maksimov and K . T . Poroshin, ibid. 1961,186; Chem.
Abstr. 55, 20984 (1961).
[35] H . A. Bruson, Org. Reactions 5,79 (1949).
[36] P . F. Butskus and G. I . Denis, Zhur. Obschei Khim. 30, 1317
(1960); Chern. Abstr. 55, 405 (1961); see also Chem. Abstr. 55,
1460, 24583 (1961).
[37] H . Fasold, G. Steinkopf, and F. Turba, Biochem. Z. 335, 1
(1961).
[38] J . Goerdeler and A . Holst, Angew. Chern. 71, 775 (1959).
[39] T. Wieland and W . Schafer, Liebigs Ann. Chem. 576, 104
(1 952).
[40] F. C . Mdntire, J. Arner. chern. SOC.69, 1377 (1947).
[41] J . C . Sheehan and W . J . Grendu, J . Amer. chern. SOC.84,
2417 (1962).
360
[41 a1 E. Dane, F. Drees, and P . Konrad, German Patent Application (Apr. 27th, 1961), Farbenfabriken Bayer.
[42] L. Birkofer and A . Ritter, Angew. Chem. 68, 461 (1956);
Liebigs Ann. Chem. 612, 22 (1958).
[43] K. Riihlmann, J. prakt. Chem. 28/, 86 (1959).
[44] K . Riihlmann, J . prakt. Chem. 2 8 / , 315 (1959).
[45] K . Riihlmnnn and G . Michael, 2. Naturforsch. 156, 811
(1960); K . Riihlmann, Chem. Her. 94, 1876 (1961).
[46] L. Birkofer and A. Rifter, Chem. Ber. 93,424 (1960).
1471 T . Wieland and B. Heinke, Liebigs Ann. Chern. 599, 70
(1956).
[48] G . W. Anderson and R . Paul, J. Amer. chem. SOC.80,4423
(1958).
[49] L. Birkofer, W. Konkel, and A . Ritter, Angew. Chem. 7 1 ,
701 (1959); Chern. Ber. 94, 1263 (1961).
[50] F. A. Henglein and W . Knoch, Makromolekulare Chem. 28,
10 (1958).
Angew. Chem. internut. Edit. 1 Vol. 2 (1963) I No. 7
cillin V [51], may be split off under particularly mild
conditions, e.g. with p-toluenesulfonic acid in boiling
benzene or with trifluoroacetic acid at room temperature. Esterification of amino acids with t-butanol in the
usual manner is not successful. N-Acylamino esters are
obtained by alkylating with t-butyl acetate using an acid
catalyst,preferably perchloric acid [52], or with isobutene
in the presence of a small amount of conc. sulfuric acid
[53]. These routes may also be used for esterifying free
amino acids, when, in view of salt formation, more perchloric acid [54]or sulfuric acid [ 5 5 ] is needed. In these
reactions the t-butyl cation probably acts as an alkylating agent. A special advantage of the t-butyl esters
is their stability towards not too strong bases. For
example, the free esters may be distilled without formation of diketopiperazine.
Similar lability to acids is shown by p-methoxybenzyl
esters [15]; these may be obtained, for example, from
carbobenzoxyamino acids and anisyl alcohol by eliminating water with dicyclohexylcarbodiimide.Another
residue easily removed by acids is the phthalimidomethyl
group proposed by Nefkens [56]. This is introduced by
treating anions of N-acylamino acids with phthalimidomethyl chloride (12). It may also be removed with
hydrazine. The contrary effect, resistance to even strong
acids, is achieved by p-nitro-substitution of the benzyl
residue. The p-nitrobenzyl esters of amino acids and
peptides are substantially more stable towards HBr in
glacial acetic acid than the benzyl esters [57]. p-Nitrobenzyl esters have been made by the action of p-nitrobenzyl bromide on amino acids in the presence of base
[57,58], followed by removal of the group protecting
the nitrogen. The free esters are now more readily
obtainable by refluxing the free amino acids with p nitrobenzyl alcohol and benzenesulfonic acid in carbon
tetrachloride, leading the condensate over silica gel to
dry it [59].
Methyl esters, which have already played a large part
in peptide chemistry, are made efficiently and conveniently by treating the free amino acids with a
solution of thionyl chloride in methanol at room tem-
____
[51] J. C . Sheehan and K . R . Henery-Logan, J . Amer. chem. SOC.
79, 1263 (1957).
[52] E. Taschner, B. Liberek, Cz. Wasielewski, and J . F. Biernut,
Angew. Chem. 71, 743 (1959); E. Taschner, C. Wasielewski, and
J. F. Biernat, Liebigs Ann. Chem. 646, 119 (1961).
[53] G. W. Anderson and F. M . Cullahan, J. Amer. chem. SOC.
82, 3359 (1960).
[54] E. Taschner, A. Chimiak, B . Bator, and T . Sokolowska, Liebigs Ann. Chem. 646, 134 (1961).
[55] R . W. Roeske, Chem. Industrie 1121 (1959).
1561 G. H . L. Nefkens, Nature (London) 193, 974 (1962).
[57] R . Schwyzer and P. Sieber, Helv. chim. Acta 42,972 (1959).
[58] H . Schwarz and K . Arakawa, J. Amer. chem. SOC.81, 5691
(1959).
[59] J . E. Shields, W . H . McGregor, and F. H. Carpenter, J. org.
Chemistry 26, 1491 (1961).
Angew. Chem. internat. Edit.
Vol. 2 (1963) No. 7
perature [60,61]. The laborious and not always fully
satisfactory use of HC1 is thus unnecessary. In order to
esterify acylated amino acids, catalytic quantities of
Lewis acids can be used according to Taschner. Sulfuryl
chloride has proved best for making the methyl [62] and
benzyl [63] esters. N-Acylamino acids can also be
alkylated with methyl or ethyl formate or acetate in
the presence of sulfuric acid 1641. This alkylation must
proceed differently to the t-butylation mentioned above
[52], perhaps via a protonated adduct ( I 3 ) and intramolecular 0-alkyl transfer.
Methods of Coupling
Use of Chlorides
The classical methods of coupling using the chlorides of
protected amino acids keep recurring today in several
variations. The chlorides are produced directly within
the reaction mixture and react at once with the amino
components. Heslinga and Awns [65] found that on
heating acylamino acids or peptides with amino ester
hydrochlorides and a-chlorovinyl ethyl ether (14a) or
a,a-dichloroethyl ethyl ether (14b), good yields of
H,c-&
II
0
+ CI-C-CHRLNHA~
II
0
peptides are obtained. It is assumed that retacion of
(14a) or (14b) with the acylated amino acid leads via an
unstable intermediate (I5) to the acid chloride, which
at once reacts further with the basic reactant. A more
readily available compound, viz. dichloromethyl methyl
ether gives the same results, according to Rieche [66].
Tosyl- and phthalylamino acids also react with excess
reagent to give the acid chlorides, a method which is of
preparativc value; under these conditions, carbo[60] M . Brenner and W . Huber, Hclv. chim. Acta 36, 1109 (1953).
[61] R . A . Boissonnas, St. Guttmatrn, P.-A. Jaquenoud, and J.-P.
Wuller, Helv. chim. Acta 38, 1491 (1955).
[62] E. Taschner and C. Wasielen~ski,Liebigs Ann. Chem. 640,
136 (1961).
