Anthranilic acid-containing cyclic tetrapeptides: At the crossroads of conformational rigidity and synthetic accessibility

Article abstract of DOI:10.1039/c6ob00693k

Each amino acid in a peptide contributes three atom units to main-chains, hence natural cyclic peptides can be 9, 12, 15, .... i.e. 3n membered-rings, where n is the number of amino acids. Cyclic peptides that are 9 or 12-membered ring compounds tend to be hard to prepare because of strain, while their one amino acid homologs (15-membered cyclic pentapeptides) are not conformationally homogeneous unless constrained by strategically placed proline or d-amino acid residues. We hypothesized that replacing one genetically encoded amino acid in a cyclic tetrapeptide with a rigid β-amino acid would render peptidomimetic designs that rest at a useful crossroads between synthetic accessibility and conformational rigidity. Thus this research explored non-proline containing 13-membered ring peptides 1 featuring one anthranilic acid (Anth) residue. Twelve cyclic peptides of this type were prepared, and in doing so the viability of both solution- and solid-phase methods was demonstrated. The library produced contained a complete set of four diastereoisomers of the sequence 1aaf (i.e. cyclo-AlaAlaPheAnth). Without exception, these four diastereoisomers each adopted one predominant conformation in solution; basically these conformations feature amide N-H vectors puckering above and below the equatorial plane, and approximately oriented their N-H atoms towards the polar axis. Moreover, the shapes of these conformers varied in a logical and predictable way (NOE, temperature coefficient, D/H exchange, circular dichroism). Comparisons were made of the side-chain orientations presented by compounds 1aaa in solution with ideal secondary structures and protein-protein interaction interfaces. Various 1aaa stereoisomers in solution present side-chains in similar orientations to regular and inverse γ-turns, and to the most common β-turns (types I and II). Consistent with this, compounds 1aaa have a tendency to mimic various turns and bends at protein-protein interfaces. Finally, proteolytic- and hydrolytic stabilities of the compounds at different pHs indicate they are robust relative to related linear peptides, and rates of permeability through an artificial membrane indicate their structures are conducive to cell permeability.

Full text of DOI:10.1039/c6ob00693k

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Biomolecular Chemistry
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Anthranilic acid-containing cyclic tetrapeptides:
at the crossroads of conformational rigidity and
synthetic accessibility†
Cite this: DOI: 10.1039/c6ob00693k
Dongyue Xin and Kevin Burgess*
Each amino acid in a peptide contributes three atom units to main-chains, hence natural cyclic peptides
can be 9, 12, 15, …. i.e. 3n membered-rings, where n is the number of amino acids. Cyclic peptides that
are 9 or 12-membered ring compounds tend to be hard to prepare because of strain, while their one
amino acid homologs (15-membered cyclic pentapeptides) are not conformationally homogeneous
unless constrained by strategically placed proline or D-amino acid residues. We hypothesized that repla-
cing one genetically encoded amino acid in a cyclic tetrapeptide with a rigid β-amino acid would render
peptidomimetic designs that rest at a useful crossroads between synthetic accessibility and conformation-
al rigidity. Thus this research explored non-proline containing 13-membered ring peptides 1 featuring one
anthranilic acid (Anth) residue. Twelve cyclic peptides of this type were prepared, and in doing so the via-
bility of both solution- and solid-phase methods was demonstrated. The library produced contained a
complete set of four diastereoisomers of the sequence 1aaf (i.e. cyclo-AlaAlaPheAnth). Without excep-
tion, these four diastereoisomers each adopted one predominant conformation in solution; basically
these conformations feature amide N–H vectors puckering above and below the equatorial plane, and
approximately oriented their N–H̲ atoms towards the polar axis. Moreover, the shapes of these confor-
mers varied in a logical and predictable way (NOE, temperature coefficient, D/H exchange, circular
dichroism). Comparisons were made of the side-chain orientations presented by compounds 1aaa in
solution with ideal secondary structures and protein–protein interaction interfaces. Various 1aaa stereo-
isomers in solution present side-chains in similar orientations to regular and inverse γ-turns, and to the
most common β-turns (types I and II). Consistent with this, compounds 1aaa have a tendency to mimic
various turns and bends at protein–protein interfaces. Finally, proteolytic- and hydrolytic stabilities of the
compounds at different pHs indicate they are robust relative to related linear peptides, and rates of per-
meability through an artificial membrane indicate their structures are conducive to cell permeability.
