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Proton affinity of dipeptides containing alanine and diaminobutyric acid
International Journal of Mass Spectrometry 378 (2015) 151–159
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International Journal of Mass Spectrometry journal homepage: www.elsevier.com/locate/ijms
Proton affinity of dipeptides containing alanine and diaminobutyric acid$ Patrick Batoon, Jianhua Ren * Department of Chemistry, University of the Pacific, 3601 Pacific Avenue, Stockton, CA 95211, USA
A R T I C L E I N F O
A B S T R A C T
Article history: Received 1 May 2014 Received in revised form 11 July 2014 Accepted 14 July 2014 Available online 18 July 2014
Thermochemical properties, including the proton affinity, the gas-phase basicity, and the protonation entropy, were determined for the acetylated unnatural amino acid, 2,3-diaminobutyric acid, AcDab and two isomeric dipeptides containing alanine (Ala) and Dab AcAlaDab and AcDabAla. The experiments were carried out using a triple quadrupole mass spectrometer. The extended Cooks kinetic method was applied to determine the values of the proton affinity. The proton affinities for the three compounds were determined to be 235.1 2.0 kcal/mol (AcDab), 244.7 2.0 kcal/mol (AcAlaDab), and 242.0 2.0 kcal/mol (AcDabAla). The gas-phase basicities (GB) and the protonation entropies (DpS) for these peptides were determined accordingly. The results suggested that the dipeptides were more basic than the single amino acid by about 7 kcal/mol, and the C-terminal Dab peptide, AcAlaDab was more basic than the N-terminal Dab peptide, AcDabAla by about 2.5 kcal/mol. Computational studies were carried out via a series of steps including conformational search using the MMFF force field, followed by geometry optimization and energy calculations at the B3LYP/6-311++G(2d,p)//B3LYP/6-311+G(d) level of theory. Theoretically predicted proton affinities were in reasonably good agreement with the experiments. The higher basicity of the C-Dab dipeptide was likely due conformational effect that stabilized the charged C-Dab peptide more efficiently than the N-Dab peptide. ã 2014 Elsevier B.V. All rights reserved.
Keywords: Gas-phase basicity Unnatural amino acid Dab Lysine homologues Kinetic method
1. Introduction Proteins, as the principal mechanical machinery of nature, can range from peptides such as Ghrelin, used as a signaling molecule for the stimulation of hunger [1], to large and highly ordered protein complexes such as DNA polymerase to carry out the synthesis of DNA [2]. Many studies have been carried out to understand and determine the structures, functions, and roles of large proteins and peptides at the biological level [3–5], but the fact that still remains is the lack of fundamental knowledge on the chemical and physical properties of smaller peptides resembling the active sites of proteins. For many proteins, acidic and basic residues can be found buried deep within the protein’s structure, implicating an important catalytic or structural function [6]. Additionally, it has been found that some of these buried residues exhibit drastic shifts in pKa values caused by a variety of factors such as charge–charge
$ This article is dedicated to Professor Veronica Bierbaum for her great contributions in the fundamentals of gas-phase ion chemistry. * Corresponding author. Tel.: +1 209 946 2393; fax: +1 209 946 2607. E-mail address: jren@pacific.edu (J. Ren).
http://dx.doi.org/10.1016/j.ijms.2014.07.025 1387-3806/ ã 2014 Elsevier B.V. All rights reserved.
interactions, charge–dipole interactions, and desolvation (the Born effect) [7]. In general, the most common buried residues that have ionizable side chains are cysteine (Cys), histidine (His), and tyrosine (Tyr), because they are intrinsically uncharged at pH 7 [8]. However, by simply placing any highly acidic or basic residue within a buried location of a protein, the residue could exhibit a pKa shift [9]. One such example of a buried residue is lysine (Lys) 115 in the protein acetoacetate decarboxylase. Positioned within the hydrophobic interior of th e protein, Lys 115 was found to exhibit an unusually perturbed pKa value of 7.5; whereas the normal pKa of Lys was 10.5. The reduced pKa value of Lys 115 allowed the residue to retain its nucleophilic property as a neutral residue and readily forms a Schiff-base intermediate with an incoming substrate at physiological pH [10]. The exact factors of stabilization or destabilization of buried ionizable residues were extremely difficult to establish. The perturbed pKa values were highly conformation dependent. When attempted to measure the pKa values of buried residues by titration, the results obtained were largely from solvated residues in the unfolded proteins [11]. This problem has become considerably complex in the study of model peptides mimicking buried sites. Due to the size and solvent accessibility of small peptides, the “buried” conformation was difficult to emulate in solution, and
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the results might not reflect the pKa perturbation of a buried residue. To solve this problem, one may study the model peptides in the gas-phase. Studies have shown that solution-phase conformations of proteins could be retained in the gas-phase under mass spectrometry conditions [12]. Extensive mass spectrometric studies on peptides and single amino acids have been carried out by establishing proton affinities to gain better understanding of the molecule’s intrinsic ability of stabilizing a protonation site [13–23]. Additionally, there have been a number of studies on peptides and single amino acids that involved measuring the gas-phase acidity [24–33]. Our previous studies have demonstrated that conformations could significantly influence the gas-phase acidity of oligopeptides. For the series of cysteine (Cys)-containing peptides, the N-Cys peptides were significantly more acidic than the C-Cys peptides [34–36]. Computational studies suggested that the deprotonated N-Cys peptides with four residues and longer existed in a helical conformation, while the deprotonated C-Cys peptides preferred a globular conformation [35,36]. The stronger acidity of the N-Cys peptides was mainly due to the favored interaction with the helix macro-dipole that stabilized the N-terminal thiolate anion. In the case of di- and tri-peptides that were too short to form an a-helix, the greater acidity of the N-Cys peptides was likely due to the more compact conformation of the deprotonated N-Cys peptides in which the thiolate anions were stabilized via efficient intramolecular hydrogen-bonding interactions [34]. We have begun to systemically investigate conformational effects on the proton affinity of peptides by using a series of model oligopeptides containing a basic probe. One such a probe was diaminobutyric acid (Dab). Dab was chosen as a basic probe due to its homologous nature to the natural amino acid lysine (PA = 238.0 kcal/mol) [37], but with lower basicity (PA = 233.2 kcal/mol) [21]. The weaker basic Dab allowed a larger variety of molecules to be used as reference bases to determine the proton affinity of Dab-containing peptides. Although considered an “unnatural” amino acid, Dab can be found as the free amino-acid in the legume genus Lathyrus and has been shown to exhibit neurotoxic effects by inhibiting the urea cycle, inducing chronic ammonia toxicity through the blood that elevated glutamate levels in the brain [38]. The two N-oxalic acid derivatives of Dab (2-amino-4[(carboxycarbonyl)amino]butanoic acid and 4-amino-2-[(carboxycarbonyl)amino]butanoic acid) can also be found in Lathyrus and have proven to be highly toxic though different metabolic pathways [39]. In this paper, we report the determination of the proton affinity and related thermochemical properties of acetylated Dab (AcDab) and two isomeric Dab-alanine dipeptides, AcAlaDab and AcDabAla.
[(Schem_1)TD$FIG]
The structures of AcDab and the two peptides are shown in Scheme 1. We calculated the theoretical proton affinities and the conformations of these molecules. 2. Experimental 2.1. Peptide synthesis All peptides were synthesized using standard solid phase peptide synthesis (SPPS) [40]. The experiments were carried out in a manual synthesis apparatus comprised of disposable 10 mL peptide synthesis vessels (Polypropylene reaction vessel, Torviq, Niles, MI) mounted onto a mechanical agitator (Model 75 WristAction Shaker, Burrel Scientific, Pittsburgh, PA). Wang resin was used as the solid support to yield the carboxyl group at the Cterminus. The general procedure can be described as the following. Wang resin (0.33 mmol, loading capacity 1.1 mmol/g) was placed in the peptide synthesis vessel. Dichloromethane was passed through the vessel to swell the resin. Coupling of the first amino acid was done by adding a 3-times excess mole ratio of a Fmoc-amino acid and O-benzotriazole-N,N,N0 ,N0 -tetramethyluronium-hexafluoro-phosphate (HBTU) and six times excess N, N-diisopropylethylamine (DIPEA) with 4 mL dimethylformamide (DMF). The solution was capped under nitrogen gas, and then shaken for 5 h. Deprotection of the coupled resin was carried out using 4 mL of 20% piperidine in DMF for 5 min, drained, and repeated for an additional two times. Subsequent coupling (2–3 h)–deprotection cycles were carried. When the last amino acid was coupled and deprotected, the peptide was acetylated by mixing a 5 mL solution of 20% DIPEA and 20% acetic anhydride in DMF. Cleavage of the peptide was carried out by incubating the resin in 5 mL of “Reagent B” cleavage cocktail (88% TFA, 5% phenol, 5% H2O, and 2% triisopropylsilane (TIPS reagent)) for 2–3 h. The flow-through was collected and then concentrated under nitrogen gas until the volume was less than 1 mL. A mixture of diethyl ether and hexane was added to the concentrate and precipitated in 4 C for at least 1 h. The precipitate was pelleted at 3500 rpm for 20 min using a centrifuge (HERMLE Labnet Z206A). Solvent was decanted, and the peptide pellet was dissolved in 5–10 mL of Milli-Q water. The solution was passed through a CHROMAFIL Xtra CA-20/ 25 syringe filter and lyophilized overnight to obtain a solid form of the peptide. The yielded peptide was sufficiently pure for mass spectrometry measurements. The sequence of the peptide was confirmed by the MS/MS sequence analysis technique. All synthesis reagents, including the Wang resin, Fmoc-amino acids, and coupling reagents were purchased from Chem-Impex International Inc. (Wood Dale, IL), and were used without further purification. 2.2. Mass spectrometry measurements
NH2 O H3C
OH
N H
O
AcDab NH2
NH2 H N
H3C O
O
O
CH3
N H
OH O
H3C
N H
H N O
AcDabAla
AcAlaDab Scheme 1.
