Download REVIEW Formation and Instability of o

ANALYTICALBIOCHEMISTRY
178,1-7
(1989)
REVIEW
Formation and Instability of o-Phthalaldehyde
Derivatives of Amino Acids
M. C. Garcia Alvarez-Coque,’
and C. Mongay Fernhdez
M. J. Medina Hernhdez,
Departamento
Facultad
de Q&mica
Anulitica,
de Q&mica,
Universidad
o-Phthalaldehyde
reacts
with
amino
acids in the
presence
of a thiol to give highly
fluorescent
l-alkylthio-2-alkyl-substituted
isoindoles.
However,
the instability
of the derivatives
limits the general
utility
of
the reaction.
Mechanistic
descriptions
of isoindole
formation
and degradation
proposed
in the last years,
which
permit
a better
understanding
of the factors
affecting
isoindole
stability,
are presented.
The use of
alternative
thiols and o-phthalaldehyde-like
reagents
is also reviewed.
0 1989 Academic
Press. Inc.
One of the most sensitive fluorogenic reagents available for the determination of amino acids is o-phthalaldehyde (OPA)’ when used in conjunction with high-performance liquid chromatography. The reagent has some
convenient properties: it rapidly forms fluorescent derivatives (h,, = 340 nm, h,, = 455 nm) at room temperature,
is nonfluorescent itself, and when present in excess does
not break down or react to form fluorescent by-products.
However, the derivatives formed are somewhat unstable
and this severely limits the general utility of the reaction.
The reaction of OPA with amino acids in the presence
of 2-mercaptoethanol at pH N 9.5 was first reported by
Roth in 1971 (1). Roth considered the possibility that
the same species was produced regardless of the nature
of the primary amine, as in the case of the ninhydrin
reaction. However, differences in quantum yields of the
amine derivatives suggested that the same fluorophore
was not obtained in each case.
In addition, the thiol compound was originally
thought to function simply as a reducing agent, but Si’ To whom correspondence
should be sent.
’ Abbreviations
used: OPA, o-phthalaldehyde;
NAC,
N-acetyl-Lcysteine;
OAB,
acetobenzaldehyde;
OBB,
o-benzoylbenzaldehyde;
NDA, naphthalene-2,3-dicarboxaldehyde.
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reserved.
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de Valencia,
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Valencia,
Spain
mons and Johnson showed that the thiol actually becomes part of the final product. Based on data collected
from proton NMR, ir and mass spectral analysis of several crystallized OPA-amine-thiol derivatives, these authors concluded that the fluorescent products were l-alkylthio-2-alkyl-substituted
isoindoles (I) (2-5). However, long reaction times (15 min), bulky alkyl thiols,
and low temperatures of crystallization (OOC)were used
to permit precipitation of the fluorescent derivatives and
these conditions are not typical of those normally used
in OPA-derivatization
of primary amines in HPLC applications. Normally, on-line HPLC procedures are carried out over a much shorter period of time (less than 1
min) and at or above room temperature. Therefore, it
is not necessarily valid to assume that the fluorescent
products, obtained under such vastly different conditions, are the same.
For this reason, Simpson et al. (6) performed gas chromatography-mass spectrometry analysis of methylene
chloride extracts of postcolumn OPA-derivatives of various aliphatic amines with 2-mercaptoethanol and ethanethiol. The study demonstrated that the fundamental
structure of the fluorophore (I) was consistently present
in all derivatives:
MECHANISM
FOR
ISOINDOLE
FORMATION
Simons and Johnson presented a mechanistic description for isoindole formation (5) in which OPA first reacts
with 2-mercaptoethanol and subsequently with the
amine (Scheme 1). The reaction sequence proceeds by
protonation of the imine intermediate (III), followed by
a “partial &l-like
intramolecular reaction” to give a
protonated isoindole (V). However, isoindole formation
GARCfA
ALVAREZ-COQUE
S-R’
+
WA
R’-SH
ET
AL.