Received 1st April 2016,
Accepted 29th April 2016
DOI: 10.1039/c6ob00693k
www.rsc.org/obc
vation of a strongly preferred conformation in solution also
makes it probable that the molecule will bind to the receptor
Introduction
Cyclization of linear peptides increases their proteolytic stabi- in a similar conformation, compared to other situations in
lities and rigidities. In ideal cases these structures will adopt which the compound exists in several solution conformations.
only one preferred conformation; if that occurs, less entropy Moreover, exclusion of competing conformational states
will be surrendered on interaction with biomolecular recep- reduces possibilities for off-target binding.
tors, increasing the free energies for the interactions. Obser-
Inconveniently, cyclic peptides composed of the 20 geneti-
cally encoded amino acids miss a “sweet spot” ring size where
conformational homogeneity is attained without compromis-
ing ease of syntheses. Thus, cyclic tri-1–4 A and tetra-peptides
B 5–11 are notoriously difficult to prepare because they are con-
Department of Chemistry, Texas A & M University, Box 30012, College Station,
TX 77842, USA. E-mail: burgess@tamu.edu
†Electronic supplementary information (ESI) available: Experimental procedures strained in 9- and 12-membered ring conformations. Analogs
and spectroscopic data for all compounds; H/D exchange experiments and temp-
of cyclic tetrapeptides, like the 12-membered ring system C,
may be more easily prepared but another problem arises: cis/
erature coefficient measurements; procedures and results for conformational
modeling with QMD and MacroModel, results of the overlays on turn structures,
trans amide bond equilibria introduces conformational hetero-
data of protein database mining and the procedure for QikProp calculations;
geneity.12 Cyclic pentapeptides,13–15 are easier to make than
procedures for stability studies and the PAMPA assay. See DOI: 10.1039/
c6ob00693k
cyclic tri- or tetrapeptides16 because their 15-membered rings
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are less strained, but they tend to equilibrate between confor-
mers D 1–5 containing β- and γ-turns. Certain states in the D 1–5
equilibrium can be favored if one of the amino acids has a
D-configuration, especially D-Pro,17 but most cyclic pentapeptides
and higher homologs overall do not tend to be rigid unless
further constrained (Fig. 1).18,19
Based on the observations above, there should be favored
ring sizes in peptidomimetic design where non-genetically
encoded residues replace one amino acid to give confor-
Fig. 1 (Contd).
mationally rigid 13- or 14-membered rings. Ghadiri and co-
workers, for instance,20,21 have used copper-mediated azide–
alkyne cycloadditions to give the 13-membered rings E which
were conformationally rigid.22 In other illustrative work, Fairlie
et al. substituted β-amino acids into cyclic tetrapeptides and
found some 13-membered ring systems, including F23 and G,24
that could be prepared efficiently, and were conformationally
rigid. However, that same work showed similar, but confor-
mationally heterogeneous, 13-membered ring systems.24
Anthranilic acid is readily available and more rigid than
most other β-amino acids. Several peptidic macrocycles con-
taining anthranilic acid occur in Nature, most where this unit
is one of five in a pentapeptide ring.25–38 There are also numer-
ous examples of similar hexapeptides and higher homologs
incorporating anthranilic acid,25,39–45 a few 10-membered tri-
peptide derivatives46–48 and several cyclic systems containing
the “Anth” residue and another non-encoded amino acid.49–51
However, 13-membered ring systems containing this ubiqui-
tous residue have been under-explored. Only one natural tetra-
peptide H that features Anth in a 13-membered ring has been
discovered,52 and the only 13-membered cyclic peptide con-
Fig. 1 (a) 12-Membered ring peptidic systems (e.g. cyclic tetrapeptides) taining Anth that has been synthesized is compound I, pre-
pared as part of a medicinal chemistry project.53 To the best of
our knowledge, neither H nor I have been studied in solution
are highly constrained and their conformations may be complicated by
cis-trans isomerism. (b) Cyclic pentapeptides tend to equilibrate
between similar states containing both γ and β-turns, i.e. they tend to be
conformationally heterogeneous. (c) 13-Membered rings may give only
to determine their conformational biases.
We hypothesized compounds 1 could be prepared from readily
available starting materials, and would be conformationally
one preferred conformer (e.g. E–G) but this issue has not previously
been studied for systems containing the Anth residue (e.g. for H and I).
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rigid. This paper describes how those compounds were made,
and the conformational biases of one complete set of enantio-
mers in this series. In the event the conformations of these
molecules were shown to correlate with their chiral amino acid
stereochemistries in a logical, easily understood, way that is
useful for predicting the preferred shapes of these rigid
scaffolds.
