Systematic N-methylation of oxytocin: Impact on pharmacology and intramolecular hydrogen bonding network

Article abstract of DOI:10.1016/j.bmc.2016.05.062

Oxytocin (OT) is a peptide hormone agonist of the OT receptor (OTR) that plays an important role in social behaviors such as pair bonding, maternal bonding and trust. The pharmaceutical development of OT as an oral peptide therapeutic has been hindered historically by its unfavorable physicochemical properties, including molecular weight, polarity and number of hydrogen bond donors, which determines poor cell permeability. Here we describe the first systematic study of single and multiple N-methylations of OT and their effect on physicochemical properties as well as potency at the OT receptor. The agonist EC₅₀and percent effect for OTR are reported and show that most N-methylations are tolerated but with some loss in potency compared to OT. The effect of N-methylation on exposed polarity is assessed through the EPSA chromatographic method and the results validated against NMR temperature coefficient experiments and the determination of NMR solution structures. We found that backbone methylation of residues not involved in IMHB and removal of the N-terminal amine can significantly reduce the exposed polarity of peptides, and yet retain a significant OTR agonist activity. The results of this study also expose the potential challenge of using the N-methylation strategy for the OT system; while exposed polarity is reduced, in some cases backbone methylation produces a significant conformational change that compromises agonist activity. The data presented provides useful insights on the SAR of OT and suggests future design strategies that can be used to develop more permeable OTR agonists based on the OT framework.

Full text of DOI:10.1016/j.bmc.2016.05.062

Accepted Manuscript
Systematic N-Methylation of Oxytocin: Impact on Pharmacology and Intramo-
lecular Hydrogen Bonding Network
Simone Sciabola, Gilles H. Goetz, Guoyun Bai, Bruce N. Rogers, David L. Gray,
Allen Duplantier, Kari R. Fonseca, Michelle A. Vanase-Frawley, Natasha M.
Kablaoui
PII:
S0968-0896(16)30406-0
DOI:
http://dx.doi.org/10.1016/j.bmc.2016.05.062
BMC 13046
Reference:
Bioorganic & Medicinal Chemistry
To appear in:
Received Date:
Revised Date:
Accepted Date:
19 March 2016
26 May 2016
28 May 2016
Please cite this article as: Sciabola, S., Goetz, G.H., Bai, G., Rogers, B.N., Gray, D.L., Duplantier, A., Fonseca,
K.R., Vanase-Frawley, M.A., Kablaoui, N.M., Systematic N-Methylation of Oxytocin: Impact on Pharmacology
and Intramolecular Hydrogen Bonding Network, Bioorganic & Medicinal Chemistry (2016), doi: http://dx.doi.org/
10.1016/j.bmc.2016.05.062
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Systematic N-Methylation of Oxytocin: Impact on
Pharmacology and Intramolecular Hydrogen
Bonding Network
Simone Sciabolaǂ†, Gilles H. Goetz§†, Guoyun Bai§†, Bruce N. Rogersǂ, David L. Grayǂ, Allen
Duplantierǂ, Kari R. Fonseca, Michelle A. Vanase-Frawley§ and Natasha M. Kablaouiǂ†*
AFFILIATIONS:
Worldwide Medicinal Chemistry, Pfizer Global Research & Development,
§Groton Laboratories, Eastern Point Road, Groton, CT 06340, USA
ǂNeuroscience, 610 Main Street, Cambridge, MA 02139, USA
†These authors contributed equally
*Corresponding author e-mail: Natasha.M.Kablaoui@pfizer.com
KEYWORDS: Intramolecular Hydrogen Bond, Hydrogen Bonding, Polarity, Hidden Polarity,
SFC, NMR, Oxytocin, cyclic peptide, peptide
1

ABSTRACT
Oxytocin (OT) is a peptide hormone agonist of the OT receptor (OTR) that plays an important
role in social behaviors such as pair bonding, maternal bonding and trust. The pharmaceutical
development of OT as an oral peptide therapeutic has been hindered historically by its
unfavorable physicochemical properties, including molecular weight, polarity and number of
hydrogen bond donors, which determines poor cell permeability. Here we describe the first
systematic study of single and multiple N-methylations of OT and their effect on
physicochemical properties as well as potency at the OT receptor. The agonist EC50 and percent
effect for OTR are reported and show that most N-methylations are tolerated but with some loss
in potency compared to OT. The effect of N-methylation on exposed polarity is assessed through
the EPSA chromatographic method and the results validated against NMR temperature
coefficient experiments and the determination of NMR solution structures. We found that
backbone methylation of residues not involved in IMHB and removal of the N-terminal amine
can significantly reduce the exposed polarity of peptides, and yet retain a significant OTR
agonist activity. The results of this study also expose the potential challenge of using the N-
methylation strategy for the OT system; while exposed polarity is reduced, in some cases
backbone methylation produces a significant conformational change that compromises agonist
activity. The data presented provides useful insights on the SAR of OT and suggests future
design strategies that can be used to develop more permeable OTR agonists based on the OT
framework.
2