S-R’
B
II
6
d
I?-NHz
SCHEME
nobenzylsulfide
3.
Alternate
(X) (7).
mechanism
for formation
of the a-alkylami-
1-W
a
S-R’
I
V
SCHEME
mons
1. Mechanism
and Johnson
for isoindole
formation
proposed
by Si-
(5).
is known to proceed preferentially
in alkaline solutions,
where formation of a protonated hemithioacetal
(IV) or
isoindole (V) would be unfavorable.
Sternson et al. proposed an alternative
pathway
(Scheme 2) (7), where the amine reacts with free OPA
to form a carbinolamine
(VIII) which then dehydrates
to give the highly reactive protonated imine (IX). This,
in turn, is rapidly attacked by the thiol anion to form an
a-alkylaminobenzylsulfide
(X). Intramolecular
nucleophilic attack by the resulting secondary amine on the
remaining
carbonyl group forms the isoindole ring to
give the intermediate
XI, which undergoes facile dehydration to yield the fluorescent isoindole (I).
Operationally
the derivatization
reaction is indeed
conducted by first combining
OPA with the thiol and
then adding the primary amine. Aqueous solutions of
OPA are in equilibrium
with the cyclic hydrate (VI) (a),
and in the presence of thiols a reversible pathway to the
VIII
-
2.
Mechanism
et al. (7).
6
- Hz0
I
SCHEME
Sternson
x
IX
XI
for
isoindole
formation
proposed
by
cyclic thiol addition product (VII) exists (5)) but neither
addition products VI or VII would be expected to be reactive toward a primary amine. In addition, if, as proposed by Simons and Johnson (5), the formation of X
proceeds by initial reaction of the hemithioacetal
(II)
with the primary amine to form the protonated imine
(XII) (Scheme 3), this would be followed by attack of
the thiol anion to provide the hemithioacetal
(XIII),
which will rapidly decompose in basic media (9,10), generating X. Further reaction would proceed as shown in
Scheme 2.
The plausibility
of the mechanism was further substantiated
by substituting
methyl-(o-formyl)benzoate
for OPA in the amine-thiol
condensation (7). Formation
of 1-alkylthio-2-alkylphthalimidine
supported the feasibility of thiocarbinolamine
(X) intermediacy
and the
likelihood
that cyclization is initiated by attack of the
amino moiety of X on an o-carbonyl. In the case of OPA,
such an attack would occur at an aldehydic carbon and
would be expected to proceed with ease. The final dehydration step (XI -W I) has been previously observed in
the synthesis of 1-aryl isoindoles from o-aminomethyl
benzophenones (11).
FACTORS AFFECTING
ISOINDOLE STABILITY
Early applications of the fluorogenic OPA reaction included the postcolumn
derivatization
of amino acids
separated by ion-exchange
chromatography
(12,13).
However, this approach must scrupulously avoid impurities in the reagents and mobile phase buffers, which
can contribute
to high background fluorescence (14).
Postcolumn schemes also result in some loss of both resolution and sensitivity due to mixing of the mobile phase
with diluent (1516).
More recently, precolumn derivatization
of primary
amines with the OPA-2-mercaptoethanol
reagent, followed by reverse-phase HPLC separation, has become
popular (14-18). The precolumn approach offers the advantages of improved detection limits, simplification
of
the chromatographic
system, and reduced analysis time.
Unfortunately,
precolumn OPA approaches present the
problem of derivative instability,
with glycine, alanine,
lysine, and ornithine derivatives being particularly
unstable (2,4,5,19-25). Thus, careful timing of the reaction
or even instrumental
automation
is required to ensure
acceptable analytical precision.
o-PHTHALALDEHYDE
DERIVATIVES
When isoindoles are generated using equimolar concentrations of all reactants (amine, OPA, and thiol), or
when the amine is present in excess, degradation slows
drastically. In contrast, addition of excess OPA subsequent to isoindole formation results in rapid degradation
(26,27).
