- Email: [email protected]
The effect of n-alkylation on the gas phase basicities of small peptides measured by the kinetic method
Mass Spectrometry
ELSEVIER
and Ion Processes
International Journal of Mass Spectrometry and Ion Processes 146/147 (1995) 339- 347
The effect of N-alkylation on the gas phase basicities of small peptides measured by the kinetic method Igor A. K a l t a s h o v , Catherine C. Fenselau* Department o[' Chemistry and Biochemistry, University of Maryland Baltimore Count.v, 5401 Wilkens Ave., Baltimore. MD 21228, USA
Received 31 January 1995; accepted 13 March 1995
Abstract
Gas phase basicities for a series of N-alkylated peptides were found by the kinetic method using amines and guanidines as reference bases. The basicity value of one of these peptides was then remeasured using the rest of the derivatized peptides as reference bases. Both values appear to be in a good agreement. This suggests that structurally similar peptides (with unaltered topology of intramolecular hydrogen bonding) can be used as reference bases in the kinetic method experiments. Keywords: Biomolecules; Computer simulations; Gas phase basicities; Kinetic method; Metastable ions
of M and B [3,4]:
I. Introduction
The kinetic method, introduced almost two decades ago by Cooks and co-workers [1,2], has been proved to be a very valuable experimental technique in measurements of gas phase basicities/proton affinities. In this method, the process under study is the decomposition of a proton-bound dimer, formed by a c o m p o u n d of interest, M, and a reference base, B:
M . . . H +...B
kl MH + + B <~ k 2 M + BH +
(1) (2)
The relative abundances of MH + and BH + ions in a decomposition spectrum of the dimer reflect the difference in proton affinities (PAs)
ln([MH+]/[BH+]) = ln(kl/k:) = ln(QMH .... B/QBH+..M) + (PA(M) - P A ( B ) ) / R T e n -
(3)
where Teff represents the effective temperature of the dimer, and QMH~...B and QBH~...M represent the partition functions for activated complexes in reactions (1) and (2), respectively. Entropy (Q) and enthalpy (PA) terms in Eq. (3) can be combined to give the free energy term, gas phase basicity (GB). Expression (3) may then be rewritten simply as ln([MH+]/[BH+]) = ln(kl/k2)
~"~Dedicated to the memory of Professor Alfred O. Nier. * Corresponding author. 0168-1176/95/$09.50 © 1995 Elsevier Science B.V. All rights reserved S S D I 0168-1176(95)04196-6
=(GB(M) - GB(B))/RTef f
(4)
340
LA. Kaltashov, C.C. Fenselau/International Journal of Mass Spectrometry and Ion Processes 146/147 (1995) 339 347
The kinetic method is readily applicable to thermally liable, non-volatile compounds while consuming only small amounts of material. The use of this method has dramatically extended the range of compounds for which thermochemical data are now available. It has recently been applied to measurement of the gas phase basicities/proton affinities of biological compounds: nucleic acids [5,6], amino acids [7-10] and small peptides [11-13]. In recent years, a lot of interest has been drawn to studies of the effect of various structural characteristics of peptides on their gas phase basicities. The factors mentioned are amino acid composition of the peptide [14,15], peptide chain length [11], and position of the most basic amino acid residue in the peptide chain [12]. Also, some studies have been carried out to investigate the effect of the N-derivatization of amino acids on their gas phase basicities/proton affinities [16]. The results of the latter study indicate that Nmethylation of phenylalanine leads to a moderate increase in its proton affinity (only by about 1-2 kcal mol-1), as opposed to the dramatic increase in proton affinity in the series NH3, NHzCH3, NH(CH3)2, N(CH3)3 [17]. In the present, study we investigate the influence of N-alkylation of a pentapeptide FGGFL on its gas phase basicity. We have also found that the gas phase basicity increase in the series of N-alkylated peptides is enthalpic, rather than entropic.
2. Experimental
2.1. Materials The pentapeptides F G G F L and N-methylF G G F L were synthesized by the BioPolymer Laboratory (University of Maryland, Baltimore, MD, USA). Sodium borate and sodium borohydrate were purchased from Sigma (St. Louis, MO, USA). Reagent grade formalde-
hyde and acetaldehyde and organic reference bases di-sec-butylamine, N,N-diethylmethylamine, tripropylamine, N,N-di-isopropylethylamine, tributylamine, 1,1,3,3-tetramethylguanidine (TMG), 1,5-diazobicyclo[4.3.0]non5-ene (DBN) and 1,8-diazobicyclo [5.4.0]undec-7-ene (DBU) were purchased from Aldrich (Milwaukee, WI, USA). All chemicals were used without further purification.
2.2. Methods Three N-alkylated peptides (N-ethylFGGFL; N,N-dimethyl-FGGFL and N,Nethylmethyl-FGGFL) were synthesized using a procedure similar to that of Rice et al. [18]. In the present work, 50 #1 of 1 M sodium NaBH4 was added to a solution of peptide in 0.2 M sodium borate buffer, quickly followed by the addition of 50 #1 of 2.4 M formaldehyde or acetaldehyde. The additions of NaBH4 and acetaldehyde were repeated 5-10 times at 10 min time intervals. The sample was then acidified with acetic acid to pH < 4 and desalted using a Bakerbond SPE solid phase extraction column (J.T. Baker, Inc., Phillipsburg, NJ, USA). The reaction was monitored by HPLC and the products characterized using a Kratos Kompact MALDI III time-of-flight mass spectrometer (Kratos Analytical/Shimadzu Scientific Instruments, Inc., Columbia, MD, USA). All kinetic method measurements were performed on a JEOL (Tokyo, Japan) H X l l 0 / HX 110 four-sector mass spectrometer (EBEB geometry). Proton-bound dimers were generated in an FAB source using about 1 #l of sample dissolved in 0.1% aqueous TFA mixed with about 1 #1 of glycerol/monothioglycerol matrix. The proton-bound dimer formed by a compound of interest and a reference base was mass-selected by the first two sectors of the mass spectrometer. The products of the decomposition of the metastable dimer in the third field-free region were
I.A. Kaltashov, C.C. Fenselau/lnternational Journal o]" Mass Spectrometry and Ion Processes 146/147 (I995) 339 347
analyzed by the third (electric) sector (E2). The relative abundances of the decomposition products were deduced from the resulting M I K E spectra using the ratio of areas of fragment peaks. Normally, 10 scans were summed for each M I K E spectrum. All spectra were recorded using the JEOL MP7000 data system. Molecular dynamics/energy minimization studies were performed using the CHARMm22/ QUANTA4.0macromolecular modeling program [19] (Molecular Simulations, Inc., Waltham, MA, USA) on a Silicon Graphics workstation (Silicon Graphics, Inc., Mountain View, CA, USA).