INTRODUCTION
The pro-social effects of the peptide hormone oxytocin (OT) have been studied in clinical trials
for over 30 years.1 These small and focused studies have shown the positive effects of OT on
social bonding,2 trust,3 generosity,4 and emotional recognition,5 and additionally its alleviation of
the symptoms of schizophrenia,6 post-traumatic stress disorder (PTSD),7 and autism spectrum
disorder (ASD).8 The poor oral bioavailability of oxytocin requires intravenous or intranasal
administration both in OT clinical investigations as well as for its use in the induction of labor
(Pitocin). Injectable drugs are better controlled for dosage, however they have a higher barrier to
patient compliance due to the necessity of either self-injections or going to a clinic for treatment.
Intranasal dosing of peptides9 is non-invasive and is proposed to facilitate privileged access
across the blood brain barrier (BBB), but it is difficult to ensure accurate dosing and is
unpleasant for the patient. Oral delivery typically improves both compliance and dose control;
however, with the low oral bioavailability for OT typical for peptide drugs10 this is not a viable
route of administration.
The poor bioavailability of peptide drugs has been attributed both to pre-systemic enzymatic
degradation and poor cell permeability through the intestinal mucosa. General strategies to
address both of these issues have been previously reviewed.11 In general, the high number of
hydrogen bond donors inherent in the peptide backbone as well as the polar side chains that are
often necessary to interact with molecular targets disfavor passage through the hydrophobic
interior of the membrane. Both in natural and synthetic systems, one tactic that has been used to
improve permeability of peptides is to mask hydrogen bond donors through reversible
intramolecular hydrogen bonds (IMHBs), which form in non-polar environments such as plasma
membranes. Thus the effective polarity of a peptide can be altered by its immediate environment
(i.e. solvent) via changes in its three dimensional structure. The potential for peptides to access
3

multiple conformations allows them to optimize a wide range of properties like aqueous
solubility, microsomal stability and receptor binding, without compromising their ability to fold
into highly lipophilic and membrane permeable conformations. This is illustrated by the cyclic
peptide natural product Cyclosporin A (CsA) which can populate distinct conformational states
depending on the polarity of its environment (Figure 1).12 CsA indeed is a remarkable peptide
that has sufficient polarity to bind specifically to its biological partner yet is situationally able to
hide its polarity through IMHBs to facilitate significant permeation through lipophilic
membranes. CsA is highly permeable as measured by the EPSA13 method (EPSA = 70) and the
oral bioavailability of CsA is significantly higher than most peptides, varying from 10% to
89%.14
Figure 1. a) Unbound (CSD code: DEKSAN). All amide NHs are either methylated or
participating in intramolecular H-bond. As CsA does not contain peripheral polar side chains, the
major contributors to poor permeability have been neutralized. b) Solution structure of CsA
bound to Cyclophilin (PDB code: 1CYA), the amide polar NH’s fold out toward the solvent.
Several groups have applied this strategy in synthetic peptides to generate cyclic peptides with
improved cell permeability.15 Lokey and coworkers have used conformational analysis and N-
methylation strategies, generating a library of small, cyclic hexapeptides with few polar side
chains to identify highly permeable substrates.16 Kessler and coworkers have developed cyclic
4

peptides with improved pharmacokinetic profile based on hexapeptides that bind to somatastatin
and melanocortin using similar tactics.17 In each of these examples, backbone nitrogens were
either N-methylated or involved in IMHBs.
Unlike the peptides reviewed in these studies which contain mostly lipophilic side chains, OT
and many known bioactive peptides have polar side chains and are less likely to be passively
permeable. Naturally occurring OT has no backbone N-methylation, contains multiple polar side
chains, and has an appended acyclic tail (Figure 2). Although structural studies have shown the
presence of two IMHBs within the cyclic portion of OT18 (Figure 2), its exposed polarity as
measured by the EPSA method13b remains in the high range (EPSA = 153) which has been
associated with poor passive cell permeability.13a
Figure 2. Structure of oxytocin with backbone amides numbered according to amino acid
sequence. Multiple literature accounts report two IMHBs between the NH of the Asn5 and the
C=O of the Tyr2 and between the NH of the Tyr2 and the C=O of the Asn5.
To investigate the extent to which the exposed polarity of OT could be modulated through
backbone structural changes we have synthesized a set of single and double N-methylated OT
5

analogs and studied their structural and physicochemical properties, as well as their
pharmacological modulation of the OT receptor. We found that backbone methylation of
residues not involved in IMHB can significantly reduce the exposed polarity of peptides when
compared to naturally occurring OT, and yet retain a significant OTR agonist activity.
RESULTS
SAR of methylated oxytocin derivatives
A systematic N-methyl walk was conducted, exploring the effects of each single N-methylation
on EPSA, the functional potency and the intrinsic agonist activity evaluated by intracellular Ca2+
increase in CHO cells stably expressing human OTR (Table 1). The EPSA values for each OT
N-methylation varies from -13 to 7, with negative numbers indicating a decrease in exposed
polarity compared to OT and the positive numbers indicating an increase in exposed polarity. If
all backbone nitrogen atoms were in equivalent local environments, each N-methylation should
reduce the polarity of OT by the same amount; however, a pattern emerges that some N-
methylations reduce polarity while others increase it.
Analogs [MeCys6]OT (7), [MeLeu8]OT (8) and [MeGly9]OT (9) with single N-methylation in
the 6, 8, or 9 positions, retain sub-nanomolar potency, while methylation of the N-terminal
amine, compound [MeCys1]OT (2), is also tolerated. Other than the Cys6, methylation of the
other backbone amides in the cyclic portion of oxytocin result in a more significant loss of
potency, with N-methylation at the Tyr2 (3), Ile3 (4), and Asn5 (6) resulting in a 1000-fold
reduction in potency, and N-methylation of the Gln4 (5) essentially eliminating agonist activity.
Each singly methylated analog that shows functional activity is a full agonist (>94% OTR
agonist activity). N-methylations at the Cys1, Ile3, and Cys6 result in marked reduction in the
6