O OH CH3
All mass spectrometry experiments were performed using a triple quadrupole mass spectrometer interfaced to an electrospray ionization (ESI) source (Varian 320L, Agilent Technologies, Santa Clara, CA). The sample solution was prepared by dissolving the peptide and a reference base into a 1:1 (v:v) solution of methanol: water to achieve a final concentration of about 104–105 M. The sample solution was directly infused into the ESI source at a flow rate of 10 mL/min. Data acquisition was controlled by the MS Workstation software package (version 6.9) in the positive ion mode. The ESI needle voltage was maintained at 4.2–4.8 kV. The capillary voltage was set between 25 and 50 V. The drying gas pressure was set to 14 psi, and the temperature was set between 150 and 250 C, respectively. The nebulizing gas was set between 35 and 50 psi. Nitrogen was used as the nebulizing and drying gas. The hexapole
P. Batoon, J. Ren / International Journal of Mass Spectrometry 378 (2015) 151–159
[(Schem_2)TD$FIG]
ion guide chamber operated at a pressure of about 1 mTorr nitrogen gas. The capillary voltage and drying gas temperature were adjusted within the specified range to maximize the signal of the proton-bound dimer. The ions generated in the ESI source were presumed to be thermalized by multiple collisions with the nitrogen molecules in the ion guide chamber. CID experiments were performed by isolating the proton-bound dimer with the first quadrupole [43_TD$IF](MS1) with a peak width of 1.2 (instrument parameter). The isolated ion was allowed to undergo collisions with argon atoms leaked into the collision chamber which was held at a pressure of 0.4 mTorr. The product ions were analyzed with the third quadrupole [4_TD$IF](MS2) with a peak width of 1.0 and a dwell time of 0.2 ms. Product ion spectra of the proton-bound dimer were recorded at several collision energies to determine all possible product ions representing the protonated peptide or the protonated reference. Product ion intensities were then measured in selected reaction monitoring (SRM) mode for ions representing the protonated peptide or the protonated reference base. The SRM acquisition was carried out continuously for 2 min and repeated two times in one day. Multiple measurements were carried out on different days with a relative uncertainty of around 5%. All CID experiments were performed at four different collision energies corresponding to the center-of-mass energies (Ecm) of 1.0, 1.5, 2.0, and 2.5 eV, respectively. The center-of-mass energies were calculated using the equation Ecm = Elab[m/(M + m)], where Elab is the laboratory frame collision energy, m is the mass of argon, and M is the mass of the proton-bound dimer ion. 2.3. CID bracketing experiments Bracketing experiments were carried out by isolating the proton-bound dimer in Q1 and obtaining the product ion mass spectra in Q3 at 1.0, 1.5, 2.0, and 2.5 eV collision energies in the center of mass frame (Ecm) for 30 s. The collision gas (argon) pressure was set to 0.4 mTorr to retain at least 10% of the protonbound dimer precursor ion at the highest collision energy. Bracketing experiments primarily yielded two product ions representing the protonated peptide and the protonated reference base, respectively. The product ion with the higher peak intensity was determined to have the higher proton affinity. Bracketing experiments were performed with all given reference bases and made it possible to qualitatively estimate the proton affinities of the peptides. 2.4. Extended Cooks kinetic method The gas-phase basicities and proton affinities of AcDab, AcADab and AcDabA were determined using the extended Cooks kinetic method [20,41–43]. The gas-phase basicity (GB) is the Gibbs free energy change for the reaction, MH+(g) ! M(g) + H+(g), typically at 298 K. The proton affinity (PA) and the protonation entropy (DpS) are the enthalpy and entropy changes for the reaction, respectively. All three thermodynamic values are related by the fundamental Gibbs free energy equation (Eq. (1)): GBPep ¼ PAPep T Dp SPep (1) Thermochemical determinations are carried out by isolating a proton-bound dimer between the peptide (Pep) and a reference base (B), [PepHB]+, into the collision cell of the mass spectrometer. The proton-bound dimer is accelerated through the chamber pressurized with argon at about 0.4 mTorr and undergoes collision induced dissociation. The expected product ions represent the two competitive dissociation pathways of the proton-bound dimer that yields the protonated peptide (PepH+) and the protonated reference base (BH+), shown in Scheme 2.
153
[17_TD$IF]Scheme 2.
The ion abundance ratio [PepH+]/[BH+] is presumed to represent the rate constant ratio kP/kB. The natural logarithm of the rate constant ratio is a function of the free energies of activation of these reactions and is related by a linear equation (Eq. (2)), where R is the ideal gas constant and Teff is the “effective temperature” of the activated proton-bound dimer. The effective temperature is an empirical parameter that depends on several experimental variables and properties of the proton-bound dimers [44–49]. The equation can be transformed to include the relative enthalpy and entropy of activation (Eq. (2)). Assuming no reverse activation barriers, the relative free energy and enthalpy of activation becomes equal to the relative GB and PA between the peptide and the reference base, respectively, (Eq. (3)). Similarly, the relative activation entropy D(DSz) becomes equal to difference in protonation entropy D(DS) (Eq. (4)). If the reference bases used are structurally similar, the assumption can be made that D(DS) will remain constant between the different dissociation pathways for dimers of different reference bases. In addition, the average protonation entropy of all reference bases, DpSAvg can be used to replace the term, DpSB. After all amendments to Eq. (2) have been made, Eq. (2) is transformed into Eq. (5). DGzb DGzP DHzB DGzP DSz kP ln ¼ þ ¼ kB RTeff RTeff R
(2)
DHzB DHzP ¼ PAPep PAB
(3)
D DSz D DS ¼ Dp SPep Dp SB
(4)
PepHþ PAPep PAB D DS ¼ þ R RTeff BHþ
(5)
ln
In order to achieve proper statistical treatment of the uncertainty, the average proton affinity of all reference bases PAAvg is introduced into Eq. (5) and this yielded Eq. (6) [50,51]. After rearrangement, Eq. (6) can be transformed into a functional linear equation (Eq. (7)). PepHþ D DS PAPep PAAvg PAB PAAvg (6) ln þ ¼ R RTeff RTeff BH PepHþ PAPep PAAvg D DS 1 ln þ ¼ PAB PAAvg þ RTeff R RTeff BH
(7)
The proton affinity values are extracted in two steps. First, a plot of the ln ([PepH]+/[BH]+) vs PAB PAAvg is made. The slope [45_TD$IF]represents 1/RTeff and the y-intercept represents [(PAPep PAAvg)/RTeff D(DS)/R]. If dissociation of [PepHB]+ is carried out at different collision energies, a set of different slopes and y-intercepts can be obtained. Using this set of data, a plot of [(PAPep PAAvg)/RTeff D(DS)/R] vs 1/RTeff is made. The slope of the plot represents PAPep PAAvg, and the y-intercept represents D(DS)/R. Because PAAvg and R are known values, PAPep and D(DS) can be obtained. The value of DpSPep can be determined using the equation D(DS) = DpSPep DpSAvg. The gas-phase basicity of the
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Table 1 Thermochemical values for the reference bases[7_TD$IF]. Reference [15_TD$IF]base
Abbreviation
PAa [8_TD$IF]kcal/mol
GBa kcal/mol
DpSb cal/mol/K
[9_TD$IF]AcDab Asparagine Glutamine Piperidine 1,3-Propanediamine 1,6-Hexanediamine 1,5-Pentanediamine Average
Asn Gln Pip 1,3DAP 1,6DAHx CAD
222.0 224.0 228.0 235.9 238.9 238.9 231.3
213.1 215.0 220.0 224.7 226.1 226.1 220.8
29.9 30.2 26.8 37.6 43.0 43.0 35.1
[10_TD$IF]AcAlaDab/AcDabAla Histidine 1,7-Heptanediamine 1,6-Hexanediamine 1,5-Pentanediamine 1,4-Butanediamine N,N-Dimethyl-1,3-propanediamine Average
His 1,7DAHp 1,6DAHx CAD 1,4BDA DMPDA
238.0 238.6 238.9 238.9 240.3 245.0 240.0
227.1 225.8 226.1 226.1 228.1 233.1 227.7
36.6 43.0 43.0 43.0 41.1 39.9 41.1
a
Obtained from the most recent data compiled in the NIST Chemistry WebBook [37]. Derived from the relationship [18_TD$IF]DG = DH–T(DS), where T = 298 K. It is assumed that each PA and GB value has an uncertainty of [1_TD$IF]2.0 kcal/mol K, and each D [12_TD$IF] pS value has an uncertainty of 2.0 cal/mol K. b
peptides, GBPep, 298 K can be determined using the Gibbs free energy relation (Eq. (1)). The uncertainties of the linear regression were calculated using orthogonal distance regression (ODR) using the ODRPACK suite of programs [52]. The uncertainty of the average proton affinity (PAAvg) was calculated as the root sum square of the random and systematic errors. We assume that the proton affinity of each reference base has an uncertainty of (2.0 kcal/mol). For a set of six reference bases, the random error was treated as the averaged uncertainty of the reference bases (2.0 kcal/mol) divided by the square root of the number of the reference acids, p (2.0/ 6) = 0.82 kcal/mol, and the systematic error was assigned as p 2.0 = 1.4 kcal/mol. The root sum square of the random and p systematic errors yielded (0.822 + 1.42) = 1.6 kcal/mol. 2.5. Computational method The input structures of the peptides were constructed using the Spartan’11 suite of programs (Wavefunction, Irvine, CA) for which the site of proton attachment was chosen to be on the basic sidechain of the peptide. Merck molecular force field (MMFF) conformational search calculations were carried out using the same software suite. The 200 lowest energy conformers were saved, and AM1 level geometry optimization immediately proceeded. Of the pool of AM1 optimized conformers, the 20 lowest energy structures were subjected to HF/3-21G(d) optimization and frequency calculations to obtain a more accurate energy ladder. From which the 10 lowest energy conformations were further subjected to geometry optimization and frequency calculations at the B3LYP/6-311+G(d) level of theory. Single point electronic energies were obtained at B3LYP/6-311++G(2d,p) level using the geometries calculated at the B3LYP/6-311+G(d) level. The calculated enthalpy at 298 K was determined using the sum of the electronic energy (e0) and the thermal correction to enthalpy (DHcorr). All quantum level calculations (HF, B3LYP) were carried out using the Gaussian’09 software suite [53]. Proton affinity values were calculated by an isodesmic reaction scheme with ethylamine (PA = 217.97 kcal/mol) as the reference base (Eq. (8)). In order to evaluate the effect of different basis sets, the lowest energy conformations of the neutral and the protonated peptides were further optimized and calculated at the B3LYP/6-311++G(d,p) level. The resulting energetics were used to calculate the proton affinity using Eq. (8).
