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Mechanisms of Aspartimide
Formation: The Effects of
Protecting Groups, Acid, Base,
Temperature and Time
James P. Tam, Mark W.
Riemen and R. B. Merrifield
The. Rockefeller University
Factors affecting aspartimide formation, such as protecting groups, acidity,
basicity, and temperature, were studied
using the model tetrapeptide, Glu-AspGly-Thr. The aspartyl carboxyl side chain
in this tetrapeptide was either free or
protected as a benzyl or cyclohexyl ester.
Our results showed that the cyclohexyl
ester led to far less aspartimide formation
during acidic or tertiary amine treatment
than the corresponding benzyl ester. The
rate constants of aspartimide formation in
HF-anisole (9: 1, vlv) for the tetrapeptz'de
protected as the benzyl ester werejozmd to
be 6.2 x 10-6 and 73.6 x 10-6 s- at -15°
and 0° C respectively. These values were
about three times faster than the corresponding free- or cyclohexyl ester-protected tetrapeptide. Little difference was
seen when the studies were carried out at
room temperature. The cyclohexyl protected tetrapeptide gave only 0.3% aspartimide in diisopropylethylamine treatment in 24 h, a 170-fold reduction of
imide formation when compared with the
benzyl protected tetrapeptide. Thus, using
the cyclohexyl ester for aspartyl protection, our studies showed aspartimide for-
marion could be significantly reduced to
less than 2% under standard peptide synthesis conditions. Furthermore, with these
model peptides, the mechanism of acid
catalyzed aspartimide was studied in a
range of HF concentrations. In dilute HF
cleavage conditions ( HF:dimethylsulfide
1:3, vlv ), the mechanism was found to be
of the AAc2 type, with the rate of aspartimide formation increasing very slowly
with increasing acid concentration. In
concentrated HF solutions (HF >70% by
volume), the rate of aspartimide formation
increased rapidly with the increase in acid
concentration. However, from model
studies, the mechanism of aspartimide formation in concentrated HF was AAc2
rather than AAcl.
A central problem in the synthesis
of aspartic acid-containing peptides is
aspartimide fonnation under acidic or
basic conditions (2,4, 12-13,16-18,20,
23,28,31-32,34,39,41 ). The subsequent
ring opening of the five membered aamino-succinimide ring by aqueous
bases provides largely the wrong isomer containing a P-am ide linkage, and
the net effect is an a- to 13-amide bond
isomerization (Figure 1).
The acid or base catalyzed aspartimide formation is sequence dependent. The amino acid residue, x, immediately following the aspartyl
residue in sequence (Asp-x), exerts a
significant influence on the rate of
imide formation. Ring closure is sensi-
tive to steric and electronic factors.
Thus, amino acids in this position, containing either electron donating or nonbulky side chains, are conducive to the
cyclization side reaction.
Sequences such as Asp-Gly, AspSer, Asp-Asn or Asp-His, when protected with the nonnal benzyl esters,
have been reported to produce extensive imide formation (1,7,9,12,18,20,
Since base catalyzed imide formation has been shown to follow the
biomolecular mechanism BAc2 (7,9),
base catalyzed aspartimide formation
under normal peptide synthesis conditions is believed to follow a similar
mechanism. The proposed BAc2 mechanism is consistent with the observed
rate of aspartimide formation with
respect to the structural effect of the
protecting groups. For example, electron withdrawing protecting groups
such as phenacyl or nitrobenzyl esters
are more susceptible to imide formation than the benzyl protecting group
(43,51 ), rendering these protecting
groups unattractive in long syntheses.
On the other hand, electron donating or
sterically hindered groups such as the
tert-butyl ester have been found to be
much more resistant to base-catalyzed
imide formation. These results agree
well with the BAc2 mechanism, in
~hich the immediate c?~plex is negatively charged. Thus, tmide fonnation
should be accelerated by electronegative, and retarded by electropositive groups. Furthennore, since the
rate-controlling process is bimolecular
one would expect steric retardation of
the side reaction from substituents
close to the reaction center. A protecting group with electropositive and
sterically hindered structures such as
the tert-butyl ester would be most
suitable for aspartyl protection. However, the use of tert-butyl ester protection would necessitate a-amino protecting groups which are removed by
methods other than trifluoroacetic acid.
A protecting group that retains in part
the steric hindrance and the electron
donating properties of the .tcrt-butyl
group, but is stable to trifluoroacetic
acid, would be extremely desirable.
Most observations of acid catalyzed
aspartimide formation are in concentrated, strong acids such as HF or
TFMSA-TFA solutions. Under these
conditions, the observed rate of aspartimide fonnation is usually fast. The
proposed mechanism for the cycliza-
tion is of the unimolecular AAc 1 type,
in analogy with side reactions observed
for glutamyl residues (11 ). However, it
is now known that aspartimide formation also occurs over a wide range of
acidity. Mild acids, such as trifluoroacetic acid, hydrochloric acid, or dilute
HF, will catalyze aspartimide formation, albeit at a slower rate than strong,
concentrated acids. It is likely that the
mechanism of acid catalyzed aspartimide formation is of the bimolecular
AAc2 type in low to moderate acid
concentrations, and
AAcl only in the concentrated acid.
These mechanistic considerations remain to be established. In this paper,
the acid catalyzed aspartimide formation is examined by acid-rate profile.
Since the AAc2 mechanism is influenced by steric and polar factors, a
bulky and electron donating ester will
be more resistant to aspartimide formation than the benzyl ester (6,47). In
most synthesis of small peptides, the
difference is relatively small. However,
in syntheses of large peptides and
proteins, it can be large enough to be
Based on all these considerations,
secondary alkyl esters appear potentially able to fulfill these criteria. They are
also compatible with the usual solid
phase protecting scheme, Na-tertbutoxycarbonyl- and benzyl side chain
protection. Since quantitative data
from solvolysis experimems and acidolytic stabilities of secondary alkyl
esters (33,49), carbamates (5,21 ,25,36)
and phenolic ethers are available, a rational choice of the best secondary
alkyl ester can be made on this basis.
From all indications, the cyclohexyl
(s;;Hex) ester possesses the proper acid
stability for the synthesis of large peptides and fulfills our requirements for a
new protecting group for aspartic and
glutamic acids (47). The cyclopentyl
(6) and cycloheptyl esters (50) have
been chosen as protecting groups for
- NH-CH-g-NH-
I 2
- NH-CH-C0 H
Figure 1. Aspartimide formation and
rearrangement of aspartyl pep tides.
Vnl. I. No. I (1988)
a-, P·
aspartic acid, based on similar logic.
The nonreactive nature of the cyclohexyl carbonium ion generated in HF,
as shown by the production of fewer
alkylated side products of tyrosine relative to those from its benzyl derivative,
prompted us to consider this as an additional benefit of the cyclohexyl ester.
In this paper we describe the effects of
cyclohexyl ester as a side chain protecting group, and of temperature and
time of hydrogen fluoride treatment on
aspartimide formation in peptides containing cyclohexyl-, benzyl- or unprotected aspartyl residues. Finally, the
mechanism of acid catalyzed aspartimide formation in dilute to concentrate HF is examined.