3. Results and discussion
To determine the gas phase basicities of intact and N-alkylated pentapeptides FGGFL, the following set of structurally similar reference bases was used: di-sec-butylamine (GB = 223.6 kcal tool -l [17]), N,N-diethylmethylamine (GB = 222.2 kcal mol -L [17]), tripropylamine (GB = 226.8 kcal tool -1 [17]), N,N-di-isopropylethylamine (GB = 227.7 kcal tool -I [17]), tributylamine (GB = 228.2 kcal tool i [17]), T M G (GB = 234.8 kcal tool -l [20]), DBN (GB = 237.4 kcal tool -1 [20]) and DBU (GB = 239.6 kcal mol -I [20]). Fig. 1 shows typical M I K E spectra of the decompositions of metastable proton-bound dimers formed by F G G F L and its N-methyl analog with tributylamine as the reference base. For each peptide derivative, the ratio of intensities of the protonated reference peak (BH +) versus the protonated peptide peak (MH +) was calculated, and the logarithm of this ratio was plotted as a function of the gas phase basicities of reference bases (Fig. 2). The plot for each of the five peptides studied results in a straight line with a correlation coefficient r2>~ 0.990. The effective temperatures Teff of the metastable dimers, deduced from the
341
slopes of the graphs, are very close to each other for all the peptides in the series, and lie in the range 1025-1100 K. The interception of each graph with the zero point abscissa gives the value of gas phase basicity for each peptide: 232.3 kcal tool -~ for intact FGGFL, 233.9 kcal tool -~ for N-methyl-FGGFL, 235.0 kcal mo1-1 for N-ethyl-FGGFL, 235.8 kcal mol -I for N,N-dimethyl-FGGFL and 236.4 kcal mo1-1 for N,N-ethylmethylFGGFL. It can be seen from this series that the consecutive methylation of the pentapeptide increases its gas phase basicity by 1.6-1.9 kcal mol -l At the same time, N-ethylation leads to an increase in the gas phase basicity by 2.5-2.7 kcal mo1-1 . Most structural changes in molecules (peptides in particular) alter the intramolecular hydrogen bonding and thus the proton stabilization. This leads to a change in protonation entropies as well as effective temperatures of the dimers, which is why the set of reference bases used in kinetic method experiments has to consist of structurally similar molecules. Cheng et al. [4] showed that even protonation of rather small peptides (triglycines and tetraglycines) had a very large entropic effect due to intramolecular charge stabilization (self-solvation). Longer peptide chains favor the formation of intramolecular hydrogen bonds, so the protonation entropy term is different for peptides of different size (even if they have the same amino acid composition, e.g. Gly3 and Gly4) [4]). It has also been shown that varying the chain length may alter the effective temperature of metastable dimers [11,12]. A difference in amino acid composition also leads to a different peptide protonation entropy, since amino acids themselves vary in protonation entropy (e.g. lysine and histidine [9]). The structure of the peptides may also be altered by modification of their functional groups, such as alkylation of their N-terminals. As shown above, N-alkylation of peptides increases their gas phase basicity. At the
342
I.A. Kaltashov, C.C. Fenselau/International Journal
I00
of MassSpectrometry
and Ion Processes 1461147 (1995) 339-347 rx 1
_r
a
90
80
3
70
40
!il 2
60
20 I
1
0
50
I00
150
200
250
300
350
400
450
550
500
600
650
700
_*JZ
m
rx 1
10EJX5
90 -.
70 5’
50-
I!
40
30
II
I
186
. 150
200
250
300
350
400
450
Fig. 1. MIKE spectra of ions produced by metastable decomposition of proton-bound FGGFL (m/z 540), and (b) tributylamine and N-methyl-FGGFL (m/z 554).
503
550
600
650
dimers formed by (a) tributylamine
1
7oo m/z
(m/z 186) and
2!
I.A. Kaltashov, C.C. Fenselan/International Journal of Mass Spectrometry and Ion Processes 146/147 (1995/ 339 347
Ill
343
~i
ill
U
0 4.
.$.
-2 ~ !
-3
~
~% '
m
*= F G G F L • MeFGGFL • EtFGGFL • (Me)2FGGFL • (Et,Me)FGGFL
II
-4
=A
--1//// 222
224
~J 226
230
232 234 236 GB(B), k c a l / m o l
228
238
240
242
Fig. 2. Plot of the logarithms of the relative rates of metastable dissociation of proton-bound dimers formed by N-alkylated peptides and small organic reference bases (amines and guanidines) as a function of gas phase basicity.