PepHþ þ Bethylamine ! Pep þ Bethylamine Hþ
(8)
3. Results 3.1. Relative proton affinity of AcDab, AcAlaDab and AcDabAla The relative proton affinities of AcDab and the two peptides, AcAlaDab and AcDabAla were bracketed against a set of reference amine bases (B) with known values of the proton affinity (PA) and the gas-phase basicity (GB). The reference amine bases alone with their thermochemical properties are listed in Table 1. The relative proton affinity was estimated by comparing the ion intensities of the protonated peptide and the protonated reference amine base resulting from the dissociation of the proton-bound dimer, [AcDabHB]+, [AcAlaDabHB]+, or [AcDabAlaHB]+, under the CID conditions. The results suggested that the proton affinity of AcDab was between that of piperidine (Pip 228.0 kcal/mol) and 1,3propanediamine (1,3DAP, 235.9 kcal/mol), and the proton affinity of both peptides would be between that of 1,4-butanediamine (1,4BDA, 240.3 kcal/mol) and N,N-dimethyl-1,3-propanediamine (DMPDA, 245.0 kcal/mol). Four representative CID spectra are shown in Fig. 1. The relative ion abundances of the protoned peptides and protonated 1,4BDA clearly showed the trend of the proton affinity: AcAlaDab, AcDabAla > 1,4BDA. The fact that the ion abundance of 1,4BDAH+ was closer to that of AcDabAlaH+ than to that of AcAlaDabH+ implied that AcAlaDab had a higher proton affinity than AcDabAla. 3.2. Quantitative proton affinity of AcDab, AcAlaDab and AcDabAla The quantitative value of the proton affinity was determined based on the linear equation described in Section 2 (Eq. (7)). The natural logarithms of the CID product ion ratios (branching ratios) measured at four collision energies, 1.0, 1.5, 2.0, and 2.5 eV (Ecm) are shown in Table 2. All secondary fragment ions were counted into the intensities of the corresponding precursor ions and were included in the data analysis. The corresponding thermo–kinetic plots of the branching ratios at the four collision energies against the relative proton affinity of the reference bases, PAB PAAvg, are shown in Fig. 2a. Linear regression yielded four sets of slopes (X) and intercepts (Y), and the results are summarized in Table 3. Each slope represented the value of 1/RTeff at the specific collision energy. Each intercept represented the term [(PAPep PAAvg)/
P. Batoon, J. Ren / International Journal of Mass Spectrometry 378 (2015) 151–159
[(Fig._1)TD$IG]
155
Fig. 1. CID spectra obtained at 1.5 eV (Ecm) for the proton-bound heterodimer anions of [1_TD$IF](a1) [AcDabHPip]+, (a2) [AcDabH1,3DAP]+, (b) [AcAlaDabH1,4BDA]+, and (c) [AcDabAlaH1,4BDA]+.
RTeff D(DS)/R]. To derive the value of PAPep, the obtained intercepts (Y) and the slopes (X) were used to construct a new thermo–kinetic plot, shown in Fig. 2b. Linear regression of each plot yielded a slope of PAPep PAAvg and an intercept of D(DS)/R. The results for AcDab and for the two peptides are shown in Table 4. Combined with the known values of PAAvg (Table 1), the values of PAPep for AcDab and for both peptides were determined, shown in Table 5. The protonation entropy (DpSPep) was determined using the relation D(DS) = DpSPep DpSAvg, where DpSAvg (Table 1) represented an average entropy of the reference bases. The results are shown in Table 5. The gas-phase basicity (GBPep) of each of the compounds was derived using the thermodynamic relation shown in Eq. (1). The results are shown in Table 5. The uncertainties were calculated by weighted orthogonal distance regression (ODR) using the ODRPACK suite of programs [52]. Here we use the AcAlaDab system to illustrate the procedure. Table 2 Values of [13_TD$IF]ln ([PepH+]/[BH+]) measured at four collision energies (Ecm) with different references bases[14_TD$IF]. Peptide
Reference [15_TD$IF]base
1.0 eV
1.5 eV
2.0 eV
2.5 eV
AcDab
Asn [16_TD$IF]Gln [17_TD$IF]Pip [18_TD$IF]1,3DAP [19_TD$IF]1,6DAHx [20_TD$IF]CAD
5.994 2.053 1.191 -0.061 -1.049 -1.722
5.880 1.984 0.580 -0.724 -0.917 -1.623
5.750 1.851 0.204 -0.745 -0.776 -1.474
5.522 1.829 -0.063 -0.698 -0.627 -1.306
[21_TD$IF]AcAlaDab
His [2_TD$IF]1,7DAHp [23_TD$IF]1,6DAHx [24_TD$IF]Cad [25_TD$IF]1,4BDA [19_TD$IF]DMPDA
2.854 3.744 3.578 3.037 1.406 -2.725
2.341 3.654 3.474 2.802 1.241 -2.835
2.029 3.637 3.428 2.631 1.197 -2.840
1.689 3.627 3.446 2.437 1.222 -2.802
[120_TD$IF]AcDabAla
His [2_TD$IF]1,7DAHp [28_TD$IF]1,6DAHx [24_TD$IF]Cad [29_TD$IF]1,4BDA [30_TD$IF]DMPDA
1.702 3.486 2.837 1.998 0.305 -3.677
1.344 3.400 2.701 1.838 0.253 -3.677
1.121 3.409 2.604 1.798 0.347 -3.602
0.925 3.430 2.448 1.774 0.478 -3.434
For each of the plots of ln ([PepH]+/[BH]+) vs PAB PAAvg, the uncertainty used in all x-axis values was 2.0 kcal/mol, and in all yaxis values was 0.05. The uncertainties (with 95% confidence) in the slopes (X) and in the intercepts (Y) are shown in Table 3. The corresponding uncertainties were carried into the plot of Y vs X. The resulting uncertainty in the slope (PAPep PAAvg) was 0.940 and in the intercept (D(DS)/R) it was 0.826. Combined with the 1.6 kcal/mol uncertainty estimated for PAAvg, the uncertainty p for PAPep would be (0.942 + 1.62) = 1.9 kcal/mol. It should be pointed out that the uncertainties obtained from the kinetic measurements were relative values. They did not include the absolute error in the overall calibration of the proton affinity scale of the references. Considering about 2 kcal/mol uncertainties for the reference bases, we assigned an uncertainty of 2.0 kcal/mol for the measured proton affinity and the gas-phase basicity, and 2.0 cal/mol K for the protonation entropy, Table 5. 3.3. Computational results The conformations of the neutral and the protonated peptides were calculated via a series of steps including a conformational search followed by geometry optimizations and energy calculations at different levels of theory. From this the 10 lowest energy conformations were selected for further geometry and energy calculations at the B3LYP/6-311+G(d) level of theory. The relative contributions of the set of low energy conformations were calculated based on the Boltzmann distribution. The results are shown in the Supplementary Information. All peptide species, neutral and protonated, had three or fewer conformations with population greater than 5%. All structures with contributions more than 5% are shown in the Supplementary Information, and the lowest energy structures are shown in Fig. 3. The dashed lines and the numbers labeled on the structures indicated the distances between the corresponding atoms. These distances were used to estimate the hydrogen-bonding interactions. The set of the selected lowest energy conformations were subjected to single point energy calculation at the B3LYP/6-311++G(2d,p) level of theory, and the resulting energetic values were used to calculate the proton affinities of the peptides according to Eq. (8). The proton
156
[(Fig._2)TD$IG]
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Fig. 2. [2_TD$IF](a) Plots of ln ([PepH+]/[BH+]) against PAB [3_TD$IF] PAAvg (kcal/mol) from the dissociation of [Pep[4_TD$IF]HB]+ at four collision energies (Ecm), 1.0, 1.5, 2.0 and 2.5 eV. [5_TD$IF](b) Plots of (PAPep PAAvg)/RTeff D(DS)/R against 1/RTeff, where the [6_TD$IF]R2 values for the three systems are AcDab 0.89, AcAlaDab 0.92, and AcDabAla 0.74.
affinities were calculated using the lowest energy conformation (Table 5) as well as using the weighted average of the set of low energy conformations (Supplementary Information). The two sets of proton affinity values were comparable, and they were in good agreement with the experimental results. In order to evaluate the effect of different basis sets on the calculated proton affinity, the proton affinities were also calculated at B3LYP/6-311+G(d) and B3LYP/6-311++G(d,p) levels of theory. The results are shown in the Supplementary Information. Although the absolute values were different (by up to 3.8 kcal/mol) among
the results from the three levels, the relative proton affinity between the two peptides was about the same at all three levels. 4. Discussion The CID bracketing experiments suggested that the relative proton affinity followed the trend: AcDab < AcDabAla < AcAlaDab. By using the extended Cooks kinetic method, the proton affinities of the three compounds were determined to be 235.1 2.0 kcal/mol for AcDab, 244.7 2.0 kcal/mol for AcAlaDab
P. Batoon, J. Ren / International Journal of Mass Spectrometry 378 (2015) 151–159
157
Table 3 Results of linear regression according to [12_TD$IF]Eq. (X), the first set of the thermokinetic plots. [32_TD$IF]Ecm 1.0 eVb AcDab AcAlaDab AcDabAla
a b
[3_TD$IF]1/RTeff Ya [34_TD$IF]1/RTeff Ya [35_TD$IF]1/RTeff Ya
0.339 0.999 0.923 1.982 0.946 1.108
1.5 eVb
0.008 0.529 0.108 0.256 0.162 0.383
0.321 0.881 0.8973 1.779 0.913 0.976
2 eVb
0.008 0.584 0.136 0.321 0.176 0.415
0.303 0.827 0.873 1.680 0.884 0.946
2.5 eVb
0.009 0.620 0.154 0.363 0.186 0.439
0.283 0.802 0.842 1.603 0.841 0.937
0.009 0.628 0.176 0.416 0.193 0.456
Y = (PAPep [36_TD$IF] PABAvg)/RTeff D(DS)/R. The uncertainties were calculated by weighed orthogonal distance regression (ODR) using the ODRPAC suite of program.