Results and discussion
Syntheses via iterative precipitations
Couplings to anthranilic acid are not facile because the aro-
matic amine is deactivated via resonance. However, Scheme 1
describes how solution-phase syntheses of several compounds
were achieved using a large excess of Anth and a high concen-
tration of all agents; if high concentrations were not used then
epimerization was competitive with product formation. Use of
a relatively weak base (N-methyl morpholine) and of the
superior, though more expensive, coupling additive HOAt,54
was also beneficial in this step.
Early in this study we realized the physiochemical pro-
perties of peptides containing anthranilic acid facilitated their
isolation. Thus, coupling three amino acids to the Anth unit
gave products that precipitated from dichloromethane/hexanes
with sufficient purities to use in the next steps. In fact, the
only chromatography needed in the “Boc-approach” to the
cyclic systems shown in Scheme 1 was to isolate the cyclized
product. Serine and tyrosine residues in the compounds pre-
pared were protected with benzyl groups. Glutamic acid side-
chain protection was achieved using a tert-butyl ester, which
withstands selective deprotection of the N-Boc functionality
with 4 M HCl.55
Scheme S2 (ESI†) shows a similar solution-phase approach
to the same types of products but using Cbz protected amino
acids and Nα-deprotection via hydrogenolysis. In one of these
syntheses the first amino acid added was H-Glu(OtBu) where
successive deprotection under acidic conditions, as in
Scheme 1, might have eroded the yield of this product, but
N-deprotection via hydrogenolysis circumvented this potential
problem. A complementary solid phase Fmoc-approach was
also established (Scheme S3, ESI†), based on 2-chloro-trityl
polystyrene resin and involves cleavage from the support then
cyclization in the final step.
Scheme 1 Boc approach to products 1.
perturbation from side-chain interactions. The ideal system to
study might have been the simple peptide 1aaa (or cyclo-AlaA-
laAlaAnth). However, there was insufficient dispersion of the
Conformational analyses
One goal in this study was to elucidate the intrinsic confor- 1H NMR peaks in that particular compound to facilitate con-
mational biases of the stereoisomeric scaffolds 1 with minimal venient conformational analyses. Consequently, we decided to
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study one enantiomeric series of the compounds 1aaf (or cyclo-
AlaAlaPheAnth).
One-dimensional 1H NMR of the 1aaf series all showed
sharp, resolved peaks for the side-chains and for the amide
protons. All the amino acid amide NH
into doublets via coupling to the CH
̲
resonances were split
protons (see below,
̲
Table 1). These observations imply that the compounds do not
equilibrate between different conformations on the NMR time-
scale, and that in solution each exists predominantly in one
form. Temperature coefficient data56,57 and rates of H/D
exchange58,59 were also measured. Neither set of data were par-
ticularly informative, except that they indicate there are no
“endo-cyclic” H-bonds, consistent with the conformations
deduced from NMR and calculations that are described
immediately below. NOE data were collected for all the com-
pounds, and no cis-amide bonds were present (CαH–CαH
cross-peaks absent).
Two methods were used to deduce the predominant confor-
mations of compounds 1 in polar solvents. First, the molecules
1aaa were simulated in a medium of dielectric 46.7 represent-
ing DMSO (and 80 representing water, see ESI†) using the
quenched molecular dynamics (QMD) technique.60,61 QMD
simulations are valuable because they are not bias by NOE
data which over-represents some conformations due to the
NOE effect depends on 1/r6 distance relationships.62 Moreover,
QMD thoroughly explores possible local minima in a Boltz-
mann equilibrium. Second, and independently, NOE restraints
were applied in a Macromodel63 simulation of molecules 1aaf
in dielectric 46.7.
One conformational cluster (maximum RMSD of the
Cα–Cβ coordinates 0.5 Å) arose from the QMD simulations of
each of the 1aaa stereoisomers, Fig. 2a (QMD simulated
scaffold conformations are shown in black throughout
this paper). The fact that >1000 conformers (all below
3 kcal mol−1 of the lowest energy one identified) all converged
to one cluster emphatically indicates conformational rigidity.
Comparison of the QMD-generated structures with the NOE
data showed they are consistent. Similarly, the MacroModel
simulations with NMR constraints also gave one predominant
conformation for each 1aaf stereoisomer, Fig. 2b (Macro-
Model simulated scaffold conformations involving NOE
Fig. 2 Conformations simulated by: (a) QMD calculations for 1aaa
stereomers without NOE constraints; and, (b) MacroModel for 1aaf
stereomers with NOE constraints. The blue arrows approximate the
orientation of the NH bond vectors to either pointing up or below the
ring system.