Amino acid and peptide analyses
were conducted with Beckman Model
1208 or 121 amino acid analyzers. All
solvents and bulk chemicals were
reagent grade. Dichloromethane was
distilled from sodium carbonate and
stored in amber bottles. Diisopropylethylamine (Aldrich Chemical, bp 1261290 C) was distilled from calcium
hydride. Thin layer chromatography
(TLC) was run on precoated silica gel
GF plates (Analtech, 250 Jl) with the
following solvent systems: CA (chloroform:methanol, 95:5), CMA (chloroform:methanol:acetic acid, 85: 10:5)
and (chloroform:ethylacetate, 1:1).
Hoc-Aspartic Acid-~-Cyclohexyl
Ester and Hoc-Glutamic
Acid-y-Cyclohexyl Ester
From cyclohexene. Z-Asp-0Bzl45
(lOg, 0.028 mol) in 100 ml ofCH2Ch
was stirred with cyclohexene ( 10 g,
0.122 mol) and 1 ml of BF3·Et20 for
24 h at ambient temperature. After
removal of all solvents to obtain a
syrupy residue, the product was redissolved in 150 ml of ethyl acetate, and
washed with copious aqueous acid (0.1
N HCI) and base (NaHC03:NazC03
solution, pH 10). After removal of solvent, the oily diester 2 (Rf 0.6. CMA)
was dried. The diester was then
hydrogenated over 5% Pd{BaS04 ( 1 g)
in 60 ml of 95% ethanol. The progress
of the reaction was monitored by TLC
(CMA, Rf 0. 13). Asp (Oc_Hex)-OH 3
was obtained after filtration and crystallization in EtOH-HzO mixture. Con-
version to Boc-Asp(Os;;Hex)-OH was
accomplished as described, using ditertbutyldicarbonate in triethylamine
and DMSO. The overall yield was 44%
based on Z-Asp-OBzll.
From cyclohexyl bromide. To a
stirred solution of pulverized KF (7 .01
g, 0.121 mol) in dimethylformamide
(120 ml) at 55° C was added Boc-GluOBzl (10.02 g, 0.03 mol) and cyclohexyl bromide (4.0 m1, 0.033 mol). The
reaction proceeded very slowly, as
judged by TLC and three portions of
cyclohexyl bromide (4 ml) and KF (7.0
g) were added successively at 24 h intervals. The reaction was stopped after
96 h, filtered and solvents removed to
obtain a syrupy oil. The oil was redissolved in 120 ml of ethylacetate. After
filtering the insoluble salts, the filtrate
was washed three times with 80 ml of
pH 9 buffer (0.5 M KzC03:0.S M
NaHC03, l :2, v/v) and 80 ml of water.
The organic layer was dried, and
evaporated to a syrupy oil. TLC (CA)
showed a single spot. The oil was dissolved in 60 ml of 95% ethanol and
hydrogenated over Pd/BaS04 (1.26 g)
for 17 h to obtain 6.05 g of Boc-Glu
(Oc.Hex)-OH (78% yield) m.p. (DCHA
salt) 133-136° C. Rf (CA) 0.53. Anal.
(as DCHA salt, C2sHsoN206) calcd. C
65.88; H 9.80; N 5.49. Found C 65.68;
H 9.67; N 5.38.
N,N-Dimethylaminopyridine (0.41 g, 0.003
mol), cyclohexanol (10.96 ml, 0.102
mol) - Note: Must be extra pure
reagent grade, Eastman Kodak Chemical Co., to avoid impurities that would
lead to side products) and water soluble
carbodiimide (7.15 g, 0.037 mol, 1ethyl-3-(3-dimethylaminopropyl)- carbodi-imide hydrochloride) were added
successively to a stirring solution at
10° C of Boc-Asp-OBzl (1 0.95 g,
0.034 mol) in methylene chloride (70
ml, 0.48 molar). The progress of the
reaction was monitored by TLC (chloroform: acetic acid 98:2 Rf 0.76, CE Rf
0.88). It was >95% complete after 4 h.
The reaction was generally worked up
after 6 h. In some cases additional
equiv.) was added after 3 h and the
reaction allowed to proceed for another
3 h. Upon removal of the solvent, the
residue was redissolved in ethylacetate
(150 ml), washed with aqueous acid
(0.5 N HCI) and base (pH 9 buffer,
NaHC0.3/Na2C03), and dried over
magnesium sulfate. If necessary, the
crude diester was adsorbed to 20 g of
silica gel and eluted with 250 ml of
ethylacetate to remove slow moving
impurities. After evaporation of the
combined solvent, a waxy solid (m.p.
68-69° C; similarly Boc-Glu(O-Hex)OBzl m.p. 74-76° C) was obtained.
Hydrogenation of this solid over 5%
Pd/BaS04 (1.0 g, prewashed with 50
ml of 95% EtOH) in 60 ml of 95%
EtOH for 2-4 h resulted in a solid, after
workup. Longer hydrogenation time
produced Boc-Asp-OH as a side product. Crystallization was effected in
cyclohexane-hexane (1:6, v/v) to obtain Boc-Asp(O.QHex)-OH in 85%
yield. m.p. 93-95° C, TLC (CA, Rf
0.67) Anal. (C1sH2sN06). Calcd C
57 .13, H 7 .99, N 4.44; found C 57 .22,
H 8.04, N 4.36.
Direct esterification with aspartic
acid and cyclohexanol. H2S04 (50
ml) was added to ethyl ether (500 ml:
CAUTION) and cyclohexanol (270
ml). The mixture was concentrated to a
constant volume under reduced pressure and 75 g of aspartic acid was then
added. The colloidal solution was
stirred at 50° C and became homogenous after 18 h. The reaction was
stopped after 24 h by pouring the mixture into crushed ice and 2 1 of 2 N
NaOH. The biphasic solution was
separated into the upper and lower
phases. The basic aqueous layer (containing mostly Asp) was extracted
twice with 200 ml of ether. The combined organic phase (containing the a-,
[3-, and di-esters) was washed once
with 0.1 N NaOH and then twice with
water. Upon storage in cold, the Asp
(O.QHex) crystallized. The crystalline
material contained 1 to 5% of diester.
TLC in CMA 85:10:5 gave an Rf of
0.14 (diester Rf 0.44; a-ester 0.1; Asp,
0). Final purification of Asp (O.QHex)
was achieved by ion exchange chromatography. Asp( O.QHex) (15 g). was
loaded onto a Dowex SOW-X-4 (2.5 x
30 em) column. It was eluted by 0.2 M
pH 3.1 pyridine acetate buffer. The
order of elution was Asp, Asp(O~Hex)
and AspO.QHex. A broad peak of Asp
(O~Hex) was collected between fraction 22 to 35 (5 ml fractions). After
lyophilization, 12.5 g of Asp(0£Hex)
was obtained.
Syntheses of Test Pcptides 10-13
and 24
Boc-Thr(Bzl)-OCH2-resin (30 g,
0.31 mmol/g resin, based on: Picric
acid titration; nitrogen analysis; HF
cleavage of resin; amino acid analysis
after 6 N HCI hydrolysis of resin) was
obtained from potassium fluoride esterification ( 10) of chloromethyl resin
(Lab Systems copoly-(styrene-1 %-divinylbenzene) resin, 200-400 mesh,
0.32 Cl/g substitution). Preparations of
tetrapeptides 10 (1 g), 11 (5 g), 12 (5 g)
and 24 ( 10 g) were accomplished using
Boc-Glu(OBzl)-OH, Boc-Glu(0£Hex)
-OH, Boc-Asp (OBzl)-OH, Boc-Asp
(O~hex)-OH or Boc-Asp-OBzl as Glu 1
and Asp 2 to form the tetrapeptide. The
essential protocol for one synthetic
cycle was: (1) De protection with trifluoroacetic acid/ methylene chloride
(1:1, v/v) for 1 and 20 min, (2) neutralization with diisopropylethylamine/
methylene chloride (1:19, v/v) for 2 x 5
min and (3) double coupling with 3
equivalent of preformed symmetrical
anhydride of Boc-amino acid for 1 h.