Table 4 Results of linear regression according to [37_TD$IF]Eq. (7), the second set of the thermokinetic plots. Compound
PAPep [38_TD$IF] PAAvga
D(DS)/Ra
D(DS) cal/mol/K
AcDab AcAlaDab AcDabAla
3.832 0.995 4.729 0.940 1.960 0.820
0.316 0.297 2.418 0.826 0.764 0.733
0.628 0.590 4.805 1.641 1.517 1.546
a The uncertainties were calculated by weighed orthogonal distance regression (ODR) using the ORDPAC suite of program [52].
Table 5 Experimental (expt) and theoretical (calc) thermochemical quantities of the compounds obtained from this work[39_TD$IF]. Compound
AcDab AcAlaDab AcDabAla
PAPep (expt) kcal/mol
DpSPap (expt)a cal/mol K
GBPep (expt)b kcal/mol
PAPep (calc)c kcal/mol
235.1 2.0 244.7 2.0 242.0 2.0
35.7 2.0 45.9 2.0 42.6 2.0
224.5 2.0 231.1 2.0 229.3 2.0
235.2 241.7 238.9
a Determined using the equation DpSPap = D(DS) + DpSAvg, where D(DS) is the entropy term ([40_TD$IF]Table 4) and DPSAvg is the average deprotonation entropy of the reference acids ([41_TD$IF]Table 1). b Derived using [37_TD$IF]Eq. (1), where T = 298 K. c Calculated using [37_TD$IF]Eq. (8), where the enthalpies for both the neutral and the protonated peptides were calculated at the B3LYP[42_TD$IF]/6-311++G(2d,p)//6-311+G(d) level of theory.
and 242.0 2.0 kcal/mol for AcDabAla and again followed the trend: AcDab < AcDabAla < AcAlaDab. Computationally predicted proton affinity values were in reasonably good agreement with the experiments. Both the experimental and the computational results suggested that proton affinity of either of the two Dab-containing dipeptides was higher than that of the isolated AcDab. The enhanced proton affinity of the dipeptide is expected as the protonation site could be better stabilized via internal solvation in the form of hydrogen-bonding and/or charge–dipole interaction, as well as the degree of polarizability. As shown in Fig. 3, the protonated amino group involved in two hydrogen-bonds (shorter than 2 Å between the two atoms) in the dipeptides, while one hydrogen-bond was observed in AcDabH+. Many studies have shown that longer peptides could stabilize a charge more efficiently than shorter analogs, and hence longer peptides often exhibited greater gas-phase acidity and basicity. The studies of the basicity of polyglycine peptides showed that the basicity of the peptides increased as the peptides were elongated [13,20,54], while the tripeptides, GlyGlyGly, GlyAlaGly and GlyGlyAla exhibited similar basicity [14]. We have observed comparable trends in the gas-phase acidity. Our previous studies on cysteinecontaining oligopeptides suggested that the gas-phase acidity of the cysteine residue (the side-chain thiol group) increased systemically as the peptide was elongated [34,35]. For example the gas-phase acidity of PolyAla-Cys peptides followed
the trend: AlaCys(NH2) < AlaAlaCys(NH2) < AlaAlaAlaCys(NH2) < AlaAlaAlaAlaCys(NH2). Computational studies showed that all of these anionic peptides (formed upon deprotonation at the thiol group) existed in randomly coiled conformations. As the peptide chain became longer, the possibility of hydrogen-bonding interactions between the thiolate anion and the nearby N H bonds also became higher, and hence, larger peptides could stabilize the negative charge more efficiently. Numerous proton affinities for a variety of dipeptides have been reported in the literature [18]. It would be interesting to compare the results from this study to those published in the literature. One notable difference is the sequence effect on basicity (or proton affinity). It has been reported that the gas-phase basicity was higher when the basic residue was located at the N-terminus, such that the relative basicity followed with HisGly > GlyHis and LysGly > GlyLys [55]. In these studies, the peptides were not acetylated and had free amino group at the N-termini. In the protonated form of LysGly or HisGly both the N-terminal amino group and the side-chain amino group could be bound to the proton via intramolecular hydrogen-bonding interaction. This would stabilize the protonated peptide and enhance the basicity of the peptides with a basic residue located the N-termini. Both the experimental and the computational results suggested that the C-Dab peptide, AcAlaDab had a higher proton affinity than the N-Dab peptide, AcDabAla by 2–3 kcal/mol. It was somehow surprising, considering that peptides with only two residues would exist in a very similar conformation. Carefully examining the lowest energy structures of the peptides (Fig. 3), one could see the subtle difference between the N-Dab and the C-Dab structures. For the two protonated peptides, the conformation of AcAlaDabH+ appeared to be more compact than that of AcDabAlaH+. The more compact structure allowed more efficient hydrogen-bonding interactions that could lower the potential energy of the peptide ion. As indicated on the structures, the overall distance between the hydrogen atoms of the NH3 group and the oxygen atoms of the carbonyl groups was shorter for AcAlaDabH+. Quantitatively AcAlaDabH+ was more stable than AcDabAlaH+ by 1.3 kcal/mol (B3LYP/6-311++G(2d,p)//B3LYP/6311+G(d)). While for the neutral peptides, AcAlaDab was less stable than AcDabAla by 1.5 kcal/mol. The difference between the structures of the two neutral peptides was not obvious, except that the distance between the nitrogen atom of the NH2 group and the nearby hydrogen of the NH bond was shorter in AcDabAla. The shorter NH distance might contribute to a more efficient hydrogen-bonding interaction. Therefore, the higher proton affinity of the C-Dab peptide might be due to the combination of the lower energy in the protonated form and the higher energy in the neutral form as compared to the isomeric N-Dab peptide. In our previous studies of the gas-phase acidity of Cyscontaining oligopeptides, we have observed that the N-Cys dipeptides, CysAla(NH2) or CysGly(NH2), were more acidic than the C-Cys dipeptides, AlaCys(NH2) or GlyCys(NH2), by about
158
[(Fig._3)TD$IG]
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Fig. 3. Lowest energy conformations for AcAlaDab, AcAlaDabH+, AcDabAla and AcDabAlaH+ obtained at the B3LYP/6–311 + G(d) level of theory. The numbers (in Å) indicate the distances between the two corresponding atoms shown in the structure.
4 kcal/mol [34]. The stronger acidity of the N-Cys peptides was largely due to the more efficient hydrogen-bonding interaction that stabilized the thiolate anions in the deprotonated N-Cys peptides. We have also observed that the N-Cys tetrapeptide, CysAlaAlaAla(NH2), was more acidic than the isomeric C-Cys analog by about 10 kcal/mol [35]. Computational studies suggested that the deprotonated N-Cys tetrapeptide existed in a one-turn helix, while the deprotonated C-Cys analog formed a coil. The favored charge-helix macro-dipole interactions could stabilize the N-terminal thiolate anion significantly, and contributed to the greater acidity of the N-Cys tetrapeptide. 5. Conclusions The proton affinities and related thermochemical quantities of the lysine analog, AcDab and two isomeric Dab-containing dipeptides were determined using the extended Cooks kinetic method with full entropy analysis. The proton affinities (PA) for the three compounds were determined to be 235.1 2.0 kcal/mol (AcDab), 244.7 2.0 kcal/mol (AcAlaDab), and 242.0 2.0 kcal/mol (AcDabAla). The gas-phase basicities (GB) and the protonation entropies (DpS) for these peptides were determined accordingly. Theoretically predicted values of the proton affinities were in reasonably good agreement with the experiments. The overall results suggested that the dipeptides were more basic than the single amino acid by about 7 kcal/mol, and the C-Dab dipeptide was more basic than the isomeric N-Dab analog by about 2.5 kcal/mol. Computational studies showed that the conformations of the two peptides were slightly different in both the neural and the protonated forms. The stronger basicity of the C-Dab peptide was
likely due to the combination of the higher energy of the neutral form and the lower energy of the protonated form of the C-Dab peptide as compared to those of the isomeric N-Dab peptides. The study provided important experimental data in helping understanding the conformational effects on thermochemical properties of peptides in a solvent-free environment. Acknowledgements This research was supported by the National Science Foundation (CHE-1301505). The authors thank Dr. Yu Zheng for helping synthesis of the compound AcDab. P. Batoon thank the graduate research support from the University of the Pacific. The instrument usage was provided by the Mass Spectrometry Facility at the University of the Pacific. Appendix A. Supplementary data Supplementary data associated with this article can be found, in the online version, at http://dx.doi.org/10.1016/j.ijms.2014.07.025. References [1] [46_TD$IF]A. Inui, A. Asakawa, C.Y. Bowers, G. Mantovani, A. Laviano, M.M. Meguid, M. Fujimiya, Ghrelin, appetite, and gastric motility: the emerging role of the stomach as an endocrine organ, [47_TD$IF]FASEB J. 18 (2004) 439. [2] T.A. Steitz, DNA polymerases: structural diversity and common mechanisms, J[48_TD$IF]. Biol. Chem. 274 (1999) 17395–17398. [3] W.T. Clark, P. Radivojac, Analysis of protein function and its prediction from amino acid sequence, Proteins 79 (2011) 2086–2096. [4] D. Lee, O. Redfern, C. Orengo, Predicting protein function from sequence and structure, Nat[49_TD$IF]. Rev. Mol. Cell Biol. 8 (2007) 995–1005.
