Peptide dissociation in solution or bound to a polymer: Comparative solvent effect

Article abstract of DOI:10.1016/j.tet.2004.08.013

Dissociation of peptide when in solution or attached to a polymer was investigated. Magnified solvation of peptide-resins occurred in solvent with similar polarity. Conversely the solubilization of peptides was not usually directly related to the medium polarity. The greater the difference between acidity and basicity of solvent and its potential to form van der Waals interaction, the stronger its solubilization strength. Solvents with similar electrophilicity and nucleophilicity usually did not solvate aggregated peptide-resins nor dissolve peptides. The peptide solubilization in water-containing mixed solvents depended on combination of acidity/basicity of both components. Some criteria for choosing suitable solvents for peptide-resin solvation or peptide solubilization could be advanced. Graphical abstract.

Full text of DOI:10.1016/j.tet.2004.08.013

Tetrahedron 60 (2004) 9417–9424
Peptide dissociation in solution or bound to a polymer:
comparative solvent effect
*
´
Luciana Malavolta and Clovis R. Nakaie
Department of Biophysics, Universidade Federal de Sa˜o Paulo, Rua 3 de Maio 100, CEP 04044-020, Sa˜o Paulo, SP, Brazil
Received 9 March 2004; revised 28 July 2004; accepted 5 August 2004
Abstract—Dissociation of peptide when in solution or attached to a polymer was investigated. Magnified solvation of peptide-resins
occurred in solvent with similar polarity. Conversely the solubilization of peptides was not usually directly related to the medium polarity.
The greater the difference between acidity and basicity of solvent and its potential to form van der Waals interaction, the stronger its
solubilization strength. Solvents with similar electrophilicity and nucleophilicity usually did not solvate aggregated peptide-resins nor
dissolve peptides. The peptide solubilization in water-containing mixed solvents depended on combination of acidity/basicity of both
components. Some criteria for choosing suitable solvents for peptide-resin solvation or peptide solubilization could be advanced.
q 2004 Elsevier Ltd. All rights reserved.
1. Introduction
simply referred to as the overall solvation power of a
solvent.1,2
Complete understanding of the phenomenon of solute–
solvent interaction has been eluding researchers for almost
two centuries. Despite the exceptional relevance of this
theme for all fields of science, this mystery has yet to be
unravelled. The findings obtained to date only reinforce the
difficulties in attaining a consensus about the rules that
might govern this interaction. Amongst the innumerable
factors that have been proposed over the years as controlling
the solvent effect upon solute molecules, the relationship
between the polarities of both components seems to be of
utmost relevancy.1 However, the so-called polarity par-
ameter is not also easy to define or quantify and has been
In a conceptual departure from the great majority of
approaches that have been applied to investigate this
physico-chemical parameter, we have focused on interpret-
ing the solute–solvent effect, deliberately using complex
polymeric materials as examples of the solute component.
Emphasis has been given to peptide resins and their
interaction with a great number of solvents of varying
polarities. This relationship has been assessed by measure-
ment of peptide-resin solvation3,4 or by determination of the
dynamics of the interior of the solvated peptide–polymer
network5 using amino-acid type spin probes.6 Starting from
the knowledge that the presence of electrophilic and
nucleophilic groups in a peptide bond (N–H and C]O
moieties, respectively) might strongly affect the interaction
of the solute with the solvent system, we have recently
proposed the 1:1 sum of Gutman’s7 solvent electron
acceptor number (AN) and solvent electron donor number
(DN) as a novel, dimensionless and more accurate polarity
scale.3,4 Due to the presence of opposite concepts within the
same parameter, the combined polarity term (ANCDN)
was recently denoted amphoteric constant or scale.8
Keywords: Peptide; Polymer; Solubility; Polarity; Solvent.
