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Determination of intrinsic hydrophilicity/hydrophobicity coefficients of amino acid side-chains using synthetic model peptides

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Title:
Determination of intrinsic hydrophilicity/hydrophobicity coefficients of amino acid side-chains using synthetic model peptides
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Kovacs, James Monroe
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English
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xiii, 152 leaves : ; 28 cm

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Subjects / Keywords:
Amino acids ( lcsh )
Peptides -- Separation ( lcsh )
High performance liquid chromatography ( lcsh )
Amino acids ( fast )
High performance liquid chromatography ( fast )
Peptides -- Separation ( fast )
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bibliography ( marcgt )
theses ( marcgt )
non-fiction ( marcgt )

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Bibliography:
Includes bibliographical references (leaves 146-152).
Thesis:
Department of Chemistry
Statement of Responsibility:
by James Monroe Kovacs.

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|University of Colorado Denver
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ocm62777068
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Full Text
DETERMINATION OF INTRINSIC HYDROPHILICITY/HYDROPHOBICITY
COEFFICIENTS OF AMINO ACID SIDE-CHAINS USING SYNTHETIC MODEL
PEPTIDES
by
James Monroe Kovacs
B.A., Goshen College, 2002
A thesis submitted to the
University of Colorado at Denver
in partial fulfillment
of the requirements for the degree of
Master of Science
Chemistry
2005


This thesis for the Master of Science
degree by
James Monroe Kovacs
has been approved
by
Douglas F. Dyckes


Kovacs, James Monroe (M.S., Chemistry)
DETERMINATION OF INTRINSIC HYDROPHILICITY/HYDROPHOBICITY
COEFFICIENTS OF AMINO AICD SIDE-CHAINS USING SYNTHETIC MODEL
PEPTIDES
Thesis directed by Professor Douglas F. Dyckes
ABSTRACT
It was the aim of this research to determine a set of coefficients representing the
intrinsic hydrophilicity/hydrophobicity of amino acid side-chains. Reversed-phase
high-performance liquid chromatography (RP-HPLC) was carried out on synthetic
model peptides to determine intrinsic coefficients. Peptide design is such that no
nearest-neighbor effects or restriction of conformational space affect the substituted
residue, which would inhibit the substituted residue from expressing its intrinsic
hydrophilicity/hydrophobicity. These intrinsic coefficients were determined at three
pH values and six mobile phase conditions. All amino acid side-chains, except the
potentially charged side-chains, were unaffected by pH, mobile phase conditions and
stationary phase functionality. In addition to determining a new
hydrophilicity/hydrophobicity scale, a quantitative measure of nearest-neighbor effect
was determined by RP-HPLC by substitution of D- and L- amino acids adjacent to a
leucine residue, a bulky hydrophobic residue. Again, as with the intrinsic
coefficients, nearest-neighbor effects are nearly the same across differing pH values,
m


mobile phase conditions and stationary phase functionality. Finally, in order to
substantiate the newly determined intrinsic hydrophilicity/hydrophobicity scale, it
was compared against seventeen published normalized coefficient scales.
This abstract accurately represents the content of the candidates thesis. I recommend
its publication.
Signed
IV


ACKNOWLEDGEMENT
I would like to acknowledge the people who have helped me with this
research. First and foremost I would like to acknowledge my advisor, Robert S.
Hodges, for his willingness to allow me to work in his lab and the countless hours of
knowledge sharing, discussions and direction. I would also like to thank members of
the Hodges Lab, both past and present. To Anthony Mehok for teaching me solid
phase peptide synthesis. To Brian Tripet for many challenging discussions. To Colin
Mant for hours of education and editing help.
Last, but most certainly not least, I would like to thank my parents, Charles
and Leora Kovacs, for their unconditional support and encouragement through all the
years of my life and education.


CONTENTS
Figures....................................................................x
Tables .;......<..........................................................xiii
Chapter
1. General Introduction High Performance Liquid Chromatogrhapy...........1
1.1 Reversed-Phase High Performance Chromatography Introduction..........3
1.2 RP-HPLC: Stationary Phase..............................................4
1.3 RP-HPLC: Mobile Phase..................................................5
1.4 RP-HPLC: Preparative Chromatography ...................................7
1.5 Goals Of This Research............................................. 8
2. Experimental...........................................................11
2.1 Materials.............................................................11
2.2 Methods..............................................................11
2.2.1 Peptide Synthesis and Cleavage......................................11
2.2.2 Purification of Crude Peptides .....................................15
2.2.3 Instrumentation.....................................................16
2.2.4 RP-HPLC Analysis of Synthetic Model Peptides .......................16
2.2.5 Mass-Spectrometry...................................................17
2.2.6 Normalization of Hydrophilicity/Hydrophobicity Scales ..............17
VI


3. Determination of Intrinsic Hydrophilicity/Hydrophobicity of Amino Acid
Side-Chains in Peptides in The Absence of Nearest-Neighbor
or Conformational Effects ....................................................18
3.1 Introduction..............................................................18
3.2 Experimental:...........:.................................................22
3.2.1 General Methods........................................................ 22
3.2.2 Analytical RP-HPLC of Synthetic Model Peptides...........................22
3.3 Results...................................................................23
3.3.1 Design of Model Peptides ................................................23
3.3.2 Elimination of Nearest-Neighbor Effects..................................25
3.3.3 RP-HPLC Retention Behavior of Model Peptides At pH 2, pH 5
and pH 7..............:.................................................31
3.3.4 Correlation of RP-HPLC Retention Behavior of Peptides under
Different Mobile Phase Conditions........................................35
3.3.5 Amino Acid Side-Chain Hydrophilicity/Hydrophobicity Coefficients.........45
3.3.5.1 Isosteric Side-Chains: n-Leu, Leu, lie.................................46
3.3.5.2 Carbon Atom Extension of Side-Chains: Non-Polar Groups
(Gly/Ala, Val/Ile, n-Val/n-Leu).........................................49
3.3.5.3 Carbon Atom Extension of Side-Chains: Polar And Charged
Groups (Asn/Gln, Asp/Glu, Om/Lys).......................................49
3.3.5.4 Carbon Atom Extension of Side-Chains: Addition of
/3-Branch Methyl Group (Ser/Thr)........................................51
3.4 Effect of pH and Mobile Phase Composition on
Hydrophilicity/Hydrophobicity of Amino Acid Side-Chains....................51
vii


3.4.1 Uncharged Side-Chains..................................................51
3.4.2 Potentially Charged Side-Chains........................................54
3.5 Conclusions............................................................ 61
4. Quantitative Determination of Nearest-Neighbor Effects in Peptides
by Reversed-Phase Liquid Chromatography of Synthetic Model Peptides
with D- and L- Amino Acid Substitutions ..................................62
4.1 Introduction....................................:.......................62
4.2 Experimental............................................................66
4.2.1 General Experimental................................................. 66
4.2.2 Analytical RP-HPLC of Synthetic Model Peptides.........................66
4.3 Results.................................................................67
4.3.1 Design of Model Peptides...............................................67
4.3.2 Nearest-Neighbor Effects on RP-HPLC Retention
Behavior of Model Peptides........................................... 69
4.3.2.1 Effect of Mobile Phase Conditions on Retention Behavior of Peptides .72
4.3.2.2 Evaluation of Nearest-Neighbor Effects by RP-HPLC
of Model Peptides.....................................................77
4.3.2.3 Hydrophilicity/Hydrophobicity Coefficients of D- and L-
Amino Acids in Presence of Nearest-Neighbor Effects ..................83
4.4 Conclusions.............................................................92
5. Intrinsic Amino Acid Side-Chain Hydrophilicity/Hydrophobicity
Coefficients Determined by Reversed-Phase Chromatography of Model
Peptides: Comparison with Other Hydrophilicity/Hydrophobicity
Scales.....................................................................93
viii
5.1 Introduction
93


5.2 Normalization of Hydrophilicity/Hydrophobicity Scales....................95
5.3 Results and Discussion ..................................................96
5.3.1 Criteria for Expectations of a Hydrophilicity/Hydrophobicity Scale......96
5.3.2 Comparison of Amino Acid Side-Chain Hydrophilicity/Hydrophobicity
Scales Generated from RP-HPLC of Peptide Mixtures ......................97
5.3.2.1 Random Coil Peptides..................................................98
5.3.2.2 a-Helical Peptides.................................................. Ill
5.3.3 Overall Correlation and Evaluation of Side-Chain
Hydrophilicty/Hydrophobicity Scales....................................116
5.3.3.1 Random Coil Peptides.............................................. 116
5.3.3.2 a-Helical Peptides...................................................124
5.3.3.3 Correlation of Intrinsic Coefficients with Those Derived
from Non-Peptide Models...............................................136
5.4 Conclusions.............................................................141
6. Summary And Future Directions.......................................... 142
6.1 Summary.................................................................142
6.2 Future Directions.................................................... 144
References...................................................................146
ix


FIGURES
2.1 Scheme of solid phase synthesis using standard Fmoc chemistry...............12
2.2 Activation of Fmoc amino acid using a phosphonium reagent.................13
2.3 Deprotection and cleavage scheme using standard Fmoc chemistry............14
3.1 Elimination of nearest-neighbor effects to determine intrinsic
hydrophilicity/hydrophobicity coefficients..................................27
3.2 Effect of gradient rate on RP-HPLC elution profile of diastereomeric
peptide pairs at pH 2.0....................................................30
3.3 Plot of tR of peptides in 20 mM TFA mobile phase system versus
tR in 20 mM H3PO4 mobile phase system......................................36
3.4 Plot of tR of peptides in 20 mM H3PO4 (pH 2) mobile phase system
versus tR in 10 mM NaH2PC>4 (pH 7; no salt) mobile phase system............38
3.5 RP-HPLC elution profiles of peptide mixtures at pH 2 (top) and
pH 7 (bottom).......................................................... 40
3.6 Plot of tR of peptides in 10 mM NaHaPC>4 (pH 5) mobile phase
system versus 10 mM NaH2P04 (pH 7) mobile phase system.....................43
3.7 Plot of tR of peptides in 10 mM NaH2P04 (pH7) mobile phase system
versus 10 mM NaH2P04 (pH 7) containing 50 mM NaCl (A) or
50 mM NaClC>4 (B) mobile phase systems.....................................44
3.8 Correlation of hydrophilicity/hydrophobicity coefficients
(AtR(Giy)) obtained at pH 2, pH 5 and pH 7.................................53
3.9 Effect of mobile phase conditions on magnitude of
hydrophilicity/hydrophobicity coefficients of potentially charged
acidic (D,E) and basic (0,K,R,H) residues...................................55
x


3.10 Relationship between peptide net charge and effect of mobile
phase counterion at pH 2 (TFA anion; Panel A) and pH 7
(Cl", CIO4' anions; Panel B)..............................................57
3.11 Effect of pH on the magnitude of hydrophilicity/hydrophobicity
coefficients of potential positively charged residues.....................60
4.1 Demonstration of nearest-neighbor effects in a model peptide system.......70
4.2 Plot of AtRp.L) of peptides in 20 mM H3PO4 (pH 2) versus
20 mM TFA (pH 2) (A) and 10 mM NaH2P04 (pH 7) versus
10 mM NaH2P04 (pH 7) with 50 mM NaCl or 50 mM NaC104 (B)...................82
4.3 Plot of hydrophilicity/hydrophobicity coefficients (AtR(Giy>)
determined at pH 2,20 mM H3PO4 versus pH 7 10 mM NaH2PC>4
plus 50 mM NaC104; panel A, L- diastereomers;
panel B, D- diastereomers...................................................87
4.4 Plot of hydrophilicity/hydrophobicity coefficients (AtR(Giy))
determined at pH 2, comparing D- versus L- diastereomeric pairs............90
4.5 Plot of hydrophilicity/hydrophobicity coefficients (AtR(Giy))
determined at pH 7, comparing D- versus L- diastereomeric pairs............91
5.1 Peptide sequences used to determine
hydrophilicity/hydrophobicity scales........................................99
5.2 Helical nets of model alpha-helical synthetic peptides
used to determine hydrophilicity/hydrophobicity scales.....................113
5.3 Plot of normalized hydrophilicity/hydrophobicity coefficients
determined by Meek and Meek & Rossetti versus Kovacs et al. at pH 2........118
5.4 Plot of normalized hydrophilicity/hydrophobicity coefficients
determined by Guo et al. and Browne et al. versus Kovacs et al. at pH 2....120
5.5 Plot of normalized hydrophilicity/hydrophobicity coefficients
determined by Wilce et al. versus Kovacs et al. at pH 2....................121
5.6 Plot of normalized hydrophilicity/hydrophobicity coefficients
determined by Kovacs et al. in different mobile phases at pH 7.............123
XI


5.7 Plot of normalized hydrophilicity/hydrophobicity coefficients
determined by Meek and Guo et al. versus Kovacs et al. at pH 7............125
5.8 Plot of normalized hydrophilicity/hydrophobicity coefficients
determined by Sereda et al. versus Kovacs et al. at pH 2 .................128
5.9 Plot of normalized hydrophilicity/hydrophobicity coefficients
determined by Liu & Deber versus Kovacs et al. and
Liu & Deber versus Sereda et al. at pH 2..................................130
5.10 Plot of normalized hydrophilicity/hydrophobicity coefficients
determined by Sereda etal. (pH 2) versus Monera et al. (pH7).............132
5.11 Plot of normalized hydrophilicity/hydrophobicity coefficients
determined by Monera et al. Tripet et al. versus Kovacs et al. at pH 7...134
5.12 Plot of normalized hydrophilicity/hydrophobicity coefficients
determined by Monera et al. versus Tripet et al. at pH 7.................135
5.13 Plot of normalized hydrophilicity/hydrophobicity coefficients
determined by Fauchere & Pliska and Eisenberg & McLachlan
versus Kovacs et al. at pH 7.............................................137
5.14 Plot of normalized hydrophilicity/hydrophobicity coefficients
determined by Abraham & Leo and Bull & Breese versus
Kovacs et al. at pH 7....................................................140
xii


TABLES
3.1 RP-HPLC peptide retention time data in 20 mM H3PO4, pH 2 at 25C...........29
3.2 RP-HPLC peptide retention time data in various mobile phases at 25C.......32
3.3 Hydrophilicity/hydrophobicity coefficients determined at 25C
by RP-HPLC of model peptides............................................... 48
4.1 RP-HPLC peptide retention data in various mobile phase conditions
at 25C.................................................................... 71
4.2 Effects of mobile phase conditions........................................74
4.3 Nearest neighbor effects determined at 25C by RP-HPLC of
model peptides...............................................................78
; £
4.4 RP-HPLC hydrophilicity/hydrophobicity coefficients in various
mobile phases at 25C.................................................... 85
5.1 Twenty normalized hydrophilicity/hydrophobicity scales
determined by various methods.............................................. 100
xiii


1. General Introduction High-Performance Liquid
Chromatography
It should be noted that at the beginning of each results chapter (chapters 3,4
and 5) a more specific introduction will be included. This general introduction will
serve as an introduction to high-performance liquid chromatography and the multiple
facets in which it is employed. While the introductions in each results chapter serve
to give background and introduce more specifically the topics relevant to the material
presented.
Since its inception about four decades ago, high-performance liquid
chromatography (HPLC) has been widely used as a separation tool for peptides and
proteins. HPLC is much more useful than early forms of chromatography due to its
increased throughput, high resolving power and reproducibility. Early forms of
chromatography utilized large particle packings in glass columns and solutes were
eluted with large volumes of mobile phase, reducing throughput, resolution and
reproducibility. These large columns were hand packed, allowing for large variances
in column-to-column reproducibility. The large, generally agarose-based, particles
used in classical chromatography are not resistant to high pressures and subsequently
only very slow mobile phase flow-rates can be employed, leading to long elution
times. In addition, the large particle sizes also result in poor solute resolution. It
1


should also be noted that such materials are unsuited for peptide separations. The
materials used in HPLC are of much smaller particle sizes (generally based on porous
silica), requiring high performance pumping systems to force mobile phase through a
column packed with such particles. These particles are resistant to the resulting high
pressures produced by the flow of mobile phase, hence the early term high-pressure
liquid chromatography for this chromatographic approach. The high pressure
produced in HPLC also require the particles (or packings) to be packed into
stainless steel cylinders rather than glass. The small particle size of HPLC packings,
coupled with high mobile phase flow rates, has produced a tremendous improvement
in efficiency of solute resolution compared to traditional chromatography. In
addition, since the overall physical size of the HPLC column is much smaller, sample
sizes are also much smaller and are better resolved due to the decrease in eluent
volume used. The packing material or stationary phase, used in HPLC is, as noted
above, generally silica gel or a derivatized silica gel. Today the general trend is a
reduction in particle size to increase the efficiency of the chromatography. Based on
differing column packings, HPLC techniques are separated into various types,
including reversed-phase chromatography (RP-HPLC), size exclusion
chromatography and ion-exchange chromatography. This introduction and following
thesis will focus on RP-HPLC, an HPLC mode which has no precedent in classical
chromatography and which has revolutionized the ability to carry out efficient peptide
separations.
2