3
Table 1 Comparison of experimentally observed JNH-α coupling con-
stants with those calculated using the NMR constrained structures simu-
lated in Fig. 2b
3
constraints are grey throughout). None of the JNH-α coupling
constants were above 9 Hz (see Table 1 below); only couplings
>9 Hz are indicative of unambiguous calculated dihedral
angles, hence none were used as constraints in the Macro-
Model calculations.
Overlays of the conformations generated using the
approaches described above, i.e. without and with NOE con-
straints, showed close agreement (RMSD 0.18–0.34 Å for the
Cα–Cβ coordinates; Fig. 3). Moreover, even though the fit of
these overlays was based on Cα–Cβ vectors, it is clear that the
ring structures also are very similar.
Ala(R1)
Ala(R2)
Phe (R3)
Exp’la
Calc.b
Exp’la
Calc.b
Exp’la
Calc.b
LLL
DLL
LDL
DDL
5.0
7.7
7.2
5.2
6.2–6.7
7.6–8.2
6.8–8.0
5.6–6.3
—
7.8–8.3
6.6–7.9
7.9–8.9
6.6–7.1
7.0
7.9
7.7
5.6
6.5–7.8
8.0–9.0
6.9–8.1
6.6–7.1
8.9
8.8
—
a Directly from NMR spectra. b Based on the structures simulated from
the NOE data, and calculated using the Poulson form of the Karplus
equation.64
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Fig. 4 “North south orientations” of the amide N–H vectors in stereo-
mers of 1 correlate with the amino acid configurations. L-Amino acid
N–H vectors point South, and while the D-isomers give North-aligned
local N–H vector orientations, when drawn in the orientations shown.
Circular dichroism (CD) data for the 1aaf stereomers are
shown in Fig. 5 (solid lines). Unsurprisingly, significantly
greater molar elipticities were observed for these compounds
compared with related linear peptides (dotted lines), indicative
of more conformational ordering in the cyclic systems. More-
over, there are logical trends in the data. For instance, the LLL-
1aaf stereomer (red line) has negative maxima at ca. 195 and
215, and a maximum at ca. 230 nm. Substitution of two
L-amino acids in LLL-1aaf giving DDL-1aaf is accompanied by
near complete inversion of the CD maxima and minima. The
two possible “intermediate” diastereomers, i.e. DLL-1aaf and
LDL-1aaf, in which only one L-amino acid of LLL-1aaf is
replaced, show shallower peak intensities. The CD spectrum of
Fig. 3 Overlays of 1 simulated structures without (black, 1aaa) and with
(grey, 1aaf) NOE constraints are within 0.18–0.34
Å based on 6
coordinates.
Comparisons of favored conformations for stereomers of
1 revealed a simple correlation. When drawn with the ring
on the equator, and the Anth residue on the West, then
L-amino acids point their N–H vectors South, while N–H
vectors for D-amino acids are North-oriented, irrespective of
the other amino acid stereochemistries. Thus the cyclic 13-
membered ring scaffold is constrained to one highly predict-
able conformation per stereoisomer. This observation held
for all the compounds prepared in this study based on simi-
larities in NOE cross-peaks, i.e. for cyclo-DGlu′-DAla-LPhe-
Anth, cyclo-LVal-LSer′-LTyr′-Anth, cyclo-DTyr′-DSer′-DVal-Anth,
cyclo-LVal-LSer-LTyr-Anth, cyclo-LPhe-DAla-LPhe-Anth, cyclo-
DPhe-LAla-LGlu′-Anth, cyclo-DPhe-LAla-LGlu-Anth (see ESI†);
those observations imply the conformations are governed by
the scaffold and the side-chain variables are less significant
(Fig. 4).
DL
̲
̲L-1aaf
̲
(red) than it is to DD
̲
(blue) the inverse is true; this implies the amino acid opposite
Table 1 compares NH–CαH coupling constants directly
from NMR spectra with those calculated from the MacroModel
simulations involving NOE constraints. In some cases the
coupling was obscured, and in several cases the true J-values
were marginally (by 1.2 Hz at most) outside the calculated
range, but the rest were consistent with the values inferred
from simulations.
Fig. 5 CD spectra of compounds 1aaf (solid lines) and closely related
linear peptides (dashed lines).