Cooper et al. (28) indicated that when the OPA-2-mercaptoethanol amino acid derivatives were injected into
the HPLC column immediately after the reaction was
carried out, they were observed to be stable, including
the glycine, ornithine, and lysine isoindoles. Only the diacidic amino acids, aspartic and glutamic, were shown to
undergo a significant loss of fluorescence. The reason for
the enhanced stability may be the removal of excess
OPA from the derivatives during the chromatographic
process, or that immobilization of the OPA-2-mercaptoethanol amino acid derivatives on the reverse phase of
the HPLC column retards degradation.
Several studies on the degradation kinetics of isoindoles suggested that the process occurs via at least two
parallel routes, described by a linear dependence of the
observed rate constant, kobs,vs OPA concentration (27):
hbs
= h
+
MOPAl,
where b and kl are the apparent rate constants for the
uncatalyzed and OPA-catalyzed processes, respectively.
The OPA independent degradation pathway appears unimportant in the presence of a moderate excess of OPA.
The destabilization of OPA depends strongly on thiol
structure (26). Because $ is much less variable than kl ,
stability differences related to structural variations may
almost solely be due to inhibition of the OPA dependent
process (27).
In the presence of an excess of OPA, a nonlinear decrease in decay rate with increasing thiol concentration
was observed (27,29). This observation is contrary to
previous studies in which excess 2-mercaptoethanol was
reported to have no effect on derivative stability. In one
of these studies (30), however, no excess OPA was present when thiol concentration was varied, so that the primary destabilizing influence was absent. In the second
(26), the maximum thiol concentration examined was
too low. The observed supression of the rate of OPA decomposition at high thiol concentration is probably due
to the formation of one or more OPA-thiol derivatives
which decreases the free OPA concentration.
Several avenues are therefore available for maximizing isoindole stability when using precolumn OPA-derivatization; the control of thiol or OPA concentration,
and the use of an appropriate thiol. Use of a minimum
OPA concentration is the most obvious route for in situ
stabilization, Very large excesses should indeed be
avoided, but the need for rapid and quantitative deriva-
OF AMINO
3
ACIDS
tive formation imposes some limits, particularly in samples where widely varying substrate concentrations are
encountered. Thus, finally, the use of an appropriate
thiol seemsto be the best way to achieve stabilization.
EFFECT OF THIOL
STRUCTURE
ISOINDOLE
STABILITY
ON
Jacobs et al. (27) studied a series of 4-aminobutyric
acid derivatives and diverse thiols and observed that
stability clearly increases with increased branching
of the side-chain (Y to the thiol function (-C(CH&
> -CH(CH&
> -CH2C!HB > -CHB). This trend lends
credence to the assumption that steric bulk of the thiol
is a dominant factor in determining derivative stability.
Mercaptoethanol
has become more extensively utilized than other thiols for the OPA-derivatization
of
amino acids. Several researchers have investigated the
possibility of enhancing isoindole stability by varying
the structure of the thiol compound, but for some thiols
(dithiothreitol,
ethanethiol, and 2-methyl-2-propanethiol) the fluorescence intensity achieved is weaker than
that obtained with 2-mercaptoethanol, and others result
in nonfluorescent compounds (methylmercaptoacetate
and mercaptosuccinic acid) (20,22,31).
Simons and Johnson proposed ethanethiol as a substitute for 2-mercaptoethanol, due to the improved stability of ethanethiol-amino acid-derived isoindoles (4,22).
Recently Stobaugh et al. (32) checked the higher stability of these derivatives. However, ethanethiol is more
volatile than 2-mercaptoethanol and one of the major
disadvantages of the latter, its pervasive odor, is increased in ethanethiol.
Arylthiols (benzylmercaptan, thiophenol, and triphenylmethylmercaptan)
(33), which are less malodorous,
have also been investigated. However, benzylmercaptan
does not produce fluorescent products, thiophenol seems
to form a different derivative, and the formation of fluorescent isoindoles from the latter is too slow. Other thi01s proposed are 3-mercaptopropionic acid (34,35) and
thioglycerol(36), but neither has received extensive use.