Amino acid analysis (HCI: HOAc:
phenol, 2:1:1, v/v/v; 120° C, 24 h) of
aU peptide resins after the completion
of the syntheses revealed that
Glu:Asp:Gly:Thrratios were 1:1:1:1 (±
Peptide 13 was obtained from peptide-resin 10 by hydrogenolysis (l M
concentration of Pd(0Ac)2 in dimethylformamide at 30° C for 24 h).
Phenol (0.1%) was added to the solution to prevent imide formation. The
yield was 21%, and <1% of aspartimide 15 was detected by ion-exchange chromatography. The crude
product was precipitated from ethylacetate-hexane. In the absence of
phenol, 3.9% of aspartimide 15 was
detected at 30° C, 48% at 50° C.
However, the cleavage yield at 50° C
was raised to 70%. The crude product
in all cases contained approximately
30-40% of Boc-Glu-Asp-Giy-Thr
Tritluoroacetic Acid Stability of
Cyclohexyl and Benzyl Esters
Boc-Glu(OBzl)-OH and
Boc-Glu(O.QHex)-OH (l mmol each)
were dissolved separately in 40 ml of
trifluoroacetic acid at 55° C. Boc-AlaOH (0.1 mmol) was included as the internal standard. At various time intervals, 1 ml aliquots of each solution
were withdrawn, evaporated to dryness, dissolved in pH 2.2 citrate buffer
and analyzed immediately for Glu or
Asp on a Beckman 120B AA-15
column. The rate of acidolytic Joss of
the protecting group was calculated according to the equation of lnXo/lnX1 kt, where Xo is the concentration of
either benzyl or cyclohexyl ester at the
beginning of the reaction, Xt is the concentration of the ester at time t, and k is
the rate constant.
Deprotection and cleavage of
amino acids and peptides in HF. The
deprotection of the side chain protected
amino acids, or the cleavage of the
resin-bound amino acids and peptides
to the free, unprotected amino acids
and peptides were carried out in a
fluorocarbon HF-Reaction Apparatus
(Type I, Peptide Institute, Japan). A
typical procedure was as follows: Peptide-resin (1 00 mg, 0.31 mmol/g of
peptide) was charged with 0.5 ml of
anisole (10% v/v) and then chilled by
dry ice-acetone bath to -78° C for 10
min. HF (4.5 ml, 90% v/v) was then
added and the temperature was quickly
brought up to the desired temperature
by the appropriate solvent bath (-15°,
0° or 25° C). After the appropraite time
treatment (0.5 ·- 4 h), HF was rapidly
removed under high vacuum at -10° C
to 0° C. The peptide-resin was extracted thrice with dry ether (3 ml) to
remove the remaining anisole, dried in
high vacuum, extracted with 10-25%
HOAC-H20 (v/v). The aqueous HOAc
mixture was collected and lyophilized
to obtain the peptide.
Cooling baths. Cooling baths of
-15° C could be attained using an
NaCl-ice mixture (23:77 w/w); NaCl:
H20: acetone (12:2:50, w/v/v) or carbon tetrachloride slush. The baths were
precooled by dry ice-acetone bath to 15° C and insulated with a layer of a
highly porous material such as glassfibers or styrofoam chips encased in
another beaker. To maintain the temperature, small pieces of dry ice were
added. Alternatively, a low temperature bath (Ultra Kryomat TK30,
MeOH as circulating solvent) was
used. Deprotection of protected amino
acids were as follows: A mixture of 2-5
J.lrnol each of protected amino acids:
Boc-Ser-(Bzl)-OH, Boc-Thr(Bzl)-OH,
Boc-Tyr (2,6-Ch-Bzl)-OH, Boc-Lys
(2-Cl-Z)-OH, Aoc-Arg(Tos)-OH, BocCys(4-Me-Bzl)- OH, Boc-His(Tos)OH, Boc-Asp(OBzl)-OH, Boc-Glu
(Bzl)-OH, Boc-Val-OH and Boc-AlaOH, was treated with HF:anisole (9:1,
v/v) at -15° C for 1 h, -15° C for 2 h, 0°
C for 1 hand 25° C for 1 h. Separately,
two samples of this mixture were
hydrolyzed in 6 N HCl at 120° C for 24
Vol. I, No. 1 (1988)
Table 1. Jon-Exchange Chromatography of a:- and ~-cyclohexyl Esters or Aspartic Acid
pH 3.202
Elution Time (min)
Asp(O.QHex) 3
Asp-O_gHex 4
dissolved in 20 ml of pH 2.2 citrate
buffer and analyzed by ion exchange
chromatography (AA-15 column). The
following peak (detection 0.2%) were
observed, Leu (178 min), Asp (42
min), Leu-Asp ( 156 min), but Leu-DAsp (lit. 109 min) was not detected.
AA-15 (0.9 x 54 em) flow rate 66 ml/h
pH of buffer
Synthesis of Cyclohexyl Esters
h and 48 h. The results were quantitated by amino acid analysis on Beckman 121. Boc-Ala-OH and Boc-VaiOH were used as the internal standards.
Attempts to Trap the Aspartic
Acylium Ion
To a stirred solution of Boc-GluAsp-Giy-Thr-OH 13 ( 10 mg) or resin
25 (150 mg, 0.9 mmol/g substitution,
prepared from esterification of phenol
in KF-KHC03-NMP with 2-bromopropionyl resin) ( 19) in tetrahydrofuran
(1.0 ml) was added HF (9.0 ml). These
mixtures were stirred for 1 and 2 h at
25° C. After the usual workup, the 10%
aqueous acetic acid filtrate was analyzed by ion-exchange chromatography from the 1 h treatment. The
chromatogram indicated that 65% of
the peptide was recovered. However,
67% of this material was the imide of
H-Glu-Asp-Gly-Thr-OH, 15. From the
2 h treatment. 37% of the tetrapeptide,
H-Giu-Asp-Giy-Thr-OH was recovered and 82% of this material represented the imide 15. The resulting
resins were then washed with piperidine-dimethylformamide (1: 1 v/v, 3 x 5
min) and trifluoroacetic acid-methylene chloride (1: 1, 3 x 5 min). Amino
acid hydrolysis of the resins (12 N
HCl:phenol:HOAc, 2:1:1, v/v) gave
Asp (1): Gly (1.03): Thr (0.92).
Quantitative Analyses for Imide 15
The crude peptides I 0-13 and 24,
after cleavage from the resins, extraction with 10-25% HOAc-HzO and
lyophilization, were dissolved in water.