HPLC can be applied to many biological molecules, including peptides and
proteins [1]. Peptide or protein samples are generally detected by UV detection at
210 nm, where the peptide bond absorbs. The peptide bond ranges in absorbance
from approximately 200 to 230 nm; however, the absorbance is measured at 210 nm
due to the UV cutoff of the mobile phase used in HPLC of peptides and proteins [2].
Specific amino acids (phenylalanine, tyrosine and tryptophan) may also be detected at
260 nm-280 nm due to their side chains containing aromatic groups.
1.1 Reversed-Phase High Performance
Chromatography Introduction
RP-HPLC is the most widely used technique used in the analysis and
separation of peptides and proteins [1]. RP-HPLC is used in a wide variety of
separation and analysis roles due to the wide range of mobile phase and stationary
phase choices and combinations. In this thesis, RP-HPLC was the mode of
chromatography used to study the hydrophilicity/hydrophobicity of amino acids and
for the preparation of crude synthetic peptides. The defining characteristics of RP-
HPLC are that of a non-polar stationary phase and an increasingly non-polar aqueous
mobile phase. Thus, solutes are eluted from this hydrophobic stationary phase in
order of increasing solute hydrophobicity based on increasing organic modifier
concentration in the mobile phase.
3


1.2 RP-HPLC: Stationary Phase
As mentioned above, RP-HPLC uses non-polar, hydrophobic stationary
phases. Most commercial reversed-phase columns for peptide separations are silica-
based. This is due to the high stability and high efficiencies of silica-based packings.
Though most manufacturing processes are proprietary, the general reaction scheme
for derivatizing the silica packing is to react the free hydroxyl groups (silanols) on the
surface of the silica with a silane that contains an hydrophobic ligand. It is well
known that silica-based reversed-phase columns are not stable at alkaline pH, due to
dissolution of the silica at pH values above neutral. Such drawbacks of silica-based
columns have been overcome with the advent of more thermal and chemically stable
silica packings. In this thesis, the neutral pH studies were conducted using an extra
dense bonding of C8 alkyl chains (XDB reversed-phase column). This column was
designed to shield the silica support from dissolution by neutral mobile phase
systems. A wide variety of bonded phases are used in peptide separations by RP-
HPLC, the most common being C4, C8 and Cl 8, where the number represents the
number of methylene groups in the alkyl chain of the bonded silane. Another
parameter that is considered when choosing a stationary phase is that of particle size.
It is known that peptide separation improves as particle size decreases [1]. Most
commercial columns are in the order of 5-8 micron particle size; smaller particle sizes
are available but do not give a beneficial degree of increased peptide separation in
4


trade for higher back pressures and concerns about column clogging [3]. In
accordance with the above considerations, the columns used in this thesis are a C8
and Cl 8 column with a pore size of 80-100 angstroms and a particle size of 5
microns.
1.3 RP-HPLC: Mobile Phase
The mobile phase refers to the solvent system with which solutes are eluted
from the column. Mobile phase characteristics can have large effects on peptide
separations; these include mobile phase pH, solvent composition, flow rate and
gradient rate. When choosing a mobile phase system, it is important to optimize these
four parameters to ensure the best separation, resolution and meaningful results.
Since RP-HPLC allows high resolution at acidic and neutral pH, pH values of 2, 5
and 7 were used in this research. Before the current availability of RP-HPLC
packings stable at neutral pH, the vast majority of researchers used acidic pH mobile
phase systems since reversed-phase columns are generally more stable at low pH [1].
Indeed, such acidic mobile phases are still employed for the majority of peptide
separations. Lower pH Systems also suppress undesirable ionic interactions (due to
negatively charged, underivatized silanol groups) with basic residues. Solutes in RP-
HPLC are eluted based on the order of increasing solute hydrophobicity. Solutes bind
to the stationary phase based on hydrophobic interactions between the Solute and the
hydrophobic stationary phase; the aqueous mobile phase is thus made increasingly
5


non-polar through the addition of a water-miscible organic solvent, most commonly
acetonitrile, which is more non-polar than water. The addition of organic eluents may
be introduced in two ways; one being that of isocratic elution and the other is by
gradient elution, the latter being by far the most common elution method for peptides.
Isocratic elution is carried out by adding a set amount of organic solvent at time zero
and not varying the percentage over the duration of the run. Gradient elution is
carried out by a gradual addition of organic solvent over the duration of the run, this
gradient being expressed as a percentage of organic solvent per minute. Unlike small
organic molecules, peptides display mainly an adsorption/desorption (on/off) mode
of interaction with the stationary phase, and partition very little. Due to the wide
range of hydrophobicities in peptide mixtures, it is hard to optimize an isocratic
elution for separation. Thus, peptides can be separated isocratically only over a very
narrow range of acetonitrile concentrations due to their much narrower partitioning
windows [4]. Taking this knowledge into account, gradient elution was utilized in
both preparative and analytical RP-HPLC. Peptides are initially loaded onto the
column in the absence of or in the presence of a low concentration of aqueous organic
modifier. As the concentration of the organic solvent is increased, mixture
components are eluted from the column in order of increasing peptide
hydrophobicity. A decrease in gradient rate improves the separation of components,
albeit at the expense of some peak broadening. Optimization of gradient rate is
dependent on the types of mixtures that are being separated.
6


The mobile phase solvents used in RP-HPLC consist of water, non-polar
water-miscible solvent and mobile phase additives. Trifluroacetic acid (TFA) is the
most widely used mobile phase additive in RP-HPLC due to its volatility and UV
transparency along with the hydrophobic trifluoroacetate anion (more than that of
phosphate), which is able to interact with positively charged residues, resulting in
improved solute resolution [1]. An added benefit of using TEA is that, at the
resulting low pH (~pH 2), the positively charged residues are more able to interact
with the mobile phase; also, ionization Of any free silanols is suppressed at this low
pH, thus avoiding any undesirable electrostatic interactions with positively charged
peptide groups. At higher pH values, such silanol groups will interact with the
positively charged residues (positively charged side-chains or free a-amino group)
and salts such as sodium perchlorate (NaClO/t) are added to neutralize the effect of
such free silanol interactions [1]. Recently in our laboratory, it has been shown that
the perchlorate anion is more effective than the trifluroacetate anion as an ion-pairing
reagent for RP-HPLC of peptides [5]. NaC104 is also desirable because of its
relatively high solubility in aqueous solutions of organic solvents used in RP-HPLC
[6].
1.4 RP-HPLC: Preparative Chromatography
The previous sections have dealt with analytical RP-HPLC whereas this
section will deal with preparative scale RP-HPLC to purify crude synthetic peptide
7


samples. In purifying crude synthetic peptide samples, a shallow gradient is normally
optimal. By using a shallow gradient, it is possible to separate out closely-related
peptide impurities produced during solid phase peptide synthesis. Generally,
preparative chromatography is carried out using larger columns than analytical scale
columns, where analytical columns are generally 2.1 mm internal diameter (I.D.). By
using a larger diameter column (e.g., 4.6 mm I.D.) it is possible to inject a large
sample load. During preparative RP-HPLC runs, fractions were collected in order to
detect the desired peptide product. Fraction analysis was run using an analytical scale
HPLC instrument, followed by pooling and lyophilization of pure peptide fractions.
1.5 Goals Of This Research .
It is the aim of this research and thesis to determine the intrinsic
hydrophilicity/hydrophobicity coefficients of amino acid side chains. Intrinsic
coefficients are those that represent the actual hydrophihcity/hydrophobicity values of
amino acid side chains in the absence of any interactions which may diminish the full
expression of such values. Over one hundred such scales have been determined [7].
Previously published scales were determined by many different means, each having
possible inherent errors associated with the coefficients [8]. By designing a model
peptide, it is possible to eliminate such errors. Factors which may be eliminated by
constructing a model peptide are nearest neighbor effects and restriction of
conformational freedom about the peptide bond, thus giving an equal representation
8


to each amino acid residue. Nearest neighbor effects have to be eliminated because
each amino acid residue has the potential to interact with its neighbors, thus either
increasing or decreasing the observed (apparent) hydrophilicity/hydrophobicity of
the amino acid residue; therefore, the true intrinsic hydrophilicity/hydrophobicity of
the amino acid residue is masked and the determined value is either falsely inflated or
under-represented. Similarly, the restriction of conformational freedom about the
peptide bond may distort intrinsic hydrophilicity/hydrophobicity coefficients. For
instance if one side chain restricts the ability of the adjacent side chain to be fully
exposed to the reversed-phase matrix, the adjacent side chain may not be able to
express fully its intrinsic hydrophilicity/hydrophobicity. This restriction of
conformational freedom can be overcome by designing a model peptide where the
substitution site (where all twenty naturally occurring amino acid residues will be
substituted) is adjacent to a glycine residue, thereby allowing no restriction of
rotational freedom between \p of residue i (substitution site) and of residue i +1,
where refers to rotations about the C^-C single bond and refers to rotations about
the CcrN single bond, thus allowing the substituted residue to express fully its
intrinsic hydrophilicity/hydrophobicity. To assure equal representation of each amino
acid, our model peptide system consisted of twenty peptides of the same sequence
except for the amino acid variation at the substitution site. At the substitution site,
each of the twenty naturally occurring amino acid residues were substituted, resulting
in twenty peptides with an equal representation of all twenty amino acid residues.
9


Since in nature not all amino acid residues are equally represented [9], this model
system overcomes the possible errors in using native peptides.
In order to validate the new hydrophilicity/hydrophobicity scale determined
by RP-HPLC of our model peptides, it is necessary to compare this new scale with
previously reported scales. Thus, the new scale will be compared to many existing
and widely used scales to determine the validity of the new scale. When the scale has
been compared and validated it will be useful in predictive studies in the field of
proteomics.
10


2. Experimental
2.1 Materials
All chemicals and solvents used were reagent grade unless otherwise
specified. Phosphoric acid was obtained from Caledon Laboratories (Georgetown,
Ontario, Canada). TFA was obtained from Hydrocarbon Products (River Edge, NJ,
USA); NaCl and NaC104 were obtained from Sigma-Aldrich (St. Louis, MO, USA).
HPLC grade acetonitrile was obtained from Fisher Scientific (Pittsburgh, PA, USA).
The water used throughout the experiments was de-ionized water purified by an E-
pure water filtration device from Bamstead/Thermolyne (Dubuque, LA).
Fluoroenyloxymethylcarbonyl (Fmoc) amino acids and resins were purchased from
Novabiochem (San Diego, CA).
2.2 Methods
2.2.1 Peptide Synthesis and Cleavage
Peptides were synthesized using solid-phase peptide synthesis methodology
usinga4-(2,4-dimethoxyphenyl-Fmoc-aminomethyl)-phenoxyacetamido-norleucyl
MBHA resin with conventional Fmoc chemistry (Figs 2.1,2.2 and 2.3). Elongation
of the peptide chains were carried out in polypropylene reaction vessels where
protected amino acids were added manually.
11


p
I
Rj O
Fmoc -nCCOH +
H
linker
support
P
I
Ri o
H
Attach to linker
|Fmoc{-NCCO-
H
P
I
Ri O
linker
support
Deprotect alpha amino
protection group
H2NCco-
H
R2 9
linker
support
--- H | || -----------
IFmoclNCCO' activating group
H -----------
P
I
R2 O
Couple next protected
amino aicd
1 O
H
|Fmoc|-NCC-NH-CHCoH linker || support
H
P
I
r2 o
p
I
Ri o
Repeat deprotection and coupling
H
|Fmoc|-(NCCVNH-CHCoH linker || support
H n
Ri. O Rl O
I 11 H | ||
Cleave
(H,NCC^-NC---COH + support
. H H -----
Fig 2.1 Scheme of solid phase synthesis using standard Fmoc chemistry. The abbreviation R denotes
amino acid side chains and P denotes side-chain protecting group.
12


R O
rz-1 H | || A n
1 Fmoc InCCOH nr
H
3 Amine
Protected Amino Acid
''
H
R O
I
|Fmoc|NCH-C 6
Y3P-OBt PF6-
Coupling Reagent
(phosphonium)
~\
K. OBt

o=py3
o
PyBOP
Phosphonium Reagent
(coupling agent)
P
I
R O
=--1_H I H
Fmoc NCH-C
OBt
Activated Fmoc Amino Acid
Fig 2.2 Activation of Fmoc amino acid using a phosphonium reagent. R denotes an amino acid side
chain. P denotes side-chain protecting group.
13


ch3
I
H3C-C-CH3
ch, r
I 3 1 o
HjC-CCH3 TPA
U-l- ___________I
0
s. /v\ f12 h2 h . ..
C^H HjCO-^-Cj-NHcC-NH-C CNCHC
NH O O
CH,
'-------------------> 7
Rink amide MBHA resin
Fig 2.3 Deprotection and cleavage scheme using standard Fmoc chemistry. R denotes a resin bead,
Nle denotes norleucine and TFA denotes trifluoroacetic acid.
14


The side-chain protecting groups used were: Arg (Pbf), Lys (Boc), Om (Boc), Trp
(Boc), Asn (Trt), Gin (Trt), Cys (Trt), His (Trt), Asp (OBut), Glu (OBut), Ser (But),
Thr (But) and Tyr (But), where Pbf denotes 2,2,4,6,7-pentamethyldihydrobenzofuran-
5-sulfonyl, Boc denotes tert. butoxycarbonyl, Trt denotes trityl, OBut denotes butoxy
and But denotes butyl. During the elongation step of synthesis the resin was washed
with methylene chloride and N,N-dimethylformamide. After the final amino acid
was added, the peptides were N-terminally acetylated using acetic anhydride. Most
peptides were cleaved from the resin using 95% trifluoroacetic acid (TFA), 2.5%
water and 2.5% triisopropylsilane (TIS) for 120 minutes at room temperature.
Peptides containg methionine were cleaved Using 95% TFA, 2.5% TIS and 2.5%
methyl sulfide and peptides Containing cysteine were cleaved using 94.5% TFA, 1.5%
TIS, 1.5% H2O and 1.5% ethanedithiol (EDT). Crude peptides were washed from the
resin with clean TFA. The crude peptides were then precipitated out with cold
diethyl ether in a seven-fold excess and rinsed with cold diethyl ether twice. Crude
peptides were then dissolved in 50% acetonitrile, 50% water and then lyophilized.
2.2.2 Purification of Crude Peptides
Crude peptides were purified by RP-HPLC with a Kromasil analytical C18
column (150mm x 2.1mm I.D., 5-/rai particle size, 100-A pore size; Hichrom, Ltd.
Berkshire, UK) with a linear AB gradient (1.0% B/min.) at a flow rate of 0.3 ml/min,
where solvent A is aqueous 0.2% TFA and solvent B is 0.2% TFA in acetonitrile.
15