[63] E. Taschner and C. Wasielenlski, Liebigs Ann. Chem. 640,
139 (1961).
[64] E. Taschner and C. Wasielewski, Liebigs Ann. Chem. 640,
142 (1961).
1651 L. Heslinga and J. F. Arens, Recueil Trav. chim. Pays-Bas
76, 982 (1957).
[66] A . Rieche and H . Gross, Chem. Ber. 92, 83 (1959).
361
benzoxyamino acids are converted into N-carbamic anhydrides [67]. Mixed anhydrides (16) of acylamino
acids and orthoformates can be isolated from the
reaction between two moles of amino acid and one mole
of dichloromethyl alkyl ether in a medium which binds
HCI. In the reaction with amino esters, only one of the
aminoacyl residues reacts to form a peptide bond.
After further lengthening of the chain, the trityl residue
can be split off with acid. The azide is then made with
nitrous acid. The use of t-butyloxycarbonylhydrazinein
combination with the carbobenzoxy group, which can
be removed by catalytic hydrogenation, to protect the
amino-end has also been recommended [72]. Here, too,
the coupling can be effected with dicyclohexylcarbodiimide [72], or by the anhydride method [73]. The formation of azides from hydrazides and nitrous acid
is occasionally accompanied by a more or less extensive side-reaction, involving evolution of N20 from
I1 11
1
Use of Hydrazides and Azides
the primary N-nitrosohydrazide to form the aniide.
This side-reaction can be averted by using higher proton
and nitrite concentrations, or by nitrosation in an
organic solvent with esters of nitrous acid or with
nitrosyl chloride [74].
Hydrazides of peptides which are slow to react may be
formed very easily from thiophenyl esters [75]. Since
the azide group can also be introduced by the mixed
anhydride method [76,77], this process will perhaps
also be of use in peptide chemistry. The principle of
aminoacyl insertion has been recently extended by
Brenner and coworkers [78] to hydrazine derivatives.
N,N'-Diaminoacylhydrazines, in which one amino
group is acylated (17), undergo rearrangement in the
presence of organic acids or bases to give peptide
The use of azides for coupling according to Curtius has
emerged in the course of recent years as the only method
in which no racemization of the activated peptide
components takes place. Several authors have therefore
concerned themselves with the development of this
procedure. One particular aim was to avoid the difficult
hydrazinolysis of many long-chain peptide esters.
Carbobenzoxyhydrazides had been recommended earlier for this purpose [70]; tritylhydrazine has now been
suggested for introducing the azide [71]. Starting from
the hydrazine derivative and a trifluoroacetylamino
acid, treatment with dicyclohexylcarbodiimide or
carbonyldiimidazole yields the tritylhydrazide of the
trifluoroacetylamino acid
or of the trifluoroacetylpeptide; the trifluoroacetyl group is then split off with
alkali.
hydrazides (18). This is the inverse of the isomerization
observed by Kiirtz and Niemunn [79] of hydrazides of
acylamino acids into diacyl hydrazines. For aminoacylation, it is preferable to react the acylhydrazines
with N-carboxyamino acid anhydrides in acetic acid.
The peptide hydrazides can then be lengthened either
by the methods outlined here or by the classical route
via the azides, or oxidized with hypobromous acid to
peptides with free carboxyl groups.
When oxidized with N-bromosuccinimide (NBS) in the
presence of an amino acid or peptide ester, the hydra-
The reaction product from dimethylformamide and
phosgene, viz. the chloride of N,N-diniethyliminoformyl chloride [(CH3)2N=CH -Cl]CI [68], can also
serve as a chlorinating agent for acylamino acids [69].
With this reagent, peptide bonds may be formed at
0°C between acylamino acids and amino or peptide
esters.
-
F,C-CO-NH-CHR
-
+ HzO
H2N-CHR -
1
----
1 HzO
H,N@-CHR
-
- -- - - -CO--NH-NH-C(GjHs),
- - -
0Ho
-
- CO-NH-NH-C(C6Hs),
t Ho
- _.- - . -
-
CO-NH-NH~
[67] K . Podusku and H . Gro.\c, Chem. Ber. 94, 527 (1961).
[ 6 8 ] H . H . Bosslzurd, R. Mory , M . Sthmrd, and H . Zollinger,
Helv. chim. Acta 42, 1653 (1959).
[69] M. Zuoral and Z . Arnold, Tetrahedron Letters No. 14, 9
(1960).
[70] K . Hofmann, A . Lindenmunw, M . Z . Mugee, and N . H . Khan,
J. Amer. chem. SOC.74,470 (1952).
[71] F. Weygand and W . Steghch, Chem. Ber. 92, 313 (1959).
362
[72] R . Schwyzer, Angew. Chem. 7 1 , 742 (1959); Chimia 14, 366
(1960).
[73] R . A. Boissonnus, S . Gurttncrwii, and P . A . Jaquenoud, Helv.
chim. Acta 43, 1349 (1960).
[74] J . Honzl and J . Rudingw, Collect. czechoslov. chem. Commun. 26, 2333 (1961).
[75] T . Wieland and B. Heifrke, Liebigs Ann. Chem. 615, 184
(1958).
[76] P. R . Bhanduri, Ph.D. thesis, Universitlt Mainz (1953).
[77] J . Weinstock, J . org. Chemistry 26, 3511 (1961).
[78] M . Brenner and W. Hq/Pr, Helv. chim. Acta 44, 1794, 1798
(1961); R . Weber, W . Hofer, W . Heer, and M . Brenner, Helv.
chim. Acta 44, 2154 (1961).
[79] A. N . Kurtz and C. Nienrtznw, J . Amer. chem. Soc. 83, 3309
(1961).
Angew. Chem. intrmat. Edit. 1 Vol. 2 (1963)
I No. 7
zides of carbobenzoxyamino acids or peptides form
peptides in very good yield and with negligible racemization [80]. The reaction may also be used for making
polypeptides from tripeptide hydrazides. N o details of
the mechanism of the reaction, which perhaps proceeds
via an acyldiazonium salt (19), are yet known.
0
I1
4 I NH-CHR~-C-NII-NII~
ester of aIanine without racemization to yield the
dipeptide ester derivative. N-(p-Nitrobenzoxycarbony1)histidine gives the lactam (21), which acts as an acylating agent; the P-thiolactam from N-carbobenzoxy-3,3dimethylcysteine (22) has the same effect.
2 NES
(19)
fl
Z~~N-CIIR~--CO~R
* AcNH-CIfRCC-NH-CHH2--C0~R
Use of Unsaturated Compounds
Compounds with double bonds which readily undergo
addition reactions can add o n acylamino acids, giving
reactive intermediate products. If peptides are to be
made, the intermediates are not isolated but are condensed in the reaction mixture with the basic components.
To clarify the reaction mechanism of the activation of
carboxyl groups by carbodiimides, experiments were
made in the absence of the components to be coupled.
On applying the customary method used with these
reagents [8I ] to several carbobenzoxyamino or phthalylamino acids [82,83], symmetrical anhydrides were
isolated: these are known to yield peptides with amino
esters. It is thus not unlikely that such anhydrides are
also formed as intermediates in peptide syntheses with
dicyclohexylcarbodiimide [84].