Small aliquots were applied to an AA15 column (Beckman, 54 x 0.9 em) and
eluted with pH 3.20 citrate buffer at
59° C. The elution time and color yield
(CY) of the peptides were (1) H-GiuAsp-OH Gly-Thr-OH, 49 min, (2) HGlu-Asp-Giy-Thr-OH, 70 min, CY =
Vol. I, No. I (1988)
0.86 x CY Leu and (3) H-Giu-AspGly-Thr-OH, 130 min, CY = 0.80 x CY
Leu. Results are shown in Table 1).
Reverse phase (Cts) high pressure liquid chromatography was carried out on
a (0.4 x 30 em) column, with elution by
90% of aqueous phase [containing
0.1% H3P04] and 10% acetonitrile.
The tetrapeptide, H-Giu-Asp-Giy-ThrOH and the imide 15 (r.t. 21.8 min)
were separated from the anisylated
tetrapeptides 17 and 18 (r.t.: 48 min
and 56 min). Evidence for these assignments were: Amino acid analysis of the
acid hydrolysate from these two
sample peaks (48 min and 56 min)
revealed the absence of Glu, but Asp,
Gly and Thr were found in equal ratios
(± 1.5% ). UV analysis of the peak at 48
min and 56 min showed max at 275
nm. Quantitative TLC (n-butanol: pyridine-acetic acid-water: 65:50:10: 40
v/v) in which all four products were
separated: tetrapeptide (Rf 0.18), imide
15 (Rf 0.29), 18 (Rf 0.5) and 17 (Rf
0.6), substantiated the appearance of
each product during all the time course
Studies of Racemization Using
Manning-Moore Procedures
Boc-Asp(CkHex)OH 4 (36.01 mg)
was deprotected with HF/anisole (5 ml,
9:1, v/v) at 0° C for 1 h. HF was
evaporated, and the residue extracted
with ether. It was dissolved in 5%
HOAc. After lyophilization of the 5%
HOAc solution, a white solid was obtained. A portion of this lyophilized
solid (3.55 mg) was added to a stirring,
buffered tetrahydrofuran solution (0.15
ml) of Boc-Leu-OSu (hydroxysuccinimide ester, 18.0 mg, 7.1 J.lmol;
NaHC03 1.3 mmol/ml). The mixture
was stirred for 1 h. After evaporation of
the solvent, deprotection by trifluoroacetic acid for 50 min, and removal of
the solvent, the resulting residue was
Three approaches to the synthesis of
Boc-Asp(()&Hex)-OH have been developed using a-diprotected aspartic
acid as starting material. The acid
catalyzed esterification ofZ-Asp-OBzl
with cyclohexene was carried out in
BF3·Et20, since both the benzyloxycarbonyl and benzyl ester groups were
stable to small amounts of this acid
catalyst (Figure 2). Hydrogenolytic
removal of both benzyl-based protecting groups, followed by reprotection of
the a-amino group, gave the desired
Boc-Asp({kHex)-OH in 44% yield.
This method has the flexibility of introducing the desired a-amino protecting
group at the last step and would be
useful for groups that are very acid or
base sensitive. Boc-Asp(O&Hex)-OBzl
could be obtained from a displacement
reaction of the carboxylate salt of the
commercially available Boc-Asp-OBzl
(2) with cyclohexyl bromide (Figure
3). When the reaction was attempted
with either Cs+, Ag+or hindered amine
salts of 2, extensive aspartyl anhydride
formation was observed. The cyclohexyl bromide was less reactive than
expected, allowing this competing side
reaction to occur. The best reagent
found was potassium fluoride, but the
reaction had to be carried out for a long
time (5 days) and only a moderate yield
was obtained. After hydrogenolytic
removal of benzyl ester from 3, a 42%
yield of 4 was obtained. However, the
potassium fluoride procedure worked
satisfactorily with Boc-Glu-OBzl, producing Boc-Glu-(O&Hex)-OBzl. Little
anhydride was detected during the
course of this reaction. After hydrogenolysis, 78% Boc-Glu (O&Hex)-OH
was obtained.
An alternate approach which alleviates the steric problem is activation
of the carboxylic acid and use of cyclohexanol as the nucleophile (Figure 4).
The production of the diester 3 using
dicyclohexylcarbodiimide activity was
Figure 2. Synthesis of Boc-Asp (O,~;Hex)-OH from cyclohexene by acid catalysis.
Boc-Aap-OBzl _ _ _K;.;.F;._--4•
Figure 3. Synthesis of Boc-Asp (O~Hex)-OH by displacement of cyclohexyl bromide.
Figure 4. Synthesis of Boc-Asp(O,Hex) by carbodiimide condensation of cyclohexanol.
H-Asp-OH _ __.,.. NH
£_Hex, R2 = H
H, R2 = £_Hex
R2 = £_Hex
FigureS. Synthesis of Asp(O.cHex) from aspartic acid.
slow and led to significant amounts of
N-acylurea byproducts 5. The formation of the symmetrical anhydride or
the use of an additive such as 1hydroxybenzotriazole (HOBt) did not
accelerate the reaction or alter the
amount of side products. It has been
reported that 4-dimethylaminopyridine
is a powerful acylation accelerating
reagent that also suppresses the formation of N-acyl urea (14,27). When 1020 mol% of this catalyst in methylene
chloride was used, the esterification
proceeded extremely rapidly and was
complete in 1 h. However, theN-acyl
urea side product still accounted for 35% of the yield. It was nonsuppressible, even with additives such as
HOBt, or by maintaining a low temperature. In order to avoid the need for
extensive chromatographic purification, a water soluble carbodiimide was
used, since the side product 6 could be
removed in an aqueous workup. The
diester 3 was thus obtained in 90%
yield. After hydrogenolytic removal of
the a-benzyl ester, Boc-Asp-(O~Hex)­
OH 4 was obtained as a solid in 85%
yield. Similarly, Boc-Glu(O~Hex)-OH
was obtained in 82% yield. To test for
racemization that might occur during
this preparation, Boc-Asp-(O~Hex)­
OH was treated with HF to remove all
the protecting groups. The free Asp
was shown to be free from racemization using the Manning and Moore procedure (19,24). This synthetic procedure is efficient and gives high yields
and produces pure products.
A practical and direct laboratory
synthesis of Boc-Asp(O~Hex)-OH (4)
was also undertaken, starting from
aspartic acid and cyclohexanol, using
concentrated sulfuric acid as the catalyst (Figure 5) (3). The reaction
proceeded much too slowly at room
temperature and required elevated tern.
perature (50° C) for satisfactory yield.
After 24 h, the yield of desired product
1 was found to be 75% based on the
ion-exchange chromatography analysis
of the reaction mixture. Fractional
crystallization of the correct product in
the presence of the starting material
(Asp), the a-isomer (AspO~Hex) and
the diester 9 was found to be difficult,
and 35% of Asp(O~Hex) was obtained
by ion-exchange chromatography. The
product 7, when examined under analytical ion-exchange chromatography
(r.t. 124 min), was found to be free of
Asp (r.t. 29 min) and its diester, but
contained 0.5 to 1.5% of Asp-O~Hex 8
Table 2. Deprotection of Benzyl and Cyclohexyl Esters of Aspartic and Glutamic Acid in TFA at
55° c
k(10" 7 x s" 1)
t 1/2 (h)
Boc-Giu (OBzi)-OH
Table 3. HF Cleavage of Boc-Asp(O~Hex)-OH
TempeC) Time (h)
(388 min). Boc-Asp(O~Hex) was then
prepared from Asp(0£Hex) with in DMF using
triethylamine as base.