Peptide purity was verified by analytical RP-HPLC and electrospray mass
spectrometry.
2.2.3 Instrumentation
RP-HPLC runs were carried out on an Agilent 1100 Series liquid
chromatograph from Agilent Technologies (Little Falls, DE, USA).
2.2.4 RP-HPLC Analysis of Synthetic Model Peptides
Determination of relative hydrophobicity/hydrophilicity of each peptide
analog was performed by reversed-phase HPLC (RP-HPLC) on either a Zorbax
Eclipse XDB-C8 column (150 mm x 2.1 mm I.D., 5-fim particle size, 80-A pore size;
Agilent Technologies, Palo Alto, CA), pH 5 & 7 or a Kromasil Cl8 column (150mm
x 2.1mm I.D., 5-/xm particle size, 100-A pore size; Hichrom, Ltd. Berkshire, UK), pH
2, with a linear AB gradient (0.25% B/min) at a flow rate of 0.3 ml/min at 25 C
using an Agilent 1100 Series HPLC system. Solvent systems are as follows; pH 2:
solvent A is aqueous 20 mM H3PO4 and solvent B is 20 mM H3PO4 in acetonitrile, or
solvent A is aqueous 20 mM TFA and solvent B is 20 mM TFA in acetonitrile; pH 5:
solvent A is aqueous 10 mM NaH2PC>4 and solvent B is 10 mM Na^PCL in aqueous
50% acetonitrile; or solvent A is aqueous 10 mM NaH2P04 plus 50 mM NaCl and
solvent B is 10 mM NaH2P04 plus 50 mM NaCl in aqueous 50% acetonitrile; or
solvent A is aqueous 10 mM NaH2P04 plus 50 mM NaC104 and solvent B is 10 mM
16


NaH2P04 plus 50 mM NaC104 in aqueous 50% acetonitrile; pH 7: solvent A is
aqueous 10 mM NaH2P04 and solvent B is 10 mM NaH2P04 in aqueous 50%
acetonitrile; or solvent A is aqueous 10 mM NaH2P04 plus 50 mM NaCl and solvent
B is 10 mM NaH2PC>4 plus ,50 mM NaCl in aqueous 50% acetonitrile; or solvent A is
aqueous 10 mM NaH2P04 plus 50 mM NaC104 and solvent B is 10 mM NaH2P04
plus 50 mM NaC104 in aqueous 50% acetonitrile.
2.2.5 Mass Spectrometry
Correct mass confirmation of synthetic peptides was determined by
electrospray mass spectrometry using a Mariner Biospectrometry Workstation mass
spectrometer (PerSeptive Biosystems, Inc. Framingham, MA).
2.2.6 Normalization of Hydrophilicity/Hydrophobicity
Scales
For comparison of hydrophilicity/hydrophobicity scales the
hydrophilicity/hydrophobicity coefficients for a scale were manipulated. Thus, the
average of each set of coefficients was set as zero and the standard deviation was set
at 1.
17


3. Determination of Intrinsic Hydrophilicity/
Hydrophobicity of Amino Acid Side-Chains in
Peptides in the Absence of Nearest-Neighbor or
Conformational Effects
A version of this chapter has been submitted for publication. J.M. Kovacs,
C.T. Mant, R.S. Hodges, Biopolymers (Peptide Science), submitted.
3.1 Introduction
Thp concept of hydrophobicity has been a topic of much study in all aspects of
science, particularly in the fields of biology and chemistry [8]. Thus, for example, the
hydrophobic effect, as exemplified by the relative hydrophilicity/hydrophobicity of
amino acid side-chains and how they interact, is considered to be the most important
factor underlying the hierarchical structure and stability of proteins [10]. As noted by
Wilce et al. [11], manifestations of this hydrophobic effect are evident in many facets
of protein structure, including stabilization of protein globular structure in solution,
the presence of amphipathic structures induced in peptides or membrane proteins in
lipid environments, and protein-protein interactions associated with protein subunit
assembly, protein-receptor binding and other intermolecular biorecognition processes.
Thus, the quantitative evaluation of the magnitude of the hydrophilic/hydrophobic
contribution of a specific amino acid side-chain to such processes remains an
important challenge.
18


With a detailed knowledge of the contribution of individual amino acids to the
hydrophobic effect, the prediction of the overall three-dimensional structure of a
protein from its amino acid sequence alone may one day become a reality. Indeed,
hydrophobicity values (also described as coefficients or indices), obtained from a
variety of sources and methods, have been used widely for the prediction of protein
secondary structure (p-sheet, a-helix and turns) in globular proteins [7,12-14],
periodicities in residue distributions [7], antigenic sites [15-18], interior-exterior
regions [7] and membrane-associated regions [19-21]. Such indices have also been
utilized to simulate the sequence of signal peptides [22], to study quantitative
structure/activity relationships in polypeptide hormones [23] and to aid the rational de
novo design of biologically active peptides, e.g., antimicrobial peptides [24,25].
The measurement of amino acid side-chain hydrophilicity/hydrophobicity has
been carried out by a number of approaches, both chromatographic [26-38] and non-
chromatographic [39-50], which, as described in an excellent review by Biswas et al.
[8], may be generally divided into five different categories: partitioning (particularly
liquid-liquid) [40,42-44,46,48,49]; accessible surface area calculations [39,41,44,
45,48]; site-directed mutagenesis [51-55]; physical property measurements, e.g.,
measurement of surface tension of amino acid solutions [56], solvation free energy of
amino acids [57] and apparent heat capacity of peptides [58]; and chromatographic
techniques, almost invariably reversed-phase high-performance liquid
chromatography (RP-HPLC) [26-39]. A consensus on the values and ranking of
19


hydrophilicity/hydrophobicity values, however, has still not been obtained based as
they are on markedly different techniques and the nature of the solute, i.e.,
peptides/proteins or amino acids and derivatives thereof. Certainly, we believe that
despite the general usefulness of hydrophilicity/hydrophobicity scales previously
reported, a definitive determination of relative intrinsic
hydrophilicity/hydrophobicity of side-chains has yet to be achieved. Intrinsic
hydrophobicity implies the maximum possible hydrophilicity or hydrophobicity of
side-chains in a polypeptide chain in the absence of any nearest-neighbor effects (i ->
i+1 side-chain interactions or restriction of conformational freedom about the peptide
bonds, CqtC or CorN, on either side of residue i) and/or any conformational effects of
the polypeptide chain that prevent full expression of the side-chain
hydrophilicity/hydrophobicity. Such an hydrophobicity scale should be the
fundamental starting point for truly meaningful predictive applications and
understanding the parameters that decrease the intrinsic hydrophobicity.
In their review, Biswas et al. [8] noted that chromatographic methods,
particularly RP-HPLC, have shown much promise as generators of amino acid side-
chain hydrophilicity/hydrophobicity scales, based on the premise that the non-polar
stationary phase characteristic of this HPLC mode mimics a biological membrane
[59]. Using this RP-HPLC-based approach, most researchers have carried out
regression analysis of a random collection of peptides to relate peptide
hydrophobicity to peptide retention behavior [11,26-31, 34, 36-38]. The preferred
20


approach of our laboratory is to apply RP-HPLC to the separation of mixtures of
synthetic model peptides with just single amino acid substitutions in a defined peptide
sequence. We believe that such an approach eliminates such concerns as the relative
frequency with which a particular amino acid appears compared to others in a random
collection of peptides. In addition to the application of side-chain coefficients
generated from such model peptides to the prediction of peptide retention behavior
during RP-HPLC (becoming increasingly important for the rational design of
separation protocols for complex peptide mixtures characteristic of proteomic
applications [60-66]), this approach has enabled the design of a peptide/stationary
phase model of ligand-receptor interactions [35,67,68] as well as the ability to
predict potential antigenic sites on the surface of proteins [18].
The present study uses a novel approach to the determination of intrinsic
hydrophilicity/hydrophobicity of amino acid side-chains using RP-HPLC of synthetic
model peptides. Thus, we have applied RP-HPLC to the separation of mixtures of de
novo designed model peptides with the sequence Ac-XD,L-GAKGAGVGL-amide,
where X is substituted by Gly, the 19 D- and L- amino acids plus 3 other L- and D-
amino acids (norvaline, norleucine and ornithine). From the observed retention
behavior of these model peptides, we have obtained intrinsic
hydrophilicity/hydrophobicity values of the amino acid side-chains at pH 2, pH 5 and
pH 7 (the latter in the presence and absence of salts).
21


3.2 Experimental
3.2.1 General Methods
General methodology can be found in chapter 2 of this thesis.
3.2.2 Analytical RP-HPLC of Synthetic Model Peptides
Linear AB gradient (0.25% acetonitrile/min) at a flow-rate of 0.3 ml/min and
a temperature of 25C.
Mobile phase 1: Eluent A is 20 mM aq. H3PO4, pH 2, and Eluent B is 20 mM
H3PO4 in acetonitrile, denoted pH 2/H3PO4 system.
Mobile phase 2: Eluent A is 20 mM aq. TFA, pH 2, and Eluent B is 20 mM
TFA in acetonitrile, denoted pH 2/TFA system.
Mobile phase 3: Eluent A is 10 mM aq. NaH2P04, pH 5, and Eluent B is
Eluent A containing 50% acetonitrile, denoted pH 5/no salt system.
Mobile phase 4: Eluent A is 10 mM aq. NaH2PC>4, adjusted to pH 7 with
NaOH, and Eluent B is Eluent A containing 50% acetonitrile, denoted pH 7/no salt
system.
Mobile phase 5: same as mobile phase 4 but also both eluents containing 50
mM NaCl, denoted pH 7/NaCl system.
Mobile phase 6: same as mobile phase 4 but also both eluents containing 50
mM NaClC>4, denoted pH 7/NaC104 system.
22


3.3 Results
3.3.1 Design of Model Peptides
In order to determine truly intrinsic hydrophilicity/hydrophobicity values for
amino acid side-chains in peptides/proteins, several criteria must be met: (1) the
model peptide sequence should have no tendency to form any type of secondary
structure (a-helix, (5-sheet or P-tum) in any environment (aqueous or hydrophobic)
which could restrict the interaction of the substitution site with the hydrophobic
matrix during partitioning of the peptide between the mobile phase and stationary
phase during RP-HPLC [1]; (2) the peptide should be of sufficient length to ensure
multi-site binding [1]; (3) the peptide should be of sufficient overall hydrophobicity
to allow the substitution of all 20 naturally occurring amino acid side-chains while
maintaining satisfactory retention behavior; (4) the distribution of amino acid side-
chains should be such that there is no clustering of hydrophobic side-chains which
may minimize the contribution of the substituting amino acid side-chain; (5) the
peptide should be long enough to maintain satisfactory retention behavior on
substituting the 20 amino acids but not so long as to diminish the full expression of
the hydrophilicity/hydrophobicity of the substituted amino acid due to a chain length
effect (generally for peptides >15 residues) on peptide retention times [71]; (6) the
substitution site should be next to a residue that has a side-chain minimal in terms of
size and hydrophobicity, thus allowing the substituting amino acid to express its true
intrinsic hydrophilicity/hydrophobicity; and (7), there should be no nearest neighbor
23


effects (i to i + 1 interactions with the substituting residue) such effects can be
eliminated if there is free rotation about the 0 and 0 angles between the substituting
residue and its nearest neighbor.
The sequence chosen to reflect the above criteria in determining the intrinsic
hydrophihcity/hydrophobicity of 23 amino acid side-chains (20 naturally occurring
amino acids in peptides/proteins plus norvaline, norleucine and ornithine) was Ac-
XD,L-G-A-K-G-A-G-V-G-L-amide. This sequence contains four Gly residues spread
periodically throughout the sequence to ensure that the peptide has no secondary
structure tendencies [72,73]. The substitution site (denoted X) is adjacent to a Gly
residue to ensure that there is unrestricted dotation about the 0 and 0 angles between
the substitution site and the residue next to it, where 0 refers to rotations about the
CqtC single bond and 0 refers to rotations about the C^N single bond. In order to
demonstrate complete freedom of rotation about this peptide bond, CcrC and Co-N, all
23 amino acids were substituted in both the D- and L-configuration since the D- and
L- diastereomers should have identical retention behavior if there is free rotation
about the 0 and 0 angles. In addition, since the guest site is the N-terminal residue,
there is no restriction in its interaction with the reversed-phase matrix. The N-
terminus was acetylated and the C-terminus was amidated to eliminate potential
effects of a positively charged a-amino group or negatively charged carboxyl group,
respectively, on the hydrophihcity/hydrophobicity of the side-chains in the peptide.
A single Lys residue was incorporated into the model peptide sequence to ensure
24


peptide solubility over a wide pH range. The four hydrophobes in the peptide
sequence (2 Ala, 1 Val, 1 Leu) were distributed throughout the peptide sequence to
ensure no clustering of hydrophobes and subsequent creation of a preferred
hydrophobic binding domain. Finally, the 10-residue length of the model peptide was
selected as the minimum size to meet all design requirements (overall hydrophobicity,
random coil structure, no clustering of hydrophobic residues and no chain length
effect).
3.3.2 Elimination of Nearest-Neighbor Effects
It was our hypothesis that nearest-neighbor effects (i to i +1 interactions)
would affect the full expression of the hydrophilicity/hydrophobicity of an amino acid
side-chain at position i and that this effect would be dependent upon the side-chain at
position i and i + 1. Thus, as noted above, to ensure that there was free rotation about
the peptide bond between the substituting residue at position i and the residue at
position i + 1, Gly was selected for the i + 1 position of the peptide sequence shown
above in section 3.1. It was also our hypothesis that if there is complete rotational
freedom about the peptide bond, bonds CVC and CqtN, the difference in retention
time between all diastereomeric peptide pairs (Xd- and Xl- peptides) would approach
zero. On the other hand, if there was a restriction in conformational space between
the residues at positions i and i + 1, the difference in retention time of the Xd- and Xl-
peptides would be significantly different from zero. To demonstrate such a situation,
20 D- and L- amino acid substitutions were made in the sequence Ac-Xd,l*L-(j-A-K-
25


G-A-G-V-G-amide, with the substitution site now adjacent (i + 1) to a bulky Leu
residue instead of the Gly residue of our model peptide sequence Ac-Xd,l-G-A-K-G-
A-G-V-G-L-amide. It is important to note that the amino acid composition of these
two peptide sequences (i.e., whether the substitution site is next to a Gly or Leu) are
identical.
Fig. 3.1 shows representative RP-HPLC elution profiles at pH 2 of the D-
Ile/L-Ile peptide pairs of the peptide sequences where the substituted N-terminal He
residue is adjacent either to a Leu residue (Panel A) or a Gly residue (Panel B).
26


Absorbance at 210 nm
Fig 3.1 Elimination of nearest-neighbor effects to determine intrinsic
hydrophilicity/hydrophobicity coefficients. Column: KromasilC18. Conditions: linear AB
gradient (0.25% CH3CN/min, starting from 2% CH3CN) at a flow-rate of 0.3 ml/min, where
eluent A is 20 mM aq. TFA and eluent B is 20 mM TFA in CH3CN; temperature, 25C.
Panel A: representative RP-HPLC elution profile at pH 2.0 of two peptides of the same
sequence (Ac-X-L-G-A-K-G-A-G-V-G-amide), where position X contains L-Ile or D-fle
adjacent to a Leu residue. Panel B: RP-HPLC elution profile of two peptides of the same
sequence (Ac-X-G-A-K-G-A-G-V-G-L-amide), where position X contains L-Ile or D-Ile next
to a Gly residue.
27