EPSA value from OT (EPSA>9), indicating a decrease in exposed polarity. N-methylations at
the Tyr2, Gln4 and Asn5 show no change or, in the case of 6, an increase in EPSA (negative
delta value) indicating an increase in exposed polarity. Methylations on the acyclic portion of OT
(compounds 8 and 9) result in a moderate reduction in EPSA value.
The results of the N-methylation SAR on OT suggest that the NH’s on the Tyr2, Gln4, and Asn5
are involved in intramolecular H-bonds in OT, while those in positions Cys1, Ile3, and Cys6 are
not involved in IMHBs; methylation at the latter positions should therefore remove an H-bond
donor without disrupting the existing IMHB network. As expected, methylation of the backbone
nitrogens in the acyclic portion of OT has minimal impact on the IMHB network. These results
are in agreement with the literature reports of OT IMHB networks, however, the EPSA data does
suggest involvement of the NH of the Gln4 in an IMHB, a finding we explored further via NMR
experiments.
Table 1. Potency, activity, and EPSA values for oxytocin and N-methylated analogs.
Compounds were tested at least twice in each assay and the value reported is the average of those
values. EC50 data are means ± SEM of at least three independent experiments conducted as
duplicates determinations.
EC50
(nM)
EPSA
(OT)
EPSA
([Mpa]OT)
%agonist
Name
ID
EPSA
oxytocin (OT)
[MeCys1]OT
[MeTyr2]OT
[MeIle3]OT
[MeGln4]OT
[MeAsn5]OT
[MeCys6]OT
[MeLeu8]OT
[MeGly9]OT
0.04±0.01
4.77±2.10
23.93±13.42
14.74±8.43
>1000ǂ
102±5
102±3
98±2
153
140
154
144
152
160
144
150
150
0
-13
1
1
2
3
4
5
6
7
8
9
100±5
N.D.
-9
-1
7
25.99±10.22
0.99±0.63
0.52±0.22
0.47±0.23
100±4
95±8
-9
-3
-3
97±5
102±2
7

174.86±53.75
6.98±4.44
81±26
78±4
94
135
153
137
140
N.D.
149
135
132
130
125
127
127
131
-18
0
[MeCys1,MeCys6]OT
[MeTyr2,MeCys6]OT
[MeIle3,MeCys6]OT
10
11
12
13
14
15
16
17
18
19
20
21
22
-16
-13
N.D.
-4
15.57±0.72
10.79±2.13
5.52±2.19
0.04±0.01
0.29±0.19
7.98±1.70
>1000ǂ
83±3
90±9
96±3
106±7
84±8
97±13
N.D.
86±6
84±1
86±6
[MeCys6,MeLeu8]OT
[MeCys6,MeGly9]OT
[MeLeu8,MeGly9]OT
[Mpa1]OT
-18
-21
-23
-28
-26
-26
-22
0
-3
-5
-10
-8
-8
-4
[Mpa1,MeCys6]OT
[Mpa1, MeTyr2,MeCys6]OT
[Mpa1, MeIle3,MeCys6]OT
[Mpa1, MeCys6,MeLeu8]OT
[Mpa1, MeCys6,MeGly9]OT
[Mpa1, MeLeu8,MeGly9]OT
4.23±2.25
2.41±1.39
0.59±0.26
ǂEC50 data for compound 5 and 19 did not show any signal. Compound 12 was run only once due to sample

limitations. Mpa = Mercaptopropionic acid. The functional agonist activity at the OT receptor was evaluated by
intracellular Ca2+ increase in CHO cells stably expressing human OTR.
Multiple N-methylations should further reduce the exposed polarity of OT. While some of the
single N-methylations maintain sub-nanomolar agonist activity at OTR, double N-methylations
erode the potency and intrinsic activity to a greater extent over OT (analogs 10 to 15).
Compound [MeTyr2,MeCys6]OT (11) maintains single digit nanomolar potency (7 nM), but
intrinsic activity is eroded (78%), and there is no improvement in EPSA over OT presumably
due to disruption of IMHB at Tyr2. Compound [MeLeu8,MeGly9]OT (15), is also single digit
nanomolar and remains a full agonist, but it, too, has roughly equivalent EPSA as OT. As
expected from single methylated compounds, the biggest changes in EPSA for dimethyl analogs
are found in [MeCys1,MeCys6]OT (10), [MeIle3,MeCys6]OT (12), and [MeCys6,MeLeu8]OT
(13). These results are consistent with the IMHB network inferred from the N-methylations at
the residues that are not involved in the IMHB network (Cys1, Ile3, and Cys6) and a noncyclic
residue (Leu8). The potencies of the compounds also track with the single methylated SAR,
except for 11, which is surprisingly more potent than 3.
8