Chemical Stability of the
Cyclohexyl Esters of Aspartic and
Glutamic Acids
The cyclohexyl esters of aspartic
and glutamic acids are stable to prolonged TFA treatment (Figure 6). The
rate constants k 1 for acidolytic loss of
kHex esters at 55° C in ne~ TFA were
determined to be 1.9 x 10- s- 1 for Asp
(O.c.Hex) and 2.4 X 10"7s" 1 for Glu
(O~Hex). These rate constants indicate
84- to 88-foJd more stability in TFA
than their respective benzyl esters
(Table 2). They are consistent with the
acid stability of Tyr(~Hex), which is
100 times more stable than Tyr(Bzl)
(10). Thus, the repetitive loss of the
cyclohexyl ester protecting group per
synthetic cycle due to 0.5 h of TFA
treatment will be about 0.0002%,
making this protecting group one of the
most stable in the TFA-HF protecting
group strategy. The added acid stability
should be useful in the synthesis of
large polypeptides or proteins and
prevent acidolytic loss of the side chain
protecting group during long synthesis.
Both aspartyl- and glutamyl-cyclo-
• · !loc-Asp(OBzl
, • 90
Time (h)
Figure 6. Acidolytic loss of benzyl and cyclohexyl esters in trinuoroacetic acid at 55° C.
hexyl ester protecting groups were
completely removed by treatment with
HF:anisole (9: 1, v/v) for 1 hat 0° Cor
2 h at -15° C (Table 3 ). They could also
be conveniently removed by 1 M
TFMSA-thioanisole-TFA in 1 h at
0° C. However, the reaction was extremely sluggish in HOAc-HBr-TFA
(1:1:1, v/v), and only 78% of the esters
were removed after 18 h. The rate of
acidolytic removal of .c.Hex ester in HF
or TFA could be conveniently quantitated by ion-exchange chromatography, since the end products, Asp (r.t.
92 min) and Glu-Asp-Giy-Thr (r.t. 73
min), were well separated from their
starting materials, Asp(O~Hex) (r.t.
189 min) and Glu-Asp(O.c.Hex)-GlyThr (r.t. 489 min). The observed acidolytic deprotection rates of the .c.Hex
esters in HBr or HF were consistent
with those of kHex carbamate observed
by McKay and Albertson (21 ), Blaha
and Rudinger (5) and Munakata et al.
(25). They are consistent with the expected properties of the cyclohexyl
ester that it could be removed more
favorably by the A-1 mechanism in
strong acid.
The cyclohexyl esters were more
stable towards nucleophile than the
corresponding benzyl ester. This
property is particularly useful with
glutamic acid, since the y-protected
benzyl ester is prone to intramolecular
cyclization to fonn pyrolidone-1-carboxylic acid (pyroglutamic acid) neutalization and coupling. This side reaction leads to significant termination of
peptide chain and is a serious problem
in long peptide synthesis. The use of ycyclohexyl glutamic acid is expected to
greatly minimize this side reaction.
Several recent syntheses have been
successful using this strategy. The cyclohexyl esters were also completely
stable towards hydrogenation. The
hydrogenolytic stabilities improve the
usefulness of the cyclohexyl ester with
regard to semi-synthesis and fragment
Table 4. Aspartimide formation from Boc-Giu(ORI)-Asp(OR2)·Giy·Thr(Bzi)-OCHz-Polystyrene
Resin in Trialkyamine at 25° C
10 R1=R2:;;:Bzl
Model Peptides and Method of
Earlier attempts to synthesize the
decapeptide fragment, residues 114123 of human growth hormone, using
Asp(OBzl) for the Asp-Gly sequence,
gave rise to extensive imide formation
(51). Therefore, the effects of the
cyclohexyl ester relative to the benzyl
ester were examined on tetrapeptide
119-123, (Glu-Asp-Gly-Thr) from this
fragment. The suitability of this
tetrapeptide as a model has already
been investigated in detail (51). Since
the essential Asp-Gly sequence is not
located at the N- or C-terminus, it is a
more reasonable peptide model than
other simpler sequences ( 1,8). Furthermore, the ion-exchange chromatographic behavior of this peptide, its
imide and its f3-peptide isomer are well
characterized (51) (Figure 7). Aspar-
11- peptidel
Aspartimide Formation (%)
11 R1:;;:Bzl, R2:cHex
12 R=R=cHex
24 h
24 h
> 1.4
Table 5. Aspartimide Formation from Pep tides 10, 11, 12, 13, and 14 in HF
TempCOC) Time(h)
Aspartimide (%)
timide formation during HF cleavage
or base treatment was easily quantitated. By overloading the column, a
sensitivity of 0.1% for the detection of
aspartimide could be obtained. The
placement of a glutamic acid residue at
the N-tenninus of this model tetrapeptide did not interfere with the analyses
of aspartimide, and, in addition, it allowed examination of three possible
side reactions of glutamic acid.
For the purpose of comparison. five
tetrapeptides were synthesized. Peptides 10, 11, 12, and 14 (Figure 8) were
prepared on a chloromethyl-styrene-divinylbenzene support (50) using identical synthetic conditions. However, 10
contained benzyl ester protecting
groups on both Glu and Asp, 11 had a
benzyl on Glu and a cyclohexyl on
Asp, and 12 had cyclohexyl on both
Glu and Asp. Peptide 13 was obtained
from 10 by catalytic hydrogenolysis
(37). (Conditions to minimize aspartimide formation during hydrogcnolysis
are discussed in Experimental section.)
Peptide 14 was similar to 10 except
Time (min)
l'igure 7. Ion-exchange chromatographic
analyses of HF cleavage of Boc-Giu(OBzl).
Asp(OR)·Gly-Thr(Bzl)·OClh·resin for quantitation of a-, p-pcptides and imide. Lower
panel: Dilute sample; middle panel: 50 X more
· concentrated than the lower panel for the detection
of ~-peptide; top panel: Standards at same concen·
Figure 8. Model peptides 10-14 for the aspartimide study.
that it contained a f3-peptide bond at the
Asp-Giy sequence instead of the usual
a-peptide bond linkage. Peptide 14
was synthesized as a control peptide to
test whether aspartimidc could be
formed via the acylium intermediate,
since a-carboxylic acid or ester is
known to be resistant to such formation.
Trialkylamine Treatment
Treatment with triethylamine (TEA)
in CH2Cl2 for 24 h at 25° C led to
100% imide formation from the b'enzyl
ester-containing peptide 10 and 14%
from the cyclohexyl ester peptide 11
(Table 4). Treatment with a more
hindered base, diisopropy1ethy1amine
(DIEA), reduced aspartimide formation to 51% for benzyl and only 0.3%
for cyclohexyl. These values would be
equivalent to 72 cycles of sequential
base neutralization of 20 min each in a
normal double coupling solid phase
synthesis. This gives >1.4% and 0.7%
imide per step for the benzyl ester
protecting group using TEA and DIEA,
respectively, but only 0.17% and
0.004% per step for the cyclohexyl
ester. These data confirm that neutralization with a hindered base is
beneficial in the reduction of aspartimide formation in the presence of
benzyl esters (28,32). DIEA was 40fold better than TEA and the cyclohexyl group was 180-fold better than
the benzyl.
