Anal. Chem. 1989, 61, 2631-2637
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Rota-Microspeciation of Aspartic Acid and Asparagine BBla Nosz&l* Department of Inorganic and Analytical Chemistry, L. Eotuos University, Muzeum krt. 4/B, Budapest H-1088, Hungary
Peter SBndor Central Research Institute of Chemistry, Pf. 17, Budapest H-1575, Hungary
Rotamer analysis and methods for the determlnation of microspecies concentration were combined. Relationships between the protonation constants of rotational isomers and bulk (macro- and micro-) constants were deduced. Populatlons for 9 rotamers of asparagine and 15 rotamers of aspartlc acld were estimated by uslng pH dependence of 'H and ''C NMR coupling constants. The -COOH and -CONH, groups proved to be practically equlvaient In the formation of rotamer populations. A close correlation was found between rotamer population data and other NMR parameters. Protonation constants for each rotational isomer were calculated, inciuding rota-microconstants for the acidic reglon of aspartlc acld, where 12 microforms coexlst, six of whlch are Isomers both from protonatlon and rotatlonai point of view. Such rota-microconstants could not be determlned previously, due to thelr overiapplng equllibrla.
INTRODUCTION Aspartic acid and asparagine exist in solution in several forms of protonation and intramolecular rotation. The isomeric products of protonation and rotation always occur in the presence of each other, due to their fast, continuous interconversion. These coexisting species produce composite analytical signals; however they act individually in specific biochemical processes. This is why the determination of their concentration, e.g. an appropriate type of speciation is important, but it needs indirect methods. Speciation aims a t determining not only the total concentration of a given component in the sample but also its distribution among different species. Microspeciation distinguishes between the different isomeric products of protonation (I 1, whereas rota-microspeciation, in addition, must specify the position of the flexible moieties of the molecule. A sine qua non of the above types of speciation is information on the equilibrium constants. Simple speciation uses thermodynamic macroconstants. Microspeciation is based upon microconstants (microscopic protonation or dissociation constants) or group constants (1,2), whereas rota-microspeciation requires rota-microconstants. This paper presents the determination of the rotameric and microscopic forms and the relevant equilibrium constants of the title compounds. These closely related amino acids are equally important in biochemical interactions and metal complexation processes ( 3 ) . Asparagine is one of the best possible examples to study protonation processes of different rotamers. I t takes up protons in well-separated steps, but its rotamer populations have not yet been fully elucidated ( 4 ) . The bulky, polar -CONH, side-chain does not associate with protons but can influence either the proton-binding ability of other groups or the population of the rotamers. The proton-binding equilibria of aspartic acid are more composite processes. Solutions of aspartic acid contain five 0003-2700/89/0361-2631$01.50/0
protonation microspecies and, thus, 15 rota-microspecies. For the two carboxylates of aspartic acid, which are of similar basicity, protonation constants have been published (5)but were derived only from empirical relationships. We therefore performed experiments to evaluate more reliable microconstants. Several valuable contributions have been made toward the characterization of aspartic acid rotamer populations in the basic and neutral pH region (6-10). However, no attempt has been made for the rotamer analysis in the most complicated acidic region, where 12 rota-microspecies coexist. We have also estimated the concentration of all these rota-microforms, including the six doubly isomeric (protonation and rotation) species.
EXPERIMENTAL SECTION Amino acids were of analytical reagent grade (banal, Hungary) and used without further purification. All the potentiometric titrations were done with a Radiometer pHM 64 digital research pH meter and ABU 12 automatic buret, attached to an Ingold 1040523059 combined electrode. In all solutions, ionic strength was kept constant at 2.0 mol dm-3 by NaC1. This was also the internal filling of the combined electrode. The titrant used was carbonate-freeNaOH in 0.1-2.0 mol dm-3 concentrations. Amino acids were dissolved in hydrochloric acid solutions. Concentrations were identical with those of the titrants. For pH calibration the following standard solutions were used 0.01 mol dm-3 HCl + 0.09 mol dmm3KCl (declared pH = 2.07); 0.05 mol dm-3potassium acid phthalate (pH = 4.008); 0.01 mol dm-3 Na2B407.10H20(pH = 9.180);0.025 mol dm-3 Na2C03+ 0.025 mol dm-3 NaHC03 (pH = 10.012). The temperature was kept at 25.0 f 0.1 " C by ultrathermostat. The NMR solvent was DzO. To obtain conand vertible pH and pD values by means of analytical qpH) , potentiometric amino acid titrations were also carried data ( l l ) all out in D,O. Most NMR spectra were run on a Varian XL-100/15 spectrometer operating at 100.1 MHz for 'H and 25.16 MHz for 13C. The proton spectra of the extremely acidic samples (pH = 0.0) were recorded on a Varian XL-400 instrument since the P-methylene protons proved to be magnetically equivalent at 100 MHz. Chemical shifts were measured relative to internal tertbutyl alcohol (1.22 ppm for 'H and 32.50 ppm for 13C). Accurate NMR data of the ABX type proton spectra were obtained by iterative calculations using the LAOCN3 program (12). The rms errors of the calculated "best fit" spectra were less than 0.03 Hz,and the probable errors for individual data points were better than 0.03 Hz. RESULTS AND DISCUSSION Protonation Equilibria of Asparagine and Aspartic Acid. Asparagine and aspartic acid have two and three binding sites for protons, respectively. Figures 1 and 2 show their major protonation pathways. Published values (13)for the separately occurring amino and carboxylate protonations of asparagine are log K1 = 8.79 and log K , = 2.09 at T = 293 and 1.0 M/dm3 ionic strength. Our analogous pH-metric stepwise macroconstants at 298 K and 2.0 M/dm3 ionic strength in H 2 0 were found to be log K1 = 8.73 0.02 and log K , = 2.02 f 0.04,where uncertainties are standard deviations.