During peptide synthesis, N-(3-Dimethylaminopropyl)N'-t-butylcarbodiimide [85] takes up water to give a
urea which is soluble in dilute acids and thus can be
removed readily. This also holds for other basic carbodiimides, Sheehan [86] having shown that it applies
particularly for N-ethyl-N'-3-(dimethylaminopropyl)carbodiimide. Carbodiimides are obtained from the
ureas by dehydration with tosyl chloride and triethylamine. They may be methylated in ether to give the
equally useful quaternary methylammonium salts. The
dehydrating action of carbodiimides was used by
Sheehan [87,88] for the synthesis of some amino acid
derivatives that are themselves reactive. N-Tritylserine
gives a p-lactone (20) which reacts with the methyl
[80]Y. Wolman, P. M . Gallop, and A. Patchornik, J. Amer.
chem. SOC.83, 1263 (1961).
[81] F. Zetzsche and H . Lindlar, Ber. dtsch. chem. Ges. 71, 2095
(1938); F. Zetzsche and A . Fredrick, ibid. 72, 363 (1939).
[82] H . Schiissler and H . Zahn, Chem. Ber. 95, 1076 (1962).
1831 I. Muramatsu and A. Hagitani, J. chem. SOC. Japan 80,1497
(1959); Chem. Abstr. 55,6394(1961); I . Muramatsu, J . chem. SOC.
Japan 82, 83 (1961); Chem. Abstr. 56, 10273 (1962).
[84] J. C. Sheehan and G. P. Hess, J. Amer. chem. SOC.77, 1067
( 1955).
[85] German Pat. 1070639 (Dec. 1959), Farbenfabriken Bayer,Inventors: H . B. Konig and F. Moosmiiller; Chem. Abstr. 55, 11 324
(1961).
[86] J. C. Sheehan, P. A . Cruickshnnk, and G . L. Boshart, J. org.
Chemistry 26,2525 (1961).
[87] J. C.Sheehan, Ann. N. Y. Acad. Sci. 88, 665 (1960); Chern.
Abstr. 55, 25779 (1961).
[88] J. C. Sheehan, K . Hasspacher, and Y. L. Yeh, J. Amer. chem.
SOC. 81, 6086 (1959).
Angew. Chem. internat. Edit. / Vol. 2 (1963) / No. 7
R
H 5 C s C I I2-(tC-N€I-C-C
f 221
II
I
II,C-c-s
I
CH,
=O
I
Cyanamide, which perhaps reacts in its tautomeric form
as carbodiimide, and also unsymmetrically dialkylated
cyanamides bring about peptide synthesis from carbobenzoxyamino acids and amino esters on heating at
100 "C without solvent for two hours, yields of 60 to 70 %
being obtained [89]. In the latter case too, acylated
isourea derivatives may be the actual carriers of the
acyl group.
Special interest has been attracted by the introduction of
a new reagent for making peptides by Woodward and
coworkers [90]. Yields of 73 to 93 %, with negligible
racemization, are quoted for 16 examples of syntheses
of di- and tripeptides using N-ethyl-5-phenylisoxazoliuni-3'-sulfonate (23). In the mechanism suggested for
this interesting reaction, the hydrogen next to the
positive nitrogen is supposed to be lost as a proton to
a base, whereupon an oxoketenimine (24) is formed by
opening of the ring [91]. The protected amino acid adds
onto this at once, and the actual reactant, the O-aminoacylenol f25/, is formed by migration of the acyl group.
Use of Activated Esters
One of the first types of compounds of this nature, viz. a
cyanomethyl ester [92], has also been used for a tri[89] G. Losse and H . Weddige, Angew. Chern. 72, 323 (1960);
Liebigs Ann. Chem. 636,144 (1960).
[90] R. B. Woodward, R . A . Olofson, and ii. Mayer, J. Amer.
chem. SOC.83, 1010 (1961).
[91] R . B. Woodwardand R. A . Olofson, J. Arner. chem. SOC. 83,
1007 (1961).
[92] R . Schwyzer, M . Feurer, B. Iselin, and H . Kagi, Helv. chim.
Acta 38, 80 (1955).
363
peptide synthesis without isolation of the intermediate
products [93]. In addition, it was found that treatment
with HBr in glacial acetic acid hydrates the nitrile
carbon and thus gives rise to aminocarbonylmethyl
esters H2N -CHRCOOCHz-CONH2, which have no
acylating activity. In the paper cited, p-nitrophenyldipeptide esters are also used as activated dipeptides.
For example, carbobenzoxyamino acids are first treated
with aminoacyl-p-nitrophenols by means of the anhydride method or dicyclohexylcarbodiimide, forming
the p-nitrophenyl esters of the carbobenzoxy-dipeptides,
which react further without isolation with another
amino ester.
The p-nitrophenyl esters may be obtained from acylamino acids and the corresponding sulfite OS(0Ar)Z or
phosphite P(OAr)3 [94] as well as from their components
using dicyclohexylcarbodiimide [95-971. Heating of the
acids with di-p-nitrophenyl carbonate OC(OAr)2 [98] is
also successful. Numerous other phenyl esters have
also been made by the sulfite and phosphite methods
[94]. The p-methylsulfonylphenyl esters have recently
been used [99] on account of the resistance of this group
later on, when the N-protecting group has to be split
off by catalytic hydrogenation. Phenyl esters of acylated
amino acids [loo], e.g. the cc-phenyl ester of carbobenzoxyglutamic acid, have proved suitable for the synthesis
of a-glutamyl peptides [loll, although they are much less
active. 2,4,6-TrichlorophenyI esters of several N-protected amino acids have proved useful acylating agents for
the synthesis of dipeptides at room temperature [102].
Although all peptide syntheses with activated amino
esters had previously been carried out in presence of
bases, Weygund and Steglicli [I031 found that activated
esters, particularly the thiophenyl esters of carbobenzoxyamino acids, reacted with free amino acids to
form peptides when heated in glacial acetic acid. The
zwitterionic forms of the amino acids seem to be partly
abolished by the action of the solvent on both polar
groups. The time necessary for reaction and the yield
are strongly dependent upon steric factors. Carbobenzoxyglycylglycine is formed in 60 minutes in 92 %
yield, but the reaction of the thiophenyl ester of carbobenzoxyleucine with alanine yields only 43 % peptide in
10.5 hours. The nitrogen of the amino acid component
[93] M . Goodman and K . C . Stueben, J. Amer. chem. SOC.81,
3980 (1959).
[94] B. Iselin, W . Rittel, P . Sieber, and R . Schwyzer, Helv. chim.
Acta 40, 373 (1957).
[95] M . Rothe and F.- W . Kunitz, Angew. Chem. 68, 414 (1956);
Liebigs Ann. Chem. 609, 88 (1957).
[96] D . F. Elliot and D. W. Russel, Biochem. J. 66, 49 P (1957).
I971 M . Bodanszky and V . Du Vigneaud, J. Amer. chem. SOC.
81, 5688 (1959).
[98] T . Wieland, B. Heiitke, K . Vugeler, and H . Murimoto, Liebigs Ann. Chem. 655, 189 (1962).
[99] R . Schwyzer and P . Sieber, Helv. chim. Acta 41,2190 (1958).
[loo] T . Wieland and W . Schafer, Angew. Chem. 63, 146 (1951);
T . Wielund and F. Jaenicke, Liebigs Ann. 55’9, 125 (1956);
see [103].
[I011 E. Klieger and H . Gibian, Liebigs Ann. Chem. 655, 195
(1962).