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Contents lists available at ScienceDirect
International Journal of Mass Spectrometry journal homepage: www.elsevier.com/locate/ijms
Proton affinity of dipeptides containing alanine and diaminobutyric acid$ Patrick Batoon, Jianhua Ren * Department of Chemistry, University of the Pacific, 3601 Pacific Avenue, Stockton, CA 95211, USA
A R T I C L E I N F O
A B S T R A C T
Article history: Received 1 May 2014 Received in revised form 11 July 2014 Accepted 14 July 2014 Available online 18 July 2014
Thermochemical properties, including the proton affinity, the gas-phase basicity, and the protonation entropy, were determined for the acetylated unnatural amino acid, 2,3-diaminobutyric acid, AcDab and two isomeric dipeptides containing alanine (Ala) and Dab AcAlaDab and AcDabAla. The experiments were carried out using a triple quadrupole mass spectrometer. The extended Cooks kinetic method was applied to determine the values of the proton affinity. The proton affinities for the three compounds were determined to be 235.1 2.0 kcal/mol (AcDab), 244.7 2.0 kcal/mol (AcAlaDab), and 242.0 2.0 kcal/mol (AcDabAla). The gas-phase basicities (GB) and the protonation entropies (DpS) for these peptides were determined accordingly. The results suggested that the dipeptides were more basic than the single amino acid by about 7 kcal/mol, and the C-terminal Dab peptide, AcAlaDab was more basic than the N-terminal Dab peptide, AcDabAla by about 2.5 kcal/mol. Computational studies were carried out via a series of steps including conformational search using the MMFF force field, followed by geometry optimization and energy calculations at the B3LYP/6-311++G(2d,p)//B3LYP/6-311+G(d) level of theory. Theoretically predicted proton affinities were in reasonably good agreement with the experiments. The higher basicity of the C-Dab dipeptide was likely due conformational effect that stabilized the charged C-Dab peptide more efficiently than the N-Dab peptide. ã 2014 Elsevier B.V. All rights reserved.
Keywords: Gas-phase basicity Unnatural amino acid Dab Lysine homologues Kinetic method
1. Introduction Proteins, as the principal mechanical machinery of nature, can range from peptides such as Ghrelin, used as a signaling molecule for the stimulation of hunger [1], to large and highly ordered protein complexes such as DNA polymerase to carry out the synthesis of DNA [2]. Many studies have been carried out to understand and determine the structures, functions, and roles of large proteins and peptides at the biological level [3–5], but the fact that still remains is the lack of fundamental knowledge on the chemical and physical properties of smaller peptides resembling the active sites of proteins. For many proteins, acidic and basic residues can be found buried deep within the protein’s structure, implicating an important catalytic or structural function [6]. Additionally, it has been found that some of these buried residues exhibit drastic shifts in pKa values caused by a variety of factors such as charge–charge
$ This article is dedicated to Professor Veronica Bierbaum for her great contributions in the fundamentals of gas-phase ion chemistry. * Corresponding author. Tel.: +1 209 946 2393; fax: +1 209 946 2607. E-mail address: jren@pacific.edu (J. Ren).
http://dx.doi.org/10.1016/j.ijms.2014.07.025 1387-3806/ ã 2014 Elsevier B.V. All rights reserved.
interactions, charge–dipole interactions, and desolvation (the Born effect) [7]. In general, the most common buried residues that have ionizable side chains are cysteine (Cys), histidine (His), and tyrosine (Tyr), because they are intrinsically uncharged at pH 7 [8]. However, by simply placing any highly acidic or basic residue within a buried location of a protein, the residue could exhibit a pKa shift [9]. One such example of a buried residue is lysine (Lys) 115 in the protein acetoacetate decarboxylase. Positioned within the hydrophobic interior of th e protein, Lys 115 was found to exhibit an unusually perturbed pKa value of 7.5; whereas the normal pKa of Lys was 10.5. The reduced pKa value of Lys 115 allowed the residue to retain its nucleophilic property as a neutral residue and readily forms a Schiff-base intermediate with an incoming substrate at physiological pH [10]. The exact factors of stabilization or destabilization of buried ionizable residues were extremely difficult to establish. The perturbed pKa values were highly conformation dependent. When attempted to measure the pKa values of buried residues by titration, the results obtained were largely from solvated residues in the unfolded proteins [11]. This problem has become considerably complex in the study of model peptides mimicking buried sites. Due to the size and solvent accessibility of small peptides, the “buried” conformation was difficult to emulate in solution, and
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the results might not reflect the pKa perturbation of a buried residue. To solve this problem, one may study the model peptides in the gas-phase. Studies have shown that solution-phase conformations of proteins could be retained in the gas-phase under mass spectrometry conditions [12]. Extensive mass spectrometric studies on peptides and single amino acids have been carried out by establishing proton affinities to gain better understanding of the molecule’s intrinsic ability of stabilizing a protonation site [13–23]. Additionally, there have been a number of studies on peptides and single amino acids that involved measuring the gas-phase acidity [24–33]. Our previous studies have demonstrated that conformations could significantly influence the gas-phase acidity of oligopeptides. For the series of cysteine (Cys)-containing peptides, the N-Cys peptides were significantly more acidic than the C-Cys peptides [34–36]. Computational studies suggested that the deprotonated N-Cys peptides with four residues and longer existed in a helical conformation, while the deprotonated C-Cys peptides preferred a globular conformation [35,36]. The stronger acidity of the N-Cys peptides was mainly due to the favored interaction with the helix macro-dipole that stabilized the N-terminal thiolate anion. In the case of di- and tri-peptides that were too short to form an a-helix, the greater acidity of the N-Cys peptides was likely due to the more compact conformation of the deprotonated N-Cys peptides in which the thiolate anions were stabilized via efficient intramolecular hydrogen-bonding interactions [34]. We have begun to systemically investigate conformational effects on the proton affinity of peptides by using a series of model oligopeptides containing a basic probe. One such a probe was diaminobutyric acid (Dab). Dab was chosen as a basic probe due to its homologous nature to the natural amino acid lysine (PA = 238.0 kcal/mol) [37], but with lower basicity (PA = 233.2 kcal/mol) [21]. The weaker basic Dab allowed a larger variety of molecules to be used as reference bases to determine the proton affinity of Dab-containing peptides. Although considered an “unnatural” amino acid, Dab can be found as the free amino-acid in the legume genus Lathyrus and has been shown to exhibit neurotoxic effects by inhibiting the urea cycle, inducing chronic ammonia toxicity through the blood that elevated glutamate levels in the brain [38]. The two N-oxalic acid derivatives of Dab (2-amino-4[(carboxycarbonyl)amino]butanoic acid and 4-amino-2-[(carboxycarbonyl)amino]butanoic acid) can also be found in Lathyrus and have proven to be highly toxic though different metabolic pathways [39]. In this paper, we report the determination of the proton affinity and related thermochemical properties of acetylated Dab (AcDab) and two isomeric Dab-alanine dipeptides, AcAlaDab and AcDabAla.
[(Schem_1)TD$FIG]
The structures of AcDab and the two peptides are shown in Scheme 1. We calculated the theoretical proton affinities and the conformations of these molecules. 2. Experimental 2.1. Peptide synthesis All peptides were synthesized using standard solid phase peptide synthesis (SPPS) [40]. The experiments were carried out in a manual synthesis apparatus comprised of disposable 10 mL peptide synthesis vessels (Polypropylene reaction vessel, Torviq, Niles, MI) mounted onto a mechanical agitator (Model 75 WristAction Shaker, Burrel Scientific, Pittsburgh, PA). Wang resin was used as the solid support to yield the carboxyl group at the Cterminus. The general procedure can be described as the following. Wang resin (0.33 mmol, loading capacity 1.1 mmol/g) was placed in the peptide synthesis vessel. Dichloromethane was passed through the vessel to swell the resin. Coupling of the first amino acid was done by adding a 3-times excess mole ratio of a Fmoc-amino acid and O-benzotriazole-N,N,N0 ,N0 -tetramethyluronium-hexafluoro-phosphate (HBTU) and six times excess N, N-diisopropylethylamine (DIPEA) with 4 mL dimethylformamide (DMF). The solution was capped under nitrogen gas, and then shaken for 5 h. Deprotection of the coupled resin was carried out using 4 mL of 20% piperidine in DMF for 5 min, drained, and repeated for an additional two times. Subsequent coupling (2–3 h)–deprotection cycles were carried. When the last amino acid was coupled and deprotected, the peptide was acetylated by mixing a 5 mL solution of 20% DIPEA and 20% acetic anhydride in DMF. Cleavage of the peptide was carried out by incubating the resin in 5 mL of “Reagent B” cleavage cocktail (88% TFA, 5% phenol, 5% H2O, and 2% triisopropylsilane (TIPS reagent)) for 2–3 h. The flow-through was collected and then concentrated under nitrogen gas until the volume was less than 1 mL. A mixture of diethyl ether and hexane was added to the concentrate and precipitated in 4 C for at least 1 h. The precipitate was pelleted at 3500 rpm for 20 min using a centrifuge (HERMLE Labnet Z206A). Solvent was decanted, and the peptide pellet was dissolved in 5–10 mL of Milli-Q water. The solution was passed through a CHROMAFIL Xtra CA-20/ 25 syringe filter and lyophilized overnight to obtain a solid form of the peptide. The yielded peptide was sufficiently pure for mass spectrometry measurements. The sequence of the peptide was confirmed by the MS/MS sequence analysis technique. All synthesis reagents, including the Wang resin, Fmoc-amino acids, and coupling reagents were purchased from Chem-Impex International Inc. (Wood Dale, IL), and were used without further purification. 2.2. Mass spectrometry measurements
NH2 O H3C
OH
N H
O
AcDab NH2
NH2 H N
H3C O
O
O
CH3
N H
OH O
H3C
N H
H N O
AcDabAla
AcAlaDab Scheme 1.