Abbreviations: Abbreviations for amino acids and nomenclature of peptide
structure follow the recommendations of IUPAC-IUB (Commission on
Biochemical Nomenclature (J. Biol. Chem. 1971, 247 997). Other
abbreviations are as follows: AN, electron acceptor number; BHAR,
benzhydrylamine resin; Boc, tert-butyloxycarbonyl; DCM, dichloro-
methane; DIC, diisopropylcarbodiimide; DIEA, diisopropylethylamine;
DN, electron donor number; DMF, N,N0-dimethylformamide; DMSO,
dimethylsulfoxide; HF, hydrogen fluoride; HOBt, 1-hydroxybenzotriazole;
HFIP, hexafluoroisopropanol; HPLC, high-performance liquid chromato-
graphy; iPrOH, isopropanol; MBHAR, methylbenzhydrylamine resin;
MeCN, acetonitrile; MeOH, methanol; NMP, N-methylpiperidine; PAM,
4-(oxymethyl)-phenylacetamidomethyl-resin; PEG, poly(ethylene glycol);
PIP, piperidine; TBTU, 2-(1H-benzotriazole-1-yl)-1,1,3,3-tetramethyl-
uronium tetrafluoroborate; TEA, triethylamine; TFA, trifluoroacetic acid;
TFE, trifluoroethanol; THF, tetrahydrofuran.
Hence, the present study aimed to pursue this approach of
evaluating solvent effect upon peptide chains attached to a
polymeric matrix. However, the solvent effect upon peptide
chains that are free in homogeneous solution was also
investigated. Needless to say, both approaches have
enormous relevance. Improving the many solid-phase
support processes has been crucial not only in the synthesis
* Corresponding author. Tel.: C55-11-5539-0809; fax: C55-11-5575-
9617; e-mail: clovis.biof@epm.br
0040–4020/$ - see front matter q 2004 Elsevier Ltd. All rights reserved.
doi:10.1016/j.tet.2004.08.013

9418
L. Malavolta, C. R. Nakaie / Tetrahedron 60 (2004) 9417–9424
of peptides9 and other macromolecules10–11 but also for the
unique solid-phase based combinatorial strategy that
allowed the generation of peptide libraries12 and develop-
ment of new therapeutic drugs through solid-phase organic
synthesis.13 In respect to the attempt at investigating
solubilization and aggregation phenomenon of peptide
segments in solution, the physico-chemical findings could
be further extended to any other homogeneous or hetero-
geneous types of macromolecule interactions. Special
attention might be also given to well known degenerative
disorders induced by pronounced peptide aggregation in
physiological conditions such as those seen in
Alzheimer’s,14–16 prion-related diseases17 and type 2
diabetes mellitus.18
amide], the 12–24 fragment22 of the Ab amyloid peptide
responsible for formation of amyloid fibril plaques in the
nervous system, inducing the appearance of Alzheimer’s
disease.23 Both peptide sequences were deliberately
assembled in a very highly substituted (2.6 mmol/g)
methylbenzhydrylamine resin (MBHAR).24 These last two
resins have peptide contents of 82 and 72%, respectively,
which will magnify the effect of peptide chains on the
overall resin solvation behavior. The solvation properties of
polymers were estimated by measuring, under microscopy,
the swelling of dry and swollen beads25,26 in 31 single or
mixed solvents that encompass almost the entirety of the
polarity scale.
Following previously established rules for polymer
solvation as a function of the solvent polarity,3,4,8 the
areas of maximum solvation for the lesser polar resins 1 and
2 in terms of solvent polarity values (ANCDN) are located
at !20 and G25, respectively (Fig. 1(A) and (B)). The
presence of a greater number of polar peptide bonds in the
peptide resins 3 and 4 shifted their areas of maximum
solvation for solvents having polarity values of about 40–50
(Fig. 1(C) and (D)). These results confirm that polymeric
materials achieve maximum solvation in solvents with
polarities similar to that of their backbone,3,4,8,27 thus
revealing a clear relationship between peptide-resin
solvation and polarity of the medium.
The main assumption addressed in this work is related to the
application of electron acceptor and electron donor proper-
ties, either of the solvent or of the solute components. The
strategy has its origins in the findings of previous studies,3,4
in which mixed solvents composed of strong electrophilic
solvents, such as trifluoroethanol (TFE), or strong nucleo-
philic solvents, such as dimethyl sulfoxide (DMSO) and
dimethylformamide (DMF), were unable to dissociate
aggregating peptide sequence attached to a polymeric
backbone. As a consequence, swelling of beads was less
than that predicted by its polarity value. In this sense, we
rationalize that even single solvents, when characterized by
rather small differences between AN and DN values, might
also be poor solvating agents, not only in peptide-resin
solvation but also in dissociation of peptide chains in
solution. Typically, acetonitrile (MeCN), acetone and
isopropanol (iPrOH) would be representative of this class
of solvents, as their AN/DN values are 18.9/14.1; 12.5/17.0
and 33.5/36.0, respectively.7 The maximum difference
between both properties for the three solvents is therefore
not higher than 4.8 (MeCN). In complement, these two
distinct peptide chain dissociation processes (peptide-resin
solvation and peptide solubilization in solution) are herein
evaluated in solvent systems having as great a difference as
possible between acidity and basicity. In addition, other
factors such as the polarity of the media, potential of the
solvent to induce van der Waals interactions, effect of water
and urea in the medium, pH and the strength of peptide-
chain aggregation are also considered.