[lo21 G . Kupryszewski, Rocrniki Chern. 35, 595 (1961); Chem.
Abstr. 55, 27 121 (1961).
[lo31 F. Weygand and W . Steglich, Chem. Ber. 93,2983 (1960).
364
is acylated in a secondary reaction. Steric hindrance
affects the thiophenyl esters of phthalylamino acids
most unfavorably [28]. To reduce steric hindrance as
much as possible, activated vinyl esters have been made
from some acylamino acids by transvinylation with
vinyl acetate and palladium chloride [104]. The acetaldehyde produced by the condensation with amino esters
is taken up by the solvent, cyanoacetic ester. Some dipeptide esters are formed in this way in more than 80 %
yield. A new activated ester introduced into peptide
chemistry by Nefkens and T k w r [lo51 is the hydroxyphthalimide “ester” (26) of carbobenzoxyamino acids.
This ester is obtained in moderate to good yields by the
action of dicyclohexylcarbodiimide on carbobenzoxyamino acids and N-hydroxyphthalimide, which is made
from hydroxylamine and the phthalylating agent [27]
N-ethoxycarbonylphthalimide (7).
0
0
I
AcNEt-CHH’--C02H
f
(26)
-1120
The activated amino acids that occur in nature, i.r. 2’or 3’-amino esters containing terminal adenosine groups
from specific, soluble ribonucleic acids [106], have
hitherto only been prepared enzymatically. In order to
understand the ease of aminolysis of these esters, Zuchuu
and Karuu [lo71 and W i e h d , Merz, and Pfleiderer [I081
synthetized amino esters of several cyclic glycols and
compared their rates of reaction with hydroxylamine
with that of amino esters o f some nucleotides. It turned
out that either a free adjacent hydroxyl group or an
oxygen atom in the ring have an accelerating effect. The
valine ester of cis-3,4-dihydroxytetrahydrofuran(27),
the most active compound, still reacted significantly
slower with hydroxylamine than 2’- (or 3’-)valyladenosine-5-phosphoric acid (28) ; hence it must be
I
IIzN-CH
I
H3C-CH
I
CH3
OZC-CH-CH-CH~
I
1
H2N
CH3
concluded that the purine nitrogen, and perhaps also
the phosphoric acid, have an additional effect. This
effect is shown in the comparison of the rates of cleavage
[lo41 F. Weygandand W. Steglich, Angew. Chem. 73,757 (1961).
11051 G . H . L. Nefkens and G . J. Tesrer, J. Amer. chem. SOC.83,
1263 (1961).
[lo61 H . G . Zachau, G . Acs, and F. Lipmann, Proc. nat. Acad.
Sci. U S A . 44, 885 (1958).
[lo71 H . G. Zachau, Chem. Bcr. 5’3,1822 (1960); H. G. Zachau
and W . Karuu, Chem. Ber. 93, 1830 (1960).
[lo81 T. Wieland, H . Merz, and G. Pfleiderer, Chem. Ber. 93,
1816 (1961).
Angew. Chem. internat. Edit. I Vol. 2 (1963) No. 7
with hydroxylamine of l-~~-alanyl-2-phosphorylglycol
Use of Azoles
(29), alanylglycol, and the methyl ester of alanine [log].
In the teichoic acids (30) of bacterial cell walls, which
Nothing has been reported about the mode of action of
are D-alanyl esters of poly(glycero1 phosphate) or polyimidazole (see above), yet it may be supposed that an
(ribityl-l,5-diphosphate), reactivities of the esterified
imidazolide of the amino acid is an intermediate. After
alanine residues were observed which corresponded
these energy-rich compounds [115] - which can be
approximately to that of the activated amino acids of
coupled without isolation according to Paul and Anderthe cell [ 1101.
son [48] - became readily accessible by the smooth
HzC-CHz
I
NH2
I
I
IT203P0 CFC-CH-CII,
(29)
II
0
0
I/
0
-CHzU-C
ll
I
OH
HTC H - C H z U P U C H z I
I
0
OH
I
O = C-C H-N I l z
I
CIIS
(30)
Use of Phosphorus Derivatives
Diphenylphosphoryl chloride has been added [ l l l ] to
the numerous phosphorus derivatives proposed as
components for activating amino acids.
Phosphorus pentoxide has been introduced as a reagent
for peptide synthesis by Schrumm and Wissmann. The
method was tried out on several peptides: protected
amino acids and amino esters were dissolved in diethyl
phosphite and heated with tributylamine and PzO5 on a
steam bath [112]. This process gives racemic tripeptides
but is particularly suitable for manufacturing large quantities of dipeptides, as very concentrated solutions can
be used. It is supposed that ethyl polyphosphates formed
from P2O5 and diethyl phosphite are the actual condensing agents. Derivatives of phosphorous acid recommended as being useful for peptide bonding include,
in addition to the tetraethyl pyrophosphite customary
before, bis-a-phenylene pyrophosphite - prepared from
pyrocatechol and phosphorus trichloride [113] - and
diethylethylene pyrophosphite [18]. In peptide syntheses with these reagents, an equivalent quantity of
imidazole has an accelerating effect. Anderson [114] has
therefore synthetized ethylene P-imidazoly1phosphonite
(31) and used it in place of the reaction mixture as a
FN-(-rHa
Nd
0-CH2
(31)
condensing agent. He obtained yields about 10 %higher
than those obtained by the pyrophosphite method
without imidazole.
I1091 Z . A. Shabarowa, N . A . Hughes, and J. Baddiley, Biochern.
J. 83, 216 (1962).
[110] A . R. Archibald, J. J. Armstrong, J. Baddiley, and J. B. Hay,
Nature (London) 191,570 (1961).
[ill] A . Cosinatos, J. Photaki, and L. Zervas, Chem. Ber. 94,
2644 (1961).
[112] B. F. Erlanger and N . Kokowsky, J. org. Chemistry 26,
2534 (1961).
[I131 P . C . Crafts, J . H. H. Markes, and H. N . Rydon, J. chern.
SOC.(London) 1959,3610.
[114] G. W. Anderson, Ann. N. Y. Acad. Sci. 88, 676 (1960).
Angew. Chem. internal. Edit. / Vol. 2 (1963) 1 No. 7
reaction of N,N’-carbonyldiimidazole [I 161with protected peptides and amino acids; the discoverers of this procedure applied themselves to working out the optimum
conditions [117]. The ethyl ester of carbobenzoxyglycylL-phenylalanylglycine,which is - as mentioned later the most frequently used test compound forracemization
studies, was obtained in 90 ‘i: yield, along with 5 %
of the DL-tripeptide. During the formation of the acylimidazole, an excess of carbonyldiimidazole was
avoided, and water was excluded, The coupling components, e.g. salts of amino acids, may then be added
in the presence of water, but use of esters without water
is recommended.