*
0 1989 American Chemical Society
ANALYTICAL CHEMISTRY, VOL. 61, NO. 23, DECEMBER 1, 1989
2632
CONHZ
CCNH2
CCNH2
coo-
COO'
COCH
Am'
--K1
HAsn
L
Table I. Protonation Macroconstants for Amino Acids at 298 K and 2.0 mol dm-3 Ionic Strength"
log K i log K2 log K3
HZAsn*
T---
Figure 1. Major protonation processes of asparagine.
a
aspartic acid
asparagine
a-alanine
@-alanine
9.53 f 0.02 3.65 f 0.02 1.96 f 0.02
8.73 f 0.02 2.02 f 0.04
9.74 f 0.01 2.35 f 0.01
10.14 f 0.03 3.60 f 0.01
Uncertainties are standard deviations.
COOH
Table 11. Protonation Microconstants for Aspartic Acid Given by Edsall et al. (Column I) and Determined in This Work by Method 1 (111) and 2 (11)"
SH2 'H,N.
ccoFH2 H2N .CH
COO-
_KL,
y
CH
too.
y
y 2
*H,N
coo' ASP
C"2 'H3N. CH
- CH
COO' ASP*
Y
coo'
ASP^,^
y 2 ccc4
&
HASp.
I1
I11
log k B log kBA log k A B
2.4 3.7 2.0 3.2
2.37 3.63 1.98 3.24
3.59 2.02
reference
5, 3
this work
this work
log k A
coon
' H ~ N . CH
Asp>.
I
A5pN.B
+
2
"%A ~ p r p
H ~ A S ~ +
1
"For methods see text. Estimated uncertainties are around 0.05 log k units.
Figure 2. Major protonation processes of aspartic acid.
In the case of aspartic acid, K z and K , are composites of microconstants (14)
K , = kA + kB K,K, = kAkAB= kBkBA The superscripts A and B on microconstant k denote the functional group protonating in the given process, while the subscript (if any) indicates the group attached to the proton. Indices A and B denote a- and @-carboxylates,respectively. Two examples of microconstants are given below (see also Figure 2)
(3)
(4)
Several combined pH-metric-spectroscopic methods for the determination of microconstants have been discussed elsewhere (15-17). However, no such method could be used for aspartic acid, due to the similar spectroscopic character of the two carboxylates and the small number of intervening atoms between them. Edsall's early work (5)for the estimation of microconstants by using semiquantitative substituent effect data was based on pK values of esters and other derivatives. The utilization of these constants is only recommended with caution (3),since the size and the H-bonding characteristics of the ester group are obviously different from those of the carboxyl. To minimize the ambiguity of the aspartic acid microconstants, we used two further approaches: (1)Equations 1 and 2 show that in addition to K , and K,, at least one other independent piece of information is necessary in order to evaluate the microconstants. The NMR data of asparagine and aspartic acid in acidic medium are very similar. In fact, their rotamer populations are virtually the same (see Table III), indicating that the -CONH, group very closely mimics the -COOH. Thus, K , of asparagine can be considered a good approximation for kBA of aspartic acid. Microconstant kB is calculated from eq 2 as kB = K,K3/kBA. However, note that this treatment provides reliable values only for kB and kBA. The analogous microconstants for the minor
pathway of protonation (kA and kAB) cannot be determined accurately by this approach, due to the inevitable experimental errors in K , and kB, and the insignificant difference between them. (2) Aspartic acid can be regarded either as a &carboxyl derivative of a-alanine or as an a-carboxyl derivative of @alanine. In this respect we always denote the N-bound carbon a . When the aspartic acid molecule is "built up" in this way, the absolute value of basicity for both carboxylates changes, but the ratio remains equal to 18.1, as in the case of the two alanines. K , of eq 1can be divided into kA and kBaccordingly. Knowing both k A and kB, the other two microconstants kBA and kAB can be calculated from eq 2. The k A and kB microconstants derived in this way express the inherent basicity of the unmodified a- and @-carboxylates. Moreover, since they are calculated by using K,, their values reflect the interaction between the two carboxylates. The effect of protonation on the other group is also reflected in the microconstants (see kBA/kAand kAB/kBratios), by using the K3 macroconstant in the calculations. At present, this is the best approach for determination of aspartic acid microconstants. In an effort to obtain fully