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the Anth residue in the cyclic scaffold has a more profound face was implicated in the overlay. There were between
effect on the molar elipticity.
106–258 matches of <0.3 Å RMSD for each stereomer. The
term “no secondary structure” is used here to describe situ-
ations in which the region overlaid did not contain any dis-
cernable secondary structure. “Turns” refers to a turn of any
type (α, β, γ, δ) that has appropriate intrachain hydrogen
bonds, while loops and turns without any intra-ring
H-bonding interactions are classified as “bends”. Fig. 7 shows
Comparisons of the Anth-cyclic peptidomimetics with peptide
and protein structures
Exploring key orientations on secondary structures (EKOS)65
facilitates comparison of all the favored QMD simulated con-
formers with all the common ideal secondary structures based
on Cα–Cβ coordinates. For 1aaf there was only one preferred
conformer cluster for each stereomer. Not surprisingly, then,
most stereomers matched on only one secondary structure, or
none at all, i.e. they are not universal peptidomimetics.66,67
One apparent exception was LLL-1aaf which gave an acceptable
fit on γ- and type II β-turn conformations (Fig. 6); however,
γ- and type II β-turn conformations have similar side-chain
orientations that overlay well on each other (ESI Fig. S1b†). An
inverse γ-turn matched reasonably well with LDL-1aaf, and
DLL-1aaf overlaid closely with a type I β-turn.
Whereas, Fig. 6 depicts preferred overlays of select 1aaf
stereoisomers on ideal secondary structures, the EKO routine68
facilitates matching QMD generated structures of 1aaa with
crystallized protein–protein interaction interfaces based on
Cα–Cβ coordinates. Thus, preferred conformations of each
stereomer were compared with around 160 000 protein–protein
interfaces, and each match of RMSD <0.3 Å was analyzed
in terms of what secondary structure type at the inter-
Fig. 6 Preferred conformations of select stereoisomers of 1aaf overlaid
on γ-, inverse γ-, type I β-, and type II β-turns. RMSD values indicated are
for overlay of the side-chain Cα–Cβ vectors.
Fig. 7 Distribution of best overlays on PPI interface segments with
respect to secondary structure for the stereomers featured in Fig. 6.
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the distribution of overlays within those categories and some
other secondary structures. Thus the preferred 1aaa confor-
mations are strongly bias towards turns and bends, consistent
with the EKOS study presented in Fig. 6. Full data for all the
1aaa stereomers are given in the ESI (Fig. S2†).
Finer detail of the secondary structure types that were over-
laid in the EKO analysis of PPIs in the PDB is indicated by ϕ,ψ-
angles, indicative of the type of secondary structure impli-
cated. Fig. 8 shows select data for the stereomers featured in
Fig. 6, and Fig. S3† shows the complete data set. Thus, the
number of occurrences where LLL-1aaa, LDL-1aaa, and DLL-
1aaa overlaid closely with γ-turns, type I β-turns, inverse γ- and
type II β-turn conformations are consistent with the favored
conformations predicted in Fig. 6.
Physiochemical properties
Table 2 shows how HPLC was used to monitor the stability of
LLL-1aaf under aqueous conditions of pH 2,7, and 12, and, in
separate experiments, the mixture of proteases called
Pronase69 that is used to extensively hydrolyze proteins in pro-
teomics studies (any amide between two hydrophobic residues
could be cleaved by this enzyme mixture). Under neutral or
acidic conditions, and in the presence of Pronase, LLL-1aaf
did not any significant cleavage. Only some cleavage was
observed after extended expose to the aqueous pH12
conditions.
Predictions of cell permeability
Fig. 9 shows data calculated (QikProp)70,71 for the polar
surface area (PSA) and cell permeability of LLL-1aaf. The impli-
Table 2 Stabilities of LLL-1aaf under different pH conditions
Conditions
t1/2/h
pH stability of LLL-1aaf
LLL-1aaf linear LLL-aaf
pH 2.0a
pH 12.0b
pH 7.4c
>500
240
>500
Pronasec
Pronasec
No cleavage after 12 h
1.5
a 10 mM HCl. b 10 mM NaOH. c PBS buffer. 20% MeOH was added in
all cases to increase solubilities.
Fig. 8 Each dot on these plots is associated with a ϕ,ψ-bond vector of
a protein interface region that overlaid closely with a preferred confor-
mer of the 1aaa stereomer indicated.
Fig. 9 Comparison of PSA (blue) and predicted Caco-2 cell permeabil-
ities (red) for linear peptides based on aaf and a featured cyclic molecule
1aaf.