Recently, N-acetyl-L-cysteine (NAC) has been substituted for 2-mercaptoethanol in the pre- and postcolumn
OPA-derivatization
of primary amines and amino acids
with good results. Its optical activity enables the separation of enantiomers (37-40). The specific fluorescence of
the corresponding OPA-NAC derivatives is nearly identical to that of the OPA-2-mercaptoethanol
products,
the reaction is rapid, and the NAC derivatives are more
stable than either 2-mercaptoethanol or ethanethiol derivatives (including the glycine isoindole). The sensitivity achieved in the analysis of secondary amines is also
improved, presumably because the fluorophore is more
stable to hypochlorite, which is used for the oxidative
cleavage of the imino linkage (41). NAC is commercially
4
available, inexpensive,
odor.
GARCiA
ALVAREZ-COQUE
ET AL.
and presents no objectionable
EFFECT OF AMINE STRUCTURE
The stability of OPA-derived isoindoles is known to
vary depending on the nature of the amine moiety. Nakamura et ~2. (30) reported qualitative data on the influence of primary amine structure on the stability of the
OPA-2mercaptoethanol
derivatives. Later, Stobaugh et
al. (26) evaluated quantitatively the kinetic stability of
several isoindoles. Examination of the isoindole structures and the degradation rate constants revealed slight
increases in stability as the N-substituents of the isoindole became larger and when the N-substituent was additionally substituted at C-10. Still greater stability was
afforded to the isoindole by the presence of a carboxylate
in the amine substrate, with the degree of stabilization
increasing as the carboxylate approaches C-10. Thus, (Yamino acids tend to form more stable isoindoles than
other amines.
Lindroth and Mopper (18) claimed that the stabilizing
effect of the carboxylate group of the a-amino acids was
due to an electron-donating effect on the isoindole at the
C-10 position. However, the results of Stobaugh et al.
(26), together with those of other workers (42,43) indicate that increased isoindole stability is predominantly
due to steric factors.
Jacobs et at. (27) examined the stability of isoindoles
where amine structure was varied systematically. The
introduction of branch points into the amine side chain
has a mashed stabilizing effect on the resulting isoindoles, the magnitude increasing as the branch point approaches the isoindole ring (-C(CH,),
> -CH(CH&
> -CH&HB > -CH3). Simple linear extension of the side
chain without branching results in a steady twofold factor per methylene decrease in decay rate (-CH2CH2CH&H3 > -CH&H&H3
> -CH&Ha > -CHB).
The presence of the bulky groups seemed to stabilize
isoindole fluorophores, but in some cases it decreased
the rate of reaction (t-butylamine or Ly-methyl-L-tyrosine with 2-mercaptoethanol) or the fluorescence response (t-butylamine or cyclooctylamine with 2-mercaptoethanol) (30).
Cysteine and lysine form derivatives much less fluorescent than most of the other amino acids. The low fluorescence yield with cysteine is due to its sulfhydryl
group, which competes intramolecularly with P-mercaptoethanol for position 1 in the isoindole (44). Cysteine
can best be determined if previously converted to cysteic
acid, S-3-sulfopropylcysteine
or S-carboxymethylcysteine. The quenching with lysine derivatives is apparently
due to an interaction between the two isoindole groups
since fluorescence enhancement can be achieved in the
presence of a detergent such as sodium dodecylsulfate,
which favors separation of the isoindole groups (20,45).
I
‘I N-R+ kCH,ui~-S-~
cl2
XVI
SCHEME 4. Degradation mechanism for isoindoles obtained from
P-mercaptoethanol, suggested by Simons and Johnson (5).
MECHANISM
FOR ISOINDOLE DEGRADATION
Simons and Johnson characterized the degradation
products of isoindoles derived from 2-mercaptoethanol
as N-alkylphthalimidines
(XVI) (2) and pointed out two
possibilities for their formation: acid hydrolysis (4,22)
(decay of fluorophores is accelerated in aqueous solutions at low pH) and intramolecular nucleophilic attack
by the hydroxyl group of 2-mercaptoethanol (Scheme
4) (5).