The superiority of the cyclohexyl
ester over the benzyl ester was ex-
Table 6. Rate Constants, Half-lives and Activation Energies or Aspartimide Formation from Pep·
tides I 0, II and 13 in HF Treatments
Temp (°C)
k(s"1 x 106)
t 1/2 (h)
Ea (Kcal/mol}
Table 7. HF Cleavagf at -15° C for 2 h 1
Vield (%)
983 ,4
Boc-His(Tos )·OH
Boc-Giu(OBzi)-Asp{OBzi)-Giy-Thr( Bzi)-OCI-Q-R
10% anisole as co-solvent
Amino acid yields are determined by Beckman 1208 and based on the following equation. [mole% (HF)/mole % (6NHCI hydrolysis)] x 100%, and resin
cleavage yield is based on hydrolysis of the resulting resin
3Determined by ion-exchange chromatography (PA-35), pH 6.4 buffer
About 2% of 3-alkylated Tyr product
Addition of 2% aromatic thiol
H-Giu-Asp(O£Hex)-Giy-Thr-OH was not detected in ion-exchange chromatography
4 times slower for cyclohexyl, refelcting an apparent activation energy of
17.7 Kcal/mol for cyclohexyl and 15.3
Kcal/mol for benzyl (Table 6). Thus at
-15° C, the yield of aspartimide byproduct was reduced to 6.5% for the
benzyl ester (peptide 10) and to 1.7%
for the cydohexyl ester (peptide 11).
These results were independent of
whether the protecting group for GJu 1
was cyclohexyl or benzyl.
The aspartimide formation of the
protected ester tetrapeptide (10, 11,12)
were compared with the tetrapeptide in
which the aspartyl residue was unprotected (13). It has been reported that
the peptides with an unprotected aspartyl residue are resistant to the imide
formation. Our data showed that aspartimide formation in HF could arise not
only from the aspartyl esters but also
from the free acid itself (Figure 9). The
rate of aspartimide formation of the
free acid tetrapeptide (13) was comparable to the tetrapeptide with the
cyclohexyl ester protecting group (11
or 12), but was slower than tetrapeptide
(10), which contained the benzyl ester
protecting group. It is noteworthy that
the rate increases more rapidly with
temperature for the free peptide (13)
than for the ester (10-12). At -15° Cit
was 0.01 %/min for the free peptide and
increased 9- and 118-fold respectively
at 0° and 25° C. The activation energy
of aspartimide fonnation was 16.7
Kcal, a value between the benzyl ester
and the cyclohexyl ester. Thus, at the
higher temperature, there were no sig-
pected, based on mechanistic considerations. Trialkylamine aspartimide formation follows a BAc2 mechanism,
which is known to be influenced by
both the electronic and steric properties
of the leaving group (15).
Concentrated HF Treatment of the
Model Tetrapeptides
The results of high concentration
HF-anisole treatment (9:1, v/v) of the
model peptides (Table 5 and Figure 9)
allow a comparison of the effects of
Vol. I, No. I (1988)
temperature and time on aspartimide
formation in the presence of the two
protecting groups. At 25° C, imide formation was complete within 2 h, with
no measurable difference between the
two protecting groups. As the temperature was lowered, the observed imide
decreased more sharply for the cyclohexyl ester than for the benzyl ester. At
0° C, 1 h (nom1al HF reaction conditions), the peptide containing ~-benzyl­
aspartic acid gave 24% of aspartimide,
whereas the ~-cyclohexyl ester gave
4.7%. At -15° C the reaction was 3.5 to
Figure 9. Aspartimide formation from benzyl
ester peptide l 0 ( - ) and cyclohexyl ester 11
(---·) in HF treatments at various times and
nificant differences in the rates of
imide formation between the protected
peptide resins (10, 11, 12) and the free
peptide (13). These data suggest that
the esters were rapidly removed and
that the aspartimide was produced
primarily via free aspartic acid. However, at the lower temperatures (0° C
and -15° C) the rate of imide formation
from the free acid could not account for
the extent of imide found with the
esters. Earlier studies had indicated that
the removal of the benzyl protecting
groups would be completed under such
conditions in 2 min. Thus, under these
conditions, imide was primarily
formed from the esters.
Minimization of aspartimidc formation in synthetic peptides during HF
treatment required ]ow temperature.
The free acid did not cyclize readily,
and the ester was still quantitatively
removed. In addition, replacement of
the benzyl ester by the cyclohexyl ester
further reduced imide formation, arising from the ester, by a factor of 3 to 4.
The best condition examined was -15°
C for 2 h. It was shown that under these
conditions cleavage of the peptides
from the resin support went in high
yield and the deprotection of other
common side chain protecting groups
was essentially quantitative (Table 7).
It follows that the workup of the reaction mixture should be carried out
rapidly and at low temperature.
The acidity of HF cleavage mixtures
can be altered by the addition of co-solvents. Anisole was used by Feinberg
and Merrifield (11) and pyridine by
Sugano et al. (42) to reduce the acidity
of the HF deprotection mixture in order
to minimize side reactions of glutamyl
peptides, due to acylium ion formation.
In this study, we proposed that if aspartimide formation in peptides 10-13 was
due to an acylium ion, it would be
similarly reduced by addition of appropriate diluents or weak base, maintaining the SN 1 deprotection condition.
To test this hypothesis, four types of
solvents were mixed with HF for the
cleavage reaction: Anisole; phenol, pcresole tetrahydrofuran; toluene, benzene; pyridine, triethylamine. Their effects on the reduction of aspartimide
formation are shown in Table 8. Very
weak basic diluents such as benzene or
toluene (pKa < -I 0) that do not undergo hydrogen bonding or protonation
with HF allowed very little (< 20%)
cleavage of the peptides from the resin
and would be of little value as co-sol-
TableS. Aspartimide Formation in Boc-Giu(OBzi)-Asp(OBzl)-Giy-Thr(Bzl)-OCHz-Resin in Concentrated HF Solution with Different Weak Bases as Diluent
Aspartimide (mol %)1
All reactions were carried out at oo C for 1 h, and products were analyzed by
ion-exchange chromatography (see Experimental).
Table 9. Comparison of Aspartimide Formation by Model Tetrapeptides in Dilute and Concentrated HF
Model Tetrapeptide
10 (R=Bzl)
13 (R=OH)
1 For 2 h and for 4 h in parentheses at oo C
(90:100 v/v) 2
(25:75 v/v) 1
2 (5)
0.6 (1.2)
0.6 (1.4)
For 1 h at oo C
vents. Analysis of the cleavage
products, however, showed there was
no reduction of aspartimide formation.
This result was expected, since both
gave a biphasic cleavage mixture and
produced no dilution effect on the HF.
On the other hand, strong basic solvents such as pyridine and triethylamine were expected to neutralize their
respective dilution volume of HF and
considerably reduce the acidity of the
mixture. Indeed, aspartimide fonnation
was reduced three-fold when compared
with anisole. Other weak bases such as
tetrahydrofuran (pKa = -2.5) and
phenols (pKa -7 .2) were found to lie
in between these two extremes and
produced some reduction of aspartimide formation. As expected, dilution
of the HF to 80% (vol) produced even
0 -Giu(OBzl)·- f:Gilu~
Figure 10. Acid-rate profile of aspartimide and pyroglutamyl formation in HF.
Vnl. I.