comparable data, we made pHmetric measurements to determine macroconstants for the four relevant amino acids a t 298 K and 2.0 mol/dm3 ionic strength. Table I summarizes the results. The data in Table I1 indicate that the microconstants for aspartic acid are essentially the same even when different source data and methods are used and despite differences in H-bonding ability of the functional groups in the auxiliary compounds. Protonation Equilibria of Asparagine Rotamers. Figure 3 shows the staggered rotameric forms of differently protonated asparagine species. The number of theoretically possible species is limited by two factors: (1) the eclipsed rotameric forms essentially do not exist; (2) the concentration of the "inverse" single-proton-containing species, in which the carboxylate holds the proton (-COOH), and the amino group is uncharged (-NH,), is negligible, since the basicity of the amino group is several orders of magnitude greater than that of the carboxylate. By use of the convention cited previously (7, 8) t and g designate the rotamers in which the bulkiest groups are in trans and gauche positions, respectively. In h rotamers, all three carbon-bound hydrogens as well as the three bulky substituents are adjacent. Subscripts N and A occur when
ANALYTICAL CHEMISTRY, VOL. 61, NO. 23, DECEMBER 1, 1989 CONH2
K, = coo-
cooKlt
t
,
7
[H2Asn+] [ HAsn] [ H+]
COOH
K2t tN
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,
7
Rotamer mole fractions are designated by using three symbols: F, the concentration of any rota-microspecies relative to the total asparagine concentration (CASn); f , the concentration of any rota-microspecies relative to the concentration of one of the macrospecies (Asn-, HAsn, or H,Asn+); and a, the concentration of a rotameric form, regardless of the protonation stage. (at,ag,and ah comprise d species in the given t, g, or h rotameric form.) For example, the mole fraction of g can be written as
%.A
Fg
= [g]/CAsn
(17)
Examples of partial mole fractions are as follows:
H
H
Klh
h
,
7
H
hN
’K 2 h
~N,A
- K - K Am-
Figure 3.
HAsn
Staggered rotamers of
Asn-, HAsn,
H2Asn’
and
H,Asn+.
the amino and a-carboxylate groups are protonated, respectively. Protonation constants of the individual rotational isomers can then be defined as follows: (5)
(7)
The bulk macroconstants are given as [HAsn] K, = [Asn-][H+]
By the introduction of F-type mole fractions not only for rotameric forms but also for macro- (or micro)species, the relation between the f - and F-type rotamer populations can be expressed. The example for Asn- and g is as follows: FAsn-
= [Asn-1/
CAsn
(24)
From eq 18, 12, and 24, we derive
F and @ values depend on the pH; however, f values are pH-independent. From eq 25, it is clear that fg and F, are equal when FAsn-= 1. This is the case for asparagine in very basic solutions. In general, rotameric f and F values become practically identical whenever a species in a given state of protonation becomes the overwhelmingly predominant species. In that case, some simplifications become possible. At very high pH, the concentrations of HAsn and H,Asn+ are negligible
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ANALYTICAL CHEMISTRY, VOL. 61, NO. 23, DECEMBER 1, 1989
Table 111. 'H NMR Data a n d Calculated Rotamer Populations for Asparagine a n d Aspartic Acid" obsd three-bond coupling values, Hz
chemical shifts, ppm compound
pH (pD)
A
B
X
13.1 7.4 0.0 13.2 7.2 3.87 3.19 0.0
3.691 4.109 4.271 3.493 3.914 4.042 4.127 4.358
3.849 4.151 4.305 3.815 3.985 4.088 4.155 4.395
4.767 5.226 5.613 4.730 5.105 5.179 5.231 5.654
asparagine aspartic acid
35AX
35BX
8.70 7.27 6.25 9.55 7.94 7.69 7.26 6.34
4.96 4.01 4.38 4.06 3.87 3.88 4.13 4.35
rotamer DoDulations. @ JG = 2.56, JT = 13.6 g h t g h
JG = 2.4, J T = 13.3 t
0.58 0.45 0.35 0.66 0.51 0.49 0.45 0.36
0.23 0.15 0.18 0.15 0.14 0.14 0.16 0.18
0.19 0.41 0.46 0.19 0.36 0.38 0.40 0.46
0.56 0.43 0.33 0.63 0.49 0.47 0.43 0.34
0.22 0.13 0.16 0.14 0.12 0.12 0.14 0.16
0.23 0.44 0.50 0.23 0.39 0.42 0.43 0.50
The most acidic spectra were recorded a t 400 MHz, all others a t 100 MHz.
When the media is very acidic, only the doubly protonated species is present CAsn(pH