As discussed above, the solvents 21 and 22 (open circles)
are composed of a strong electron acceptor (TFE) and strong
electron donors (DMF and DMSO, respectively), and these
components tend to interact with each other rather than
disrupt closely associated peptide chains throughout the
resin network (Table 1). As a consequence, the swellings
observed for these two mixed systems were less extensive
than what had been expected based upon their polarity
values (Fig. 1(C) and (D)). Accordingly, this effect was even
less pronounced when the solutes under study were peptide-
free polymers (Fig. 1(A) and (B)).
In agreement with our initial presupposition of a self-
neutralizing effect occurring in some single solvents when
their electrophilicity and nucleophilicity powers are com-
parable, a clear lack of swelling was observed for MeCN,
acetone and iPrOH (open circles 29–31, respectively),
mainly towards peptide resins 3 and 4. Interestingly, even in
the peptide-free polymers 1 and 2, MeCN and acetone were
unable to solvate the resin matrices completely. In
conclusion, these single solvents seem to typify organic
solvents with a very weak solute solvation capacity, which
is induced by an internal self-neutralizing effect in terms of
electron acceptor/electron donor capacities. These findings
confirm that the use of the electrophilicity and nucleo-
philicity terms is advantageous in interpreting solvent effect
upon polymer-type solutes. None of the solvation data found
for mixed 21 and 22 or for single 29–31 solvents could be
explained if, for instance, other classical one-component
polarity scales, such as Hildebrand’s d parameter,28
Dimroth–Reichardt’s ET3029 or even the classical dielectric
constant 3,30 were used in this approach.
2. Results and discussion
2.1. Peptide chain dissociation bound to a polymeric
matrix
Table 1 depicts the AN, DN and (ANCDN) values for 31
single and mixed solvents, together with the swelling
degrees of the following resins: (1) benzhydrylamine resin
(BHAR), a copolymer of styrene and 1% divinylbenzene,
containing a low 0.30 mmol/g of phenylmethylamine
groups;19 (2) PAC-PEG-PS, a 0.18 mmol/g substituted
polyethylene glycol (PEG) grafted polystyrene-1% divinyl-
benzene copolymer, containing the proanthocyanidin (PAC)
spacer20 (Millipore, Bedford, CA, USA); (3) the peptide
resin (NANP)3-Nle, corresponding to the immunodominant
epitope of the sporozoite of Plasmodium falciparum malaria
parasite;21 (4) the peptide resin [VHHQKLVFFAEDV-
Of note is that the present results emphasize the low resin-
swelling capacity of MeCN, acetone and iPrOH and point

L. Malavolta, C. R. Nakaie / Tetrahedron 60 (2004) 9417–9424
Table 1. Solvent parameters and swelling degrees of resins
9419
Entry
Solvent
ANa
DNa
(ANCDN)
Resinb
1
2
3
4
1
2
3
4
5
6
7
8
Toluene
DCM
Chloroform
NMP
DMF
DMSO
TFE
EtOH
3.3
20.4
23.1
13.3
16.0
19.3
53.5
37.1
41.3
39.8
28.4
27.0
36.9
46.9
14.5
13.7
11.5
18.2
19.9
30.3
34.8
36.4
18.5
14.5
17.5
16.3
12.8
15.4
18.9
12.5
33.5
0.1
1.0
4.0
3.4
21.4
27.1
40.6
42.6
49.1
53.5
69.1
71.3
63.8
28.5
27.5
37.5
47.4
42.3
38.6
36.1
32.0
35.3
60.2
48.1
51.3
25.1
44.5
50.4
25.1
42.1
47.2
33.0
29.5
69.5
87
84
83
67
70
51
28
19
17
23
71
72
56
42
73
65
79
70
68
25
27
28
76
66
47
78
73
62
32
48
14
64
79
83
75
70
71
77
53
59
61
82
78
80
80
71
68
75
76
69
66
69
70
81
78
72
76
75
71
65
63
46
26
46
53
70
75
76
63
38
45
61
62
70
73
75
65
62
68
66
68
72
29
31
60
69
71
55
66
70
24
21
10
40
52
64
64
57
65
60
40
41
46
64
60
58
65
61
55
66
61
65
56
47
47
62
65
64
nd
nd
nd
36
40
37
27.3
26.6
29.8
0.0
32.0
30.0
24.0
0.1
9
MeOH
Formamide
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
50% TFE/Toluene
20% TFE/DCM
50% TFE/DCM
80% TFE/DCM
20% DMSO/NMP
50% DMSO/THF
65% NMP/THF
50% DCM/DMF
50% DCM/DMSO
50% MeOH/DMSO
50% TFE/DMF
50% TFE/DMSO
10% TEA/DCM
10% TEA/DMF
10% TEA/DMSO
20% PIP/DCM
20% PIP/DMF
20% PIP/DMSO
Acetonitrile
0.8
0.5
0.2
27.8
24.9
24.8
13.8
15.4
29.9
13.3
14.9
6.6
30.0
32.9
8.8
29.3
31.8
14.1
17.0
36.0
Acetone
2-Propanol
ndZnot determined.
a
Reference.4,7
b
[(Swollen volume–dry volume)/swollen volume]!100 using the following values for measured diameters of dry beads: resins: 1Z50 mm; 2Z114 mm;