The reduced rate of decay of the 2-mercaptoethanol
derivatives in borate buffer, which is assumed to complex with hydroxyl groups, compared with phosphate
buffer, supports the involvement of the hydroxyl group
in the isoindole decay (22). Furthermore, ethanethiol
forms more stable derivatives than 2-mercaptoethanol.
The identity of the degradation product is consistent
with the scheme proposed by Stobaugh et al. (26). However, the degradation mechanism of Simons and Johnson does not account for the acceleration of isoindole
degradation by increasing OPA concentration.
Nakamura et al. (30) suggested that OPA destabilizes
the isoindole ring by acting either as a dienophile or a
nucleophile, but based on schemes supposing the formation of degradation products that differ from those previously identified. Furthermore, Stobaugh et al. (26) indicated that neither of these roles is likely: for OPA to
function as a dienophile toward the isoindole ring it
would be necessary to form a product in which the aromaticity of OPA is lost, an energetically unfavorable
process. On the other hand, the aldehyde groups of OPA
should exhibit electrophilic character and would not undergo nucleophilic attack on the isoindole ring system.
Unlike many other aromatic aldehydes, OPA undergoes extensive hydration to a mixture of cis and trans
cyclic 1,3-phthalandiols (VI), approximately 80% of
OPA existing in the hydrated form in aqueous solution
(8). The occurrence of such facile equilibria and the wellknown ability of thioethers to lend anchimeric assistance in displacement reactions (46), together with some
kinetic observations, led Stobaugh et al. to postulate an
alternative degradation mechanism (Scheme 5) (26).
o-PHTHALALDEHYDE
DERIVATIVES
OF
AMINO
ACIDS
OH
S-R’
+
//
la 1
S-R’
.N-R
via
-Hz0
I
+ 0;.
-R
4-R ‘+a
S-R’
/
I
Hz0
xxv
XXIV
SCHEME
proposed
6. Autoxidation
mechanism
by Stobaugh
et al. (48).
for
isoindole
N-R
d
degradation
XIX
XVI
SCHEME
6.
2-mercaptoethanol,
Degradation
mechanism
for isoindoles
suggested by Stobaugh
et al. (26).
obtained
from
In the absence of excess OPA, the isoindole (I) forms
a sulfonium ion (XVII) via path 1. Attack by water and/
or hydroxide may occur at the methylene positions of the
sulfonium ion to regenerate I or at C-l of the isoindole
ring system to yield XVI. In the presence of excess OPA,
the isoindole (I) may rapidly equilibrate with the hemithioacetal (XVIII)
via path 2. The enhanced leavinggroup capability offered by the phthalandiol group in
XVIII
(over that of the bare hydroxyl group in I) facilitates the formation of sulfonium ion (XVII),
resulting
in a higher steady-state concentration of this species and
therefore an enhanced rate of loss of isoindole (I).
The use of 3-mercapto-1-propanol in place of 2-mercaptoethanol in the derivatization reaction provides an
isoindole derivative of improved kinetic stability because its degradation would require the formation of a
four-membered ring sulfonium ion intermediate and this
is a slower process than the formation of the threemembered sulfonium.
This mechanism, like that of Simons and Johnson (5)
is applicable only to derivatives based on hydroxythiols
(like 2-mercaptoethanol). However, the capability of
OPA to accelerate isoindole degradation is also present
in other thiols. Jacobs et al. (27) suggested that a common rate limiting process should exist, regardless of parent thiol, and since the chemistry of isoindoles is dominated by electrophilic substitutions (47), instability
must depend on nucleophilic addition of the isoindole to
unreacted OPA. However, the authors did not assign a
definite mechanism.