Stuab and Wendel [I 18,1191 later described thionyldiimidazole (32), which is readily obtained from thionyl
chloride and four equivalents of imidazole in tetrahydrofuran at room temperature and can also be used
without isolation for peptide syntheses in one-step
reactions [120]. Compound (32) reacts at 0°C with
carbobenzoxyalanine to give the imidazolide (33), even
~ N - i l - N q (32)
NZ/
\=N
H3Y
::
c 10
AcNH-CH-C-Nq
\-N
(33)
-
H3::
CJ
+ HsN-CHz-C, 40
OC2H6
R
ACNH-CH-C-NH-CHt-C,
/p
OC2H5
when the ester component - protected as its hydrochloride - is present. Peptide coupling is initiated by
addition of triethylamine. The yields are so far around
75 %, but can probably be raised. A phosphoryldiimidazole, viz. the diimidazolide of phenylphosphoric acid,
is also capable of transfering a n imidazole residue to an
acylamino acid to form its imidazolide, which can then
be treated with alanine to give a peptide [121].
Free imidazole is known to be able to catalyse hydrolysis of reactive esters [122]. This process, which is
thought to entail intermediate formation of an Nacylimidazole [123], may also be used to activate alkyl
[115] T. Wieland and G. Schneider, Liebigs Ann. Chem. 580,
159 (1953).
[I161 H. A . Staab, Liebiga Ann. Chem. 609, 75 (1957).
11171 R. Paul and G. W . Anderson, J. Amer. chern. SOC.82,4596
(1960).
11181 H. A . Staab and K . Wendel, Chem. Ber. 93, 2902 (1960).
[119] H. A . Staab, Angew. Chem. internat. Edit. I , 351 (1962).
11201 T. Wieland and K. Vogeler, Angew. Chern. 73,435 (1961).
[121] F. Cramer and H . Schaller, (’hem. Ber. 91, 1634 (1961).
[122] See M . L. Bender, Chem. Reviews 60, 53 (1960).
[123] M . L. Bender and B. W. Turnquest, J. Arner. chern. SOC.79,
1656 (1957).
365
esters of acylamino acids [124]. On stirring the methyl
ester of carbobenzoxyalanine for several hours with the
sodium salt of glycine in liquid imidazole at 80 "C, over
60 % of the carbobenzoxy-dipeptide is obtained; with
the more soluble tetraethylammonium salt the yield is
over 70 %; without imidazole, no coupling takes place.
By using the t-butyl ester of glycine under these conditions, 75580 % of the carbobenzoxy-dipeptide ester
may even be attained. In this connection, we found that
the phthalyl residue is rapidly split off on heating with
imidazole in the presence of a little water.
3,5-DimethyIpyrazolides (34) of N-acylamino acids and
peptides are significantly less reactive than the imidazolides [125]. They can be made from the acids and
3,5-dimethylpyrazole by the 'anhydride method' [126].
A further azole introduced into peptide chemistry as a
coupling reagent is 1,2,4-triazole, which is applied in
the form of N,N'-carbonyl-bis-l,2,4-triazole(35) [127].
(34)
(35)
It is prepared in the same manner as carbonyldiimidazole; addition of one mole of pyridine to capture the
acid formed in this reaction raises the yield. The compound, in which the positions of attachment of the
carbonyl group are not known exactly, reacts more
slowly in the activation step than the imidazole analogue, but faster in the coupling step. For example, the
triazolides react rapidly with amino acids or peptide
esters in dimethylformamide at room temperature,
forming peptide bonds.
Sundry Methods
Oxazolidones. The procedure of Micheel and coworkers
utilizing oxazolidones has also been further developed
recently. It may be used with tosyl-L-glutamic acid [128]
and other tosyl-L-amino acids [129] and gives dipeptides
without racemization.
Acetic anhydride and thionyl chloride (in catalytic
quantities) are now used to form the ring. Carbobenzoxyglycine has also yielded oxazolidones (36) with
paraldehyde, benzaldehyde, or chloral by dehydration
[ 1301. Chloral gives the best yield of oxazolidone. Dane
and coworkers [131] found that this aldehyde reacts
[124] T. Wielandand K. Vogeler, Angew. Chem. internat. Edit.2,
42 (1963).
[125] W. Riedand A . Czack, Liebigs Ann. Chem. 642,133 (1961).
[I261 W. Ried and K . Marquard, Liebigs Ann. Chem. 642, 141
(1961).
[127] H. C. Beyermann and W. Maassen van den Brink, Recueil.
Trav. chim. Pays-Bas 80, 1372 (1961).
[I281 F. Micheel and E l . Haneke, Chem. Ber. 92, 309 (1959).
[129] F. Micheel and H. Haneke, Chem. Ber. 95, 1009 (1962).
[130] F. Micheeland W. Meckstroth, Chem. Ber. 92, 1675 (1959).
11311 E. Dane, R. Heiss, and H. SchaJer, Angew. Chem. 71, 339
(1959); German Pat. 1079648 (Apr. 1960), Farbenfabriken
Bayer; Chem. Abstr. 55,25784 (1961).
366
even with free amino acids to give 1,3-oxazolidine-5one derivatives (37) in which the nitrogen bears the
trichlorohydroxyethyl residue. These compounds react
smoothly with amines at 20-40°C and thus with
glycine esters to give N-formyl-dipeptide esters.
OH
I
C13C-7HR
Ac
I
n
c1,c
€1
(36), li = ( ' 1 1 7 ,
1-0
€I
CsIls. CC1,
(37)
Carbonic Acid Derivatives. Mixed alkylcarbonic anhydrides still deserve consideration, although no novelty
in the method has been forthcoming. Studies of theoretical interest have been made on the mechanism of decomposition of mixed carboxylic carbonic anhydrides
(formation of ester and COz; disproportionation) using
the mixed anhydride of benzoic and n-butylcarbonic
acids as a model; the decomposition is supposed to be
base-catalysed [132]. Mixed anhydrides may be obtained from carbobenzoxy-E-aminocaproic acid by
reaction with diethyl pyrocarbonate and used in the
normal way for acylating the amino group of a second
component [133]. With a-amino acids, however, such a
synthesis is not possible [98]. Cyanuryl chloride reacts
with acylated amino acids in the presence of a tertiary
base at 0 "C to give an anhydride-like compound which
gives a dipeptide on reaction with an amino ester. A
dipeptide ester was formed in 77 % yield from carbobenzoxyalanine and glycine ester. On activation of a
peptide, however, extensive racemization occurred [ 1341.
The carbobenzoxy group can also be used for peptide
bonding : on fusion with phthalylamino acids, N-carbobenzoxyaminoesters lose carbon dioxide and give dipeptide esters [98]. The mechanism here entails initial
elimination of benzyl alcohol, leaving an isocyanatofatty ester which condenses with acylamino acids in a
familiar manner, carbon dioxide being evolved. a-Isothiocyanato-fatty esters (38) behave similarly, carbonyl
sulfide being split off [89]. They are obtained from
amino esters with carbon disulfide and alkali, the dithiocarbaminates (39) initially formed being treated
with HgCl2 to remove HzS. Isothiocyanates are
also formed from free amino acids with carbon
disulfide, but cannot be isolated. They give polypeptides, probably via intramolecular thiocarbamic
SH
I
S=C-NH-CEIR~-CO~K
-HzS
S=C=N-CHR~-CQR
H
[I321 E. J. Longosz and D.S. Tarbell, J. org. Chemistry 26, 2161
(196 1).
[133] W . Thoma and H . Rinke, Liebigs Ann. Chem. 624, 30
(1959).
[134] Th. Wieland and P. Duesberg, unpublished.