O OH CH3
All mass spectrometry experiments were performed using a triple quadrupole mass spectrometer interfaced to an electrospray ionization (ESI) source (Varian 320L, Agilent Technologies, Santa Clara, CA). The sample solution was prepared by dissolving the peptide and a reference base into a 1:1 (v:v) solution of methanol: water to achieve a final concentration of about 104–105 M. The sample solution was directly infused into the ESI source at a flow rate of 10 mL/min. Data acquisition was controlled by the MS Workstation software package (version 6.9) in the positive ion mode. The ESI needle voltage was maintained at 4.2–4.8 kV. The capillary voltage was set between 25 and 50 V. The drying gas pressure was set to 14 psi, and the temperature was set between 150 and 250 C, respectively. The nebulizing gas was set between 35 and 50 psi. Nitrogen was used as the nebulizing and drying gas. The hexapole
P. Batoon, J. Ren / International Journal of Mass Spectrometry 378 (2015) 151–159
[(Schem_2)TD$FIG]
ion guide chamber operated at a pressure of about 1 mTorr nitrogen gas. The capillary voltage and drying gas temperature were adjusted within the specified range to maximize the signal of the proton-bound dimer. The ions generated in the ESI source were presumed to be thermalized by multiple collisions with the nitrogen molecules in the ion guide chamber. CID experiments were performed by isolating the proton-bound dimer with the first quadrupole [43_TD$IF](MS1) with a peak width of 1.2 (instrument parameter). The isolated ion was allowed to undergo collisions with argon atoms leaked into the collision chamber which was held at a pressure of 0.4 mTorr. The product ions were analyzed with the third quadrupole [4_TD$IF](MS2) with a peak width of 1.0 and a dwell time of 0.2 ms. Product ion spectra of the proton-bound dimer were recorded at several collision energies to determine all possible product ions representing the protonated peptide or the protonated reference. Product ion intensities were then measured in selected reaction monitoring (SRM) mode for ions representing the protonated peptide or the protonated reference base. The SRM acquisition was carried out continuously for 2 min and repeated two times in one day. Multiple measurements were carried out on different days with a relative uncertainty of around 5%. All CID experiments were performed at four different collision energies corresponding to the center-of-mass energies (Ecm) of 1.0, 1.5, 2.0, and 2.5 eV, respectively. The center-of-mass energies were calculated using the equation Ecm = Elab[m/(M + m)], where Elab is the laboratory frame collision energy, m is the mass of argon, and M is the mass of the proton-bound dimer ion. 2.3. CID bracketing experiments Bracketing experiments were carried out by isolating the proton-bound dimer in Q1 and obtaining the product ion mass spectra in Q3 at 1.0, 1.5, 2.0, and 2.5 eV collision energies in the center of mass frame (Ecm) for 30 s. The collision gas (argon) pressure was set to 0.4 mTorr to retain at least 10% of the protonbound dimer precursor ion at the highest collision energy. Bracketing experiments primarily yielded two product ions representing the protonated peptide and the protonated reference base, respectively. The product ion with the higher peak intensity was determined to have the higher proton affinity. Bracketing experiments were performed with all given reference bases and made it possible to qualitatively estimate the proton affinities of the peptides. 2.4. Extended Cooks kinetic method The gas-phase basicities and proton affinities of AcDab, AcADab and AcDabA were determined using the extended Cooks kinetic method [20,41–43]. The gas-phase basicity (GB) is the Gibbs free energy change for the reaction, MH+(g) ! M(g) + H+(g), typically at 298 K. The proton affinity (PA) and the protonation entropy (DpS) are the enthalpy and entropy changes for the reaction, respectively. All three thermodynamic values are related by the fundamental Gibbs free energy equation (Eq. (1)): GBPep ¼ PAPep T Dp SPep (1) Thermochemical determinations are carried out by isolating a proton-bound dimer between the peptide (Pep) and a reference base (B), [PepHB]+, into the collision cell of the mass spectrometer. The proton-bound dimer is accelerated through the chamber pressurized with argon at about 0.4 mTorr and undergoes collision induced dissociation. The expected product ions represent the two competitive dissociation pathways of the proton-bound dimer that yields the protonated peptide (PepH+) and the protonated reference base (BH+), shown in Scheme 2.
153
[17_TD$IF]Scheme 2.
The ion abundance ratio [PepH+]/[BH+] is presumed to represent the rate constant ratio kP/kB. The natural logarithm of the rate constant ratio is a function of the free energies of activation of these reactions and is related by a linear equation (Eq. (2)), where R is the ideal gas constant and Teff is the “effective temperature” of the activated proton-bound dimer. The effective temperature is an empirical parameter that depends on several experimental variables and properties of the proton-bound dimers [44–49]. The equation can be transformed to include the relative enthalpy and entropy of activation (Eq. (2)). Assuming no reverse activation barriers, the relative free energy and enthalpy of activation becomes equal to the relative GB and PA between the peptide and the reference base, respectively, (Eq. (3)). Similarly, the relative activation entropy D(DSz) becomes equal to difference in protonation entropy D(DS) (Eq. (4)). If the reference bases used are structurally similar, the assumption can be made that D(DS) will remain constant between the different dissociation pathways for dimers of different reference bases. In addition, the average protonation entropy of all reference bases, DpSAvg can be used to replace the term, DpSB. After all amendments to Eq. (2) have been made, Eq. (2) is transformed into Eq. (5). DGzb DGzP DHzB DGzP DSz kP ln ¼ þ ¼ kB RTeff RTeff R
(2)
DHzB DHzP ¼ PAPep PAB
(3)
D DSz D DS ¼ Dp SPep Dp SB
(4)
PepHþ PAPep PAB D DS ¼ þ R RTeff BHþ
(5)
ln
In order to achieve proper statistical treatment of the uncertainty, the average proton affinity of all reference bases PAAvg is introduced into Eq. (5) and this yielded Eq. (6) [50,51]. After rearrangement, Eq. (6) can be transformed into a functional linear equation (Eq. (7)). PepHþ D DS PAPep PAAvg PAB PAAvg (6) ln þ ¼ R RTeff RTeff BH PepHþ PAPep PAAvg D DS 1 ln þ ¼ PAB PAAvg þ RTeff R RTeff BH
(7)
The proton affinity values are extracted in two steps. First, a plot of the ln ([PepH]+/[BH]+) vs PAB PAAvg is made. The slope [45_TD$IF]represents 1/RTeff and the y-intercept represents [(PAPep PAAvg)/RTeff D(DS)/R]. If dissociation of [PepHB]+ is carried out at different collision energies, a set of different slopes and y-intercepts can be obtained. Using this set of data, a plot of [(PAPep PAAvg)/RTeff D(DS)/R] vs 1/RTeff is made. The slope of the plot represents PAPep PAAvg, and the y-intercept represents D(DS)/R. Because PAAvg and R are known values, PAPep and D(DS) can be obtained. The value of DpSPep can be determined using the equation D(DS) = DpSPep DpSAvg. The gas-phase basicity of the
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Table 1 Thermochemical values for the reference bases[7_TD$IF]. Reference [15_TD$IF]base
Abbreviation
PAa [8_TD$IF]kcal/mol
GBa kcal/mol
DpSb cal/mol/K
[9_TD$IF]AcDab Asparagine Glutamine Piperidine 1,3-Propanediamine 1,6-Hexanediamine 1,5-Pentanediamine Average
Asn Gln Pip 1,3DAP 1,6DAHx CAD
222.0 224.0 228.0 235.9 238.9 238.9 231.3
213.1 215.0 220.0 224.7 226.1 226.1 220.8
29.9 30.2 26.8 37.6 43.0 43.0 35.1
[10_TD$IF]AcAlaDab/AcDabAla Histidine 1,7-Heptanediamine 1,6-Hexanediamine 1,5-Pentanediamine 1,4-Butanediamine N,N-Dimethyl-1,3-propanediamine Average
His 1,7DAHp 1,6DAHx CAD 1,4BDA DMPDA
238.0 238.6 238.9 238.9 240.3 245.0 240.0
227.1 225.8 226.1 226.1 228.1 233.1 227.7
36.6 43.0 43.0 43.0 41.1 39.9 41.1
a
Obtained from the most recent data compiled in the NIST Chemistry WebBook [37]. Derived from the relationship [18_TD$IF]DG = DH–T(DS), where T = 298 K. It is assumed that each PA and GB value has an uncertainty of [1_TD$IF]2.0 kcal/mol K, and each D [12_TD$IF] pS value has an uncertainty of 2.0 cal/mol K. b
peptides, GBPep, 298 K can be determined using the Gibbs free energy relation (Eq. (1)). The uncertainties of the linear regression were calculated using orthogonal distance regression (ODR) using the ODRPACK suite of programs [52]. The uncertainty of the average proton affinity (PAAvg) was calculated as the root sum square of the random and systematic errors. We assume that the proton affinity of each reference base has an uncertainty of (2.0 kcal/mol). For a set of six reference bases, the random error was treated as the averaged uncertainty of the reference bases (2.0 kcal/mol) divided by the square root of the number of the reference acids, p (2.0/ 6) = 0.82 kcal/mol, and the systematic error was assigned as p 2.0 = 1.4 kcal/mol. The root sum square of the random and p systematic errors yielded (0.822 + 1.42) = 1.6 kcal/mol. 2.5. Computational method The input structures of the peptides were constructed using the Spartan’11 suite of programs (Wavefunction, Irvine, CA) for which the site of proton attachment was chosen to be on the basic sidechain of the peptide. Merck molecular force field (MMFF) conformational search calculations were carried out using the same software suite. The 200 lowest energy conformers were saved, and AM1 level geometry optimization immediately proceeded. Of the pool of AM1 optimized conformers, the 20 lowest energy structures were subjected to HF/3-21G(d) optimization and frequency calculations to obtain a more accurate energy ladder. From which the 10 lowest energy conformations were further subjected to geometry optimization and frequency calculations at the B3LYP/6-311+G(d) level of theory. Single point electronic energies were obtained at B3LYP/6-311++G(2d,p) level using the geometries calculated at the B3LYP/6-311+G(d) level. The calculated enthalpy at 298 K was determined using the sum of the electronic energy (e0) and the thermal correction to enthalpy (DHcorr). All quantum level calculations (HF, B3LYP) were carried out using the Gaussian’09 software suite [53]. Proton affinity values were calculated by an isodesmic reaction scheme with ethylamine (PA = 217.97 kcal/mol) as the reference base (Eq. (8)). In order to evaluate the effect of different basis sets, the lowest energy conformations of the neutral and the protonated peptides were further optimized and calculated at the B3LYP/6-311++G(d,p) level. The resulting energetics were used to calculate the proton affinity using Eq. (8).