3Z87 mm; 4Z94 mm.
out to the need for caution in the application of some types
of column chromatography in which these solvents are
routinely used as a mobile phase.
(ANKDN) parameters of most of the solvent systems used
in this study.
Differing from what was observed with peptide resins, no
clear correlation was observed between degree of
solubilization and polarity of solvent represented by the
(ANCDN) or by any other scales (Fig. 2). This lack of
correlation was also observed when the hydrophobicity of
each sequence (values of 8.2, 45.7, 27.8 and 37.5 for
peptides A–D, respectively), calculated from their amino-
acid composition as previously reported,33 was plotted
against the polarity of the solvents (figure not shown). These
findings stress the difference, in terms of dependence upon
the polarity of the medium, between peptide chains that are
free in solution and those that are bound to a polystructural
network. The fundamental aspect distinguishing these two
situations is the fact that, when attached to a polymer
backbone, the overall degree of freedom, as well as the
intra- or interchain association propensity of peptide chains,
is, perforce, affected by the nature and structure of the
neighboring polymeric environment. Conversely, in the
case of peptide chains free in solution, they are character-
ized by having a higher range of mobility and a complex set
of structural characteristics that influence the type and
intensity of their intra- or interchain associations.
2.2. Peptide solubilization
Paralleling previous correlation studies comparing peptide-
resin solvation and polarity of the medium, solubility of four
model peptide sequences was determined in 18 single or
mixed solvents (Table 2). Differing from the resin solvation
approach, water (single or mixed) was deliberately
involved, together with other solvent systems previously
applied in the evaluation of insolubility problems related to
peptides and other macromolecules. The peptides coded A
and B are those attached to resins 3 ([NANP]3-Nle) and 4
(VHHQKLVFFAEDV), respectively. They were cleaved
from the resin, purified conventionally by HPLC until
homogeneous. The vasoactive bradykinin (RPPGFSPFR,
BK) and the hydrophobic VVLGAAIV-amide segment31
corresponding to the 291–298 fragment of the murine H-2K
protein32 were also introduced in this investigation as
peptides C and D, respectively. Experimentally, a 10 mg/
mL solution of each peptide was centrifuged for 1 h at
14,000 rpm and the supernatant and the precipitate were
lyophilized until a constant weight was attained. In addition
to values of the percentages of solubilization of each
peptide, Table 2 also displays the AN, DN, (ANCDN) and
Despite the lack of an acceptable relationship with the
polarity of the medium, the solubilization factor of peptides

9420
L. Malavolta, C. R. Nakaie / Tetrahedron 60 (2004) 9417–9424
2.3. Effect of self-neutralizing (or heterogeneous)
solvents
Initially, the results shown in Table 2 demonstrated that, in
close agreement with their low capacity for disruption of
peptide chains when bound to resins (low bead solvation),
those single solvents denoted self-neutralizing, such as
MeCN, acetone and iPrOH, also failed to dissolve peptides
in solution, regardless of the sequence. However, their
solubilization capacity changed profoundly when they were
mixed with water, a strong electrophilic (or hydrogen
bonding donor) solvent, characterized by an AN number of
54.8 (Table 2). Except for the strong aggregating peptide D,
where the addition of water did not significantly increase
their solubilization properties, the peptides were almost
entirely dissolved in MeCN, acetone and iPrOH when
cosolvated with 50% (v/v) water. These results may be
attributable to the increased difference between AN and DN
values (higher electrophilicity) in the mixed solvents
resulting from the presence of water molecules (Table 2,
ANKDN term). This may therefore favor the interaction of
aqueous mixed solvents over that of single solvents, in
which the difference in this physico-chemical parameter is
quite low.