same time, it is intuitively reasonable that the addition of rather small alkyl groups (such as methyl or ethyl) to the N-terminus of a peptide will have a minimal steric effect on the topology of the peptide in the gas phase. This hypothesis is supported by the results of molecular modeling studies of the protonated peptides F G G F L , N-methyl-FGGFL and N,Nethylmethyl-FGGFL. Calculations were carried using the CHARMmZ2empirical force field [19]. The structure of F G G F L was built in QUANTA4.0 using the CHARMm22 library of standard L-amino acid residues. This starting linear conformation was then energy minimized (method of steepest descent, 200 steps) to remove possible steric overlaps, followed by molecular dynamics. This included heating the molecule from 0 to 750 K over 1 ps (in 10 -3 ps time steps), equilibrating it at 750 K for 1 ps and then running the dynamics simulation at
750 K for 10 ps. The resulting structure was then energy minimized again (method of steepest descent, 5000 steps) to give a r.m.s. gradient below 0.1 kcal mo1-1. The final structure (see Fig. 3(a)) is cyclic and shows strong hydrogen bonding between the Nand C-terminals (dRo----- 1.67 /~, angleN_H_O ---- 138 °, angleR_o_c = 166°). Another rather strong hydrogen bond that stabilizes the "extra proton" is that between the N-terminal of the peptide and the Gly 2 carbonyl oxygen (dHo = 1.75 A, angleN_H_o = 154 °, angleH_o c -~ 121°). The only amine hydrogen atom not involved in hydrogen bonding was then replaced with a methyl group, thus giving a starting cyclic conformation of N-methyl-FGGFL. The molecular dynamics/energy minimization cycle was then repeated, again giving the cyclic structure (Fig. 3(b)), and showing a
344
I.A. Kaltashov, C.C. Fenselau/lnternational Journal of Mass Spectrometry and Ion Processes 146/147 (1995) 339-347
Phe I ~
(~
~
Leu5
a
Gly2 ~-J ~ k . )
"~ Gly3
Leu 5
Phe t
b
.(
~
Phe4
Gly3
L°u'
C
Fig. 3. Gas phase conformations of the peptides (a) FGGFL, (b) N-methyl-FGGFL, and (c) N,N-ethylmethyl-FGGFL, generated by CHARMm22. Dotted lines represent hydrogen bonds between the N- and C-terminals.
strong hydrogen bond between the N- and Cterminals (dHo = 1.81 ,~, angleN_H_o = 128 °, angleH_o_ c = 113°), and between the N-terminal and Gly 2 carboxyl oxygen (dHo = 1.74 A, angleN_H_o = 147 °, angleH_o_c = 116°). Interestingly, a cyclic structure also resulted from a starting linear conformation of Nmethyl-FGGFL, showing again a rather strong hydrogen bond between the N- and Cterminals (dHo = 1.81 A, angleN_H_O = 128 °, angleij_o_c = 113°), although no hydrogen bond between N-terminal and Gly 2 carboxyl oxygen was formed. The total free energies of these two conformations were essentially the same (-181.1 kcal mo1-1 and -182.7 kcal tool -1, respectively). The N-methyl group was then converted to an ethyl group and the remaining amine hydrogen atom (excluding that in the hydrogen bond) was replaced with a methyl group, giving a starting cyclic conformation of N,N-ethylmethyl-FGGFL. A molecular dynamics/energy minimization cycle did not disrupt the hydrogen bonding between the N- and C-terminals of the peptide (dHo = 1.72 •A, angleN_H_O---- 141 °, angleH_o_c---- 103°), yielding a cyclic conformation with total free energy -128.2 kcal mo1-1 (Fig. 3(c)). The dynamics/minimization cycle was repeated four times, always yielding the cyclic conformation (with total free energies in the range 123-130 kcal mol-1). The linear starting conformation of N,N-ethylmethyl-FGGFL did not yield, however, the cyclic structure. Nevertheless, this linear conformation appeared to be energetically much less favorable (only -108.9 kcal mol -l) than the cyclic one. It appears that interterminal hydrogen bond formation plays a key role in the stabilization of charges (protons) in the series of peptides studied, providing very close similarity in the folding topology. Therefore, it is reasonable to assume that the entropy of protonation (as well as effective temperatures of metastable dimers) should be similar for all peptides in the series. If these assumptions are
I.A. Kaltashov, C.C. Fenselau/International Journal of Mass Spectrometry and Ion Processes 146/147 (1995J 339 347
345
Fxl
100-
BO80 ~
?0 5G8
~
60
~
50-
.,J4
40
554 20
500
550
G00
650
700
750
800
850
900
950
1000
1050
II~
m/z
Fig. 4. MIKE spectrum of ions produced by a metastable decomposition of the proton-bound dimer formed by N-methyI-FGGFL (m/z 554) and N,N-dimethyl-FGGFL (m/z 568).
correct, one should be able to use a set of Nalkylated peptides as reference bases in kinetic method measurements. To test this hypothesis experimentally, we attempted to verify the value of the gas phase basicity of N-methyl-FGGFL using the GB values of FGGFL, N-ethyl-FGGFL, N , N dimethyl-FGGFL and N,N-ethylmethylF G G F L found in earlier experiments (with amines and guanidines as reference bases). Fig. 4 shows a typical M I K E spectrum of the decomposition of a metastable proton-bound dimer formed by two peptides. The logarithms of the ratios of the intensity of protonated Nmethyl-FGGFL (MH +) to the intensities of other peptides in the series (BH +) were plotted against the gas phase basicities of these peptides found in the earlier measurements (Fig. 5). The plot results in a straight line with a correlation coefficient r 2 = 0.989.