Methylation at the N-terminal amine 2 results in a loss of potency but with a large change in
exposed polarity as measured by EPSA. Replacement of the Cys1 with Mpa1
(mercaptopropionic acid) completely removes the N-terminal amine and should further reduce
exposed polarity. Previous reports have shown that the N-terminal amine can be completely
removed from OT with no loss of potency or intrinsic activity.19 We also found that [Mpa1]OT
(16) is equipotent to OT. Additionally, the removal of the N-terminal amine greatly reduces the
exposed polarity of the peptide. Moreover singly and doubly methylated [Mpa1]OT analogs (16
to 22) not only have consistently lower EPSA, but they are also up to 10x more potent than the
corresponding Cys1 peptides that maintain the N-terminal amine. While this is encouraging for
finding a good mix of both efficacy and permeability, it should be noted that most of these
peptides are partial agonists (intrinsic agonist activity of 79-90%), while OT itself is a full
agonist.
Table 2. Overall scoring system for N-methylations in OT. Average improvement in ΔEPSA
based on the type of modification to an amide/amine. The qualitative assessment ranges from (-)
no change to (+++) highest change in delta EPSA, which indicates largest decrease in exposed
polarity.
improvement
in ΔEPSA
qualitative
assessment
methylation type
N-terminal methylation
N-terminal deletion
-13
-18
-10
1
++
+++
++
-
Cyclic N-methylation (no IMHB)
Cyclic N-methylation (IMHB)
Non-cyclic N-methylation
-3
+
The EPSA data for the analogs in Table 1 suggest a clear IMHB pattern that is optimal for the
cyclic portion of OT: the backbone nitrogens on the Tyr2, Gln4, and the Asn5 residues are
9

involved in IMHBs while the Ile3 and Cys6 backbone nitrogens are oriented out of the ring. The
backbone nitrogens on the Leu8 and Gly9 are not involved in the overall IMHB network of OT.
Due to the lack of involvement of the Ile3 and Cys6 backbone nitrogens in the overall IMHB
network, methylation at those two positions gives the most pronounced reduction in exposed
polarity, and these analogs retain some activity. Table 2 summarizes the changes in EPSA upon
methylation of the backbone nitrogens or removal of the N-terminal amine of OT; most striking
is the difference between the backbone nitrogens in the cyclic portion that are involved in
IMHBs compared to those that are not.
Temperature Coefficient Data for Methylated OT analogs
1
Temperature dependent H chemical shifts of backbone amide protons can determine whether or
not a proton is involved in an IMHB in a particular solvent. Protons that shift upfield to a lower
ppm value at a rate greater than -4.5 ppb/K upon increase in temperature are considered not
involved in IMHBs, while those that are involved in IMHBs maintain a relatively constant ppm
value (0 to -2.5 ppb/K) due to less exposure to the solvents used in the NMR experiment.16a
Table 3 gives the NMR temperature coefficient (TC) data for oxytocin and selected OT analogs,
reported in ppb/K. The residues that show a shift of less than 2.5 (bold in table) are indicative of
IMHBs. Consistent with the EPSA analysis and literature data for OT, Tyr2 and Asn5 show
shifts of less than 2.5 (IMHB), and our data shows an additional IMHB at the Cys1. In contrast
to the prediction from the EPSA analysis of OT, the TC data does not indicate that the Gln4
amide hydrogen of OT (Compound 1) is involved in a specific IMHB; however, methylation at
the 4-position (Compound 5) disrupts the entire IMHB network. The disruption of the other
IMHBs in 5 is likely responsible for the negative impact of N-methylation on EPSA; compound
5 is also the only monomethylated OT analog that fails to show OTR agonist activity in the
10

functional assay. Methylation at the 2-position in 11, which consistently shows an IMHB in both
the TC and EPSA analysis, does not completely disrupt the hydrogen bonding network; both the
IMHBs at the Cys1 and the Asn5 are still implied by the TC shifts. In fact, except for 5, all other
analogs tested maintained some if not all of the IMHB network found in OT. Unlike OT, an
IMHB at the Gln4 is detected in 12 and was further validated by NMR solution structure.
Table 3. Temperature coefficient (TC) (ppb/K) data derived from 1H NHR spectra collected at
different temperatures. Values underlined/bolded indicate strong IMHB involvement.
Additional experimental information here.
ID
1
Name
Cys1 Tyr2
Ile3
-6.4
-3.5
-8.4
-5
Gln4 Asn5 Cys6 Leu8 Gly9
Oxytocin (OT)
-3.3
N-Me
-4.5
-7.9
-4.9
-4.9
-2.6
-5.7
-5.3
-2.5
N/A*
-1.8
-1.2
-4
-1.9
-3.3
-1.6
-1.5
-6.3
-3
[MeGln4]OT
[MeTyr2,MeCys6]OT
[MeCys6,MeLeu8]OT
[MeIle3,MeCys6]OT
[Mpa1,MeLey8,MeGly9]OT
5
N-Me
N-Me
N-Me
-5.4
N-Me
11
13
N/A*
-6.9
-2
N/A*
N/A*
N-Me
N-Me
-4.5
-5.7
12
22
-2.4
0.6
-2.2
-2.3
2.5
-9.3
-4
-10.1
N-Me
N-Me
*Not applicable due to signal broadening.
NMR-Derived Solution Conformation of OT and 12
The solution structures of 1 and 12 in DMSO-d6 were obtained using 2D NMR and are shown in
3
Figure 3. NOESY cross-peack intensities and JHN-H coupling constants were used to obtain
interproton distance and dihedral information. The corresponding restraints were then applied to
run a molecular dynamics simulated annealing protocol which generated an ensemble of low
energy conformations. The IMHBs that are simulated to be within optimal bonding distance are
highlighted in green, and support the IMHBs found with the TC method and with previously
11