From Fig. 3.1, when the substitution is made adjacent to a Leu residue (Panel A), the
D-/L- peptide pair was well resolved (by 7.8 min) at a gradient rate of 0.25%
acetonitrile/min; in contrast, when the substitution is made adjacent to a Gly residue
(Panel B), the D- and L-substituted peptides were inseparable, even at this shallow
gradient rate. Indeed, from Table 1, only 2 out of 23 substitutions of D- and L- amino
acids made adjacent to a Gly residue showed even a subtle separation at a gradient
rate of 0.25% acetonitrile/min: 0.5 min between the D- and L-Asp peptides and 0.3
min between the D- and L-Trp peptides (Fig. 3.2, top panel). At a more standard
gradient rate of 1% acetonitrile/min, these peptide pairs could not be resolved (Fig.
3.2, bottom panel). Thus, based on our criteria, the results presented in Table 3.1 and
Figs. 3.1 and 3.2 show that when the substituting residue is adjacent to a Gly residue,
there is complete freedom of rotation about the bonds on either side of the peptide
bond, bonds CqtC and C<*-N, between residue X and Gly. In contrast, profound
nearest neighbor effects were observed for all 20 amino acid substitutions (D- and L-)
adjacent to a Leu residue, varying by as much as 7.8 min between the Ac-Ileo-Leu-
peptide and the Ac-IleL-Leu-peptide (Fig. 3.1A) (Kovacs et al., unpublished results).
28


Table 3.1: RP-HPLC peptide retention time
data in 20 mM H3P04, pH 2 at 25 C
Amino Acid Substitution3 tR(min)b XL XD At(D-L)C
Tip 67.5 67.8 0.3
Phe 64.3 64.3 0.0
n-Leu 59.8 59.8 0.0
Leu 58.6 58.6 0.0
He 56.5 56.5 0.0
Met 51.3 51.3 0.0
n-Val 50.6 50.6 0.0
Tyr 50.6 50.6 0.6
Val 49.0 49.0 0.0
Pro 44.6 44.6 0.0
Cys 43.3 43.3 0.0
Ala 38.8 38.8 0.0
Glu 38.8 38.8 0.0
Thr 38.0 38.0 0.0
Asp 37.4 37.9 0.5
Gin 35.7 35.7 0.0
Ser 35.2 35.2 0.0
Asn 35.2 35.2 0.0
Gly 35.2 35.2 0.0
Arg 30.2 30.2 0.0
His 28.2 28.2 0.0
Lys 28.2 28.2 0.0
Om 27.6 27.6 0.0
3 The L- and D- amino acid substitutions at
position X in the peptide sequence
Ac-X-G-A-K-G-A-G-V-G-L-amide.
n-Leu, n-Val and Om denote norleucine,
norvaline and ornithine, respectively.
b Conditions: shown in Fig. 3 for 20 mM H3PO4
mobile phase system. tR denotes peptide
retention time.
c At^Lj is the difference in retention time between
D- and L- substituted peptides.
29


Absorbance at 210 nm
Fig 3.2 Effect of gradient rate on RP-HPLC elution profile of
diastereomeric peptide pairs at pH 2.0. Column: Kromasil Cl 8.
Conditions: same as Fig. 3.1 but with gradient rates of 0.25%
CHsCN/min (top) and 1% CH3CN/min (bottom) starting from 2%
CH3CN. L-Asp, D-Asp, L-Trp, or D-Trp substitutions were made at
position X of the peptide sequence Ac-X-G-A-K-G-A-G-V-G-L-amide
and were the only substitutions that could be partially resolved when
adjacent to a Gly residue.
30


3.3.3 RP-HPLC Retention Behavior of Model Peptides
at pH 2, pH 5 and pH 7
Having shown clearly that when the N-terminal amino acid substitution site is
adjacent to a Gly residue, the side-chain chain is able to express fully its intrinsic
hydrophilicity or hydrophobicity on interacting with the hydrophobic reversed-phase
matrix, see Fig. 3.1, the L-amino acid substituted model peptides were now subjected
to RP-HPLC under six mobile phase conditions: 20 mM H3PO4 or 20 mM TFA at pH
2; 10 mM PO4 buffer at pH 5; and 10 mM PO4 buffer at pH 7, containing no salt, 50
mM NaCl or 50 mM NaC104. Note that addition of salts (generally 50-100 mM) to
mobile phases over a pH range of ca. 4-7 has generally been designed, for silica-
based packings, to suppress negatively charged silanol interactions with positively
charged solutes [5]. The retention data for the 23 peptides are shown in Table 3.2.
From Table 3.2, it can be seen that the observed retention times of the
peptides varied depending on mobile phase pH and composition. Thus, the retention
times of the 23 peptides are all greater in the pH 2/TFA system compared to the pH 2/
H3PO4 system due to interaction of the mobile phase anions (phosphate and TFA')
with the positively charged groups in the model peptide sequence. Hence, the
peptides are retained longer in the presence of the hydrophobic TFA' anion compared
to the hydrophilic phosphate anion.
31


Table 3.2: RP-HPLC peptide retention time data in various mobile phases at 25C
Amino Acid Substituti ona 20 mM H3PO4 tR pH 2b 20 mM TFA tR pH 5b 10 mM PO4 Buffer tR pH 7b, 10 mM PO + 50 mM No Salt NaCl tR tR 4 Buffer + 50 mM NaC104 tR
Tip 67.5 73.5 73.0 72.0 71.3 79.9
Phe 64.3 70.2 69.9 69.0 68.4 77.0
n-Leu 59.8 65.7 65.4 64.7 64.2 72.8
Leu 58.6 64.4 63.9 63.3 62.9 71.3
lie 56.5 62.5 62.0 61.5 61.1 69.2
Met 51.3 56.8 56.2 55.4 55.6 63.0
n-Val 50.6 56.3 55.7 55.4 55.2 63.0
Tyr 50.6 55.8 55.0 54.5 54.3 61.3
Val 49.0 54.5 53.8 53.5 53.3 60.8
Pro 44.6 50.1 49.2 48.8 48.7 56.1
Cys 43.3 48.7 47.7 47.4 47.4 54.4
Ala 38.8 43.9 43.1 43.0 42.4 49.6
Glu 38.8 43.9 39.3 38.2 37.9 39.1
Thr 38.0 43.4 42.6 43.0 42.4 48.7
Asp 37.4 42.7 38.8 38.2 37.5 38.6
Gin 35.7 41.7 40.4 39.6 39.9 46.2
Ser 35.2 41.1 39.8 39.6 39.5 45.7
Asn 35.2 40.5 39.8 39.6 39.3 45.4
Gly 35.2 41.1 39.8 39.1 38.3 46.2
Arg 30.2 41.7 36.1 43.0 42.4 52.6
His 28.2 41.1 34.7 42.5 43.0 49.6
Lys 28.2 43.9 36.1 38.0 36.3 49.6
Om 27.6 40.5 33.0 35.5 36.3 48.3
a The L- amino acid substitutions at position X in the peptide sequence
Ac-X-G-A-K-G-A-G-V-G-L-amide. n-Leu, n-Val and Om denote norleucine,
norvaline and ornithine, respectively.
b Conditions: pH 2, shown in Fig. 3.3; pH 5, shown in Fig. 3.4;
pH 7, shown in Fig. 3.5.
c tR denotes peptide retention time.
t
32


An interesting observation from Table 3.2 lies in the retention behavior of the
peptides at pH 5 and pH 7 in the absence of salt. Thus, while the retention times of
19 of the peptides decrease slightly (average 0.5 min) between pH 5 and pH 7, the
four peptides with substitutions by positively charged residues (Ora, Lys, His and
Arg) show significant retention time increases at pH 7 compared to pH 5 (2.5 min, 1.9
min, 7.8 min and 6.9 min, respectively). A previous study by Sereda et al. [75]
demonstrated that the pKa values of even highly basic side-chains were dramatically
decreased in the hydrophobic environment characteristic of RP-HPLC, i.e., partial
deprotonation (neutralization) of side-chains such as Lys and Arg (with pKa values of
~10 and ~12, respectively, in the free amino acid), thus increasing their observed
relative hydrophobicity. In the case of His (with a pKa value of ~6 in the free amino
acid), the side-chain is likely entirely de-protonated at pH 7. In addition, there is an
increase in the concentration of the HPO4 anion as the pH increases which could
more efficiently neutralize the positively charged side-chains in the peptides, thereby
increasing their hydrophobicity. The role of this anion in ion-pairing will be
discussed later where the pH was varied from 5 to 8.5.
In contrast to the addition of 50 mM NaCl to the mobile phase at pH 7, which
had little effect on peptide retention times (average decrease for 23 peptides was 0.3
min), addition of 50 mM NaClCLj at this pH value generally significantly increased
peptide retention times. Compared to Cl', the perchlorate anion is extremely effective
at ion-pairing to positively charged peptide groups [75], thus increasing overall
33


peptide hydrophobicity of 18 peptides with a net charge of+1 by an average of 7.1
min; increasing hydrophobicity of peptides with a net charge of +2 (Arg, Lys and
Om) by 9.6 min, 11.6 min and 12.8 min, respectively; and increasing hydrophobicity
of peptides with a zero net charge (Asp and Glu) by just 0.4 min and 0.9 min,
respectively.
From Table 3.2, the difference in retention time between the most hydrophilic
and hydrophobic substitutions varies from 33 min to 41 min depending on pH and
composition of the mobile phase. It should be emphasized that we employed a
shallow gradient rate of 0.25% acetonitrile/min to maximize differences between the
peptides. The Tip-substituted peptide was the most hydrophobic peptide and the Om-
substituted peptide was the most hydrophilic peptide under five of the six conditions
studied. In the case of 10 mM PO4 buffer, pH 7, containing 50 mM NaC104, the
peptides with positively charged amino acid substitutions (Om, Lys and Arg) became
much more retentive due to the strong ion-pairing properties of the perchlorate anion;
concomitantly, the Asp-substituted peptide was the least retentive under these
conditions.
34


3.3.4 Correlation of RP-HPLC Retention Behavior of
Peptides under Different Mobile
Phase Conditions
From Fig. 3.3, when the retention times of 19 of the 23 peptides (with the
exception of those with positively charged substitutions, Om, Lys, His and Arg) in
the pH 2/H3PO4 system are plotted against those obtained in the pH 2/ TFA system,
there is an excellent correlation (r = 0.999) of the respective series of data.
35


Fig 3.3 Plot of tR of peptides in 20 mM TFA mobile phase system versus tR in 20 mM H3PO4
mobile phase system Column: Kromasil Cl 8. Conditions: linear AB gradient (0.25%
CH3CN/min) at a flow-rate of 0.3 ml/min, where eluent A is 20 mM aq. TFA or 20 mM aq.
H3PO4 and eluent B is 20 mM TFA or 20 mM H3PO4, respectively, in CH3CN starting from
2% CH3CN; temperature, 25C. Data are taken from Table 3.2. The single letter code
represents the L-amino acid substitutions at position X of the peptide sequence shown in Fig
3.2. Closed circles denote data used in the correlation plot (y = 1.0136x + 4.9733, correlation
coefficient r = 0.999); open circles represent results from positively charged peptide residues;
nL, nV and O denote norleucine, norvaline, and ornithine, respectively.
36


Such a correlation shows that the change in counterion hydrophobicity from an
hydrophilic phosphate anion to an hydrophobic TFA'anion, despite the increase in
overall peptide retention times in the TFA system due to ion-pairing with the single
Lys residue in the sequence, does not affect the relative hydrophilicity/hydrophobicity
of the 19 uncharged side-chains. In contrast, this change in counterion
hydrophobicity does affect the hydrophilicity/hydrophobicity of the positively
charged residues by making their relative hydrophobicities increase in the presence of
TFA compared to H3PO4; hence the increased retention times of the peptides
substituted with Om, Lys, His or Arg relative to the other 19 peptides (Fig. 3.3; Table
3.2). .
Fig. 3.4 now correlates peptide retention behavior at pH 2 (H3PO4 system) and
pH 7 (no salt). There is an excellent correlation (r = 0.999) for the 17 peptides
substituted at position X with neutral side-chains, demonstrating that pH has no effect
on the relative hydrophilicity/hydrophobicity of these side-chains. In contrast, the
increase in pH from 2 to 7 is increasing the relative hydrophobicity of Om, Lys, His
and Arg, likely (as described above) to partial (Om, Lys, Arg) or complete (His)
deprotonation of these residues at the higher pH value, i.e., the positive charge on
these side-chains is diminished or eliminated or an increased neutralization of the
positive charge by the increased concentration of the anions H2PO4' and HPO42' is
occurring as the pH is increased from 2 to 7.
37


tR in 10 mM NaH 2PO4 (pH 7)
Fig 3.4 Plot of tR of peptides in 20 mM H3P04 (pH 2) mobile phase system versus tR
in 10 mM NaH2P04 (pH 7; no salt) mobile phase system. Columns: Rromasil C18
(pH 2) and Zorbax XDB C8 (pH 7). Conditions: pH 2, linear AB gradient (0.25%
C^CN/min) at a flow-rate of 0.3 ml/min, where eluent A is 20 mM aq. H3PO4 and
eluent B is 20 mM H3PO4 in CH3CN starting from 2% CH3CN; pH 7, linear AB
gradient (0.25% CH3CN/min) at a flow rate of 0.3 ml/min where eluent A is 10 mM
aq. NaH2P04, pH 7, and eluent B is eluent A containing 50% CH3CN; temperature,
25C. Data are taken from Table 3.2. The single letter code represents the L-amino
acid substitutions at position X of the peptide sequence shown in Fig 3.2. Closed
circles denote data used in the correlation plot (y = 1.006 0.0376, correlation
coefficient r = 0.999); open circles represent results from positively charged peptide
residues; nL, nV and O denote norleucine, norvaline, and ornithine, respectively.
38


In contrast, the relative hydrophobicities of the acidic side-chains of Asp and Glu are
decreasing with an increase in pH 2, due to deprotonation of these residues at pH 7
(the pKa of these side-chains is ~4.0) i.e., they become negatively charged. Although
not shown here, a similar plot of retention times at pH 2 versus pH 5 also indicated
deprotonation of these acidic side-chains at pH 5.0.
Fig. 3.5 presents elution profiles of the 23 peptides at pH 2 (H3PO4) and pH 7
(no salt) for easy visualization of the effect of pH on relative retention behavior of the
peptides and, hence, relative hydrophilicity/hydrophobicity of the side-chains of die
substituted amino acids. Note that the elution order of the peptides substituted with
neutral side-chains are similar (i.e., correlate well) at these two pH values, albeit
small differences are observed, such variations possibly due to selectivity differences
between the Kromasil Cl8 column used at pH 2 and the Eclipse XDB-C8 column
used at pH 7. Clearly, from Fig. 3.5, the major changes in observed relative
hydrophilicity/hydrophobicity occur for the peptides substituted with potentially
charged side-chains: hence, the greater observed hydrophobicity of the peptides
substituted with Om, Lys, His or Arg with a rise in pH from 2 to 7 (Table 3.2) and the
concomitant decrease in peptide hydrophobicity of the peptides substituted with Asp
or Glu.
39


Y
OA
Fig 3.5 RP-HPLC elution profiles of peptide mixtures at pH 2 (top) and pH 7 (bottom). Columns: pH
2, Kromasil C18; pH 7, Zorbax XDB C8. Conditions: pH 2, linear AB gradient (0.25% CH3CN/min)
at a flow-rate of 0.3 ml/min, where eluent A is 20 mM aq. H3PO4 and eluent B is 20 mM H3PO4 in
CH3CN, starting at 2% CH3CN; pH 7, linear AB gradient (0.25% CH3CN/min) at a flow-rate of 0.3
ml/min, where eluent A is 10 mM aq. NaH2P04 (pH 7) and eluent B is eluent A containing 50%
CH3CN; temperature, 25C. Peaks are denoted by the one-letter codes of the L-amino acids
substituted into position X of the peptide sequence shown in Fig 3.2. Shaded peaks denote peptides
containing potentially positively charged residues (K,R,H,0) or potentially negatively charged residues
(D,E).
40