The value of the gas phase basicity of Nmethyl-FGGFL found in this experiment is 234.4 kcal mo1-1 . This is 0.5 kcal mo1-1 higher than that found using small organic reference bases (see the second plot in Fig. 4). This approach appears to provide values with accuracy comparable to those in the literature for many small organic reference bases [17]. Another interesting observation is the striking difference in effective temperatures Terr between the dimers formed by two peptides (~ 575 K) and those formed by a peptide and a small organic reference base (1025-1100 K). The effective temperatures of metastable ions are believed to reflect both the instrumental parameters [2] and the amount of internal energy possessed by the dimers [11,12]. The lifetime 7- of a metastable ion with internal energy E can be very roughly approximated by Kassel's equation for s
346
I.A. Kahashov, C.C. Fenselau/International Journal of Mass Spectrometry and Ion Processes 146/147 (1995) 339-347
[] F G G F L • +
amine8
derivatives and
guanidines
0
"~'-1 ra -2
-3 -4
/
-5
222
224
226
228
230
232
234
236
238
240
242
GB(B), keal/mol Fig. 5. Plot of the logarithms of the relative rates of metastable dissociation of proton-bound dimers formed by N-methyl-FGGFL and the four other N-alkylated peptides in the series, and by N-methyI-FGGFL and four small organic reference bases (amines and guanidines) as functions of gas phase basicity.
classical oscillators [21]:
7- -= 1/k(E) -- A(1 - Eo/E) -(s-l)
(5)
where E0 is the dissociation threshold and A is a constant. For the decomposition of a metastable ion to be observed, its lifetime 7should be close to its flight time from the ion source to the third field-free region of the mass spectrometer, which can be estimated as t = L ( m / 2 e V ) 1/2, where L is the flight path, m is the mass of the metastable ion, and eV is its kinetic energy. The estimation of the internal energy of the metastable ions may then be made by equating t and 7-. In a case where a reference base is changed from a small organic molecule to a peptide in a metastable dimer, the values of both m and s values would increase. This should lead
then to a decrease in the internal energy E of a dimer. In addition, the overall internal energy distribution of peptide/amine dimers might be different from that of peptide/peptide dimers, which could affect the effective temperatures.
4. Conclusions Gas phase basicities for a series of N-alkylated peptides were found using the kinetic method: 232.3 kcal mol -l for intact FGGFL, 233.9 kcal mol -Z for N-methyl-FGGFL, 235.0 kcal mol -l for N-ethyl-FGGFL, 235.8 kcal mo1-1 for N,N-dimethyl-FGGFL and 236.4 kcal mo1-1 for N,N-ethylmethyl-FGGFL. Molecular modeling studies indicate that the
I.A. Kahashov, C.C. Fenselau/lnternational Journal o['Mass Spectrometry and Ion Processes 146,'147 { 1995) 339 347
topology of charge stabilization remains essentially unaltered in a set of N-alkylated peptides. This suggests that the entropies of protonation are close to each other, so that it is possible to use these peptides as reference bases in the kinetic method studies. A second set of kinetic method measurements was then carried out using peptides as reference bases to verify the gas phase basicity of N-methyl-FGGFL. The GB value found in this experiment is in a good agreement with that found when small organic reference bases (amines and guanidines) were used. This suggests that structurally similar peptides (i.e. peptides with similar topology of intramolecular hydrogen bonding) can be used as reference bases in gas phase basicity measurements by the kinetic method. Acknowledgments The authors are very grateful to Dr. Martha M. Vestling, Dr. Yetrib Hathout and Dr. Daniele Fabris (University of Maryland Baltimore County) for very helpful discussions. This work was supported by a grant from the National Science Foundation. References [I] R.G. Cooks and T.L. Kruger, J. Am. Chem. Soc., 99 (1977) 1279.
347
[2] S.A. McLuckey, D. Cameron and R.G. Cooks, J. Am. Chem. Soc., 103 (1981) 1313. [3] S.A. McLuckey, R.G. Cooks and J.E. Fulford, Int. J. Mass Spectrom. Ion Phys., 52 (1983) 165. [4] X. Cheng, Z. Wu and C. Fenselau. J. Am. Chem. Soc., 115 (1993) 4844. [5] F. Greco, A. Liguori, G. Sindona and N. Ucella, J. Am. Chem. Soc., 112 (1990) 9092. [6] A. Liguori, A. Napoli and G. Sindona, Rapid Commun. Mass Spectrom., 8 (1994) 89. [7] R.A.J. O'Hair, J.H. Bowie and S. Gronert, Int. J. Mass Spectrom. Ion Processes, 117 (1992) 23. [8] Z. Wu and C. Fenselau, Rapid Commun. Mass Spectrom., 6 (t992) 403. [9] Z. Wu and C. Fenselau, Rapid Commun. Mass Spectrom., 8 (1994) 777. [10] G. Bojesen and T. Breindahl, J. Chem. Soc., Perkin Trans. 2, (1994) 1029. [11] Z. Wu and C. Fenselau, J. Am. Soc. Mass Spectrom., 3 (1992) 863. [12] Z. Wu and C. Fenselau, Tetrahedron, 49 (1993) 9197. [13] I.A. Kaltashov, D. Fabris and C. Fenselau, J. Phys. Chem., 99 (19951 in press. [14] G.S. Gorman and I.J. Amster, J. Am. Chem. Soc., 1t5 (1993) 5729. [15] J.W. McKiernan, C.E.A. Beltrame and C.J. Cassady. J. Am. Soc. Mass Spectrom., 5 (1994) 718. [16] S. Campbell, E.M. Murzluff, M.T. Rogers, J.L. Beauchamp, M.E. Rempe, K.F. Schwinck and D.I. Lichtenberger, J. Am. Chem. Soc., 116 (1994) 5257 [17] S.G. Lias, J.F. Liebman and R.D. Levin, J. Phys. Chem. Ref. Data, 13 (1984) 693. [18] R.H. Rice. G.E. Means and W.D. Brown, Biochim. Biophys. Acta, 492 (1976) 316. [19] B.R. Brooks, R,E. Bruccoleri, B.D. Olafson, D.J. States, S. Swaminathan and M. Karplus, J. Comput. Chem., 4 (1983) 187. [20] E.D. Raczynska, P.-C. Maria, J.-F. Gal and M. Decouzon, J. Phys. Org. Chem., 7 (1994) 725. [21] P.J. Robinson and K.A. Holbrook, Unimolecular Reactions, Wfley-Interscience, London, 1972.