Angew. Chem. internat. Edit. { Vol. 2 (1963) 1 No. 7
anhydrides (40). N-(Phenylthiocarbony1)-amino acids
(HsC~S-CO-NH-CHR-CO~H) and -peptides [135] as
well as N-(phenyloxycarbony1)- and N-(0-nitrophenyloxycarbony1)-amino acid derivatives [1361 also exhibit
this readiness to polymerize.
Other Mixed Anhydrides. Actually all activated derivatives of acylamino acids may be considered in principle as mixed anhydrides. A recent review [6] of
methods of peptide coupling is written from this point
of view, but the finer distinctions will be retained here.
Diphenyl ketene reacts very rapidly with acylated amino
acids at -15 OC to form mixed anhydrides of diphenylacetic acid. Owing to steric hindrance, only the acylamino portion is able to react with amino esters to form
peptides [137]. Benzenesulfonic acid can be introduced
easily as an anhydride component by treating acylamino acids with benzenesulfonyl chloride. Such anhydrides have also been used for peptide syntheses [138],
but they seem to offer no particular advantage, as has
been confirmed recently using toluenesulfonic acid as
one anhydride component [139]. The use of toluenesulfonyl chloride for esterification with slow-reacting
alcohols, in which a mixed anhydride is formed as an
intermediate, has been recommended since 1955 [140].
Methyl sulfonyl chloride has also been used to make a
peptide bond [141].
Passerini Reacfion. Recently, Ugi [142] has reported a
remarkable application of the Passerini reaction in the
presence of an amine, two peptide links being formed
simultaneously. A striking example is the reaction of
t-butyl isocyanoacetate (41), which is obtained from
the t-butyl ester of N-formylglycine by dehydration with
phosgene and triethylamine, by analogy to the iso-
nitrile synthesis from formamide with phosphorus
oxychloride in pyridine [143]. The ester (41) reacts at
room temperature within five hours in a multicentre
process with phthalylglycine, benzylamine, and acetaldehyde (a Schiff-base) via an 0-acylpeptide (42) to
form the t-butyl ester of phthalylglycyl-(N-benzyl-DLalany1)glycine (43) in 73 % yield. Although the middle
amino acid becomes racemic in the process, this
elegant reaction may attain importance in peptide
chemistry. In the absence of‘an amine, the analogous
reaction gives rise to a depsipeptide [144].
Degenerate Peptides. The class of compounds known as
depsipeptides, also called peptolides [1451, which includes several interesting natural products, has been
reviewed recently by Schemjakin [146]. In addition to
the principles of synthesis described therein, which
Schemjakin has lately followed for the synthesis of
optically active depsipeptides [147], a special route has
been described by which a glycolic acid residue may be
introduced specifically into an ester bond by way of
diazoacetylamino acid derivatives [148,149]. Ried and
coworkers have studied the synthesis of other peptidelike compounds. The so-called endo-thiopeptides (44)
were obtained by condensation of acylaminothionic
esters (45) with salts of amino acids [150]; imidopeptides (46) are formed in a similar manner [151] from
iminoesters (47).
Syntheses of Cyclic Peptides
Attempts to prepare cyclic peptides with definite
structures have been the aim of one branch of peptide
chemistry. Systematic studies on the effect of dilution,
which is known to be important in this field, have been
made by Rothe and coworkers [152], albeit on a “degenerate” model. Cyclization of E-aminocaproyl-Eaminocaproyl chloride, which was set free from its
hydrochloride by triethylamine in dimethylformamide,
gave the yields shown in Table 1 after two hours’
reaction. The yields depend upon the dilution.
1
(43)
[135]J. Noguchi and T. Hayakawa, J. Amer. chem. SOC.76,2846
(1954).
11361 Y . Ishizuka,Nippon Kagaku Zasshi 77,1426 (1956);Chem.
Abstr. 53, 5149 (1959).
[137]G. Losse and E. Demuth, Chem. Ber. 94, 1762 (1961).
11381 E. Tuschner, Collect. czechoslov. chem. Commun. 24, 26
(1959);see also Chem. Abstr. 52, 16236 (1958).
11391 D.Theodoropoulos and J . Garapoulos, J. org. Chemistry 27,
2091 (1962).
11401 J. H . Brewster and C. J. Ciotti, J. Amer. chem. SOC. 77,
6214 (1955).
[141]F. C. McKay, cited by N . F. Albertson 161.
[142]I . Vgi, Angew. Chem. internat. Edit. 1,s (1962).
Angew. Chem. internat. Edit. / Vol. 2 (1963) / No. 7
[I431 I. Vgi and R . Meyr, Chem. Ber. 93,239 (1960).
11441 I . Vgi and U.Fetzer, Angew. Chem. 73,621 (1961).
[145]H . Gibian and K . Liibke, Angew. Chem. 72,523 (1960).
11461 M. M . Schemjakin, Angew. Chem. 72,342 (1960).
11471 M . M . Schernjakin et al., Doklady Acad. Nauk SSSR 140,
387 (1961); Chem. Abstr. 56, 536 (1962).
[I481 R . Schwyrer and J . P . Carridn, Helv. chim. Acta 43, 2101
(1960).
[149]H.Gibianand K.Liibke,LiebigsAnn.Chem.644,130(1961).
[150] W. Ried and W. von der Emden, Angew. Chem. 72, 268
(1960);Liebigs Ann. Chem. 642,128 (1961).
[l5l] W. Ried, W. Stephan, and W.von der Emden, Chem. Ber.
95, 728 (1962).
[152] M. Rothe, H. Brunig, and G‘. Eppert, J. prakt. Chem. 280,
323 (1959).
367
Table 1. Yields from cyclization of
E-aminocaproyl-E-aminocaproylchloride,
Concentration
Yield
[mole/ll
[%I
0.001
0.004
37
30
23
0.008
5
0.002
The concentration of 1 millimole per litre found here
to be optimum is also used by most workers on similar
problems. All the recognized coupling methods of
peptide chemistry may in principle be used for cyclization. Of the activated esters, thiophenyl esters [75],
cyanomethyl and p-nitrophenyl esters [153], carboxythiomethyl esters, and the p-methylsulfonylphenyl esters
mentioned above [99] have been used. The protection
of the amino group necessary during synthesis must be
such that release is possible without attack on the
activating group [99]. Ring closure without protection
of the amino group and without special activation of the
carboxyl group can be attained with dicyclohexylcarbodiimide [154] or ethoxyacetylene [1551. A more
than one-hundred-fold excess of diimide permits
formation of the four possible polymycins from the
corresponding preformed decapeptides [1561. In the
presence of bulky substituents (tosyl group) near to the
point of linkage, ring closure is not successful [157]. In
the dimeric cyclization of the p-nitrophenyl ester of DLphenylalanylglycylglycine [99], the choice of a reaction
partner is made in an interesting manner. Schwyzer
and Tun Kyi [158] synthetized the authentic stereoisomeric cyclohexapeptides (L,L; D,D; D,L) by the azide
method and found that dimerization gave rise exclusively
to the meso-compound (D,L). Thus, only L-tripeptide
molecules had combined with D-tripeptide molecules to
form the hexapeptide ring.
Racemization during the Formation of Peptides
[*I
Although free amino acids are very resistant to racemizing influences, a certain lability may be observed
in a number of derivatives. Amino esters or N-acylamino acids are easily racemized by bases 11591. In
principle, three possible types of racemization are to be
distinguished :
[153] R . Schwyzer and P . Sieber, Helv. chim. Acta 41, 2186
(1958); R . Schwyrer and B. Gorup, Helv. chim. Acta 41, 2199
(1958).