PepHþ þ Bethylamine ! Pep þ Bethylamine Hþ
(8)
3. Results 3.1. Relative proton affinity of AcDab, AcAlaDab and AcDabAla The relative proton affinities of AcDab and the two peptides, AcAlaDab and AcDabAla were bracketed against a set of reference amine bases (B) with known values of the proton affinity (PA) and the gas-phase basicity (GB). The reference amine bases alone with their thermochemical properties are listed in Table 1. The relative proton affinity was estimated by comparing the ion intensities of the protonated peptide and the protonated reference amine base resulting from the dissociation of the proton-bound dimer, [AcDabHB]+, [AcAlaDabHB]+, or [AcDabAlaHB]+, under the CID conditions. The results suggested that the proton affinity of AcDab was between that of piperidine (Pip 228.0 kcal/mol) and 1,3propanediamine (1,3DAP, 235.9 kcal/mol), and the proton affinity of both peptides would be between that of 1,4-butanediamine (1,4BDA, 240.3 kcal/mol) and N,N-dimethyl-1,3-propanediamine (DMPDA, 245.0 kcal/mol). Four representative CID spectra are shown in Fig. 1. The relative ion abundances of the protoned peptides and protonated 1,4BDA clearly showed the trend of the proton affinity: AcAlaDab, AcDabAla > 1,4BDA. The fact that the ion abundance of 1,4BDAH+ was closer to that of AcDabAlaH+ than to that of AcAlaDabH+ implied that AcAlaDab had a higher proton affinity than AcDabAla. 3.2. Quantitative proton affinity of AcDab, AcAlaDab and AcDabAla The quantitative value of the proton affinity was determined based on the linear equation described in Section 2 (Eq. (7)). The natural logarithms of the CID product ion ratios (branching ratios) measured at four collision energies, 1.0, 1.5, 2.0, and 2.5 eV (Ecm) are shown in Table 2. All secondary fragment ions were counted into the intensities of the corresponding precursor ions and were included in the data analysis. The corresponding thermo–kinetic plots of the branching ratios at the four collision energies against the relative proton affinity of the reference bases, PAB PAAvg, are shown in Fig. 2a. Linear regression yielded four sets of slopes (X) and intercepts (Y), and the results are summarized in Table 3. Each slope represented the value of 1/RTeff at the specific collision energy. Each intercept represented the term [(PAPep PAAvg)/
P. Batoon, J. Ren / International Journal of Mass Spectrometry 378 (2015) 151–159
[(Fig._1)TD$IG]
155
Fig. 1. CID spectra obtained at 1.5 eV (Ecm) for the proton-bound heterodimer anions of [1_TD$IF](a1) [AcDabHPip]+, (a2) [AcDabH1,3DAP]+, (b) [AcAlaDabH1,4BDA]+, and (c) [AcDabAlaH1,4BDA]+.
RTeff D(DS)/R]. To derive the value of PAPep, the obtained intercepts (Y) and the slopes (X) were used to construct a new thermo–kinetic plot, shown in Fig. 2b. Linear regression of each plot yielded a slope of PAPep PAAvg and an intercept of D(DS)/R. The results for AcDab and for the two peptides are shown in Table 4. Combined with the known values of PAAvg (Table 1), the values of PAPep for AcDab and for both peptides were determined, shown in Table 5. The protonation entropy (DpSPep) was determined using the relation D(DS) = DpSPep DpSAvg, where DpSAvg (Table 1) represented an average entropy of the reference bases. The results are shown in Table 5. The gas-phase basicity (GBPep) of each of the compounds was derived using the thermodynamic relation shown in Eq. (1). The results are shown in Table 5. The uncertainties were calculated by weighted orthogonal distance regression (ODR) using the ODRPACK suite of programs [52]. Here we use the AcAlaDab system to illustrate the procedure. Table 2 Values of [13_TD$IF]ln ([PepH+]/[BH+]) measured at four collision energies (Ecm) with different references bases[14_TD$IF]. Peptide
Reference [15_TD$IF]base
1.0 eV
1.5 eV
2.0 eV
2.5 eV
AcDab
Asn [16_TD$IF]Gln [17_TD$IF]Pip [18_TD$IF]1,3DAP [19_TD$IF]1,6DAHx [20_TD$IF]CAD
5.994 2.053 1.191 -0.061 -1.049 -1.722
5.880 1.984 0.580 -0.724 -0.917 -1.623
5.750 1.851 0.204 -0.745 -0.776 -1.474
5.522 1.829 -0.063 -0.698 -0.627 -1.306
[21_TD$IF]AcAlaDab
His [2_TD$IF]1,7DAHp [23_TD$IF]1,6DAHx [24_TD$IF]Cad [25_TD$IF]1,4BDA [19_TD$IF]DMPDA
2.854 3.744 3.578 3.037 1.406 -2.725
2.341 3.654 3.474 2.802 1.241 -2.835
2.029 3.637 3.428 2.631 1.197 -2.840
1.689 3.627 3.446 2.437 1.222 -2.802
[120_TD$IF]AcDabAla
His [2_TD$IF]1,7DAHp [28_TD$IF]1,6DAHx [24_TD$IF]Cad [29_TD$IF]1,4BDA [30_TD$IF]DMPDA
1.702 3.486 2.837 1.998 0.305 -3.677
1.344 3.400 2.701 1.838 0.253 -3.677
1.121 3.409 2.604 1.798 0.347 -3.602
0.925 3.430 2.448 1.774 0.478 -3.434
For each of the plots of ln ([PepH]+/[BH]+) vs PAB PAAvg, the uncertainty used in all x-axis values was 2.0 kcal/mol, and in all yaxis values was 0.05. The uncertainties (with 95% confidence) in the slopes (X) and in the intercepts (Y) are shown in Table 3. The corresponding uncertainties were carried into the plot of Y vs X. The resulting uncertainty in the slope (PAPep PAAvg) was 0.940 and in the intercept (D(DS)/R) it was 0.826. Combined with the 1.6 kcal/mol uncertainty estimated for PAAvg, the uncertainty p for PAPep would be (0.942 + 1.62) = 1.9 kcal/mol. It should be pointed out that the uncertainties obtained from the kinetic measurements were relative values. They did not include the absolute error in the overall calibration of the proton affinity scale of the references. Considering about 2 kcal/mol uncertainties for the reference bases, we assigned an uncertainty of 2.0 kcal/mol for the measured proton affinity and the gas-phase basicity, and 2.0 cal/mol K for the protonation entropy, Table 5. 3.3. Computational results The conformations of the neutral and the protonated peptides were calculated via a series of steps including a conformational search followed by geometry optimizations and energy calculations at different levels of theory. From this the 10 lowest energy conformations were selected for further geometry and energy calculations at the B3LYP/6-311+G(d) level of theory. The relative contributions of the set of low energy conformations were calculated based on the Boltzmann distribution. The results are shown in the Supplementary Information. All peptide species, neutral and protonated, had three or fewer conformations with population greater than 5%. All structures with contributions more than 5% are shown in the Supplementary Information, and the lowest energy structures are shown in Fig. 3. The dashed lines and the numbers labeled on the structures indicated the distances between the corresponding atoms. These distances were used to estimate the hydrogen-bonding interactions. The set of the selected lowest energy conformations were subjected to single point energy calculation at the B3LYP/6-311++G(2d,p) level of theory, and the resulting energetic values were used to calculate the proton affinities of the peptides according to Eq. (8). The proton
156
[(Fig._2)TD$IG]
P. Batoon, J. Ren / International Journal of Mass Spectrometry 378 (2015) 151–159
Fig. 2. [2_TD$IF](a) Plots of ln ([PepH+]/[BH+]) against PAB [3_TD$IF] PAAvg (kcal/mol) from the dissociation of [Pep[4_TD$IF]HB]+ at four collision energies (Ecm), 1.0, 1.5, 2.0 and 2.5 eV. [5_TD$IF](b) Plots of (PAPep PAAvg)/RTeff D(DS)/R against 1/RTeff, where the [6_TD$IF]R2 values for the three systems are AcDab 0.89, AcAlaDab 0.92, and AcDabAla 0.74.