ELSEVIER
and Ion Processes
International Journal of Mass Spectrometry and Ion Processes 146/147 (1995) 339- 347
The effect of N-alkylation on the gas phase basicities of small peptides measured by the kinetic method Igor A. K a l t a s h o v , Catherine C. Fenselau* Department o[' Chemistry and Biochemistry, University of Maryland Baltimore Count.v, 5401 Wilkens Ave., Baltimore. MD 21228, USA
Received 31 January 1995; accepted 13 March 1995
Abstract
Gas phase basicities for a series of N-alkylated peptides were found by the kinetic method using amines and guanidines as reference bases. The basicity value of one of these peptides was then remeasured using the rest of the derivatized peptides as reference bases. Both values appear to be in a good agreement. This suggests that structurally similar peptides (with unaltered topology of intramolecular hydrogen bonding) can be used as reference bases in the kinetic method experiments. Keywords: Biomolecules; Computer simulations; Gas phase basicities; Kinetic method; Metastable ions
of M and B [3,4]:
I. Introduction
The kinetic method, introduced almost two decades ago by Cooks and co-workers [1,2], has been proved to be a very valuable experimental technique in measurements of gas phase basicities/proton affinities. In this method, the process under study is the decomposition of a proton-bound dimer, formed by a c o m p o u n d of interest, M, and a reference base, B:
M . . . H +...B
kl MH + + B <~ k 2 M + BH +
(1) (2)
The relative abundances of MH + and BH + ions in a decomposition spectrum of the dimer reflect the difference in proton affinities (PAs)
ln([MH+]/[BH+]) = ln(kl/k:) = ln(QMH .... B/QBH+..M) + (PA(M) - P A ( B ) ) / R T e n -
(3)
where Teff represents the effective temperature of the dimer, and QMH~...B and QBH~...M represent the partition functions for activated complexes in reactions (1) and (2), respectively. Entropy (Q) and enthalpy (PA) terms in Eq. (3) can be combined to give the free energy term, gas phase basicity (GB). Expression (3) may then be rewritten simply as ln([MH+]/[BH+]) = ln(kl/k2)
~"~Dedicated to the memory of Professor Alfred O. Nier. * Corresponding author. 0168-1176/95/$09.50 © 1995 Elsevier Science B.V. All rights reserved S S D I 0168-1176(95)04196-6
=(GB(M) - GB(B))/RTef f
(4)
340
LA. Kaltashov, C.C. Fenselau/International Journal of Mass Spectrometry and Ion Processes 146/147 (1995) 339 347
The kinetic method is readily applicable to thermally liable, non-volatile compounds while consuming only small amounts of material. The use of this method has dramatically extended the range of compounds for which thermochemical data are now available. It has recently been applied to measurement of the gas phase basicities/proton affinities of biological compounds: nucleic acids [5,6], amino acids [7-10] and small peptides [11-13]. In recent years, a lot of interest has been drawn to studies of the effect of various structural characteristics of peptides on their gas phase basicities. The factors mentioned are amino acid composition of the peptide [14,15], peptide chain length [11], and position of the most basic amino acid residue in the peptide chain [12]. Also, some studies have been carried out to investigate the effect of the N-derivatization of amino acids on their gas phase basicities/proton affinities [16]. The results of the latter study indicate that Nmethylation of phenylalanine leads to a moderate increase in its proton affinity (only by about 1-2 kcal mol-1), as opposed to the dramatic increase in proton affinity in the series NH3, NHzCH3, NH(CH3)2, N(CH3)3 [17]. In the present, study we investigate the influence of N-alkylation of a pentapeptide FGGFL on its gas phase basicity. We have also found that the gas phase basicity increase in the series of N-alkylated peptides is enthalpic, rather than entropic.
2. Experimental
2.1. Materials The pentapeptides F G G F L and N-methylF G G F L were synthesized by the BioPolymer Laboratory (University of Maryland, Baltimore, MD, USA). Sodium borate and sodium borohydrate were purchased from Sigma (St. Louis, MO, USA). Reagent grade formalde-
hyde and acetaldehyde and organic reference bases di-sec-butylamine, N,N-diethylmethylamine, tripropylamine, N,N-di-isopropylethylamine, tributylamine, 1,1,3,3-tetramethylguanidine (TMG), 1,5-diazobicyclo[4.3.0]non5-ene (DBN) and 1,8-diazobicyclo [5.4.0]undec-7-ene (DBU) were purchased from Aldrich (Milwaukee, WI, USA). All chemicals were used without further purification.
2.2. Methods Three N-alkylated peptides (N-ethylFGGFL; N,N-dimethyl-FGGFL and N,Nethylmethyl-FGGFL) were synthesized using a procedure similar to that of Rice et al. [18]. In the present work, 50 #1 of 1 M sodium NaBH4 was added to a solution of peptide in 0.2 M sodium borate buffer, quickly followed by the addition of 50 #1 of 2.4 M formaldehyde or acetaldehyde. The additions of NaBH4 and acetaldehyde were repeated 5-10 times at 10 min time intervals. The sample was then acidified with acetic acid to pH < 4 and desalted using a Bakerbond SPE solid phase extraction column (J.T. Baker, Inc., Phillipsburg, NJ, USA). The reaction was monitored by HPLC and the products characterized using a Kratos Kompact MALDI III time-of-flight mass spectrometer (Kratos Analytical/Shimadzu Scientific Instruments, Inc., Columbia, MD, USA). All kinetic method measurements were performed on a JEOL (Tokyo, Japan) H X l l 0 / HX 110 four-sector mass spectrometer (EBEB geometry). Proton-bound dimers were generated in an FAB source using about 1 #l of sample dissolved in 0.1% aqueous TFA mixed with about 1 #1 of glycerol/monothioglycerol matrix. The proton-bound dimer formed by a compound of interest and a reference base was mass-selected by the first two sectors of the mass spectrometer. The products of the decomposition of the metastable dimer in the third field-free region were
I.A. Kaltashov, C.C. Fenselau/lnternational Journal o]" Mass Spectrometry and Ion Processes 146/147 (I995) 339 347
analyzed by the third (electric) sector (E2). The relative abundances of the decomposition products were deduced from the resulting M I K E spectra using the ratio of areas of fragment peaks. Normally, 10 scans were summed for each M I K E spectrum. All spectra were recorded using the JEOL MP7000 data system. Molecular dynamics/energy minimization studies were performed using the CHARMm22/ QUANTA4.0macromolecular modeling program [19] (Molecular Simulations, Inc., Waltham, MA, USA) on a Silicon Graphics workstation (Silicon Graphics, Inc., Mountain View, CA, USA).