This unique effect of the water molecule is entirely
governed by its overall hydrogen bonding property.
However, due to its simple structure, it is, for instance,
unable to promote the number of van der Waals (hydro-
phobic) interactions necessary for disruption of strongly
aggregated chains, as can be seen for peptide D in water
(Table 2). In this context, the further interpretation of
solubilization data from more structured segments will be of
great relevance in elucidating some rules that may control
solubilization of peptides and macromolecules in general.
Many authors34,35 have made mention of the tendency of
these self-neutralizing solvents (mainly MeCN) to induce
b-sheet strands rather than disordered or a-helix-type
conformations in most peptide sequences, usually leading
to aggregated states. However, none of these reports
interpreted these findings in the light of the AN and DN
concepts as detailed herein.
Relevant again for column chromatographic application,
MeOH presented much higher peptide solubility power than
did MeCN or iPrOH, both typifying organic solvents often
applied in HPLC studies. Although this solubilization
property increased proportionately with increased water in
the mixture, it must be remembered that, as previously
stated, these two weak solvating agents (MeCN and iPrOH)
presented poor polymer solvation capability. This under-
scores the need for caution when these solvent systems are
to be used in such experiments.
Figure 1. Swelling of resins (1), BHAR, 0.3 mmol/g [A], (2), PAC-PEG-
PS, 0.18 mmol/g [B], (3), (NANP)3-Nle-MBHAR, 2.6 mmol/g [C] and (4),
VHHQKLVFFAEDV-MBHAR, 2.6 mmol/g [D] as a function of solvent
polarity (ANCDN) values.
2.4. Effect of strong electrophilic or nucleophilic solvents
showed some correlation with the Lewis acid and Lewis
basic properties of the medium, as represented by the AN
and DN terms, respectively. In this context, the expected
complementary participation of other factors, mainly the
van der Waals forces, seems to be crucial for better
evaluating the solubilization factor of each peptide
sequence.
In general, only solvents comprising the strong electron
donor DMSO (DN of 29.8) or the strong electron acceptor
hexafluoroisopropanol (HFIP; AN of 88.0)36 seemed able to
completely dissolve aggregation sequences such as peptide
D. In this case, solubilization percentages of 84 and
80%, respectively, were achieved. In contrast, the less

L. Malavolta, C. R. Nakaie / Tetrahedron 60 (2004) 9417–9424
Table 2. Solvent parameters and solubility of individual peptides
9421
Solvent
Parameter
(ANCDN)
Solubility of Peptide (%)
ANa
DNa
(AN-DN)
A
B
C
D
1. H2O pH 3.0 and 9.0
2. MeCN
3. 50% MeCN/H2O
4. Acetone
5. 50% Acetone/H2O
6. iPrOH
7. 50% iPrOH/H2O
8. MeOH
9. 50% MeOH/H2O
10. TFE
11. 50% TFE/H2O
12. HFIP
13. 50% HFIP/H2O
14. DMSO
15. 50% DMSO/H2O
16. 3.0 M Urea
17. 6.0 M Urea
54.8
18.9
36.9
12.5
33.7
33.5
44.2
41.3
48.1
53.5
54.2
88.0
71.4
19.3
37.1
nd
18.0
14.1
16.1
17.0
17.5
36.0
27.0
30.0
24.0
0.0
9.0
0.0
9.0
29.8
23.9
nd
72.8
33.0
53.0
29.5
51.2
69.5
71.2
71.3
72.1
53.5
63.2
88.0
80.4
49.1
60.9
nd
36.8
4.8
100
0
100
0
100
0
100
100
100
100
100
100
100
100
100
100
100
100
8
76
0
84
0
100
100
100
100
100
100
100
100
100
100
80
100
0
100
0
100
0
88
92
0
0
0
0
20.8
-4.5
16.2
-2.5
17.2
11.3
24.1
53.5
45.2
88.0
62.4
-10.5
13.2
nd
11
10
25
33
26
20
60
80
70
84
32
0
88
92
100
100
100
100
100
100
96
nd
nd
nd
nd
0
ndZnot determined.