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view of this, and other observations outlined in the introduc-
tion, we suggest 13-membered systems based on the Anth
residue are at a favorable point on the crossroads between ease
of synthesis and conformational rigidity.
Predictability of conformational preferences for a set of
cyclic peptidomimetics is an attractive feature of the systems
studied here. It is possible that different side-chains to the
ones in 1aaf could perturb the predicted conformations, and
this study does not encompass the special case of systems that
contain Pro- or Gly-. Nevertheless, the data above clearly indi-
cates strong intrinsic conformational biases of the scaffolds
based on other amino acids. Moreover, stereochemistries of
the scaffolds can be manipulated so that they orient their side-
chains in ways that resemble the same orientation for regular
and inverse γ-turns, and for the two most common β-turns
(types I and II). Consistent with this, conformations of 1aaa
stereomers have a pronounced tendency to orient side-chains
in ways that some turns and bends do at PPI interfaces.
Cyclic peptides containing only secondary amide bonds do
not tend to be cell permeable.80 In fact, making cyclic peptides
cell permeable by inclusion of N-methyl groups is an area of
interest in the contemporary literature.81–83 Molecules 1 show
promise as cell permeable molecules, even though they do
not have tertiary amide N-methyl groups. We suggest this is
because the favored conformers of 1 in solution orient their
N–H vectors towards a central point, i.e. relatively insulated
from the medium around the mimic.84
Fig. 10 Calculated cell permeability rates from QikProp (blue), and
experimental data from PAMPA assays (red).
cations of this data are that LLL-1aaf has less PSA and a higher
tendency to permeate into cells than closely related linear
peptide controls. Polar surface areas <140 Å2 are generally pre-
ferable for cell permeability.72 Similarly, compounds with pre-
dicted PCaco-2 permeability rates >20 nm s−1 are usually cell
permeable; data calculated for LLL-1aaf exceeds both expect-
ations, and predict cell permeability.
The parallel artificial membrane permeability assay
(PAMPA)73–77 was used to obtain experimental data to compare
with QikProp calculations (Fig. 10). In PAMPA assays, a poly-
vinyldifluoroethene membrane is coated with mixture of a lipo-
philic hydrocarbon (here dodecane) and lipids, then the rate
of diffusion of test compounds from donor to acceptor wells
are measured over a period of time to mimic passive diffusion
into cells. There is no uniformly accepted cut-off value in
PAMPA assays above which the compounds are considered to
be cell permeable, mainly because there is some variance in
the membrane composition used. However, the following
illustrative data for cell permeable pharmaceuticals have been
obtained by others using exactly the method described here78
(Papp in 106 cm s−1): testosterone (13), propranolol (10),
warfarin (1.0), furosemide (0.16), methotrexate (0.016). These
data may serve as a reference for the data collected and shown
in Fig. 10. That data suggests compounds based on the aaf
core will tend to be cell permeable. Membrane permeability
was reduced for the compounds with more polar side-chains,
vsy, and for the linear control, LLL-H-aaf-OH as expected. This
assertion is supported by another study that suggests Papp of
around 1 × 106 cm s−1 correlate with cell permeability.79
Overall, while tens of naturally occurring cyclic peptides
containing Anth have been discovered, the potential of this
simple residue in designs of conformationally rigid protein
interface mimics has hitherto been under-appreciated.
Conflict of interest
The authors declare no competing financial interests.
Acknowledgements
Financial support for this project was provided by the National
Institutes of Health (GM087981), the Robert A. Welch foundation
(A-1121), DoD BCRP Breakthrough Award (BC141561), CPRIT
(RP150559), and the High Impact Research (HIR) (UM.C/625/1/
HIR/MOHE/MED/17 & UM.C/625/1/HIR/MOHE/MED/33) from
the Ministry of Higher Education, Malaysia. We thank Dr Lisa
M. Perez and Dr Howard Williams for useful discussions. TAMU/
LBMS-Applications Laboratory provided mass spectrometric
support. The NMR instrumentation at Texas A&M University was
supported by a grant from the National Science Foundation
(DBI-9970232) and the Texas A&M University System.
Others have noted79 that data from PAMPA assays tends to
be less than those from Caco2 assays, so the difference in the
absolute values from QikProp (calculated Caco2 rates) and
PAMPA are unsurprising. Moreover, there is a reasonable corre-
lation between the relative rates in both sets of data (Fig. 10).
Conclusion
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