reported data on OT.18a-f, 20 However, other structural differences caused by the N-methylation of
backbone nitrogens are revealed. The OT structure (Figure 3a) shows a type II -turn involving
the sequence Tyr-Ile-Gln-Asn, with a hydrogen-bond between the NH of the Asn5 and C=O of
the Tyr2. A second hydrogen bond is observed between the NH of Tyr2 and the C=O of the
Asn5. The C-terminal linear chain contains a second -turn driven by the hydrogen-bonding
between the C=O of Cys6 and both the Gly9 N-H and the terminal amide NH. This IMHB is not
detected in the TC analysis, but this conformation as well as the trans Cys6-Pro7 peptide bond
required for the interaction have been reported in previous structures.18c, 18g, 21
Figure 3. NMR structure ensembles for 1 (left) and 12 (right). Intramolecular Hydrogen Bonds
(IMHB) are highlighted in green: (a) two IMHBs between Asn5 and Tyr3 and two IMHBs in
acyclic tail in OT (1); (b) two IMHBs between Asn5 and Tyr3 plus a unique IMHB in 12
between the NH of Gln4 and the C=O of Tyr2. All the residues are labeled with their position
based on the Oxytocin sequence.
The solution structure of 12 (Figure 3b) shows that the methyls on the Ile3 and Cys6 residues are
exposed to solvent, allowing the IMHB network of OT to remain intact. Indeed, the same type II
12

-turn involving the sequence Tyr-Ile-Glu-Asn, with the first IMHB between the N-H of the
Asn5 and C=O of the Tyr2 and the second IMHB between the N-H of Tyr2 and the C=O of the
Asn5 are observed. However, as seen in the TC data, a third IMHB in the cyclic portion of 12 is
observed between the N-H of the Gln4 and the C=O of the Tyr2. Figure 4a shows an overlay of
the backbones of 1 and 12. While the backbone overlay of the two structures is very similar
(RMSD = 0.75 Å), the different torsion angles caused by the steric bulk of the N-methylated
residues leads to conformational differences at the Ile3 and Cys6. The beta-carbons of Ile3 and
Cys6 shift to avoid eclipsing the N-Me to minimize torsional strain, causing a major shift in the
directionality of the corresponding side chains (Figure 4b). The resulting gauche configuration
drastically alters the directionality of the sec-butyl group on the Ile3 and the vector of the NH of
the Gln4. This shift in the position of the N-H of the Gln4 allows the IMHB observed with the
C=O of the Tyr2.
13

Figure 4. (a) Backbone overlay of 1 (green) and 12 (grey). Major differences in backbone amide
orientation are present at the Gln4 and Cys6. Both positions are affected by alleviation of
increased steric interactions from the methylated residues in compound 12. The Gln4 NH is
shifted in 12 into a position that allows an additional IMHB with the Tyr2 carbonyl that is not
present in 1. The RMSD between the two backbone structures is 0.75 Å. (b) Overlay of Ile-Gln-
Asn portion of OT (green) and compound 12 (grey) with sidechains. N-methylation at Ile3
causes major shifts in the directionality of the sidechains and the N-H at Gln4.
DISCUSSION
We have shown that N-Me modifications of the OT backbone can be introduced to tune both the
polarity and the receptor biological response. We confirmed that the N-terminal amine in OT can
be removed to significantly lower EPSA without impacting the functional activity of the
receptor. Moreover backbone NH at Ile3 and Cys6 can be methylated to further reduce EPSA
and maintain activity. There seems to be some advantage in terms of lowering the polarity of OT
through backbone N-Methylation of the three amino acid acyclic tail as observed in 13, 20 and
21.
N-methylation of Tyr2 and Asn5 in OT led to an increase in polarity confirming the presence of
a dual IMHB at these positions. Methylation of the solvent exposed NH of Gln4 failed to lower
EPSA while dropping significantly the receptor response, suggesting a major rearrangement in
the backbone conformation and a disruption of the IMHB network as found by the NMR
temperature coefficient data for 5. Capping of the solvent exposed NH in Ile3 resulted in a large
decrease in EPSA for compounds 4, 12 and 19; unfortunately, methylation at this position led to
a local conformational change, confirmed by the NMR solution structure of 12, which negatively
14

impacted the receptor functional activity. This represents a potential caveat when using
methylation of solvent exposed backbone nitrogen to improve permeability as the corresponding
structural changes are difficult to predict with respect to their effect on the biological activity.
This report demonstrates the utility of EPSA to assess intramolecular hydrogen bonding
networks, and suggest the potential use of EPSA as a tool for influencing chemical modification
strategies to improve permeability of highly polar peptides. Goetz et al.13a studied the correlation
between EPSA and passive cell permeability on a set of 814 compounds and found that a cyclic
peptide with an EPSA value below 80 has a much greater chance of being permeable (72%) than
a compound with a higher EPSA. Cyclosporine A has a measured EPSA of 70. Although the N-
methylation strategy employed herein successfully lowered the EPSA values for some of the OT
analogs up to 15-20%, this was not enough to reach the proposed permeability threshold.
Additional polarity-reducing strategies would need to be applied to OT in order to develop a
reasonable orally bioavailable therapeutic version. Modifications to the acyclic tail in OT as well
as optimizing the physicochemical properties of the remaining side chains are just a few
strategies that could further be pursued in the future.
In conclusion our N-methylation study of oxytocin represents the first systematic analysis of the
effects of single and multiple N-methylations of OT on potency and intrinsic activity at the
oxytocin receptor. While this methylation study was directed towards lowering the polarity of
oxytocin for the possible development of an orally bioavailable OTR agonist, it also explores
new SAR for this biologically important neurohypophysial hormone.
15