a
Reference.4,7
electrophilic TFE (AN of 53.5) displayed much lower
solubilization power in comparison with HFIP (20%). This
significant difference in solubilization capacity between
TFE and HFIP seems to be clearly due to the difference in
their electrophilicity (AN increased from 53.5 to 88.0,
respectively). However, to the strength of the van der Waals
interaction induced by the presence of a second –CF3 group
in the HFIP structure is equally relevant. The appropriate
combination of both effects (hydrogen bonding and
hydrophobic or van der Waals forces) seems to play a
crucial role in the significant increase in the disruption
power of aggregated peptides, which is more pronounced
when in HFIP.
The effect of the addition of water to single solvents is very
complex but of great relevancy, as observed in the case of
Figure 2. Solubility of peptides (A), (NANP)3-Nle-amide, (B), VHHQKLVFFAEDV-amide, (C), RPPGFSPFR and (D), VVLGAAIV-amide as a function of
solvent polarity (ANCDN) values.

9422
L. Malavolta, C. R. Nakaie / Tetrahedron 60 (2004) 9417–9424
the ‘poor’ solvents discussed in the previous section. When
water was added to the strong polar organic solvents DMSO
or HFIP, the solubilization yield decreased from 84 to 32%
and from 80 to 70%, respectively (Table 2). These results
indicate that the effect of the addition of water seems to be
highly dependent upon the type of organic solvent to be
mixed (electron acceptor or electron donor) and also on
the particular characteristic of peptide sequence (degree of
aggregation). When added to DMSO, the nucleophilicity of
this polar aprotic solvent are partially neutralized by the
strong electrophilicity of the water molecule, thus partially
reducing the solubilization properties of the DMSO/water
mixture. This effect is quite similar to the previously
discussed solvation behavior of the heterogeneous mixed
solvents 21 and 22, which are composed of strongly
electrophilic and nucleophilic components (Table 1).
larger numbers of solvent systems and solute models are
currently under investigation in an attempt to further
establish rules that might not only facilitate selection of
the most suitable solvent for peptide dissolution or peptide-
resin solvation but also be extended to many other solute–
solvent interactions.
3. Conclusions
Despite the huge amount of data already existing in the
literature, the solute–solvent interaction effect has eluded
the scientific community for many decades. In a recent
investigation, we combined the electron acceptor (AN) and
electron donor (DN) parameters in order to build an
alternative solvent polarity scale. As a continuation of
this, we have, in the present study, evaluated these same
physicochemical properties in order to interpret the complex
dissociation process of peptide chains, comparing those free
in solution with those coupled to polymers.
In contrast, when electrophilic water is added to other strong
electrophilic solvents such as TFE or HFIP, a homogeneous
solution is formed and the degree of alteration in their
peptide chain dissolution potential seems very complex. For
instance, in the case of HFIP, the addition of water slightly
reduced the degree of peptide D solubilization (from 80 to
70%, respectively), whereas more significant variation
(increases) in solubilization occurred in pure TFE or in
TFE/water mixture (20 and 60%, respectively). Again, this
result shows that the more pronounced effect seen when
water was added to TFE than when it was added to HFIP
could be credited to the much stronger electrophilicity of the
latter fluorinated solvent, in combination with a higher
potential to produce van der Waals interaction with
aggregated peptide D.
After investigating model peptides and peptide-resins
solvated in a large number of solvent systems, we have
reached several conclusions. First, in contrast to improved
solvation of peptide-resins in solvents with similar
polarities, the solubilization yield of a peptide in solution
is not always directly related to the polarity of the medium.