3. Results and discussion
To determine the gas phase basicities of intact and N-alkylated pentapeptides FGGFL, the following set of structurally similar reference bases was used: di-sec-butylamine (GB = 223.6 kcal tool -l [17]), N,N-diethylmethylamine (GB = 222.2 kcal mol -L [17]), tripropylamine (GB = 226.8 kcal tool -1 [17]), N,N-di-isopropylethylamine (GB = 227.7 kcal tool -I [17]), tributylamine (GB = 228.2 kcal tool i [17]), T M G (GB = 234.8 kcal tool -l [20]), DBN (GB = 237.4 kcal tool -1 [20]) and DBU (GB = 239.6 kcal mol -I [20]). Fig. 1 shows typical M I K E spectra of the decompositions of metastable proton-bound dimers formed by F G G F L and its N-methyl analog with tributylamine as the reference base. For each peptide derivative, the ratio of intensities of the protonated reference peak (BH +) versus the protonated peptide peak (MH +) was calculated, and the logarithm of this ratio was plotted as a function of the gas phase basicities of reference bases (Fig. 2). The plot for each of the five peptides studied results in a straight line with a correlation coefficient r2>~ 0.990. The effective temperatures Teff of the metastable dimers, deduced from the
341
slopes of the graphs, are very close to each other for all the peptides in the series, and lie in the range 1025-1100 K. The interception of each graph with the zero point abscissa gives the value of gas phase basicity for each peptide: 232.3 kcal tool -~ for intact FGGFL, 233.9 kcal tool -~ for N-methyl-FGGFL, 235.0 kcal mo1-1 for N-ethyl-FGGFL, 235.8 kcal mol -I for N,N-dimethyl-FGGFL and 236.4 kcal mo1-1 for N,N-ethylmethylFGGFL. It can be seen from this series that the consecutive methylation of the pentapeptide increases its gas phase basicity by 1.6-1.9 kcal mol -l At the same time, N-ethylation leads to an increase in the gas phase basicity by 2.5-2.7 kcal mo1-1 . Most structural changes in molecules (peptides in particular) alter the intramolecular hydrogen bonding and thus the proton stabilization. This leads to a change in protonation entropies as well as effective temperatures of the dimers, which is why the set of reference bases used in kinetic method experiments has to consist of structurally similar molecules. Cheng et al. [4] showed that even protonation of rather small peptides (triglycines and tetraglycines) had a very large entropic effect due to intramolecular charge stabilization (self-solvation). Longer peptide chains favor the formation of intramolecular hydrogen bonds, so the protonation entropy term is different for peptides of different size (even if they have the same amino acid composition, e.g. Gly3 and Gly4) [4]). It has also been shown that varying the chain length may alter the effective temperature of metastable dimers [11,12]. A difference in amino acid composition also leads to a different peptide protonation entropy, since amino acids themselves vary in protonation entropy (e.g. lysine and histidine [9]). The structure of the peptides may also be altered by modification of their functional groups, such as alkylation of their N-terminals. As shown above, N-alkylation of peptides increases their gas phase basicity. At the
342
I.A. Kaltashov, C.C. Fenselau/International Journal
I00
of MassSpectrometry
and Ion Processes 1461147 (1995) 339-347 rx 1
_r
a
90
80
3
70
40
!il 2
60
20 I
1
0
50
I00
150
200
250
300
350
400
450
550
500
600
650
700
_*JZ
m
rx 1
10EJX5
90 -.
70 5’
50-
I!
40
30
II
I
186
. 150
200
250
300
350
400
450
Fig. 1. MIKE spectra of ions produced by metastable decomposition of proton-bound FGGFL (m/z 540), and (b) tributylamine and N-methyl-FGGFL (m/z 554).
503
550
600
650
dimers formed by (a) tributylamine
1
7oo m/z
(m/z 186) and
2!
I.A. Kaltashov, C.C. Fenselan/International Journal of Mass Spectrometry and Ion Processes 146/147 (1995/ 339 347
Ill
343
~i
ill
U
0 4.
.$.
-2 ~ !
-3
~
~% '
m
*= F G G F L • MeFGGFL • EtFGGFL • (Me)2FGGFL • (Et,Me)FGGFL
II
-4
=A
--1//// 222
224
~J 226
230
232 234 236 GB(B), k c a l / m o l
228
238
240
242
Fig. 2. Plot of the logarithms of the relative rates of metastable dissociation of proton-bound dimers formed by N-alkylated peptides and small organic reference bases (amines and guanidines) as a function of gas phase basicity.