EXPERIMENTAL SECTION
EPSA method
a) Chemicals and Reagents. The mobile phase modifier consists of Ammonium Formate (98%)
diluted to 20mM in HPLC grade methanol (~ 5g of Ammonium Formate per 4-L solvent). b)
Standards Stock solution and sample preparation. Each standard should be dissolved to
approximately 1mg /mL in DMSO. Store the stock DMSO solution in the freezer. Combine 100
µL of each stock solution in a single vial. This solution should be stored at 4°C and discarded
upon loss of stability. Samples are delivered in 96-well plates, at 100 µL of 3mM in DMSO. The
standards are; in order of elution: Antipyrine, Chlorpromazine, Desipramine, Pindolol,
Diclofenac, m-Nitrobenzoic acid, Bumetanide, Furosemide. c) Instrumentation. The SFC/MS
used in this experiment was an Agilent-Aurora supercritical fluid chromatograph system. The
mass spectrometer used was a single quadrupole LC/MSD with an ESI source (Agilent, Palo-
Alto, CA). All data were acquired using Agilent 32-bit ChemStation™ (version B.03.01 [317]).
The effluent of the SFC was split to the MSD using a tee (Valco, Houston, TX) and PEEKsil
capillary tubing (Upchurch Scientific, Oak Harbor, WA); 50 cm long with an internal diameter
of 50 µm. d) Analysis Conditions. Analysis was performed using a 4.6mm x 250mm Chirex 3014
column (Phenomenex, Torrance, CA) with 5 µ particle and 100Å pore size. The flow rate was 5
mL/min with the outlet backpressure set to 140 bar. The injection volume was 5 L. The mobile
phase composition was varied from 5-60% in a linear gradient for 11 minutes at 5%/minute,
holding at 60% for 4 minute before returning to initial conditions kept until 16 minutes. The
column temperature was set to 40°C. Prior to reaching the UV detector, the eluent was heated to
60°C, and after the split the portion destined to the MSD was heated to 76°C. Single ion
monitoring is used along with scanning. e) Calibration. For every eight compounds a new
calibration curve is established using the calibration standards. Duplicate injections for each of
16

the eight compounds are bracketed with 4 injections of calibration standards (2 before, 2 after).
The retention times for each standard are tabulated. The %RSD for the retention time of each
standard are determined; they should not exceed 2.0%. A linear regression analysis of the
average retention times of standards is performed using the assigned EPSA values (Supplemental
material). The R2 of the resulting fit should exceed 0.99. The resulting linear regression formula
is applied to the analytes’ measured retention times to determine their EPSA values. EPSA
values are expressed as round numbers. No units. Calibration formula resulting from SI Table 1:
EPSA = 14.34*tR +29.99 (R2= 0.9995).
Temperature coefficients measurement by NMR
1D 1H-NMR spectrum of each compound in DMSO-d6 solution with a concentration of ~0.24
mM was acquired on Bruker AVANCE III 500 MHz spectrometer using standard one pulse
experiment. 32K data points were collected with a 6000 Hz sweep width, 4s repetition time and
16 scans. Spectra were processed and analyzed by using Topspin 3.0. The temperature range was
between 295-320 K. NMR spectra for representative examples are in supplemental material
(page S2-S14).
Synthesis of cyclic peptides
General: Peptides were synthesized at Chinese Peptide Company (Hangzhou, China) using
amino acids that were purchased from Novabiochem. The peptides were synthesized on Rink or
Knorr resins using standard FMOC solid phase peptide synthesis (SPPS) methods. For peptides
with methylations on residues 1-6, standard SPPS was paused at the residue to be methylated,
then the resin was treated with 2-Nitrobenaenesulfonylchloride (o-Ns-Cl) and washed, MeI and
washed, then with mercaptoethanol and washed. The remaining amino acids were added using
17

standard FMOC SPPS chemistry (Fmoc-Cys(PG)-OH, Fmoc-Tyr(PG)-OH, Fmoc-Leu-OH,
Fmoc-Ile-OH, FMOC-Pro-OH. Fmoc-Asn(Trt)-OH, Fmoc-Gln(PG)-OH). The Fmoc-MeLeu-
OH and Fmoc-MeGly-OH in the 8- and 9-positions were coupled through standard methods. All
peptides were deprotected and cleaved from the resin using
a
TFA solution
(TFA/Thioanisole/EDT/Phenol/H2O=87.5/5/2.5/2.5/2.5, 10 mL) then precipitated in cold ether.
Analytical HPLC was performed on a HP1090 system with a 4.6X150 mm SepaxGP-C18, 5
micron, 120 Å column using a gradient window of approximately 25% to 45% solvent B (varies
according to peptide) over 20 minutes at a flow rate of 1.0 mL/min (solvent A: 0.1% TFA in
water; solvent B: 0.1% TFA in 80% acetonitrile and 20% water). Preparative HPLC was run on
a Waters 4000 system connected to a Waters Delta-PakTM C18, 15 micron, 100A reversed-
phase HPLC column (25 X 200mm). Method: gradient window of solvent B of approximately
20.0% to 50.0% (varies according to peptide) over 60 minutes at a flow rate of 20.0 mL/min
(solvent A: 0.1% TFA in water; solvent B: 0.1% TFA in 80% acetonitrile and 20% water). Full
experimental details are given in the supplementary material for several examples.
Compound 2: [MeCys1]OT: c(MeCys-Tyr-Ile-Gln-Asn-Cys)-Pro-Leu-Gly-NH2 . Synthesis on
0.1 mmol Rink resin yields 32 mgs of white solid (yield: 62.7%) with a retention time of 11.261
min, 92.2% of purity by UV220. ESI-MS.(m/z)= 1021.4(100%, [M+H]+).
Compound 3: [MeTyr2]OT : c(Cys-MeTyr-Ile-Gln-Asn-Cys)-Pro-Leu-Gly-NH2 . Synthesis on
0.1 mmol Rink resin yields 42 mgs of white solid (yield: 41%) with a retention time of 11.261
min, 98.2% of purity by UV220. ESI-MS.(m/z)= 1021.4(100%, [M+H]+).
Compound 4: [MeIle3]OT: c(Cys-Tyr-MeIle-Gln-Asn-Cys)-Pro-Leu-Gly-NH2 . Synthesis on 0.1
mmol Rink resin yields 32 mgs of white solid (yield: 31%) with a retention time of 11.261 min,
97.9% of purity by UV220. ESI-MS.(m/z)= 1021.4(100%, [M+H]+).
18