Fig. 3.6 plots peptide retention times at pH 7 versus pH 5 in the absence of
salt. With the exception of peptides substituted with Om, Lys, His and Arg, the
remainder show an excellent correlation (r = 0.999) between these mobile phase
systems, demonstrating that the increase in pH on these otherwise identical buffer
systems has no effect on the relative hydrophilicity/hydrophobicity of side-chains
substituted at position X of the model peptides. The non-correlation of the Om-, Lys-
, His- and Arg-substituted peptides may be explained by the aforementioned partial
(Om, Lys, Arg) or complete (His) deprotonation and increased ion-pairing from the
increased concentration of HPO42' anions on raising the pH from 5 to 7, resulting in
longer retention times at the higher pH value. Of note here is the correlation of the
Asp- and Glu-substituted peptides with the remainder of the peptides substituted with
neutral side-chains, which may be explained by the side-chains of these acidic amino
acids being fully deprotonated (i.e., negatively charged) at both pH 5 and 7.
Fig. 3.7 highlights the relative effectiveness of Cl" ion versus CIO4" ion as ion-
pairing reagents at pH 7. Thus, when the retention times of the peptides are
correlated at pH 7 in the absence and presence of 50 mM NaCl (Fig. 3.7A), there is an
excellent correlation (r = 0.998) of all 23 amino acids, including the peptides
substituted with positively charged amino acids, concomitant with little observable
effect of NaCl on peptide retention times (Table 3.2). Such results reflect similar
conclusions by Shibue et al. [5] that the chloride ion is relatively ineffective in
41


affecting peptide retention behavior by ion-pairing with positively charged side-
chains.
42


Fig 3.6 Plot of tR of peptides in 10 mM NaH2P04 (pH 5) mobile phase system versus 10 mM
NaH2P04 (pH 7) mobile phase system Column: Zorbax XDB C8. Conditions: linear
gradient (0.25% CH3CN /min) at a flow-rate of 0.3 ml/min, where eluent A is 10 mM aq.
NaH2P04, pH 5 or pH 7, and eluent B is the respective eluent A containing 50% CH3CN;
temperature, 25C. Data are taken from Table 3.2. The single letter code represents the L-
amino acid substitutions at position X of the peptide sequence shown in Fig 3.2. Closed
circles denote data used in the correlation plot (y = 1.01 lx 0.0699, correlation coefficient r =
0.999); open circles represent results from positively charged peptide residues; nL, nV and O
denote norleucine, norvaline, and ornithine, respectively.
43


Fig 3.7 Plot of tR of peptides in 10 mM NaH2P04 (pH7) mobile phase system versus 10 mM NaH2P04
(pH 7) containing 50 mM NaCl (A) or 50 mM NaC104 (B) mobile phase systems. Column: Zorbax
XDB C8. Conditions: linear AB gradient (0.25% CH3CN/min) at a flow-rate of 0.3 ml/min, where
eluent A is 10 mM aq. NaH2P04, pH 7, containing no salt, 50 mM NaCl or 50 mM NaC104 and eluent
B is the respective eluent A containing 50% CH3CN; temperature, 25C. Data are taken from Table
3.2. The single letter code represents the L-amino acid substitutions at position X of the peptide
sequence shown in Fig 3.2. Closed circles denote data used in the correlation plot (y = 0.9948x -
0.051, correlation coefficient r = 0.998 andy= 1.0572x +4.1618, correlation coefficient r = 0.999 for
plots A and B, respectively); open circles represent results from positively charged peptide residues;
nL, nV and O denote norleucine, norvaline, and ornithine, respectively.
44


In contrast, the addition of 50 mM NaC104 to the mobile phase at pH 7 affects
the retention behavior of both the peptides substituted with three of the positively
charged side-chains (Om, Lys and Arg) and, interestingly, those substituted with
negatively charged side-chains (Asp and Glu) (Fig. 3,7B). The remainder of the
peptides, substituted with neutral side-chains, show an excellent correlation of 0.999,
indicating that the relative hydrophilicity/hydrophobicity of these side-chains is
unaffected by the addition of salt to the mobile phase. Shibue et al. [68] have
demonstrated that the perchlorate anion is a very effective ion-pairing reagent (more
effective, indeed, than trifluoroacetate) and, thus, will interact strongly with positively
charged side-chains; hence, the increase in retention time of the Om-, Lys- and Arg-
substituted peptides relative to the remainder (Fig. 3.7B; Table 3.2). Note that, at pH
7, the His side-chain is largely deprotonated (i.e., neutral) and thus is unaffected by
the addition of NaClCV The poor correlation of the Asp- and Glu-substituted
peptides in the presence of 50 mM NaC104 (Fig. 7B) is likely due to the decreased
ion-pairing of the perchlorate anion with the positively charged residues as the net
charge on the peptide was reduced to zero.
3.3.5 Amino Acid Side-Chain Hydrophilicity/
Hydrophobicity Coefficients
To determine the hydrophilicity/hydrophobicity of the substituting amino acid
side-chain at position X of the model peptide sequence, the retention time of the Gly-
45


substituted peptide was used as a reference since Gly has only an hydrogen atom as
its side-chain. Thus, the hydrophilicity/hydrophobicity coefficients of the 22 side-
chains (other than Gly) were generated from the RP-HPLC runs carried out under the
six mobile phase conditions from the difference in the retention times (AtR) of the X-
substituted peptide and the Gly-substituted peptide, i.e., AtR= tR X-substituted peptide
minus tR Gly-substituted peptide (Table 3.3). Thus, from Table 3.3, side-chains that
are more hydrophobic than Gly have positive AtR values and side-chains that are more
hydrophilic than Gly have negative AtR values.
3.3.5ol Isosteric Side-Chains: n-Leu, Leu, He
Norleucine, leucine and isoleucine are isosteric but nevertheless display
overall differences in hydrophobicity with coefficients of 24.6 min, 23.4 min and 21.3
min, respectively in the pH 2/H3PO4 system (Table 3.3). Such results are expected
since as the side-chain is more extended from the polypeptide backbone (n-Leu > Leu
> lie), it is more exposed and is thus better able to express its hydrophobicity.
Similarly, norvaline is more hydrophobic than valine with coefficients of 15.4 min
and 13.8 min, respectively, in the pH 2/H3PQ4 system (Table 3.3). As expected, these
differences are independent of pH and mobile phase conditions.
Interestingly, although n-Val and Pro have the same number of carbon atoms
in their side-chains, n-Val has a coefficient of 15.4 min compared to just 9.4 min for
Pro. This can be explained by the cyclization of the Pro side-chain to the polypeptide
46


backbone nitrogen. Thus, the Pro side-chain is closer to the backbone and less
exposed compared to the side-chain of n-Val.
47


Table 3.3: Hydrophilicity/hydrophobicity coefficients determined at 25C
by RP-HPLC of model peptides.
Amino Acid Substitutio na pH 2b 20 mM 20 raiM H3PO4 TFA AtR(Gly)C AtR(Gly) pH 5b 10 mM PO4 Buffer AtR(G]y) pH7b, No Salt AtR(Gly) 10 mM PO4 Buffer + 50 mM + 50 mM NaCl NaC104 AtR(Gly) AtR(G|y)
Tip 32.3 32.4 33.2 32.9 33.0 33.7
Phe 29.1 29.1 30.1 29.9 30.1 30.8
n-Leu 24.6 24.6 25.6 25.6 25.9 26.6
Leu 23.4 23.3 24.1 24.2 24.6 25.1
lie 21.3 21.4 22.2 22.4 22.8 23.0
Met 16.1 15.7 16.4 16.3 17.3 16.8
n-Val 15.4 15.2 15.9 16.3 16.9 16.8
Tyr 15.4 14.7 15.2 15.4 16.0 15.1
Val 13.8 13.4 14.0 14.4 15.0 14.6
Pro 9.4 9.0 - 9:4 9.7 10.4 9.9
Cys 8.1 7.6 7.9 8.3 9.1 8.2
Ala 3.6 2.8 3.3 3.9 4.1 3.4
GIud 3.6 2.8 -0.5 -0.9 -0.4 -7.1
Thr 2.8 2.3 2.8 3.9 4.1 2.5
Asp 2.2 1.6 -1.0 -0.9 -0.8 -7.6
Gin 0.5 0.6 0.6 0.5 1.6 0.0
Ser 0.0 0.0 0.0 0.5 1.2 -0.5
Asn 0.0 -0.6 0.0 0.5 1.0 -0.8
Gly 0.0 0.0 0.0 0.0 0.0 0.0
Arg -5.0 0.6 -3.7 3.9 4.1 6.4
His -7.0 0.0 -5.1 3.4 4.7 3.4
Lys -7.0 2.8 -3.7 -1.1 -2.0 3.4
Om -7.6 -0.6 -6.8 -3.6 -2.0 2.1
a The L- amino acid substitutions at position X in the peptide sequence
Ac-X-G-A-K-G-A-G-V-G-L-amide. n-Leu, n-Val and Om denote
norleucine, norvaline and ornithine, respectively.
b Conditions : pH 2, shown in Fig. 3.3; pH 5, shown in Fig. 3.4; pH 7,
shown in Fig. 3.5.
c AtR(Giy) denotes the change in retention time relative to the Gly
substituted peptide.
d The bold values denote the potentially charged residues
Asp, Glu, Arg, His, Lys, Om.
48


3.3.5.2 Carbon Atom Extension of Side-Chains: Non-
Polar Groups (Gly/AIa, Val/IIe, n-Val/n-Leu)
From Table 3.3, for the pH 2/H3PO4 system, the difference in hydrophobicity
between the n-Val and n-Leu side-chains was 9.2 min, compared to 7.5 min for Val
and He and just 3.6 min for Gly and Ala. Such differences all correlated with the
distance of the carbon atom extension from the polypeptide backbone, where the
methyl group of Ala is the P-carbon, the methyl group of He is the 5-carbon and the
methyl group of n-Leu is the s-carbon, i.e., the greater the distance of the added
carbon atom from the polypeptide backbone, the greater its hydrophobicity
contribution.
3.3.5.3 Carbon Atom Extension of Side-Chains: Polar
And Charged Groups
(Asn/Gln, Asp/Glu, Orn/Lys)
From Table 3.3, under six different mobile phase conditions, the average
increase in hydrophobicity between Asn and Gin (i.e., an increase of a CH2 group)
was ~0.6 min. Also, the increase in hydrophobicity between Asp and Glu at pH 5 and
pH 7, where the side-chain carboxyl group is negatively charged, showed a similar
value of -0.6 min. On the other hand, when the carboxyl group is protonated (i.e.,
lacking a charge), the increase in hydrophobicity from Asp to Glu at pH 2 was -1.3
min. Such results suggest that the less polar the functional group (e.g., protonated
49


Glu < deprotonated, negatively charged Glu), the greater the expression of the
hydrophobicity on extension of the side-chain by a CH2 group.
From Table 3.3, the increase in side-chain hydrophobicity on extending the
side-chain by a CH2 group from Om to Lys is very dependent on the mobile phase pH
and the ion-pairing properties of the anionic ion-pairing reagent. For example, at pH
2, in the pH 2/H3PO4 system, the change is 0.6 min; in contrast, in the pH 2/TFA
system (TFA being an hydrophobic ion-pairing reagent), this change is 3.5 min. In
the pH 5/no salt versus pH 7/no salt systems, the increase in hydrophobicity for Om
to Lys was 3.1 min and 2.5 min, respectively. On the other hand, on the addition of
salt at pH 7, this increase changed dramatically in the presence of 50 mM NaC104
versus 50 mM NaCl. Thus, no separation of the Om and Lys peptides was achieved
in the pH 7/NaCl system while the two peptides were separated by 1.3 min in the pH
7/NaC104 mobile phase. Such results demonstrate that, for Om and Lys, not only is
the hydrophobicity of the side-chain increased by the addition of the CH2 group, but
this increase in hydrophobicity also affects the charge neutralization of the side-chain
amino groups, i.e., the properties of the Om and Lys side-chain amino groups are
different (see later discussion).
50


33.5.4 Carbon Atom Extension of Side-Chains:
Addition of P-branch Methyl Group (Ser/Thr)
From Table 3.3, adding a methyl group at a P-branch location on the amino
acid side-chain, in a similar manner to extension of side-chains by insertion of a CH2
group (see above), also increases side-chain hydrophobicity. Thus, in the pH
2/H3PO4 system, there is a 2.8 min increase between Ser and Thr. However,
interestingly, the addition of a methyl group from Gly to Ala resulted in a greater
hydrophobicity increase of 3.6 min. Such a result indicates that, in the case of Thr,
the polar hydroxyl group also attached to the p-carbon of the side-chain is partially
shielding the methyl group on this same p-carbon and preventing its full hydrophobic
expression.
.' 1 '
3.4 Effect of pH and Mobile Phase Composition on
Hydrophilicity/Hydrophobicity of Amino Acid
Side-Chains
3.4.1 Uncharged Side-Chains
Fig. 3.8 plots the hydrophihcity/hydrophobicity coefficients (AtR(Giy)) of the 17
uncharged (neutral) side-chains obtained at pH 7 (no salt) versus the coefficients
obtained at the two pH 2 conditions, the pH 5 mobile phase and the two pH 7 mobile
phases containing either 50 mM NaCl or NaClC>4. The excellent correlation observed
from the plot (r = 0.997), together with the generally negligible variation of these
values in all the mobile phase systems employed (Table 3.3), point to the
51


fundamentally vital conclusion that the intrinsic hydrophilicity/hydrophobicity
characteristics of uncharged side-chains are unaffected by their environment. Also,
these results indicate that such RP-HPLC-derived coefficients are independent of
differences in the hydrophobic stationary phase (e.g., alkyl chain length, i.e., C8
versus Cl8) used to derive these values, a result which contradicts earlier work [11]
where coefficients were generated from a large collection of random peptides rather
than the de novo designed model peptides used in the present study.
52


40 i
Fig 3.8 Correlation of hydrophilicity/hydrophobicity coefficients (At^oiy)) obtained at pH 2,
pH 5 and pH 7. Columns: pH 2, Kromasil Cl 8; pH 5 and pH 7, Zorbax XDB C8.
Conditions: abscissa, 10 mM NaH2P04, pH 1, mobile phase containing no salt as described in
Fig 3.4; ordinate, mobile phase X denotes the two pH 2 systems described in Fig 3.3, the pH 5
system described in Fig 3.4 and the two pH 7 systems (10 mM NaH2P04 plus 50 mM NaCl or
50 mM NaC104) described in Fig 3.5; temperature, 25C. Data are taken from Table 3.3. The
single letter code represents the L-amino acid substitutions at position X of the peptide
sequence: Ac-X-G-A-K-G-A-G-V-G-L-amide. Data points used in the correlation plot (y =
1.0Q9x 0.272, correlation coefficient r = 0.997) exclude those obtained for potentially
positively charged (K,R,H,0) or negatively charged (D,E) residues; nL and nV denote
norleucine and norvaline, respectively.
53


3.4.2 Potentially Charged Side-Chains
As has been clearly demonstrated in the present study, and emphasized in Fig.
3.8 and in Table 3.3, any variation in retention behavior of the 23 model peptides
under all 6 mobile phase conditions employed is due to the effect of pH and mobile
phase composition on the intrinsic hydrophilicity/hydrophobicity of the ionizable
side-chains Om, Lys, His; and Arg (potentially positively charged) and Asp and Glu
(potentially negatively charged). Fig; 3.9 illustrates the variation of the side-chain
coefficients at pH 2, pH 5 and pH 7, the latter in the absence and presence of 50 mM
NaClC>4 (the mobile phase containing 50 mM NaCl is not included due to the
negligible effect of the chloride ion on side-chain hydrophilicity/hydrophobicity of
the charged residues relative to perchlorate; Table 3.3; Fig. 3.7A). The general effect
on the coefficients for the positively charged residues (O, K, R, H) at pH 2 is a
marked decrease in intrinsic hydrophilicity (i.e., increase in hydrophobicity) on
substituting the hydrophilic phosphate anion with the hydrophobic TFA" anion;
indeed, Lys would be classed as an extremely hydrophilic amino acid relative to Gly
in the pH 2/H3PO4 system and a moderately hydrophobic amino acid in the pH 2/TFA
mobile phase. In a similar fashion, a pH change from pH 5 to pH 7, in the same
phosphate-based mobile phase, decreases side-chain hydrophilicity (increases
hydrophobicity) of these amino acids quite substantially, with Arg and His being
classified as very hydrophilic relative to Gly at pH 5 but moderately hydrophobic at
pH 7 (the latter pH certainly resulting in deprotonation of His).
54