[154] T. Wieland and K . W . Ohly, Liebigs Ann. Chem. 605, 179
(1957).
[155] E. A . Morozova and S . M . Zhenodarova, Zhur. Obshchei
Khim. 31, 45 (1961); Chem. Abstr. 55, 27121 (1961).
[156] K . Vogler, R . 0.Studer, W . Lergier, and P . Lanz, Helv.
chim. Acta 43, 1751 (1960); R. 0. Studer, K . Vogler, and W.
Lergier, Helv. chim. Acta 44, 131 (1961); K . Vogler, R . 0 . Studer, P . L a m , W . Lergier, and E. Bohni, Experientia 17,223 (1961).
[157] R . 0. Studer and K . Vogler, Helv. chim. Acta45,819 (1962).
[158] R . Schwyzer and A . Tun-Kyi, Helv. chim. Acta 49, 859
(1962).
[*I Cf. F. Weygand, A . Prox, L . Schmidhammer, and W . Konig,
Angew. Chem. internat. Edit. 2, 183 (1963).
[159] A . Neuberger, Advances Protein Chem. 4,297 (1948).
368
1. Racemization of amino acids in a peptida
2. Racemization of amino-acid anhydrides
3. Racemization of activated acylamino acids on
account of ring formation.
Racemization in the peptide affects particularly amino
acids bearing electron-attracting substituents (0,S ) in
the P-position, which effect a weakening of the binding
of the cc-hydrogen atom. An example from the recent
literature is the observation by McLaren [9] that the
ethyl ester of carbobenzoxyglycyl-L-(S-benzy1)cysteine
racemizes extensively on saponification with alkali,
although the acid remains optically active under these
conditions. On treatment of the t-butyl ester of the
same peptide with alkali, it is racemized completely, with
liberation of the carboxyl group [I 141. A serine peptide,
the ethyl ester of carbobenzoxy-L-serylglycyl-L-alanine,
rearranges on merely standing for several days at
room temperature in aqueous triethylamine to give
the diastereoisomeric DL-serine peptides until a state of
equilibrium is reached [160]. The lability of amino acid
anhydrides can be attributed to the electron-attracting
property of the second acyl moiety (48) and probably
also to the possibility of the loosening of the cc-hydrogen
atom by hydrogen bond formation (49) 11591. A base
0
H O
I l l
-HN-C-CUC-R
I1
0
011
0
I1
Base_
-HN-C-CUC-R
-Ha
A1
81
R
0= C
',
H
:
\
o
oo
-HN-C
o"
= C-O=
I @ C-R
I
I
R'
may also be the actual isomerizing agent here. Although
several N-acylimino acids, in which azlactone formation
is out of the question, also become racemic as anhydrides [159], this form of racemization during peptide formation via anhydrides can nevertheless apparently be
avoided if the anhydride stage is short and is formed
at a low temperature. So far, no racemization of carbobenzoxyamino acids during activation has been observed. However, a sufficiently sensitive test has perhaps
been lacking here.
Far more rapid racemization of activated amino acids
seems to proceed during ring formation. N-Acylamino
acids, including peptides, can be cyclized to azlactones
(50) by loss of the elements of water. In such rings, loss
of the proton is greatly facilitated because an aromatic
s2
11601 E. Schnabel, Hoppe-Seylers Z. physiol. Chem. 314, 114
(1959).
Angew. Chem. intimat. Edit.
1 Vol. 2 (1963) I No. 7
system is then formed. Carbobenzoxyamino acids do
not undergo ring closure, so that this protection
of the amino group is not only advantageous on
account of the ease of removal of the protecting
group. However, a greater or lesser degree of racemization has been observed so far even during the
activation of dipeptides by almost every method (with
the exception of the azide method).
Table 2. Racemization occuring during peptide syntheses.
Method of peptide coupling
Racemization test or
peptide synthetized
Result
Mixed anhydride
(Carbonic acid derivatives) [a]
Anderson test [161]
Young test I1631
2 % DL; 77 % L
46 % D L ; 54 % L
Tetraethyl pyrophosphite
Dicyclohexylcarbodiimide
Young test
78 %
Anderson test
Young test
Anderson test
87 % L [cl
74 % L [cl
75 % L [C]
Carbonyldiimidazole
[48,117]
Anderson test
Nitrophenyl ester
[168,1691
Considerable racemization on
esterification of dipeptides by the
sulfite I1681 and carbodiimide
methods [I691 [d]
p-Nitrothiophenyl ester
11701
Cbo-Gly-L-Ala/L-Phe-Gly-OH [el
7 % DL;
I 0.5 %
DL;
[bl
87 % L
24 % DL; 56 % L
1
0 % DL [fl
Sulfur trioxide
Cho-Gly-L-AlalL-Phe-Gly-OH [el
Ethoxyacetylene
Anderson test
Phosphorazo method
Cbo-Gly-L-Phe/L-Ala [el
N-acyl-N(E)-Cbo-Lys
Phosphorus pentoxide [il
Cbo-Gly-L-Leu/Gly-OC2H5 [el
Anderson test
32 % DL;
Isoxazolium method
Anderson test
2.2 % DL; 90 % L
N-Hydroxyphthalimide
[kl
I not determined
Hydrazide oxidation
Anderson test
1 1.1%DL;63%L
0 % DL; 70 % L k 1
% DL; 80 % L
retained full rotation on
coupling [hl
15
~~
Phosgene/dimethylformamide
1691
[a] With chloroform as solvent, or on using higher temperatures during
the formation of anhydride, the racemic fraction is significantly greater.
[b] This yield was obtained by the “standard” and the “amide” methods
in the absence of quaternary salts. Considerable racemization takes
place in the “anhydride” method, especially at higher temperatures.
[c] The discrepancy is explained by the fact that Sheehan and Hess [84]
did not use the crystallization procedure which led to the detection of
small quantities of racemate by Anderson and coworkers [161,165,166].
A further valuable contribution to this question is the synthesis of the
tetrapeptide carbobenzoxy-nitro-L-arginyl-L-valyl-L-tyrosyl-L-isoleucine
as its methyl ester [I671 by all possible ways of coupling. If the carbobenzoxy-dipeptide or -tripeptide was coupled by way of the anhydride
or with carbodiimide, significantly smaller yields were always obtained,
the products being impure and not completely split by enzymes.
[d] The racemization seems to depend strongly upon the nature of the
amino acid residue. Thus, carbobenzoxy-L-(S-benzy1)cysteinyl-Ltyrosine could be esterified smoothly and without racemization by the
sulfite method, while carhobenzoxy-L-valyl-L-tyrosine gave only a
small yield, the unreacted acid also undergoing considerable racemization [1681.
[el Cbo = carbobenzoxy.
[I] Note particularly that at somewhat higher pH values than that used
here (6.5), the racemization is substantially greater.
[g] The presence of triethylammonium hydrochloride under otherwise
equivalent conditions leads to a large fraction of racemic product.
[h] The discrepancy between the two experiments is not particularly
serious, as the results of Gofdschmidtand Rosculet 11731 are only based
on rotation measurements. It is doubtful here whether small quantities
of racemate had been previously separated by crystallization, or do not
show up in the results of the determination. Comparison of two tripeptides, one made from an acyl-dipeptide and an amino ester and the
other from the acylamino acid and the dipeptide ester is not valid since
in the critical cases glycine was involved in the coupling.