same time, it is intuitively reasonable that the addition of rather small alkyl groups (such as methyl or ethyl) to the N-terminus of a peptide will have a minimal steric effect on the topology of the peptide in the gas phase. This hypothesis is supported by the results of molecular modeling studies of the protonated peptides F G G F L , N-methyl-FGGFL and N,Nethylmethyl-FGGFL. Calculations were carried using the CHARMmZ2empirical force field [19]. The structure of F G G F L was built in QUANTA4.0 using the CHARMm22 library of standard L-amino acid residues. This starting linear conformation was then energy minimized (method of steepest descent, 200 steps) to remove possible steric overlaps, followed by molecular dynamics. This included heating the molecule from 0 to 750 K over 1 ps (in 10 -3 ps time steps), equilibrating it at 750 K for 1 ps and then running the dynamics simulation at
750 K for 10 ps. The resulting structure was then energy minimized again (method of steepest descent, 5000 steps) to give a r.m.s. gradient below 0.1 kcal mo1-1. The final structure (see Fig. 3(a)) is cyclic and shows strong hydrogen bonding between the Nand C-terminals (dRo----- 1.67 /~, angleN_H_O ---- 138 °, angleR_o_c = 166°). Another rather strong hydrogen bond that stabilizes the "extra proton" is that between the N-terminal of the peptide and the Gly 2 carbonyl oxygen (dHo = 1.75 A, angleN_H_o = 154 °, angleH_o c -~ 121°). The only amine hydrogen atom not involved in hydrogen bonding was then replaced with a methyl group, thus giving a starting cyclic conformation of N-methyl-FGGFL. The molecular dynamics/energy minimization cycle was then repeated, again giving the cyclic structure (Fig. 3(b)), and showing a
344
I.A. Kaltashov, C.C. Fenselau/lnternational Journal of Mass Spectrometry and Ion Processes 146/147 (1995) 339-347
Phe I ~
(~
~
Leu5
a
Gly2 ~-J ~ k . )
"~ Gly3
Leu 5
Phe t
b
.(
~
Phe4
Gly3
L°u'
C
Fig. 3. Gas phase conformations of the peptides (a) FGGFL, (b) N-methyl-FGGFL, and (c) N,N-ethylmethyl-FGGFL, generated by CHARMm22. Dotted lines represent hydrogen bonds between the N- and C-terminals.
strong hydrogen bond between the N- and Cterminals (dHo = 1.81 ,~, angleN_H_o = 128 °, angleH_o_ c = 113°), and between the N-terminal and Gly 2 carboxyl oxygen (dHo = 1.74 A, angleN_H_o = 147 °, angleH_o_c = 116°). Interestingly, a cyclic structure also resulted from a starting linear conformation of Nmethyl-FGGFL, showing again a rather strong hydrogen bond between the N- and Cterminals (dHo = 1.81 A, angleN_H_O = 128 °, angleij_o_c = 113°), although no hydrogen bond between N-terminal and Gly 2 carboxyl oxygen was formed. The total free energies of these two conformations were essentially the same (-181.1 kcal mo1-1 and -182.7 kcal tool -1, respectively). The N-methyl group was then converted to an ethyl group and the remaining amine hydrogen atom (excluding that in the hydrogen bond) was replaced with a methyl group, giving a starting cyclic conformation of N,N-ethylmethyl-FGGFL. A molecular dynamics/energy minimization cycle did not disrupt the hydrogen bonding between the N- and C-terminals of the peptide (dHo = 1.72 •A, angleN_H_O---- 141 °, angleH_o_c---- 103°), yielding a cyclic conformation with total free energy -128.2 kcal mo1-1 (Fig. 3(c)). The dynamics/minimization cycle was repeated four times, always yielding the cyclic conformation (with total free energies in the range 123-130 kcal mol-1). The linear starting conformation of N,N-ethylmethyl-FGGFL did not yield, however, the cyclic structure. Nevertheless, this linear conformation appeared to be energetically much less favorable (only -108.9 kcal mol -l) than the cyclic one. It appears that interterminal hydrogen bond formation plays a key role in the stabilization of charges (protons) in the series of peptides studied, providing very close similarity in the folding topology. Therefore, it is reasonable to assume that the entropy of protonation (as well as effective temperatures of metastable dimers) should be similar for all peptides in the series. If these assumptions are
I.A. Kaltashov, C.C. Fenselau/International Journal of Mass Spectrometry and Ion Processes 146/147 (1995J 339 347
345
Fxl
100-
BO80 ~
?0 5G8
~
60
~
50-
.,J4
40
554 20
500
550
G00
650
700
750
800
850
900
950
1000
1050
II~
m/z
Fig. 4. MIKE spectrum of ions produced by a metastable decomposition of the proton-bound dimer formed by N-methyI-FGGFL (m/z 554) and N,N-dimethyl-FGGFL (m/z 568).
correct, one should be able to use a set of Nalkylated peptides as reference bases in kinetic method measurements. To test this hypothesis experimentally, we attempted to verify the value of the gas phase basicity of N-methyl-FGGFL using the GB values of FGGFL, N-ethyl-FGGFL, N , N dimethyl-FGGFL and N,N-ethylmethylF G G F L found in earlier experiments (with amines and guanidines as reference bases). Fig. 4 shows a typical M I K E spectrum of the decomposition of a metastable proton-bound dimer formed by two peptides. The logarithms of the ratios of the intensity of protonated Nmethyl-FGGFL (MH +) to the intensities of other peptides in the series (BH +) were plotted against the gas phase basicities of these peptides found in the earlier measurements (Fig. 5). The plot results in a straight line with a correlation coefficient r 2 = 0.989.