Hydrophilicity/Hydrophobicity Coefficients (min)
10.0
8.0
pH 2
20 mM
h3po4
pH 2 pH 5
20 mM 10 mM
TFA NaH2PQ4
pH 7 pH 7
10 mM + 50 mM
NaH2PQ4 NaC104
6.0
4.0
2.0
0.0
-2.0
-4.0
-6.0
-8.0
Fig 3.9 Effect of mobile phase conditions on magnitude of hydrophilicity/hydrophobicity
coefficients of potentially charged acidic (D,E) and basic (0,K,R,H) residues. Columns: pH
2, Kromasil C18; pH 5 and pH 7, Zorbax XDB C8. Conditions: pH 2, shown in Fig 3.3; pH 5
shown in Fig 3.4; pH 7 shown in Fig 3.5. Data are taken from Table 3.3; O denotes ornithine.
55


The addition of perchlorate ion, with its effective ion-pairing properties [69], further
enhances the hydrophobic nature of Arg relative to Gly under these conditions and
also transforms the hydrophilic nature of the Om and Lys side-chains in the absence
of salt at pH 7 to hydrophobic side-chains relative to Gly in the presence of 50 mM
NaC104.
At pH 2, the presence of the hydrophilic phosphate anion or the hydrophobic
TFA' anion has little appreciable effect on the intrinsic hydrophilicity/hydrophobicity
of the protonated (i.e., uncharged) side-chains of Asp and Glu, being classed as
moderately hydrophobic relative to Gly. These side-chains become somewhat
hydrophilic at pH 5 and pH 7 (ho salt) due to deprotonation of these side-chains and,
hence, their negative charge. The moderately hydrophilic characteristic of these two
side-chains is dramatically affected by the introduction of the negatively charged
perchlorate anion in the medium, which transforms them into extremely hydrophilic
amino acids.
The explanation for the effects of NaC104 on the retention behavior of the 23
peptides used in this study is shown in Fig. 3.1 OB, where the change in peptide
retention time at pH 7 in the absence and presence of 50 mM salt (NaCI or NaC104) is
plotted against peptide net charge. This plot clearly supports the concept that the
increase in hydrophobicity of the peptides is due to ion-pairing of the perchlorate
anion to the positively charged residues in the peptides.
56


Fig 3.10 Relationship between peptide net charge and effect of mobile phase counterion at pH 2 (TFA
anion; Panel A) and pH 7 (Cl", C104" anions; Panel B). Columns: pH 2, Kromasil Cl 8; pH 7, Zorbax
XDB C8. Conditions: pH 2, linear AB gradient (0.25% CH3CN/min) at a flow-rate of 0.3 ml/min,
where eluent A is 20 mM aq. H3PO4 or TFA and eluent B is 20 mM H3PO4 or TFA, respectively, in
CH3CN, starting at 2% CH3CN; pH 7, linear AB gradient (0.25% CH3CN/min) at a flow-rate of 0.3
ml/min, where eluent A is 10 mM aq. NaH2P04, containing no salt or containing 50 mM NaCl or 50
mM NaC104 and eluent B is the respective eluent A containing 50% CH3CN; temperature, 25C. AtR
denotes the tR of peptide in pH 2/TFA system minus tR of peptide in pH 2/H3PO4 (Panel A) or tR of
peptide in pH 7/NaCl or pH 7/NaQ04 system minus tR of peptide in pH 7/no salt system (Panel B).
The horizontal and sloping plots in Panel B are for the NaCl and NaC104 effects, respectively; nL, nV
and O denote norleucine, norvaline, and ornithine, respectively.
57


Thus, 18 of the peptides have a single positively charged Lys residue (net charge +1)
with an average increase in peptide retention with addition of 50 mM NaCICU of 7.1
min. In contrast, the three peptides with an additional positively charged residue
(Lys, Arg and Om) have a net charge of +2 and an increase in retention times of 11.6
min, 9.6 min and 12.8 min, respectively. The two peptides with a negatively charged
residue (Asp and Glu) and, thus, a net charge of zero, show only a very small increase
in peptide retention time in the presence of 50 mM NaC104 (0.4 min and 0.9 min,
respectively). The latter results suggest that the perchlorate anion is ineffective at
ion-pairing when the net charge on the peptide is zero, hence no appreciable increase
in retention time on the addition of 50 mM NaCKV The inability of the chloride
anion to ion-pair with positively charged groups is shown by the fact that retention
behavior in the presence of 50 mM NaCl is independent of net charge (zero to +2)
(Fig. 3.10B).
Fig. 3.10A illustrates the effect of the trifluoroacetate anion on peptide
retention behavior at pH 2. At pH 2, the Asp and Glu side-chains are protonated.
Thus, while the Lys-, His-, Om- and Arg-substituted peptides have a net charge of +2,
the remaining 19 peptides have a net charge of+1. The change in peptide retention
time between the H3PO4 and TFA mobile phases for the +1 peptides averaged 5.6
min, whereas the +2 peptides showed increased retention times for the Lys-, His-,
Om- and Arg-substituted peptides of 15.7 min, 12.9 min, 12.9 min and 11.5 min,
respectively.
58


Finally, Fig. 3.11 illustrates the effect of mobile phase pH on intrinsic
hydrophilicity/hydrophobicity of potentially positively charged side-chains over a
range of pH 5-8.5. From Fig. 3.11, the hydrophilicity of all four side-chains
decreases (i.e., hydrophobicity increases) with increasing pH, albeit to varying
degrees. Thus, Arg is considerably more sensitive to pH changes than Om, His or
Lys over a range of pH 5-6.5. Interestingly, the effect of pH over this range is
essentially identical for the latter side-chains, followed by a dramatic change in the
His profile between pH 6.5 and 6.75 due to essentially complete deprotonation of its
side-chain (the pKa of His is ~6.0 in the free amino acid). The profiles for Om and
Lys, similar prior to pH 6.5, diverge somewhat at values greater than pH 6.5. As
noted previously, the decrease in hydrophilicity (increase in hydrophobicity) of these
side-chains is likely due to partial deprotonation with an increase in pH, the extent of
this deprotonation being dependent on the pKa values of these amino acids under the
conditions of RP-HPLC or the increasing concentration of the HPO4 anion as the pH
is increased. Thus, the differences in the Om and Lys profiles suggest the pKa values
of these amino acid side-chains are different.
59


Hydrophilicity/Hydrophobicity
Coefficients (min)
Fig 3.11 Effect of pH on the magnitude of hydrophilicity/hydrophobicity coefficients of
potential positively charged residues. Column: Zorbax XDB C8. Conditions: pH 5-8.5,
linear AB gradient (0.25% CH3CN/min) at a flow-rate of 0.3 ml/min, where eluent A is 20
mM NaH2P04 and eluent B is 20 mM NaH2P04 in 50% CH3CN; temperature, 25 C. A
denotes arginine, 0 denotes ornithine, o denotes lysine and denotes histidine.
60


3.5 Conclusions
Intrinsic hydrophilicity/hydrophobicity side-chain coefficients of amino acids
have been determined by RP-HPLC of model synthetic peptides over a pH range of
pH 2-7 in the absence of any nearest-neighbor or peptide conformational effects. The
intrinsic values of neutral side-chains were unaffected by pH, mobile phase
composition or functional groups of the reversed-phase matrix. Only potentially
charged side-chains (Om, Lys, His, Arg, Asp, Glu) showed a variation in intrinsic
hydrophilicity/hydrophobicity with varying mobile phase environments.
61


4. Quantitative Determination of Nearest-Neighbor
Effects in Peptides by Reversed-Phase Liquid
Chromatography of Synthetic Model Peptides with
D- and L- Amino Acid Substitutions
4.1 Introduction
Although protein folding has been studied for more than 40 years, the exact
details remain extremely complex (see reviews in refs. 76-79). The classic
experiments by Anfinsen showed that in the presence of a powerful denaturant (8 M
urea), ribonuclease had no enzymatic activity or defined structure but the protein
could refold and regain its full activity and structure by removal of the denaturant.
These results led Anfinsen to conclude that the information needed to specify the
complex three-dimensional structure of a protein is contained in its amino acid
sequence [80-82]. In order to decipher the problem of protein folding, one must
unravel all the non-covalent interactions encoded in the primary amino acid sequence
that guide the initial hydrophobic collapse in an aqueous environment, subsequently
leading to a compact protein fold with well defined secondary structures. The
Levinthal Paradox clearly shows that there is insufficient time to search in a random
manner the entire conformational space available to a polypeptide chain as an
unfolded protein, i.e., the protein must fold through some directed process [83]. The
restriction of conformational space could begin with the polypeptide backbone itself.
62


Thus, Ramachandran showed that steric clashes restrict the backbone phi/psi angles
of the polypeptide chain [84]* More recently, it has been suggested that the
cumulative effects of many local side-chain backbone interactions between each
amino acid and its immediate neighbors may severely restrict the conformations
accessible to a polypeptide chain [85,86].
Specific pathways for the protein folding process can be initiated by native-
like [87] and non-native [88] hydrophobic clusters of non-polar amino acids that
maintain residual protein interactions even in experimental denaturing environments
[89,90]. Furthermore, the local amphipathicity and hydrophobic clustering pattern of
short stretches of amino acid sequence often correlate with their secondary structure,
i.e., a-helices and (1-sheets [91]. These results suggest that the early stages of protein
folding may involve transient formation of secondary structure elements or nucleation
sites stabilized by a combination of long range and local hydrophobic interactions.
Despite the aforementioned complexity of the protein folding problem, it is clear that
both backbone and side-chain forces, e.g., nearest-neighbor interactions, contribute to
protein folding, although the exact balance between these forces remains unknown
and remains a critical parameter in the eventual goal of understanding and predicting
the final three-dimensional structure of a protein from polypeptide sequence
information alone.
Two major approaches to studying the protein folding problem include the
global approach where, for instance, the effect of small polypeptide composition
63


changes on overall protein structure and stability (e.g., via site-directed mutagenesis)
are examined; and a peptide-based approach where observations of the effect of
sequence changes on interactions stabilizing higher orders of conformation
(secondary, tertiary, quaternary structure) of synthetic de novo designed model
peptides are extrapolated to proteins as a whole. The objective of the present study
was to demonstrate that we could, via a synthetic peptide-based approach,
quantitatively measure nearest-neighbor interactions (or effects) in model peptides,
since such interactions, as noted above, are known to contribute to protein folding and
overall protein stability. Thus, nearest-neighbor effects are defined as interactions
between residue i and residues in the i + 1 positions of the sequence or a restriction of
conformational freedom about the peptide bonds, bonds CqtC and C^-N, between
residues i and i 1. It was our hypothesis that these nearest-neighbor interactions
would be dependent on the type of residue at position i and i + 1. In other words, all
20 amino acid side-chains could be substituted adjacent to a Gly residue, with just a
hydrogen atom as its side-chain group, and there would be no nearest-neighbor
interactions between residue i and Gly at the i + 1 position. In contrast, substitution
of all 20 amino acid side-chains adjacent to a large, bulky and hydrophobic side-chain
such as leucine at the i + 1 position Would show significant nearest-neighbor effects
dependent on the residue at position i.
Reversed-phase high-performance liquid chromatography (RP-HPLC) has
seen extensive use as a physicochemical model of biological systems, such studies
64


frequently centered on correlating peptide [92-97] or protein [98-105] retention
behavior with polypeptide conformational stability. The assumption with such
studies is that the hydrophobic interactions between peptides and proteins with the
non-polar stationary phase characteristic of RP-HPLC [1,106,107] mimics the
hydrophobicity and interactions between non-polar residues which are the major
driving forces for protein folding and stability. In a previous paper [108], Hodges, et
al. categorized two types of sequence-dependent effects resulting in deviations from
predictable RP-HPLC retention behavior of peptides: conformational effects resulting
in an apparent reduction or enhancement of the overall hydrophobicity of a peptide as
a result of its adopting an unique conformation on interacting with the hydrophobic
stationary phase, compared to the hydrophobicity of the peptide if it existed as a
random coil; and nearest-neighbor effects resulting in sequence-dependent variability
of peptide retention behavior but independent of conformation. While the former
effects have been well studied [109], useful quantification of nearest-neighbor effects
on peptide retention behavior in RP-HPLC has proven more elusive. The present
study sets out to remedy this deficiency by observing the RP-HPLC retention
behavior of the model peptide Ac-X-Leu-Gly-Ala-Lys-Gly-Ala-Gly-Val-Gly-amide
(where position X is substituted by the 19 D- and L- amino acids and glycine) under
several mobile phase conditions. Differences in observed retention times of the D-
and L- peptide diastereomers, due to the presence of leucine adjacent to the
substitution site, have enabled us to make an initial quantification of nearest-neighbor
65


effects on peptide retention behavior and should also prove a useful first step in
extrapolating such values to the protein folding problem.
4.2 Experimental
4.2.1 General Experimental
General experimental parameters can be found in chapter 2 of this thesis.
4.2.2 Analytical RP-HPLC of Synthetic Model
Peptides
Linear AB gradient (0.25% acetonitrile/min) at a flow-rate of 0.3 ml/min and
a temperature of 25C.
Mobile phase 1: Eluent A is 20 mM aq. H3PO4, pH 2, and Eluent B is 20 mM
H3PO4 in acetonitrile, denoted pH 2/H3PO4 system.
Mobile phase 2: Eluent A is 20 mM aq. TTA, pH 2, and Eluent B is 20 mM
TFA in acetonitrile, denoted pH 2/TFA system.
Mobile phase 3: Eluent A is 10 mM aq. NaH2P04, adjusted to pH 7 with
NaOH, and Eluent B is Eluent A containing 50% acetonitrile, denoted pH 7/no salt
system.
Mobile phase 4: same as mobile phase 3 but also both eluents containing 50
mM NaCl, denoted pH 7/NaCl system.
66


Mobile phase 5: same as mobile phase 3 but also both eluents containing 50
mM NaC104, denoted pH 7/NaC104 system.
4.3 Results
4.3.1 Design of Model Peptides
The sequence chosen to quantify nearest-neighbor effects on peptide retention
behavior during RP-HPLC was Ac-X-L-G-A-K-G-A-G-V-G-amide, where position
X was substituted by the 19 D- and 19 L- amiho acids and glycine. This sequence
contains four Gly residues spread periodically throughout the sequence to ensure that
the peptide has no secondary structure tendencies [72,73]. The N-terminus was
acetylated and the C-terminus was amidated to eliminate potential effects of a
positively charged a-amino group or negatively charged a-carboxyl group on peptide
retention behavior. A single Lys residue was incorporated into the model peptide
sequence to ensure peptide solubility over a wide pH range. The four hydrophobes in
the peptide sequence (2 Ala, 1 Val, 1 Leu) were distributed throughout the peptide
sequence to ensure no clustering of hydrophobes and subsequent creation of a
preferred binding domain. The 10-residue length of the model peptide was selected
to enable full expression of the overall hydrophilicity/hydrophobicity of the peptide in
the absence of any chain-length effects (generally >15 residues) [71], which may
diminish the contribution to RP-HPLC retention behavior of substituted residues.
67


The substitution site, X, was situated next to a Leu residue since it was
hypothesized that such a bulky side-chain would maximize any nearest-neighbor
interactions on the adjacent substituted side-chain. Further, its neutral, non-polar
characteristics would eliminate any concerns of data interpretation complications, in
this initial study, due to electrostatic (i.e., positively charged or negatively charged
side-chains) or polar interactions (e.g., hydrogen bonding) affecting nearest-neighbor
effects on the substituted amino acid. Finally, D- and L- amino acids were substituted
at position X since it was hypothesized that, due to the different configuration of such
amino acid pairs, subsequent differences in conformational freedom about the X-Leu
peptide bond, bonds CqtC and Co-N, would be reflected in differences in exposure of
this substituted residue at position X and, hence, could be measured by variations in
observed RP-HPLC retention behavior. Note that, for the purposes of the present
study, only the effect of one adjacent (Leu) residue on peptide retention behavior is
under consideration (i.e., position X is the N-terminal residue) in order to simplify
data interpretation in this initial assessment of our RP-HPLC/peptide model system.
Also note that Gly was at position 3 to prevent any nearest-neighbor interactions with
Leu at position 2. Thus, only the interactions or effects caused by the amino acid at
position X and the adjacent Leu are being determined.
68