No. 1 (1988)
greater reduction of aspartimide formation. Toluene and benzene were exceptions (Table 8). It is necessary to note
that despite the beneficial effect on
reduction of aspartimide formation,
this dilution might lower the acidity of
the deprotection mixture to the extent
that some protecting groups [e.g.,
Arg(Tos)] would not be removed. Furthermore, the deprotection mechanism
at these concentrations remains SN 1
and would not reduce most alkylation
side reactions. In fact, the deprotection
HF-pyridine mixture produced much
greater alkylation products than the
HF-anisole mixture.
Dilute HF Treatment of the Model
Recently, we have advocated the
deprotection of synthetic peptides containing benzyl groups in dilute HF in
dimethylsulfide. A mechanism is produced which is predominately SN2.
When model tetrapeptides 10, 11 and
13 were treated under the SN2 deprotection condition (HF:dimethylsulfide, 25:75, v/v), aspartimide formation
(<1.5%) was detectable after 1 to 2 h
(Table 9), and increased with longer
exposure. For example, with the benzyl
ester peptide 10, 1.2% of aspartimide
formation was detectable after 1 h and
5% after 4 h. In contrast, the cyclohexyl ester peptide 11 and the free acid
peptide 13 gave only 1.8% of aspartimide after 4 h. The implication of
these results is that aspartimide formation occurs whether the aspartyl residue is free or protected in dilute HF.
To gain insight into the mechanism
of the aspartimide formation over a
wide range of HF concentrations, an
acid-rate profile of aspartimide formation was investigated. Model peptide
10 was incubated in HF:dimethylsulfide mixtures (Figure 10). For comparison, the glutamyl side reaction (formation of a pyroglutamyl residue) was
also investigated with a model dipeptide on a polystyrene resin, Boc-AlaGlu(OBzl)-OCH2-resin. A C-terminal
glutamyl residue usually generates
three times more side reaction than at
any other position.
The aspartyl and glutamyl side reactions were expected to be of either the
AAcl type, in which the acylium ion is
formed in the rate-determining step, or
the AAc2 type, in which the intramolecular attack on the amide nitrogen
by the side chain carboxylic group
Vol. I, No. I (1988)
produces a tetrahedral intermediate.
Acylium ion formation from glutamic
acid in concentrated acid is well
known. However, the corresponding
acylium ion of aspartic acid has never
been trapped or detected. Thus, the
acid-rate studies were expected to
provide evidence for the mechanism of
the acid catalyzed aspartimide fonnation. As shown in Figure 10, the acidrate profiles of side product formation
of both side reactions were similar. At
low to moderate HF concentrations, the
increase of byproduct formation was
slow with the increase in acid concentration. For example, the increase of
aspartimide formation from 25% HF to
60% HF was about 2-fold. However, at
higher HF concentrations (> 70% ),
aspartimide formation, as well as pyroglutamyl formation, increased more
rapidly, giving about 6.7% byproduct
at 75% and 17-22% at 90% HF. It can
be concluded that there is a changeover
in mechanism from dilute to concentrated acid in the aspartimide and
pyroglutamyl side reactions.
Deprotection Rates of Benzyl and
Cyclohexyl Esters in HF
The acid catalyzed aspartimide formation occurs concurrently with the
removal of the benzyl or cyclohexyl
ester protecting group in HF. Thus, it is
necessary to obtain their deprotection
rates in order to differentiate between
the contribution of the protecting group
and the free acid in aspartimide formation. It was particularly important to
measure the deprotection rates under
conditions where HF was diluted with
dimethylsulfide. In dilute HF (HF:
dimethylsufide is 25:75, v/v), where
the deprotection mechanism of the benzyl group is predominately SN2, the
rate of Asp(,PBzl) removal was slow (k
= 4.7 X w- s- 1). Asp(0£Hex) was essentially resistant to deprotectioo under
these conditions (k = 1.3 X w-6s- 1). In
concentrated HF, (HF:dimethylsulfide
90: lO, v/v, oa C) where the deprotection condition is predominately SNl,
the benzyl ester was found to be rapidly removed (k = 0.2 s- 1) and the
cyclohexyl ester was removed at a
much slower rate (k = 1.5 X 10-JS-l)
(Table 9). However, it is also useful to
compare under both sets of deprotec·
tion conditions, the ratios of deprotection to aspartimide formation. As
shown in Table 9, (k deprotection)/(k
imide formation) of the benzyl ester
was found to be 122 under dilute HF
conditions and 32 under concentrated
HF conditions. Similarly. (k deprotection)/(k imide formation) for the
cyclohcxyl ester protecting group was
also about 100 in dilute HF. However,
Asp(O~Hex) maintained the same ratio
in concentrated HF. These results suggest that the choice of benzyl ester
+<~H 2 > 2 -CO+
R = Asp-GLY-lHRR .. As~ y- THR-DH
R .. Asp-GLY- THR-OH
R "' lHR-OH
Figure 11. Glutamic acid side reactions in HF.
Peptide Research 15
protection is reasonable under SN2
deprotection conditions. The cyclohexyl ester is clearly superior under
SN I conditions.
Glutamic Acid Side Reactions
Acid treatment of the Glu-Asp-GlyThr model allowed three possible side
reactions of glutamic acid to be examined: a) pyroglutamyl fonnation, b)
anisylation of glutamic acid and c) formation of cyclo(Glu-Asp)-Gly-Thr-OH
(4) (Figure 10). None of these side
products were detected in our ion-exchange chromatographic analyses.
However, TLC and HPLC analyses of
the samples revealed the presence of
two other uv-positive products in addition to the expected free tetrapeptide
and aspartimide peptide. Amino acid
anlaysis of the uv-positive products
showed the absence of glutamic acid.
Combining this result with other
analytical data, we concluded that they
were 17 and 18 (Figure 11 ). Formation
of side products 16 and 19 would require the less likely nucleophilic attack
of the protonated a-amino group of
glutamic acid on the acylium ion 14 or
the aspartimide 15 and their occurrence
would be very unfavorable. Formation
of the anisylated glutamic acid
products 17 and 18 in HF was slow. In
2 h they were found to be< I% at -15°
C, 5-10% at oo C, but> 50% at 25° C.
However, quantitative analyses by
TLC or HPLC of the ratio of products
17 and 18 showed that it was similar to
that of the un-anisylated products. Thus
anisylation affected the quantitative
amounts of the free tetrapeptide and its
aspartimide, but not the observed relative rate of formation of aspartimide as
analyzed by ion-exchange chromatography.
Many aspects of the aspartimide
reaction have been reported and characterized. The basicity, protecting
group, and sequence dependency of the
aspartimide formation have been
thoroughly investigated by several
groups. Strong base, aprotic dipolar
solvent, protecting groups with electron withdrawing substituents, and sequences such as Asp-Gly are condi"
tions leading to aspartimide formation.
Furthermore, aspartimide formation
16 Peptide Research
has been detected under various acidic
deprotection conditions. For example,
aspartimide formation is observed
under mildly to moderately acidic conditions such as trifluoroacetic acid, HCl
and HBr, and under strongly acidic
conditions such as methanesulfonic
acid, trifluoromethanesulfonic acid,
and HF. It has also been shown that
acid catalyzed aspartimide formation
occurs from either free or protected
aspartyl residues. Despite such diverse
studies, the mechanistic aspects and the
role of protecting groups in acid
catalyzed aspartimide formation remains unresolved. Since the standard
and popular peptide synthetic methods
depend largely on acidic deprotection,
we have addressed these problems and
hope to derive practical uses from this
study. It appears that aspartimide formation is a side reaction which is difficult to avoid in peptide synthesis,
since it occurs under a wide range of
acid and base concentrations. Two
aspects of this study are particularly
relevant to peptide synthesis.