affinities were calculated using the lowest energy conformation (Table 5) as well as using the weighted average of the set of low energy conformations (Supplementary Information). The two sets of proton affinity values were comparable, and they were in good agreement with the experimental results. In order to evaluate the effect of different basis sets on the calculated proton affinity, the proton affinities were also calculated at B3LYP/6-311+G(d) and B3LYP/6-311++G(d,p) levels of theory. The results are shown in the Supplementary Information. Although the absolute values were different (by up to 3.8 kcal/mol) among
the results from the three levels, the relative proton affinity between the two peptides was about the same at all three levels. 4. Discussion The CID bracketing experiments suggested that the relative proton affinity followed the trend: AcDab < AcDabAla < AcAlaDab. By using the extended Cooks kinetic method, the proton affinities of the three compounds were determined to be 235.1 2.0 kcal/mol for AcDab, 244.7 2.0 kcal/mol for AcAlaDab
P. Batoon, J. Ren / International Journal of Mass Spectrometry 378 (2015) 151–159
157
Table 3 Results of linear regression according to [12_TD$IF]Eq. (X), the first set of the thermokinetic plots. [32_TD$IF]Ecm 1.0 eVb AcDab AcAlaDab AcDabAla
a b
[3_TD$IF]1/RTeff Ya [34_TD$IF]1/RTeff Ya [35_TD$IF]1/RTeff Ya
0.339 0.999 0.923 1.982 0.946 1.108
1.5 eVb
0.008 0.529 0.108 0.256 0.162 0.383
0.321 0.881 0.8973 1.779 0.913 0.976
2 eVb
0.008 0.584 0.136 0.321 0.176 0.415
0.303 0.827 0.873 1.680 0.884 0.946
2.5 eVb
0.009 0.620 0.154 0.363 0.186 0.439
0.283 0.802 0.842 1.603 0.841 0.937
0.009 0.628 0.176 0.416 0.193 0.456
Y = (PAPep [36_TD$IF] PABAvg)/RTeff D(DS)/R. The uncertainties were calculated by weighed orthogonal distance regression (ODR) using the ODRPAC suite of program.
Table 4 Results of linear regression according to [37_TD$IF]Eq. (7), the second set of the thermokinetic plots. Compound
PAPep [38_TD$IF] PAAvga
D(DS)/Ra
D(DS) cal/mol/K
AcDab AcAlaDab AcDabAla
3.832 0.995 4.729 0.940 1.960 0.820
0.316 0.297 2.418 0.826 0.764 0.733
0.628 0.590 4.805 1.641 1.517 1.546
a The uncertainties were calculated by weighed orthogonal distance regression (ODR) using the ORDPAC suite of program [52].
Table 5 Experimental (expt) and theoretical (calc) thermochemical quantities of the compounds obtained from this work[39_TD$IF]. Compound
AcDab AcAlaDab AcDabAla
PAPep (expt) kcal/mol
DpSPap (expt)a cal/mol K
GBPep (expt)b kcal/mol
PAPep (calc)c kcal/mol
235.1 2.0 244.7 2.0 242.0 2.0
35.7 2.0 45.9 2.0 42.6 2.0
224.5 2.0 231.1 2.0 229.3 2.0
235.2 241.7 238.9
a Determined using the equation DpSPap = D(DS) + DpSAvg, where D(DS) is the entropy term ([40_TD$IF]Table 4) and DPSAvg is the average deprotonation entropy of the reference acids ([41_TD$IF]Table 1). b Derived using [37_TD$IF]Eq. (1), where T = 298 K. c Calculated using [37_TD$IF]Eq. (8), where the enthalpies for both the neutral and the protonated peptides were calculated at the B3LYP[42_TD$IF]/6-311++G(2d,p)//6-311+G(d) level of theory.
and 242.0 2.0 kcal/mol for AcDabAla and again followed the trend: AcDab < AcDabAla < AcAlaDab. Computationally predicted proton affinity values were in reasonably good agreement with the experiments. Both the experimental and the computational results suggested that proton affinity of either of the two Dab-containing dipeptides was higher than that of the isolated AcDab. The enhanced proton affinity of the dipeptide is expected as the protonation site could be better stabilized via internal solvation in the form of hydrogen-bonding and/or charge–dipole interaction, as well as the degree of polarizability. As shown in Fig. 3, the protonated amino group involved in two hydrogen-bonds (shorter than 2 Å between the two atoms) in the dipeptides, while one hydrogen-bond was observed in AcDabH+. Many studies have shown that longer peptides could stabilize a charge more efficiently than shorter analogs, and hence longer peptides often exhibited greater gas-phase acidity and basicity. The studies of the basicity of polyglycine peptides showed that the basicity of the peptides increased as the peptides were elongated [13,20,54], while the tripeptides, GlyGlyGly, GlyAlaGly and GlyGlyAla exhibited similar basicity [14]. We have observed comparable trends in the gas-phase acidity. Our previous studies on cysteinecontaining oligopeptides suggested that the gas-phase acidity of the cysteine residue (the side-chain thiol group) increased systemically as the peptide was elongated [34,35]. For example the gas-phase acidity of PolyAla-Cys peptides followed
the trend: AlaCys(NH2) < AlaAlaCys(NH2) < AlaAlaAlaCys(NH2) < AlaAlaAlaAlaCys(NH2). Computational studies showed that all of these anionic peptides (formed upon deprotonation at the thiol group) existed in randomly coiled conformations. As the peptide chain became longer, the possibility of hydrogen-bonding interactions between the thiolate anion and the nearby N H bonds also became higher, and hence, larger peptides could stabilize the negative charge more efficiently. Numerous proton affinities for a variety of dipeptides have been reported in the literature [18]. It would be interesting to compare the results from this study to those published in the literature. One notable difference is the sequence effect on basicity (or proton affinity). It has been reported that the gas-phase basicity was higher when the basic residue was located at the N-terminus, such that the relative basicity followed with HisGly > GlyHis and LysGly > GlyLys [55]. In these studies, the peptides were not acetylated and had free amino group at the N-termini. In the protonated form of LysGly or HisGly both the N-terminal amino group and the side-chain amino group could be bound to the proton via intramolecular hydrogen-bonding interaction. This would stabilize the protonated peptide and enhance the basicity of the peptides with a basic residue located the N-termini. Both the experimental and the computational results suggested that the C-Dab peptide, AcAlaDab had a higher proton affinity than the N-Dab peptide, AcDabAla by 2–3 kcal/mol. It was somehow surprising, considering that peptides with only two residues would exist in a very similar conformation. Carefully examining the lowest energy structures of the peptides (Fig. 3), one could see the subtle difference between the N-Dab and the C-Dab structures. For the two protonated peptides, the conformation of AcAlaDabH+ appeared to be more compact than that of AcDabAlaH+. The more compact structure allowed more efficient hydrogen-bonding interactions that could lower the potential energy of the peptide ion. As indicated on the structures, the overall distance between the hydrogen atoms of the NH3 group and the oxygen atoms of the carbonyl groups was shorter for AcAlaDabH+. Quantitatively AcAlaDabH+ was more stable than AcDabAlaH+ by 1.3 kcal/mol (B3LYP/6-311++G(2d,p)//B3LYP/6311+G(d)). While for the neutral peptides, AcAlaDab was less stable than AcDabAla by 1.5 kcal/mol. The difference between the structures of the two neutral peptides was not obvious, except that the distance between the nitrogen atom of the NH2 group and the nearby hydrogen of the NH bond was shorter in AcDabAla. The shorter NH distance might contribute to a more efficient hydrogen-bonding interaction. Therefore, the higher proton affinity of the C-Dab peptide might be due to the combination of the lower energy in the protonated form and the higher energy in the neutral form as compared to the isomeric N-Dab peptide. In our previous studies of the gas-phase acidity of Cyscontaining oligopeptides, we have observed that the N-Cys dipeptides, CysAla(NH2) or CysGly(NH2), were more acidic than the C-Cys dipeptides, AlaCys(NH2) or GlyCys(NH2), by about
158
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Fig. 3. Lowest energy conformations for AcAlaDab, AcAlaDabH+, AcDabAla and AcDabAlaH+ obtained at the B3LYP/6–311 + G(d) level of theory. The numbers (in Å) indicate the distances between the two corresponding atoms shown in the structure.
4 kcal/mol [34]. The stronger acidity of the N-Cys peptides was largely due to the more efficient hydrogen-bonding interaction that stabilized the thiolate anions in the deprotonated N-Cys peptides. We have also observed that the N-Cys tetrapeptide, CysAlaAlaAla(NH2), was more acidic than the isomeric C-Cys analog by about 10 kcal/mol [35]. Computational studies suggested that the deprotonated N-Cys tetrapeptide existed in a one-turn helix, while the deprotonated C-Cys analog formed a coil. The favored charge-helix macro-dipole interactions could stabilize the N-terminal thiolate anion significantly, and contributed to the greater acidity of the N-Cys tetrapeptide. 5. Conclusions The proton affinities and related thermochemical quantities of the lysine analog, AcDab and two isomeric Dab-containing dipeptides were determined using the extended Cooks kinetic method with full entropy analysis. The proton affinities (PA) for the three compounds were determined to be 235.1 2.0 kcal/mol (AcDab), 244.7 2.0 kcal/mol (AcAlaDab), and 242.0 2.0 kcal/mol (AcDabAla). The gas-phase basicities (GB) and the protonation entropies (DpS) for these peptides were determined accordingly. Theoretically predicted values of the proton affinities were in reasonably good agreement with the experiments. The overall results suggested that the dipeptides were more basic than the single amino acid by about 7 kcal/mol, and the C-Dab dipeptide was more basic than the isomeric N-Dab analog by about 2.5 kcal/mol. Computational studies showed that the conformations of the two peptides were slightly different in both the neural and the protonated forms. The stronger basicity of the C-Dab peptide was
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