Compound 5: [MeGln4]OT: c(Cys-Tyr-Ile-MeGln-Asn-Cys)-Pro-Leu-Gly-NH2 . Synthesis on
0.1 mmol Rink resin yields 18 mgs of white solid (yield: 18%) with a retention time of 11.261
min, 97.9% of purity by UV220. ESI-MS.(m/z)= 1021.4(100%, [M+H]+).
Compound 6: [MeAsn5]OT: c(Cys-Tyr-Ile-Gln-MeAsn-Cys)-Pro-Leu-Gly-NH2 . Synthesis on
0.1 mmol Rink resin yields 50 mgs of white solid (yield: 49%) with a retention time of 11.261
min, 97.6% of purity by UV220. ESI-MS.(m/z)= 1021.4(100%, [M+H]+).
Compound 7: [MeCys6]OT: c(Cys-Tyr-Ile-Gln-Asn-MeCys)-Pro-Leu-Gly-NH2.. Synthesis on
0.1 mmol Rink resin yields 26 mgs of white solid (yield: 25%) with a retention time of 11.261
min, 92.5% of purity by UV220. ESI-MS.(m/z)= 1021.4(100%, [M+H]+ ).
Compound 8: [MeLeu8]OT: c(Cys-Tyr-Ile-Gln-Asn-Cys)-Pro-MeLeu-Gly-NH2 . Synthesis on
0.1 mmol Rink resin yeilds 20 mgs of white solid (yield: 33%) with a retention time of 11.261
min, 95.9% of purity by UV220. ESI-MS.(m/z)= 1021.4(100%, [M+H]+).
Compound 9: [MeGly9]OT: c(Cys-Tyr-Ile-Gln-Asn-Cys)-Pro-Leu-MeGly-NH2 . Synthesis on
0.1 mmol Rink resin yields 56 mgs of white solid (yield: 55%) with a retention time of
11.261min, 95.2% of purity by UV220. ESI-MS.(m/z)= 1021.4(100%, [M+H]+).
Compound 10: [MeCys1,MeCys6]OT: c(MeCys-Tyr-Ile-Gln-Asn-MeCys)-Pro-Leu-Gly-NH2.
Synthesis on 0.3 mmol Knorr resin yields 17 mgs of white solid (yield: 5%) with a retention
time of 7.648 min, 97.9% of purity by UV220. ESI-MS.(m/z)= 1035.5(100%, [M+H]+).
Compound 11: [MeTyr2,MeCys6]OT: c(Cys-MeTyr-Ile-Gln-Asn-MeCys)-Pro-Leu-Gly-NH2 .
Synthesis on 0.3 mmol Knorr resin yields 15 mgs of white solid (yield: 6%) with a retention
time of 8.767 min, 96.3% of purity by UV220. ESI-MS.(m/z)= 1035.5(100%, [M+H]+).
19

Compound 12: [MeIle3,MeCys6]OT: c(Cys-Tyr-MeIle-Gln-Asn-MeCys)-Pro-Leu-Gly-NH2 .
Synthesis on 0.3 mmol Knorr resin yields 43 mgs of white solid (yield: 17%) with a retention
time of 9.100 min, 97.7% of purity by UV220. ESI-MS.(m/z)= 1035.5(100%, [M+H]+).
Compound 13: [MeCys6,MeLeu8]OT: c(Cys-Tyr-Ile-Gln-Asn-MeCys)-Pro-MeLeu-Gly-NH2 .
Synthesis on 0.3 mmol Knorr resin yields 63 mgs of white solid (yield: 20%) with a retention
time of 9.00 min, 95.1% of purity by UV220. ESI-MS.(m/z)= 1037.1(100%, [M+H]+).
Compound 14: [MeCys6,MeGly9]OT: c(Cys-Tyr-Ile-Gln-Asn-MeCys)-Pro-Leu-MeGly-NH2 .
Synthesis on 0.3 mmol Knorr resin yields 15 mgs of white solid (yield: 9%) with a retention
time of 9.401 min, 95.7% of purity by UV220. ESI-MS.(m/z)= 1035.5(100%, [M+H]+).
Compound 15: [MeLeu8,MeGly9]OT: c(Cys-Tyr-Ile-Gln-Asn-Cys)-Pro-MeLeu-MeGly-NH2 .
Synthesis on 0.3 mmol Knorr resin yields 14 mgs of white solid (yield: 5%) with a retention
time of 8.997 min, 96.7% of purity by UV220. ESI-MS.(m/z)= 1035.5(100%, [M+H]+).
Compound 16: [Mpa1]OT: c(Mpa-Tyr-Ile-Gln-Asn-Cys)-Pro-Leu-Gly-NH2. Synthesis on 0.17
mmol Rink resin yields 34 mgs of white solid (yield: 53%) with a retention time of 11.261 min,
97.8% of purity by UV220. ESI-MS.(m/z)= 992.3(100%, [M+H]+).
Compound 17:
[Mpa1,MeCys6]OT: c(Mpa-Tyr-Ile-Gln-Asn-MeCys)-Pro-Leu-Gly-NH2 .
Synthesis on 0.3 mmol Knorr resin yields 49 mgs of white solid (yield: 20%) with a retention
time of 9.165 min, 97.4% of purity by UV220. ESI-MS.(m/z)= 1006.3(100%, [M+H]+).
Compound 18: [Mpa1,MeTyr2,MeCys6]OT: c(Mpa-MeTyr-Ile-Gln-Asn-MeCys)-Pro-Leu-Gly-
NH2. Synthesis on 0.15 mmol Knorr resin yields 18 mgs of white solid (yield: 14%) with a
retention time of 8.632 min, 95.8% of purity by UV220. ESI-MS.(m/z)= 1020.4(100%,
[M+H]+).
20