Second, optimal solubilization of peptides is strongly
dependent upon the difference between AN and DN values
of the solvent and of its ability to induce van der Waals
attraction. In addition, mixed solvents with rather equivalent
electrophilicity and nucleophilicity are not able to solvate
aggregated peptide-resins or dissolve peptide sequences.
This rule is also applicable to single solvents that present
similar AN and DN values and induce a molecular self-
neutralizing effect, thereby precluding dissociation of
peptides in solution or solvation of peptide-resins. Further-
more, this self-neutralizing effect occurring in mixed or
single solvents must be also considered for other bio-
technological applications (such as in column chromato-
graphy) since it may affect solute solubilization and resin
solvation simultaneously. Moreover, whether the addition
of the strongly electrophilic water molecule to a mixture for
peptide solubilization will be advantageous or not is clearly
dependent on the relationship between acidity and basicity
of both components. Finally, the peptide solubilization
effect of urea in the solution is sequence dependent and, in
some cases, involves an inverse correlation between
solubility and urea concentration. Therefore, in light of
the Lewis acidity and Lewis basicity properties of solvent
systems, some relevant rules could be established for the
complex processes of peptide dissolution and peptide-resin
solvation.
In the literature, a great number of studies have examined
peptide or other macromolecule solubility with the aim of
finding rules that govern the effects of solvents with weak or
strong dissociations.18,34–38 In addition to these efforts, no
clear explanation has been proffered for the apparently
random way in solvents are currently chosen. For example,
why would one consider opposite DMSO solvents to be
more suitable than HFIP/TFE solvents? We have presented
an alternative and, in some cases, consistent approach to
address this extremely complex issue of solvent effects upon
solute molecules. This approach relied mainly on the
conjugated use of AN and DN terms (sum or difference),
as well as on the potential for hydrophobic interaction, of all
solvents involved in the interaction process, and on the
specific characteristics of the peptide or peptide-resin solute
components.
Due to the great complexity of the solute–solvent inter-
action, especially in cases involving peptide or peptide–
polymer solutes, many further studies are warranted. In an
attempt to depict this complexity, we have also included, in
Table 2, dissolution data obtained when we used aqueous
urea solutions, which are often proposed for use in the
dissolution of proteins and peptides. A failure of such
solutions to dissolve peptide D was observed, as well as,
notably, an inverse relationship between solubility and urea
concentration in peptide B dissolution. Otherwise, no
correlation between solubility and media pH was observed,
suggesting the absence of an ionization effect in some
subgroups of the four peptides evaluated herein. Much
4. Experimental
All amino-acid derivatives were purchased from Bachem
(Torrance, CA, USA). Solvents and reagents were pur-
chased from Fluka (Buchs, Switzerland), Aldrich-Sigma
(Steinheim, Germany) and Advanced Chemtech
(Louisville, KY, USA). The PAC-PEG-PS resin was
acquired from Millipore (Bedford, CA, USA) and batches

L. Malavolta, C. R. Nakaie / Tetrahedron 60 (2004) 9417–9424
9423
of BHAR or MBHAR (0.3 and 2.6 mmol/g, respectively)
were synthesized in our laboratory, following guidelines
laid out in previous reports.19,26
total of 424 mg of crude peptide were obtained and, after
HPLC purification, 104 mg of pure compound remained.
ESI-MS, m/z: 1569 (theoretical); obtained (1569.2).
(c) VVLGAAIV-amide: this peptide was also synthesized in
MBHAR (2.63 mmol/g) at 0.53 mmol scale After cleavage
with HF procedure, 452 mg of crude peptide were obtained
which yielded 126 mg after HPLC purification. ESI-MS,
m/z: 740 (theoretical); 740.4 (obtained).