4.3.2 Nearest-Neighbor Effects on RP-HPLC
Retention Behavior of Model Peptides
In the previous chapter, D- and L- amino acids were substituted at position X
of a model peptide with the sequence Ac-X-G-A K- G-A-G-V-G-L-
arnide, i.e., with the same overall composition as the model peptide in the present
study but with the substitution site now adjacent to a Gly residue. Co-elution of the
D- and L- amino acid substituted peptide pairs of these peptides demonstrated that
there was complete freedom of rotation between 0 of residue i (substitution site) and
0 of residue i +1, where \p refers to rotations about the CcrC single bond and 0 refers
to rotations about the Co-N single bond. Thus, any variation in retention behavior of
the model peptides in the present study would indeed be due to the substitution site
being adjacent to a bulky Leu residue.
The D- and L- amino acid substituted model peptides, as well as the Gly-
substituted peptide, were subjected to RP-HPLC under five mobile phase conditions:
20 mM H3PO4 or 20 mM TFA at pH 2; and 10 mM PO4 buffer at pH 7, containing no
salt, 50 mM NaCl or 50 mM NaC104. Note that a very shallow gradient (0.25%
acetonitrile/min) was employed in the peptide separations in order to maximize any
differences in retention behavior of D- and L- peptide diastereomers.
Fig 4.1 illustrates elution profiles of representative D- and L- amino acid
substituted peptide pairs at pH 2 (pH 2/TFA system). The retention data for all
peptides under the five mobile phase conditions are shown in Table 4.1.
69


Fig 4.1 Demonstration of nearest-neighbor effects in a model peptide system Column: Kromasil C18.
Conditions: linear AB gradient (0.25% CH3CN/min, starting from 2% CH3CN) at a flow-rate of 0.3
ml/min, where eluent A is 20 mM aq. TFA and eluent B is 20 mM TFA in CH3CN; temperature, 25C.
Panels A, B and C are representative RP-HPLC elution profiles at pH 2.0 of peptides of the same
sequence (Ac-X-L-G-A-K-G-A-G-V-G-amide), where position X contains D- or T .-amino acid
substitution adjacent to a Leu residue. The peptides are denoted by the three letter code of the
substituted amino acid residues, e.g. D-Lys or L-Lys.
70


Table 4.1: RP-HPLC peptide retention data in various mobile phase conditions at 25C
Amino Acid Substitution3 pH 20 mM h3po4 1r(D-) 1r (L-) 2b 20 mM TFA 1r(d-) 1r Trp 69.9 66.5 78.4 74.7 71.1 67.9 73.1 69.6 83.3 79.9
Phe 71.4 63.6 79.0 71.5 72.0 64.5 74.0 65.5 84.4 77.1
Leu 64.1 57.4 73.2 66.6 66.3 59.9 -68.8 62.2 79.3 72.9
lie 64.4 56.6 72.9 65.1 65.7 58.4 68.1 60.8 78.3 71.1
Met 55.1 49.1 63.3 57.3 57.3 51.4 59.3 53.6 68.8 63.2
Val 54.6 47.9 62.8 55.8 56.8 50.5 58.8 52.2 68.1 61.5
Tyr 46.6 47.6 54.6 55.5 49.1 50.0 50.9 51.8 58.9 60.2
Pro 45.4 42.0 54.6 51.3 48.9 45.9 51.0 47.8 59.9 57.2
Cys 45.7 41.3 53.4 48.8 48.4 44.4 52.6 48.2 58.5 54.2
Glu 37.7 36.3 44.4 43.4 36.5 35.5 38.6 37.8 40.2 40.1
Ala 38.4 35.5 46.0 42.8 41.4 38.6 45.4 42.3 51.2 48.2
Asp 34.7 34.7 41.6 41.6 36.1 37.0 37.9 38.9 37.7 38.4
Thr 35.6 34.3 42.8 41.4 38.5 37.1 40.4 39.1 47.8 46.3
Gly 33.3 33.3 40.1 40.1 36.0 36.0 37.6 37.6 45.1 45.1
Ser 31.6 33.2 38.7 40.1 34.7 36.2 36.4 37.8 43.3 44.6
Gin 32.8 33.0 39.5 39.9 35.6 35.9 39.2 39.5 44.1 44.4
Asn 31.9 32.8 38.7 39.7 35.0 35.9 38.3 39.3 43.3 44.1
Arg 25.8 27.1 40.9 42.2 30.1 31.4 35.3 36.8 48.8 50.5
His 23.9 27.0 38.3 41.6 28.5 32.8 31.9 37.3 46.1 49.3
Lys 23.5 25.5 38.4 39.8 30.6 30.0 34.4 33.4 45.5 47.6
3 The D- and L- amino acid substitutions at position X in the peptide sequence Ac-X-L-G-A-K-G-A-G-V-
G-amide.
b Column: Kromasil C18. Conditions: linear AB gradient (0.25% CH3CN/min, starting from 2% CH3CN) at
a flow-rate of 0.3 ml/min, where eluent A is 20 mM aq. H3P04 or 20 mM TFA (pH 2) and eluent B is 20
mM H3PO4 or 20 mM TFA in CH3CN; temperature 25 C. tR denotes peptide retention time of the D- or
L-substituted peptide.
c Column: Zorbax XDB C8. Conditions: linear AB gradient (0.25% CH3CN/min) at a flow-rate of 0.3
ml/min, where eluent A is 10 mM aq. NaH2P04 (pH 7) with or without 50 mM NaCl or 50 mM NaC104 and
eluent B is the respective eluent A containing 50% CH3CN; temperature 25C. tR denotes peptide retention
time of the D- or L-substituted peptide.
71


Please note that the valuesin Table 4.1 represent an averaged retention determined
over at least 3 RP-HPLC runs. From Fig. 4.1 and Table 4.1 it is clear that, unlike
substitution adjacent to a Gly residue (see previous chapter), the presence of a Leu
residue adjacent to the substitution site results in significant separation of D- and L-
peptide pairs. In addition, the degree of this separation, as well as the relative order
of the D- and L- diastereomers, varies quite dramatically. Such observations are
discussed below.
4.3.2.1 Effect of Mobile Phase Conditions on
Retention Behavior of Peptides
Table 4.2 reports the effect of mobile phase additives at pH 2 and pH 7 on
retention times of the D- and L- amino acid substituted model peptides. Thus, at pH
2.0, the values shown are the change in peptide retention time between the pH 2/TFA
and pH 2/H3PO4 mobile phase systems (AtR = tR in TFA minus 1r in H3PO4), i.e., the
effect of replacing an hydrophilic phosphate anion with the relatively hydrophobic
trifluoroacetate (TFA') anion. At pH 7, the values shown are the change in peptide
time between the pH 7 (no salt) system and either the pH 7/NaCl mobile phase (AtR =
tR in presence of 50 mM NaCl minus tR in absence of salt) or the pH 7/NaClC>4
mobile phase (AtR = tR in presence of 50 mM NaClC>4 minus tR in absence of salt).
Addition of salts (generally 50-100 mM) to mobile phases over a pH range of ~4 7
has generally been designed, for silica-based packings, to suppress negatively charged
72


silanol interactions with positively charged solutes [1,106,107,26,32,110-116], with
potential selectivity effects tending to be a secondary consideration. However, our
laboratory has demonstrated how salt (specifically sodium perchlorate) addition may
offer gains in peptide selectivity at low pH [117].
From Table 4.2, the increase in anionic counterion hydrophobicity (phosphate
< TFA') at pH 2 or the addition of salt to the 10 mM NaH2P04 buffer has an
essentially equal effect on the retention time of each peptide in a diastereomeric
peptide pair, albeit the magnitude of this effect varies depending on the mobile phase
and the amino acids under consideration, specifically those with potentially positively
charged (Lys, His, Arg) and potentially negatively charged (Asp, Glu) side-chains.
At pH 2, the negatively charged anion (phosphate, TFA") of the ion-pairing
reagents H3PQ4 and TFA, respectively, affect peptide retention behavior via
interaction with positively charged groups in the peptide, the more hydrophobic TFA'
enhancing peptide retention times over that of the hydrophilic phosphate anion
[1,5,106,107,118,119]. At pH 2.0, with the exception of Lys, His and Arg, this
enhancement of peptide retention time in 20 mM TFA over that of 20 mM H3PO4 is
due to interaction of the hydrophobic TFA' ion with the single positively charged
amino group on the side-chain of the single Lys residue in the remaining 17 peptides.
73


Table 4.2 Effects of mobile phase conditions
Amino Acid Substitution3 pH 2b A(TFA-H3P04)c D L pH7b A(NaCl-No Salt)" D L pH 7b A(NaCI04-No Salt)6 D L
Tip 8.5 8.2 2.0 1.7 12.3 12.0
Phe 7.6 7.9 2.0 1.0 12.5 12.6
Leu 9.1 9.2 2.5 2.3 13.0 13.0
He 8.5 8.5 2.3 2.4 12.6 12.7
Met 8.2 8.2 2.0 2.2 11.5 11.8
Val 8.2 7.9 2.0 1.7 11.3 11.0
Tyr 8.0 7.9 1.9 1.8 9.8 10.2
Pro 9.1 9.3 2.1 1.9 11.0 11.3
Cys 7.7 7.5 4.1 3.8 10.1 9.8
Ala 7.6 7.3 4.0 3.7 9.9 9.6
Thr 7.2 7.1 1.9 2.0 9.3 9.2
Gly 6.8 6.8 1.6 1.6 9.1 9.1
Ser 7.1 6.9 1.7 .1.6 8.6 8.4
Gin 6.7 6.9 3.5 3.6 8.5 8.5
Asn 6.8. 6.9 3.3 3.4 8.3 8.2
Arg 15.1 15.1 5.2 5.4 20.4 17.4
His 14.4 14.6 3.4 4.5 17.6 16.5
Lys 14.9 14.3 3.8 3.4 14.9 17.6
Asp 6.9 6.9 1.8 1.9 1.6 1.4
Glu 6.8 7.1 2.2 2.3 3.7 4.6
a The D- and L-amino acid substitutions at position X in the peptide sequence
Ac-X-L-G-A-K-G-A-G-V-G-amide.
b Column: pH 2, Kromasil Cl 8; pH 7, Zorbax XDB C8 Conditions: pH 2, shown in
Table 1; pH 7, shown in Table 4.1.
c Denotes difference in peptide retention time between 20 mM TFA and
20 mM H3PO4 (pH2) mobile phase systems shown in Table 4.1.
d Denotes difference in peptide retention time between pH 7 mobile phase
system containing 50 mM NaCl and pH 7 mobile phase system in the
absence of salt (Table 4.1).
6 Denotes difference in peptide retention time between pH 7 mobile phase
system containing 50 mM NaC104 and pH 7 mobile phase system in the
absence of salt (Table 4.1).
74


Hence, for these peptides substituted with neutral (at pH 2.0) side-chains at position
X, the effect of the increase in hydrophobicity of the anion on peptide retention time
is similar (particularly considering the very shallow nature of the acetonitrile
gradient) within a group of peptides (i.e., D- or L- amino acid substituted peptides;
the median AtR values for the D- and L- diastereomers were 7.9 min +1.2 and 8.0
min + 1.2, respectively) or between diastereomeric peptide pairs. However, for the
peptides substituted with Lys, His or Arg, the extra positive charge on these peptides
resulted in a marked increase in retention time between the hydrophilic H3PO4 system
and the hydrophobic TFA system (approximately twice the median values for the L-
and D-peptide diastereomers noted above).
At pH 7 (Table 4.2), the addition of 50 mM NaCl to the 10 mM NaH2P04
buffer had, overall, only a relatively small effect on peptide retention time of both the
D- and L- series peptides. Again, this effect was relatively similar between D- and L-
amino acid substituted peptide pairs and within the D- and L- peptide series,
including both those substituted with potentially positively charged (Lys, Arg and
His, albeit the latter is, likely to be largely deprotonated, i.e., neutral, at pH 7) and the
potentially negatively charged Asp and Glu amino acids (both of these acidic residues
are likely to be largely deprotonated, i.e., negatively charged at pH 7). Such results
confirm earlier observations [120] that the chloride ion is relatively ineffective as an
ion-pairing reagent. In contrast, the addition of 50 mM NaC104 to the buffer at pH 7
was much more dramatic than that of 50 mM NaCl, with significant increases in
75


peptide retention time for 18 out of 20 of the pieptides for both series of peptides (the
only exceptions being the Asp- and Glu- substituted analogs). Such results for
NaC104 are consistent with the highly effective ion-pairing properties of the
perchlorate (CIO4") anion [120]. In addition, in a similar manner to the pH 2 results
where the hydrophobic TFA' anion enhanced the retention times of the Lys-, His- and
Arg- substituted peptides relative to phosphate, the presence of perchlorate at pH 7
generally shows a significant enhancement of the retention times of these peptides
over that of the remaining 17 peptides. The retention times of the D- and L- peptide
diastereomers are again essentially identical while the effect of NaCICU on retention
times within the D- and L- peptide series are similar, albeit there is evidence of some
variation which appears to correlate approximately with the
hydrophilicity/hydrophobicity of the substituted amino acid; i.e., without including
the potentially positively charged (Lys, His, Arg) and the potentially negatively
charged (Asp, Glu) residue, the effect of salt addition decreases with decreasing
hydrophobicity of the substituted side-chain, e.g., from a value of 12.3 min for the D-
Trp- substituted peptide to 8.3 min for D-Asn. A similar, albeit not as clear, effect
can also be seen at pH 2. The significantly lesser effect of the presence of NaC104 on
the retention behavior of the Asp- and Glu- substituted peptides is likely due to the
lack of ion-pairing interactions between the positively charged side-chains and the
CIO4' anion in the presence of the negatively charged side-chains.
76


4.3.2.2 Evaluation of Nearest-Neighbor Effects by RP-
HPLC of Model Peptides
Table 4.3 reports the quantitative effects of nearest-neighbor interactions of
the 20 amino acids under the five mobile phase conditions, as defined by the
magnitude of the difference in retention times between the D- and L- peptide
diastereomers, i.e., AtR(D-L> Interestingly, the AtR values are generally very similar for
all five mobile phases, i.e., the magnitude of the nearest-neighbor effect is essentially
independent of pH and mobile phase conditions. Some exceptions are apparent,
notably Glu in the pH 7/NaClC>4 mobile phase, Asp at pH 7 versus pH 2, Lys in the
pH 7/no salt mobile phase and His in the pH 7/no salt and NaClC>4 systems.
However, overall there is a distinct consistency of AtR values under all conditions.
Also from Table 4.3, there is a wide range of AtR(D-L) values for the 20 amino
acids, ranging, in the pH 2/H3PO4 system, for example, from + 7.8 min for Phe to -3.1
min for His. As noted above, this range is quite consistent for all five mobile phases.
Significantly, there is a good general correlation of AtR values with relative
hydrophilicity/hydrophobicity of the side-chains, i.e., AtR becomes generally
increasingly more positive with increasing side-chain hydrophobicity and less
positive with increasing side-chain hydrophilicity. Note that a positive AtR value
indicates that the D-amino acid substituted peptide was eluted after its L-counterpart;
in contrast, a negative AtR value indicates that the L-amino acid substituted peptide
was eluted after its D-counterpart.
77