First, it is clear that acid catalyzed
aspartimide formation occurs whether
the aspartyl residue is a free acid or a
protected ester. Further, aspartimide
formation occurs in both dilute and
concentrated HF solutions. Since the
amount of aspartimide formed in dilute
HF solution (SN2 deprotection conditions) is small, SN2 conditions are
clearly the method of choice for the
removal of benzyl protecting groups in
peptide synthesis. Further, SN2
deprotection enhances the selectivity
ratio k(deprotection)/k(imide formation) and thus allows the removal of
benzyl groups with minimal imide formation. On the other hand, in moderate
to concentrated HF solution, where the
deprotection mechanism is predominately SNl, the use of the cyclohexyl
ester as a protecting group is a better
choice. The activation energy of aspartimide formation in the cyclohexyl
ester containing model tetrapeptide (ll
was 1 Kcal higher than the corresponding benzyl ester containing tetrapeptide
(10), comparable to the free acid containing tetrapeptide 13. In light of this
fact it is necessary to note that the
removal of the cyclohexyl ester in concentrated HF solution at oo C is two orders slower than the benzyl ester.
Aspartimide fonnation can occur from
both the protonated free acid and the
cyclohexyl ester. The fact that the apparent observed rates of aspartimide
formation from the cyclohexyl ester
peptide 11 and the free acid peptide 13
are similar, leads us to believe that the
contribution from the protonated cyclohexyl ester is small. This behavior is in
strong contrast to the extensive aspar"
tim ide formation (- 50%) observed
with the HF-resistant phenacyl ester
protecting group under similar conditions.
We have also observed that using
either strong base diluents, such as
pyridine, or weak base diluents, such as
p-cresol, reduces both acidity and
aspartimide formation. However, the
cleavage mechanism remains SNL
Thus, the problems of carbocation,
alkylation and deprotection of more
acid resistant protecting groups are not
resolved by this method. This approach
is therefore not beneficial.
An alternative approach, maintaining the use of benzyl ester protection, is
deprotection in two stages, or a lowhigh HF treatment (44-46). Under the
low HF treatment (e.g., HF:dimethylsulfide, 25:75, v/v), the benzyl ester
would be removed to give the free acid.
The subsequent high HF treatment to
remove the other acid-resistant protecting groups would be expected to
produce far less aspartimide formation.
Indeed, using the typical low-high HF
procedure, model tetrapeptide 10 gave
5-8% of aspartimide, a four-fold reduction of imide formation compared to
the direct HF treatment. Similarly, lowhigh treatment of cyclohexyl peptide
13 gave only 2.8% of aspartimide formation. The cyclohexyl ester would
appear to be the protecting group of
choice if base catalyzed aspartimide
formation occurring during repetitive
trialkylamine treatments during synthesis is considered.
The second major part of our study
addresses the mechanistic aspect of the
acid catalyzed aspartimide formation.
The acid-rate profile of the aspartimide
formation (Figure 10) shows an upward break in aspartimide formation
when the HF concentration is approximately 75%. A break in product
formation usually indicates a change of
mechanism. That is clearly the case
when the similar acid-rate profile is
plotted for the dehydration side reaction of the glutamyl peptide. At HF
concentrations lower than 75%, the
dehydration reaction of the glutamyl
peptides increases slowly with acid
concentration and, thus, is AAc2. At
concentrations greater than 75% HF,
Vol. I, No. 1 (1988)
the rate of byproduct formation increases more sharply with the increased acid concentration, indicating
an AAcl mechanism. More importantly, the acylium ion intermediate of the
glutamyl residue has been identified in
this and other studies.
In analogy to the glutamic acid-rate
profile, the aspartimide formation in
low HF concentrations is expected to
be AAc2. As the acid concentration increases, the rate of byproduct formation changes at HF >75%. However, it
is not clear whether there is a change of
mechanism from AAc2 to AAC 1. Contrary to the case with glutamic acid, the
corresponding aspartyl acylium ion has
never been identified. Further, data obtained from other studies do not support the existence of an acylium ion intermediate ( 40). For example, Olah
(29) found that: ( l) a- or P-amino
acids, including aspartic acid, did not
give acylium ions in much stronger
acids than HF; for example, super acids
at 45° C. Under the same conditions yamino acids, such as glutamic acid, exhibited acylium ion production. 2)
Aspartimide formation is sequence dependent, while side reactions of
glutamyl peptides do not appear to be
sequence dependent. (3) Side reactions
of glutamyl peptides occur at slower
rates than the cyclization to aspartimide. These data argue against
acylium ion formation from aspartic
acid. The difference may be caused
by the greater charge separation of
the aspartyl acylium ion (+NH3CH(COOH)CH2CO+) as compared to
(+NH3CH(COOH)CH2CH2CO+) (29,
30). Since our model tetrapeptide contains both the a- and P-amino acid
linkage of aspartyl sequence, we conclude that acylium ion formation is not
likely to occur. To clarify the situation,
we reacted the [3-aspartyl peptide (4)
under conditions similar to other model
tetrapeptides (Table 5). It was anticipated that in HF, aspartimide could
only be formed by an AAc2 mechanism, in which either the a-benzyl ester
or the protonated a-benzyl ester a-carboxyl group was protonated. The formation of an acylium ion has not been
observed with a-carboxylic acids or
esters. Indeed, aspartimide was obtained from 14. These results, together
with literature data, appear to diminish
the likelihood of the AAc l mechanism
for aspartimide formation in strong
acid. An alternative explanation is
needed for the rate change of aspartimide formation in concentrated HF
solutions. A plausible explanation is
that aspartimide formation remains
AAc2 under these conditions, and the
rate change reflects the stability of the
dication 23 in the rate-determining step
Figure 12). Further, there is an increased tendency towards protonation
of the side chain carboxyl as the acidity
increases. Finally, it is clear from our
results that 0-protonation of the AspGly amide bond predominates over Nprotonation. Attack on the amide
nitrogen is only possible with the
Our results support the conclusion
the cyclohexyl ester is a suitable
protecting group for the synthesis of
Figure 12. Proposed AAc2 mechanism for aspartimide formation in strong acids.
Vol. 1. No. 1 (1988)
peptidcs containing aspartic acid. It
minimizes both base and acid catalyzed
aspartimide formation by either the
BAc2 or AAc2 mechanisms. From the
results of this work, the mechanism of
this side reaction is better understood
and can be better controlled. Furthermore. this study has provided insight
into the role of protecting groups in
aspartimide formation. It suggests a
protecting group strategy for peptide
synthesis which will minimize the formation of aspartimide.
This work was supported in part by
PHS grant DKOI260 and CA36544.
We thank Ms. Dolores Wilson and
Mrs. Rita Taylor for expert secretarial
We dedicate this paper to Professor
H. Yajima on the occasion of his retirement from Kyoto University.
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Address correspondence to:
James P. Tam
The Rockefeller Uni••ersity
1230 York Ave.
New York, NY 10021
Vol. 1, No. 1 (1988)