4.1. Peptide synthesis
The peptides were synthesized manually according to the
standard Boc8 protocol. The following Boc amino-acid
derivatives were used: Boc-Glu(OcHex), Boc-Asp(OcHex),
Boc-Lys(2-Cl-Z), Boc-Ser(Bzl) and Boc-His(Tos). In the
Boc chemistry, after coupling the C-terminal amino acid to
the resin, the successive a-amino group deprotection and
neutralization steps were performed in 30% TFA/DCM
(30 min) and 10% DIEA/DCM (10 min). The amino acids
were coupled using DIC/HOBt in DMF and, if necessary,
TBTU in the presence of HOBt and DIEA using 20%
DMSO/NMP as a solvent system. After a 2 h coupling
period, the qualitative ninhydrin test was performed to
estimate the completeness of the reaction. To check the
purity of the synthesized peptide sequence attached to the
resin, cleavage reactions with small aliquots of resin were
carried out with the low-high HF procedure. Analytical
HPLC, as well as LC/MS (electrospray) mass spectrometry
(Micromass, Manchester, UK) and amino-acid analysis
(Biochrom 20 Plus, Amersham Biosciences, Uppsala,
Sweden), were used to check the homogeneity of each
synthesized resin-bound peptide sequence.
(d) RPPGFSPFR (BK): this peptide was synthesized in Boc-
Arg(Tos)-PAM resin (0.6 mmol/g) at 0.5 mmol scale. After
cleavage with HF procedure, 433 mg of crude peptide were
obtained which yielded 273.4 mg after HPLC purification.
ESI-MS, m/z: 1060 (theoretical); 1059 (obtained).
4.4. Measurements of bead swellings
Before use in peptide synthesis or microscopic measure-
ment of bead sizes, most resin batches were sized by sifting
through metal sieves to lower the standard deviation of resin
diameters to about 4%. Swelling studies of these narrowly
sized populations of beads have been previously con-
ducted.3 In short, 150–200 dry and swollen beads of each
resin, allowed to solvate overnight, were spread over a
microscope slide and measured directly with an Olympus
model SZ11 microscope coupled with Image-Pro Plus
version 3.0.01.00 software. Since the sizes in a sample of
beads are log-normally rather than normally distributed, the
more accurate geometric mean values and geometric
standard deviations were used to estimate the central
value and the distribution of the particle diameters. The
resins were measured with their amino groups in the
deprotonated form, obtained by 3!5 min washes in TEA/
DCM/DMF (1:4.5:4.5, v/v/v), followed by 5!2 min
washes in DCM/DMF (1:1, v/v) and 5!2 min DCM
washings. Resins were dried in vacuum using an
Abderhalden-type apparatus with MeOH reflux.
4.2. Analytical HPLC
Analysis was performed in a system consisting of two model
510 HPLC pumps (Waters, Milford, MA, USA), an
automated gradient controller, Rheodyne manual injector,
486 detector and 746 data module. Unless otherwise stated,
peptides were analyzed on a 4.6!150 mm2 column with a
˚
300 A pore size and a 5 mm particle size (C18; Vydac,
Hesperia, CA, USA) using the solvent systems: A (H2O
containing 0.1% TFA) and B (60% MeCN in H2O
containing 0.1% TFA). A linear gradient of 10–90% B in
30 min was applied at a flow rate of 1.5 mL/min and
detection at 220 nm.
4.5. Solubility measurement of peptides
The solubility of each peptide was determined by dissolving
2.5 mg of pre-purified peptide in 0.25 mL (ca. 10 mM) of
each of the solvents described in Table 2. The solution was
centrifuged for 1 h at 14,000 rpm and the supernatant and
the precipitate were lyophilized until constant weight was
attained. Solubility data are expressed as percentages.
4.3. Preparative HPLC
Purification of peptides was carried out using solvent A
(H2O containing 0.1% TFA) or solvent B (90% MeCN in
H2O containing 0.1% TFA). A linear gradient was applied
which was dependent upon the retention time determined in
the HPLC analysis of the peptide, using the same solvent
systems. The flow rate was of 10 ml/min and the detection
of peaks was carried out at 220 nm.
Acknowledgements
The following peptides deemed requisite for solubilization
experiments were synthesized through Boc strategy:
Grants from the Brazilian governmental agencies FAPESP
and CNPq are gratefully acknowledged. L. M. is fellow of
FAPESP and C. R. N. is a recipient of research fellowships
from CNPq and FADA.
(a) (NANP)3-Nle-amide: this peptide was synthesized at
0.53 mmol scale starting from MBHAR resin
(2.63 mmol/g). The crude peptide yielded 250 mg and,
after HPLC purification, 165 mg of pure compound were
obtained. ESI-MS, m/z: 1319 (theoretical), 1320 (obtained).
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