Table 43: Nearest-neighbor effects determined at 25 C by
RP-HPLC of model peptides.
Amino Acid Substitution3 pH 2b 20 mM 20 mM H3PO4 TFA AtR(D_L)C AtR(]>.L) pH 7b, No Salt AtR(D-L) 10 mMPOa Buffer 50 mM 50 mM NaCl NaCKXj AtR(D.L) AtR(D.L)
Phe -7.8 7.5 7.5 8.5 7.3
lie 7.8 7.8 7.3 7.3 7.2
Leu 6.7 6.6 6.4 6.6 6.4
Val 6.7 7.0 . 6.3 6.6 6.6
Met 6.0 6.0 5.9 5.7 5.6
Cys 4.4 4.6 4.0 4.4 4.3
Pro 3.4 . 3.3 3.0 3.2 2.7
Trp 3.4 3.7 3.2 3.5 3.4
Ala 2.9 3.2 2.8 3.1 3.0
Glu 1.4 1.0 1.0 0.8 0.1
Thi 1.3 1.4 1.4 1.3 1.5
Asp 0.0 0.0 -0.9 -1.0 -0.7
Gly 0.0 0.0 0.0 0.0 0.0
Gin -0.2 -0.4 -0.3 -0.3 -0.3
Asn -0.9 -1.0 -0.9 -1.0 -0.8
Tyr -1.0 -0.9 -0.9 -0.9 -1.3
Arg -1.3 -1.3 -1.3 -1.5 -1.6
Lys -2.0 -1.4 -0.6 -1.0 -2.1
Ser -1.6 -1.4 -1.5 -1.4 -1.3
His -3.1 -3.3 -4.3 -5.4 -3.2
3 The D- and L- amino acid substitutions at position X in the peptide sequence
Ac-X-L-G-A-K-G-A-G-V-G-amide.
b Column and Conditions are shown in Table 4.1.
c AtRfD-L) denotes the difference in retention time between D- and L- diastereomers.
78


The varying magnitude of the AtR values shown in Table 4.3 is likely due to a
combination of side-chain characteristics (hydrophilicity/hydrophobicity, size and
structure) and the final configuration of the D- or L- amino acid when substituted
adjacent to a leucine residue. A starting point for speculation would be that, whether
a D- or L- amino acid is substituted at position X of the model peptide sequence, the
configuration of its side-chain at the end of the polypeptide chain, i.e., its orientation
relative to the bulky Leu side-chain at the i + 1 position, represents the most
energetically favorable configuration. In addition, no electrostatic interactions are
taking place between the substituted amino acid and the adjacent Leu residue.
Thus, for example, when the large, hydrophobic side-chain of D-Phe (AtR =
7.8 min in the pH 2/H3PO4 system) is substituted at position X, the most favorable
positioning of this side-chain relative to the adjacent L-Leu may be such that the
interaction of the D-Phe side-chain with the hydrophobic stationary phase is favored
more than with a L-Phe substitution, thus enhancing the apparent hydrophobicity of
the D-Phe relative to L-Phe, i.e., the L-amino acid substituted peptide is eluted prior
to its D-counterpart. Such a mechanism would also be consistent with the general
correlation of side-chain intrinsic hydrophobicity of the non-polar side-chains with
their AtR values.
An interesting anomaly from Table 4.3 is the AtR value for Trp (3.4 min in the
pH 2/H3PO4 system) which is similar to the moderately hydrophobic side-chain Ala
despite its high intrinsic hydrophobicity [109]. It is possible that, due to the bulky,
79


fused ring structure of the Trp side-chain, orientation of the D-Trp side-chain to the
hydrophobic stationary phase is such that it is unable to express its full
hydrophobicity despite the more favorable D-Trp/L-Leu interaction with the
hydrophobic matrix compared to that of L-Trp/L-Leu.
From Table 4.3, the negative AtR(D.L) values for polar and charged residues
may also be explained if it is assumed that the orientation of D-amino acids that
favors interaction of hydrophobic side-chains over that of the L-isomers also applies
to such highly polar and charged side-chains. For example, favorable orientation of a
D-Lys side-chain when adjacent to a Leu residue would bring a positively charged
side-chain in proximity to the hydrophobic stationary phase to a greater extent than if
a L-Lys residue was substituted, i.e., the overall apparent hydrophobicity of the D-
Lys substituted peptide would be less than that of its L-Lys counterpart and, hence,
would be eluted earlier.
It is interesting to note that the AtR value for Asp (0 min in pH 2/H3PO4
system) became negative, i.e., more polar, at pH 7, following deprotonation (i.e., the
acquisition of a negative charge) at neutral pH. A similar, albeit lesser, trend was also
seen for Glu between pH 2 and pH 7, which did not exhibit a negative AtR value
despite its general decreasing positive AtR value.
It was demonstrated by our laboratory [75] that an hydrophobic environment
enhanced the hydrophilic character of polar/charged side-chains. The orientation of
D-Tyr towards the hydrophobic matrix when substituted adjacent to Leu is likely
80


enhancing the hydrophilic nature of its hydroxyl group relative to its L-Tyr
counterpart, hence the overall lesser hydrophobicity of the D-Tyr-substituted peptide
causing it to be eluted prior to its L-diastereomer (AtR =-1.6 min in the pH 2/H3PO4
system). Such an enhancement in the hydrophilic character of the D-Ser side-chain (a
polar hydroxyl group on the P-carbon) relative to the L-Ser could also account for the
large negative AtR value (-1.6 min) reported in Table 4.3. In contrast, the positive
value for Thr (AtR = +1.3) may be due to masking of the polar hydroxyl group on the
P-carbon of the D-Thr side-chain by the methyl group on this same P-carbon, i.e., the
hydrophobic characteristics of the D-Thr are dominant, leading to more favored
hydrophobic interactions of this side-chain with the stationary phase relative to L-Thr.
Fig. 4.2 now plots the AtR(D.L) values of the 20 amino acids obtained in the pH
2/H3PO4 mobile phase versus the pH 2/TFA mobile phase (Fig. 4.2A) and the AtR(D-L)
values at pH 7 in the absence of salt versus those in the presence of 50 mM NaCl or
50 mM NaClC>4 (Fig. 4.2B). From Fig. 4.2A, the magnitude of the nearest-neighbor
effects at pH 2 is shown to be independent of the relative hydrophobicity of the anion
(phosphate < TFA') in the mobile phase (an excellent correlation of 0.997).
81


10.0 1
AtfioM.) in 10 mM NaH,P04 mobile phase (min)
Fig 4.2 Plot of AtR(D-L) of peptides in 20 mM H3PO4 (pH 2) versus 20 mM TFA (pH 2) (A) and 10 mM
NaH2P04 (pH 7) versus 10 mM NaH2P04 (pH 7) with 50 mM NaCl or 50 mM NaC104 (B). Column:
Kromasil Cl 8, pH 2; Zorbax XDB C8, pH 7. Conditions: linear AB gradient (0.25% CH3CN/min) at a
flow-rate of 0.3 ml/min. Panel A: eluent A is 20 mM aq. H3P04 or 20 mM TFA, pH 2 and eluent B is
the respective eluent A in CH3CN; Panel B: eluent A is 10 mM aq. NaH2P04, pH 7, containing no salt,
50 mM NaCl or 50 mM NaC104 and eluent B is the respective eluent A containing 50% CH3CN;
temperature, 25C. Data are taken from Table 4.2. The single letter code represents the L-amino acid
substitutions at position X of the peptide sequence shown in Fig 4.1, panel A. Closed circles denote
data used in the correlation plot (y = 0.9905x + 0.0599, correlation coefficient r = 0.997 and y =
1.0604x + 0.053, correlation coefficient r = 0.995 for plots in panels A and B, respectively).
82


A similar result is seen at pH 7 (Fig. 4.2B) where, despite slightly anomalous
behavior of the His value in 50 mM NaCl, the AtR values were essentially
independent of the effectiveness of the anion (C1 < CIO4") in the mobile phase (a
correlation of 0.995).
4.3.2.3 Hydrophilicity/Hydrophobicity Coefficients Of
D- And L- Amino Acids In Presence Of Nearest-
Neighbor Effects
A large number of RP-HPLC-derived hydrophilicity/hydrophobicity
coefficients of amino acid side-chains based on RP-HPLC of collections of random
peptides [26,8,11,27,30,31,34,36-38,121,122] or synthetic model peptides [32,75,33]
have been published over the past 25 years. Unlike the scales reported in the previous
chapter [109] which were determined in the absence of nearest-neighbor effects (i.e.,
intrinsic side-chain hydrophilicity/hydrophobicity was measured), these earlier scales
were, among other factors, influenced by nearest-neighbor effects such as those
described in the present study (for comparison of other scales, see next chapter).
In order to illustrate the variation in apparent side-chain
hydrophilicity/hydrophobicity which may occur even in a relatively simple model
peptide system such as that described in the present study, where amino acid
substitutions were made next to a single adjacent Leu residue,
hydrophilicity/hydrophobicity coefficients were now determined for both the D- and
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c
L- amino acid side-chains in our model peptide. The retention time of the Gly-
substituted peptide was used as a reference since Gly has only an hydrogen atom as
its side-chain. Thus, the hydrophilicity/hydrophobicity coefficients of the 19 D- side-
chains and 19 L- side-chains were generated from the RP-HPLC runs carried out
under the five mobile phase conditions from the difference in the retention times (AtR)
of the X-substituted peptide and the Gly-substituted peptide, i.e., AtR = tR X-
substituted peptide minus tR Gly-substituted peptide (Table 4.4). Thus, from Table
4.4, side-chains that are more hydrophobic than Gly have positive AtR values and
side-chains that are more hydrophilic than Gly have negative AtR values.
From Table 4.4, there is a clear difference in the side-chain coefficients
obtained from the D- and L-amino acid substituted peptides, despite the similarities in
relative hydrophilicity/hydrophobicity within the D- and L- series, i.e., most
hydrophobic to most hydrophilic side-chains. In general, side-chains more
hydrophobic than that of Gly (i.e., positive AtR values) show a greater relative
hydrophobicity when substituted as the D- amino acid than the L- amino acid; in
contrast, side-chains more hydrophilic than that of Gly (i.e., negative AtR values)
show a greater relative hydrophilicity when substituted as the D- amino acid than the
L- amino acid.
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Table 4.4: RP-HPLC hydrophilicity/hydrophobicity coefficients in various mobile phases at 25 C
Amino Acid Substitution3 Pi 20 mM H,P04 AIr (O-Oy) AtR [2 20 mM TFA AtR(IVG|y) AtR(L4J|y) PH No Salt AtR (iwjiy) AtR(Kay) 7b, 10 mM P04 Bu 50 mM NaCl AtR (D-Gly) AtR (K3y) ffer 50 mM NaC104 AtR (IMny) AtR (nay)
Tip 36.6 33.2 38.3 34.6 35.0 31.9 35.5 32.1 38.2 34.9
Phe 38.0 30.3 38.9 31.3 35.9 28.5 36.4 27.9 39.3 32.0
Leu 30.8 24.1 33.1 26.4 30.3 23.9 31.2 24.7 34.2 27.9
lie 31.1 23:3 32.8 25.0 29.7 22.4 30.5 23.2 33.3 26.0
Met 21.8 15.7 23.2 17.2 21.3 15.4 21.7 16.0 23.7 18.1
Val 21.3 14.6 22.7 15.7 20.8. 14.5 21.2 14.6 23.0 16.4
Tyr 13.2 14.2 14.5 15.3 13.0 14.0 13.3 14.2 13.8 15.2
Pro 12.1 8.6 14.5 11.2 12.8 9.9 13.4 10.2 14.8 12.1
Cys 12.4 7.9 13.3 8.7 12.4 . 8.3 15.0 10.6 13.5 9.1
Glu 4.3 3.0 4.3 3.2 0.5 -0.5 1.1 0.3 -4.9 -5.0
Ala 5.0 2.2 5.9 2.7 5.4 2.5 7.8 4.7 6.2 3.1
Asp 1.4 1.4 1.4 1.5 0.1 1.0 0.3 1.3 -7.4 -6.7
Thr 2.2 1.0 2.7 1.3 2.5 1.1 2.8 1.5 2.7 1.2
Gly 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0
Ser -1.8 -0.2 -1.4 -0.1 -1.3 0.2 -1.2 0.3 -1.8 -0.5
Gin -0.6 -0.3 -0.6 -0.3 -0.4 -0.1 1.6 1.9 -1.0 -0.6
Asn -1.5 . -0.6 -1.4 -0.5 -1.0 -0.1 0.7 1.7 -1.8 -0.9
Arg -7.6 -6.2 0.7 2.1 -6.0 -4.7 -2.3 -0.7 5.4 3.8
Lys -9.4 -6.3 -1.8 1.5 -7.5 -3.2 -5.7 -0.3 1.0 4.2
His -9.8 -7.9 -1.7 -0:3 -5.4 -6.1 -3.2 -4.2 0.4 2.6
a The D- and L-amino acid substitutions at position X in the peptide sequence Ac-X-L-G-A-K-G-A-G-V-G-amide.
bCoIumn: pH 2, Kromasil CI8; pH 7, Zorbax XDB C8. Conditions: pH 2, shown in Table 1;
pH 7, shown in Table 4.1.
c AtRflxjiy,) or AtR(K;W denotes the retention time, Ir, of the D- or L-amino acid substituted peptide minus the retention
time of the Gly substituted peptide.
85


Such variations between the D- and L- amino acids are likely due, as discussed
previously, to the different configurations assumed by the side-chains due to the
adjacent Leu residue and, hence, their relative exposure to the reversed-phase matrix.
Such exceptions to this general observation are apparent: Tyr, despite its moderately
high apparent hydrophobicity (AtR values of 13.2 mm and 14.2 min for D-Tyr and L-
Tyr, respectively), shows a higher apparent hydrophobicity for the L-amino acid,
likely, as noted above, to the response of its polar hydroxyl group to its environment
when in the D- versus L- configuration; Asp shows essentially identical coefficients
in the pH 2/H3PO4 system and pH 2/TFA system and more apparent hydrophobicity
for the L-amino acid relative to the D-isomer in all three pH 7 systems; Gin, unlike
the other four mobile phases, has positive AtR values in the pH 7/NaCl system, with
L-Gln having a larger apparent hydrophobicity (1.9 min) compared to D-Gln (1.6
min); also, Asn, in a similar manner to Gin, has positive AtR values in the pH 7/NaCl
system, with values for L-Asn and D-Asn of 1.7 min and 0.7 min, respectively.
Fig. 4.3 now correlates the hydrophilicity/ hydrophobicity coefficients of the
L- amino acids (A) and D- amino acids (B) obtained in the pH 2/H3PO4 mobile phase
versus those obtained in the pH 7/NaC104 system. From Fig. 4.3, the coefficients of
15 out of the 19 (plus Gly) D- and L- amino acids show an excellent correlation (r =
0.994 and 0.997 for the D- and L- amino acids, respectively) between the two mobile
phase systems.
86


AtRti^Gfy) in 20 mM H3PO4 (pH 2) mobile phase (min)
Ati^o-ciy) >>> 20 mM H3PO4 (pH 2) mobile phase (min)
Fig 4.3 Plot of hydrophilicity/hydrophobicity coefficients (At^oiy)) determined at pH 2,20 mM H3PO4
versus pH 7 10 mM NaH2P04 plus 50 mM NaC104; panel A, L- diastereomers; panel B, D-
diastereomers. Column: Kromasil C18, pH 2; Zorbax XDB C8, pH 7. Conditions: linear AB gradient
(0.25% CH3CN /min) at a flow-rate of 0.3 ml/min, where eluent A is 20 mM aq. H3PO4, pH2 or 10
mM aq. NaH2P04, pH 7 and eluent B is 20 mM H3PO4 in CH3CN, pH 2 or 10 mM NaH2P04 in 50%
CH3CN, pH 7; temperature 25C. Data are taken from Table 4.3. The single letter code represents the
L-amino acid substitutions at position X of the peptide sequence shown in Fig 4.1, panel A. Closed
circles denote data used in the correlation plot (y = 1.0783x + 0.4136, correlation coefficient r = 0.994
and y = 1.0575x + 0.3138, correlation coefficient r = 0.997 for plots in panels A and B, respectively);
open circles represent results from positively charged substitutions.
87