[i] The Schramm and Wissmnnn test [I741 consists of the hydrolysis of
the crystalline tripeptide followed by measurement of the rotation in
Angew. Chem. internat. Edit.
Vol. 2 (1963) No. 7
63 % L
32 % L
I extensive racemization
comparison with that of an authentic nmino acid mixture. Erlanger and
Kokowsky [112] could not reduce racemization by variation of the
coupling conditions.
[k] Performance of the “Anderson test” here [I051 was inappropriate, as
the tripeptide was made from the ethyl esters of carbohenzoxyglycine
and of L-phenylalanylglycine, so that racemization is of course not
possible.
[161l G . W . Anderson and R . W . Young, J. Arner. chem. SOC.74,
5307 (1952).
[I621 D . W . Clayton, J. A . Farrinxton, G . W. Kenner, and J. M .
Turner, J. chem. SOC.(London) 1957, 1398.
[1631 N. A . Smart, G . T . Young, and M . W . Williams, J. chern.
SOC. (London), 1960, 3902.
[163a] M . W . Williams and G . T . Young, J. chern. SOC.(London)
1963, 881.
[164] J. R . Vaughan, J. Amer. chcm. SOC.74, 7137 (1952).
[165] G. W . Anderson, J. Blodingrr, and A . D . Welcher, J. Amer.
chem. SOC. 74, 5309 (1952).
[166] G. W . Anderson and F. M , Callahan, J. Amer. chem. SOC.
80, 2902 (1958).
[t67] H. Schwarz and F. M . Bunipus, J. Amer. chern. SOC.81,
890 (1959).
[168] B. Zselin and R . Schwyzer, Helv. chim. Acta 43, 1760 (1960).
[169] K . Liibke and E. Schroder, %. Naturforsch. 16b, 765 (1961).
[170] J . A . Farrington, P . J . Hexfall, E. W . Kenner, and J. M .
Turner, J. chem. SOC. (London 19.57, 1407.
11711 H. J. Pannemann, A . F. Marx, and J. F. Arens, Recueil
Trav. chirn. Pays-Bas 78, 487 (19.59).
[172] W . Grassmunn, E. Wiinsch, and A . Riedel, Chem. Ber. 91,
455 (1958).
[173] St. Goldschmidt and G. Rosculet, Chern. Ber. 93,2327 (1960).
[174] G. Schramm and H . Wissmtmn, Chem. Ber. 91,1073 (1958).
369
In order to determine the degree of racemization, the
method worked out by Anderson [161] may be used.
Carbobenzoxyglycyl-L-phenylalanine is coupled with
the ethyl ester of glycine and the tripeptide formed is
fractionally crystallized from a 2 % solution in alcohol.
I n this way, the m-peptide precipitates first and can be
recognized by its higher melting point. In a less commonly used test for racemization [162], use is made of
the separability of carbobenzoxyglycyl-L-phenylalanylL-alanylglycine and carbobenzoxyglycyl-D-phenylalanyl-L-alanylglycine (synthetized from the dipeptides)
by countercurrent distribution. A third procedure has
been used by Young and coworkers [163] on several
coupling methods under very different conditions. It
entails the coupling of acetyl-L-leucine with the ethyl
ester of glycine. The racemic and the optically pure
dipeptides are not always obtained crystalline, however,
so that the rotation must be measured both before and
after hydrolysis, thus allowing only an approximate
estimate of the degree of racemization. Recent investigations by the same author [163a] of peptides of N-benzoylleucine, which crystallize more readily, have not
been considered in Table 2 The results obtained in tests
for the racemization occuring during the methods of
peptide bonding in general use are summarized in
Table 2. The values given were determined under the
conditions most favorable for retention of configuration.
It has already been indicated that the azide method is
the only one in which the activated peptide component
does not racemize. Table 2 shows that, apart from this
procedure, there appears to be no ideal method of
activation. The deficiency of the azide method lies in
the hydrazinolysis of polypeptide esters, which sometimes proceeds unsatisfactorily. Here the catalytic
acceleration with imidazole outlined above and the
use of a somewhat more reactive ester may be of
assistance. However, on the other hand, the dangers of
racemization during very rapid coupling should not be
exaggerated. Speedy execution of a peptide synthesis
by the anhydride method using ethyl chloroformate
at a low temperature can give good yields of pure
L-peptides by activating dipeptides. A relatively safer,
though more laborious procedure involves progressive
attachment of carbobenzoxyamino acids which are
known to racemize only with difficulty because of their
lack of ability to form azlactones. A final possibility
entails using peptides with glycine or proline at the
carboxyl end. It should be noted that not all L-amino
acids racemize to an equal extent, so that the choice
of racemization resistant oligopeptides which can be
coupled with others in a simple way may be further
increased. Much systematic work is still to be done
here. Enzymatic coupling [175] proceeds in the assurance that there is no racemization of the carboxyl end,
but this will not be considered further here.
Received, October 3rd. 1962
[A 274187 IE]
German version: Angew. Chem. 75, 539 (1963).
[175] See H. Determann, 0. Zipp, and T. Wieland, Liebigs Ann.
Chem. 651, 172 (1962).
Base-Catalysed Reactions of Ketones with Hydrogen Sulfide [11
BY PROF. DR. ROLAND MAYER, G. HILLER, MARGOT NITZSCHKE, AND D1PL.-CHEM. J. JENTZSCH
INSTITUT F’OR ORGANISCHE CHEMIE DER TECHNISCHEN UNIVERSITAT DRESDEN (GERMANY)
Monoketones react with hydrogen sulfide in the presence of basic catalysts to give geminal
dithiols or thioketones. P-Diketones are converted into monothiodiketones. a-Diketones
can be selectively reduced by hydrogen sulfide in the cold in the presence of secondary or
tertiary amines to give hydroxy- or mono-ketones.
Base-catalysed additions of hydrogen sulfide onto
carbonyl groups have been little investigated, although
they form one of the fundamental reactions of organic
chemistry. The reactions are also easily carried out
technically, and the cheap intermediate products can be
converted into heterocyclic sulfur compounds [2-41 in
[l] XVIIth Communication on Sulfur Heterocycles. - XVIth
Communication: J. Frunke and R . Mayer, J. prakt. Chem., in
the press.
[2] J. Jentzsch, J. Fabian, and R . Mayer, Chem. Ber. 95, 1764
(1962).
[3] B. Magnusson, Acta chem. scand. 13, 1715 (1959); R . Mayer
and J. Jentzsch, Angew. Chem. internat. Edit. 1, 217 (1962); J.
Jentzsch and R . Mayer, J. prakt. Chem. (4) 18, 211 (1962).
[4] H.Burreru and R . E. Lyle, J. org. Chemistry 27, 641 (1962).
370
various ways. Some new results from our work will
therefore be summarized in the following report.
The Base-Catalysed Reaction of Monoketones
with Hydrogen Sulfide
If hydrogen sulfide is passed into a solution of a monoketone in the presence of a basic catalyst, gem-dithiols
( 1 ) or in special cases thioketones (2) are formed in
varying yields.
We have recently described [2] this simple and most
productive synthesis of the gem-dithiols ( 1 ) ;hydrogen
Angew. Chem. intcrnat. Edit.
Vol. 2 (1963)
No. 7
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