Compound 19: [Mpa1,MeIle3,MeCys6]OT: c(Mpa-Tyr-MeIle-Gln-Asn-MeCys)-Pro-Leu-Gly-
NH2. Synthesis on 0.2 mmol Knorr resin yields 44 mgs of white solid (yield: 25%) with a
retention time of 7.986 min, 96.5% of purity by UV220. ESI-MS.(m/z)= 1020.2(100%,
[M+H]+).
Compound 20: [Mpa1,MeCys6,MeLeu8]OT: c(Mpa-Tyr-Ile-Gln-Asn-MeCys)-Pro-MeLeu-Gly-
NH2. Synthesis on 0.3 mmol Knorr resin yields 20 mgs of white solid (yield: 6%) with a
retention time of 7.686 min, 95.1% of purity by UV220. ESI-MS.(m/z)= 1020.3(100%,
[M+H]+).
Compound 21: [Mpa1,MeCys6,MeGly9]OT: c(Mpa-Tyr-Ile-Gln-Asn-MeCys)-Pro-Leu-MeGly-
NH2. Synthesis on 0.3 mmol Knorr resin yields 48 mgs of white solid (yield: 13%) with a
retention time of 7.322 min, 98.9% of purity by UV220. ESI-MS.(m/z)= 1020.4 (100%,
[M+H]+).
Compound 22: [Mpa1,MeLeu8,MeGly9]OT: c(Mpa-Tyr-Ile-Gln-Asn-Cys)-Pro-MeLeu-MeGly-
NH2. Synthesis on 0.2 mmol Knorr resin yeilds 31 mgs of white solid (yield: 12%) with a
retention time of 9.987 min, 98.8% of purity by UV220. ESI-MS.(m/z)= 1020.2 (100%,
[M+H]+).
In vitro functional assay
CHO hOTR NFAT Lac
21

L
°C
L
L
NMR Structure determination
Standard 2D NMR methods were used to make assignments of each proton signal and to obtain
distance and angular constraints for structure determination. DQF-COSY, TOCSY (spiN-lock
time 80 ms) and NOESY (relaxation delay 2.0 s, mixing time 200 ms) experiments were
recorded for compounds 1 and 12 with a concentration of ~4mM at 298.2K in phase-sensitive
mode with spectral width of 5000 Hz, 2048 points on F2 and 256 increments on F1.
NMR data were processed using Topspin 3.0. 2D NOE cross-peaks were characterized into
strong (1.8-2.5 Å), medium (1.8-3.5 Å) and weak (1.8-5.0 Å) based on their intensities.
3
Backbone dihedral constraints were derived from JNH-H coupling constants by Karplus
equation: 3JNH-H=Acos2()+Bcos()+C.
Restrained Molecular Dynamics
22

To determine the 3D structure for Oxytocin and [MeIle3,MeCys6]OT (12), a molecular
dynamics simulated annealing protocol was used by translating the NOE interproton distances.
The initial 3D coordinates were generated using CORINA.22 The energy minimization and
molecular dynamics calculations were performed with the program Amber10. For the
minimization stage, we ran the first 500 steps using the steepest descent algorithm, followed by
500 additional steps of conjugate gradient algorithm. The generalized Born solvation model was
used instead of explicit water23 and periodicity was turned it off. The minimized structure was
then used to run 100 molecular dynamics simulated annealing refinement cycles of 20ps each
(20000 steps, time step = 1fs). Constant temperature was used with the Berendsen temperature
coupling algorithm. A cutoff of 15Å was applied for noN-bonded interactions and the same
solvation model as for minimization was used to treat the effect of solvent via an electrostatic
continuum. The molecules were first heated from step 0 to step 5000, with the target temperature
bath maintained at 600K and starting at 0K. From step 5001 to step 18000, the system was
cooled gradually from 600K to 100K. The temperature bath was cooled linearly over this length
of time. For the final 2000 steps (from 18001 to 20000), the temperature bath was set to 0K.
23

SUPPORTING INFORMATION AVAILABLE: This material is available free of charge via the
Internet at http://pubs.acs.org.
ACKNOWLEDGEMENTS
The authors thank Chris Limberakis, David Price, and Alan Mathiewitz for enlightening
discussions around peptides and peptide handling, and Robert Stanton for critical reading and
editing of the manuscript.
ABBREVIATIONS USED: SFC (Supercritical Fluid Chromatography); Mpa (Mercaptopropionic
acid); SPPS (Solid Phase Peptide Synthesis); FLIPR (Fluorescent Imaging Plate Reader)
24

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Table of Contents Graphic
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