Stobaugh et al. observed that 1-alkylthio-2-alkyl-substituted isoindoles apparently react similarly to other
non-thio-substituted
isoindoles with respect to autoxidation (32,48), with isoindoles derived from hydroxythi01s (where degradation occurs by nonoxidative processes) being a specific case. The authors isolated and
identified four degradation products (XXIII,
XXIV,
XXV, and XXVII),
where the thio-substituted N-alkylphthalimidine (XXV) is the major product. An autoxidation mechanism that accounts for three of the observed degradation products was also proposed (Scheme
6). The extensively delocalized cation radical (XX) is
formed by electron transfer to oxygen or other radical
species present, thus initiating a chain process. Subsequently, the radical XX reacts with oxygen to form a key
intermediate, the isoindoyl peroxy radical (XXI), which
can further react by pathways previously postulated for
isoindoles, including endoperoxide formation (XXII) (49).
The nonoxidative degradation product (XXVII)
may
be derived from reaction of thiyl radicals (produced during the oxidative process described in Scheme 5) with
intact parent isoindole, via a homolytic substitution process through intermediate XXVI (Scheme 7).
NEW
OPA-LIKE
REAGENTS
Other reagents have been proposed as substitutes for
OPA. The minimal structural requirement necessary for
SCHEME
from
7.
isoindoles
Formation
(49).
of a nonoxidative
degradation
product
6
GARCiA
ALVAREZ-COQUE
a reagent to condense with a primary amine and a thiol
to form an isoindole is the presence of an o-diacylbenzene grouping in which at least one of the carbonyl
groups is aldehydic. o-Ketobenzaldehydes
such as acetobenzaldehyde (OAB) and o-benzoylbenzaldehyde
(OBB)
undergo such condensation
reactions to yield 1,2,3-trisubstituted
isoindoles, which are more resistant
to autoxidation than OPA isoindoles (7).
Isoindoles derived from OBB are significantly
more
stable than those from OAB, apparently due to a combination of steric, electronic and resonance effects exerted
by the 3-phenyl-substituent
(in OBB). The isoindole
formation rate observed for reaction of OBB was much
slower than that exhibited for OPA or OAB under the
same conditions, however. This may be explained by the
steric effect of the phenyl group, which reduces the opportunity
for nucleophilic
attack
by the secondary
amine of the a-alkyl-aminobenzyl
sulfide (X, Scheme 2),
and by resonance stabilization
of the reactant. Thus, although the isoindoles produced from OBB are considerably more stable than from OAB, their slow rate of isoindole formation from OBB makes this reagent less desirable than OAB.
Since, in general, the isoindole ring system readily undergoes autoxidation
(50) and/or electrophilic
attack at
positions
1 and 3 (51) by species such as the aldehyde
component of the OPA reagent, substitution
of thiol by
other stronger nucleophilic
species may produce a significant stability enhancement.
For this purpose, NY,
SCN-, HSO,, and CN- were studied (52), but with alanine serving as the primary amine, only HSO; and CNwere observed to give a fluorescent product. Compared
to 2-mercaptoethanol,
cyanide provided greater stability, but gave a somewhat
reduced fluorescence
with
OPA. Substitution
of naphthalene-2,3dicarboxaldehyde (NDA) in the derivatization
of amino acids with
CN- gave N-substituted
l-cyanobenz[flisoindole
derivatives, which were much more fluorescent than the OPA2-mercaptoethanol
derivatives
(52). In contrast,
NDA
and 2-mercaptoethanol
produced unstable and weakly
fluorescent derivatives.
ET
AL.
5. Simons,
S. S., Jr., and Johnson,
6. Simpson,
R. C., Spriggle,
M. J. Medina
Hernindez
thanks the “Conselleria
de Cultura,
cacio i Ciincia
de la Generalitat
Valenciana”
for the grant which
possible her collaboration
in this work.
Edumade
J. E., and Veening,
J. Chroma-
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L. A., Stobaugh,
J. F., and
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A. J. (1985)
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ACKNOWLEDGMENT
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