The value of the gas phase basicity of Nmethyl-FGGFL found in this experiment is 234.4 kcal mo1-1 . This is 0.5 kcal mo1-1 higher than that found using small organic reference bases (see the second plot in Fig. 4). This approach appears to provide values with accuracy comparable to those in the literature for many small organic reference bases [17]. Another interesting observation is the striking difference in effective temperatures Terr between the dimers formed by two peptides (~ 575 K) and those formed by a peptide and a small organic reference base (1025-1100 K). The effective temperatures of metastable ions are believed to reflect both the instrumental parameters [2] and the amount of internal energy possessed by the dimers [11,12]. The lifetime 7- of a metastable ion with internal energy E can be very roughly approximated by Kassel's equation for s
346
I.A. Kahashov, C.C. Fenselau/International Journal of Mass Spectrometry and Ion Processes 146/147 (1995) 339-347
[] F G G F L • +
amine8
derivatives and
guanidines
0
"~'-1 ra -2
-3 -4
/
-5
222
224
226
228
230
232
234
236
238
240
242
GB(B), keal/mol Fig. 5. Plot of the logarithms of the relative rates of metastable dissociation of proton-bound dimers formed by N-methyl-FGGFL and the four other N-alkylated peptides in the series, and by N-methyI-FGGFL and four small organic reference bases (amines and guanidines) as functions of gas phase basicity.
classical oscillators [21]:
7- -= 1/k(E) -- A(1 - Eo/E) -(s-l)
(5)
where E0 is the dissociation threshold and A is a constant. For the decomposition of a metastable ion to be observed, its lifetime 7should be close to its flight time from the ion source to the third field-free region of the mass spectrometer, which can be estimated as t = L ( m / 2 e V ) 1/2, where L is the flight path, m is the mass of the metastable ion, and eV is its kinetic energy. The estimation of the internal energy of the metastable ions may then be made by equating t and 7-. In a case where a reference base is changed from a small organic molecule to a peptide in a metastable dimer, the values of both m and s values would increase. This should lead
then to a decrease in the internal energy E of a dimer. In addition, the overall internal energy distribution of peptide/amine dimers might be different from that of peptide/peptide dimers, which could affect the effective temperatures.
4. Conclusions Gas phase basicities for a series of N-alkylated peptides were found using the kinetic method: 232.3 kcal mol -l for intact FGGFL, 233.9 kcal mol -Z for N-methyl-FGGFL, 235.0 kcal mol -l for N-ethyl-FGGFL, 235.8 kcal mo1-1 for N,N-dimethyl-FGGFL and 236.4 kcal mo1-1 for N,N-ethylmethyl-FGGFL. Molecular modeling studies indicate that the
I.A. Kahashov, C.C. Fenselau/lnternational Journal o['Mass Spectrometry and Ion Processes 146,'147 { 1995) 339 347
topology of charge stabilization remains essentially unaltered in a set of N-alkylated peptides. This suggests that the entropies of protonation are close to each other, so that it is possible to use these peptides as reference bases in the kinetic method studies. A second set of kinetic method measurements was then carried out using peptides as reference bases to verify the gas phase basicity of N-methyl-FGGFL. The GB value found in this experiment is in a good agreement with that found when small organic reference bases (amines and guanidines) were used. This suggests that structurally similar peptides (i.e. peptides with similar topology of intramolecular hydrogen bonding) can be used as reference bases in gas phase basicity measurements by the kinetic method. Acknowledgments The authors are very grateful to Dr. Martha M. Vestling, Dr. Yetrib Hathout and Dr. Daniele Fabris (University of Maryland Baltimore County) for very helpful discussions. This work was supported by a grant from the National Science Foundation. References [I] R.G. Cooks and T.L. Kruger, J. Am. Chem. Soc., 99 (1977) 1279.
347
[2] S.A. McLuckey, D. Cameron and R.G. Cooks, J. Am. Chem. Soc., 103 (1981) 1313. [3] S.A. McLuckey, R.G. Cooks and J.E. Fulford, Int. J. Mass Spectrom. Ion Phys., 52 (1983) 165. [4] X. Cheng, Z. Wu and C. Fenselau. J. Am. Chem. Soc., 115 (1993) 4844. [5] F. Greco, A. Liguori, G. Sindona and N. Ucella, J. Am. Chem. Soc., 112 (1990) 9092. [6] A. Liguori, A. Napoli and G. Sindona, Rapid Commun. Mass Spectrom., 8 (1994) 89. [7] R.A.J. O'Hair, J.H. Bowie and S. Gronert, Int. J. Mass Spectrom. Ion Processes, 117 (1992) 23. [8] Z. Wu and C. Fenselau, Rapid Commun. Mass Spectrom., 6 (t992) 403. [9] Z. Wu and C. Fenselau, Rapid Commun. Mass Spectrom., 8 (1994) 777. [10] G. Bojesen and T. Breindahl, J. Chem. Soc., Perkin Trans. 2, (1994) 1029. [11] Z. Wu and C. Fenselau, J. Am. Soc. Mass Spectrom., 3 (1992) 863. [12] Z. Wu and C. Fenselau, Tetrahedron, 49 (1993) 9197. [13] I.A. Kaltashov, D. Fabris and C. Fenselau, J. Phys. Chem., 99 (19951 in press. [14] G.S. Gorman and I.J. Amster, J. Am. Chem. Soc., 1t5 (1993) 5729. [15] J.W. McKiernan, C.E.A. Beltrame and C.J. Cassady. J. Am. Soc. Mass Spectrom., 5 (1994) 718. [16] S. Campbell, E.M. Murzluff, M.T. Rogers, J.L. Beauchamp, M.E. Rempe, K.F. Schwinck and D.I. Lichtenberger, J. Am. Chem. Soc., 116 (1994) 5257 [17] S.G. Lias, J.F. Liebman and R.D. Levin, J. Phys. Chem. Ref. Data, 13 (1984) 693. [18] R.H. Rice. G.E. Means and W.D. Brown, Biochim. Biophys. Acta, 492 (1976) 316. [19] B.R. Brooks, R,E. Bruccoleri, B.D. Olafson, D.J. States, S. Swaminathan and M. Karplus, J. Comput. Chem., 4 (1983) 187. [20] E.D. Raczynska, P.-C. Maria, J.-F. Gal and M. Decouzon, J. Phys. Org. Chem., 7 (1994) 725. [21] P.J. Robinson and K.A. Holbrook, Unimolecular Reactions, Wfley-Interscience, London, 1972.