Solid phase synthesis of peptides containing backbone-fluorinated amino acids

Article abstract of DOI:10.1039/c2ob26596f

Backbone-fluorinated amino acids exhibit unique conformational behaviour, and have potential utility as components of bioactive shape-controlled peptides. However, methods for the elaboration of backbone-fluorinated amino acids have thus far been limited

Full text of DOI:10.1039/c2ob26596f

View Online / Journal Homepage
Organic &
Biomolecular
Chemistry
Accepted Manuscript
This is an Accepted Manuscript, which has been through the RSC Publishing peer
Organic &
review process and has been accepted for publication.
Biomolecular
Accepted Manuscripts are published online shortly after acceptance, which is prior
to technical editing, formatting and proof reading. This free service from RSC
Publishing allows authors to make their results available to the community, in
citable form, before publication of the edited article. This Accepted Manuscript will
be replaced by the edited and formatted Advance Article as soon as this is available.
Chemistry
www.rsc.org/obc
Volume 8 | Number 3 | 7 February 2010 | Pages 481–716
To cite this manuscript please use its permanent Digital Object Identifier (DOI®),
which is identical for all formats of publication.
More information about Accepted Manuscripts can be found in the
Information for Authors.
Please note that technical editing may introduce minor changes to the text and/or
graphics contained in the manuscript submitted by the author(s) which may alter
content, and that the standard Terms & Conditions and the ethical guidelines
that apply to the journal are still applicable. In no event shall the RSC be held
responsible for any errors or omissions in these Accepted Manuscript manuscripts or
any consequences arising from the use of any information contained in them.
ISSN 1477-0520
PERSPECTIVE
FULL PAPER
Laurel K. Mydock and
AlexeiV. Demchenko
Shuji Ikeda et al.
Hybridization-sensitive fluorescent
DNA probe with self-avoidance ability
Mechanism of chemical O-glycosylation:
from early studies to recent discoveries
1477-0520(2010)8:3;1-H
www.rsc.org/obc
Registered Charity Number 207890

Organic & Biomolecular Chemistry
Page 1 of 8Organic & Biomolecular
Dynamic Article Links ►
Chemistry
View Online
Cite this: DOI: 10.1039/c0xx00000x
www.rsc.org/xxxxxx
PAPER
Solid phase synthesis of peptides containing backbone-fluorinated amino
acids†
Luke Hunter,*a,b Sharon Butlerc and Steve Ludbrookc
Received (in XXX, XXX) Xth XXXXXXXXX 20XX, Accepted Xth XXXXXXXXX 20XX
5
DOI: 10.1039/b000000x
Backboneꢀfluorinated amino acids exhibit unique conformational behaviour, and have potential utility as
components of bioactive shapeꢀcontrolled peptides. However, methods for the elaboration of backboneꢀ
fluorinated amino acids have thus far been limited to solution phase peptide coupling reactions. In this
paper, protocols are developed that allow the successful manipulation of backboneꢀfluorinated amino
10 acids using Fmocꢀstrategy solid phase peptide synthesis. To exemplify this strategy, several fluorinated
RGD peptide analogues were synthesised in moderate to good overall yields.
Introduction
Fluorinated amino acids have found a variety of applications in
peptide and protein science.1 One such application is in the
15 creation of proteins with increased stability. For example, highly
fluorinated analogues of canonical amino acids (eg. 1) have been
incorporated into “fluorousꢀcore” proteins and “fluorousꢀface”
alphaꢀhelical dimers in order to investigate the importance of
45 Fig. 1 Examples of fluorinated amino acids that have been incorporated
hydrophobic effects on protein structure and stability.1 In another
20 example, Raines and coꢀworkers have demonstrated that the
stability of the collagen triple helix is increased when the
constituent 4ꢀhydroxyproline residues are replaced with 4ꢀ
fluoroproline (2);2 the enhanced collagen stability was attributed
(in part) to interꢀstrand dipolar interactions associated with the
25 C−F bond.3 Another application of fluorinated amino acids is in
the area of protein NMR: for example, fluorinated tyrosine
analogues (eg. 3) have been employed as NMR indicators,4
taking advantage of the fact that fluorine is absent from natural
proteins. Another application of fluorinated amino acids is in the
30 conformational control of peptides.5 The highly polarised C−F
bond participates in a variety of stereoelectronic interactions with
neighbouring functional groups, and these can favour certain
molecular conformations over others.6 Taking advantage of this
concept, Seebach and coꢀworkers have incorporated αꢀfluoroꢀβꢀ
35 amino acids (eg. 4) into βꢀheptapeptides, and have demonstrated
that the configuration of the fluorinated stereocentre has a
dramatic influence on the secondary structure of the βꢀpeptide.7
Hunter and coꢀworkers have explored this concept with α,βꢀ
difluoroꢀγꢀamino acids (eg. 5);8 the different stereoisomers of 5
40 are found to have contrasting conformations that offer potentially
valuable biological applications.9
into peptides and proteins.
analogues of the canonical amino acids (eg. 1–3) can sometimes
be incorporated into analogues of natural proteins by
a
microorganism’s biosynthetic machinery, using feeding
50 experiments with bacterial strains that are auxotrophic for the
parent amino acid.10 In practice, chemical methods are also
commonly used for the elaboration of amino acids such as 1–3,
since these amino acids are readily amenable to solid phase
peptide synthesis.4 In contrast with the sidechainꢀfluorinated
55 amino acids 1–3, the backboneꢀfluorinated amino acids 4 and 5
do not resemble canonical amino acids, and hence chemical
synthesis is the only option for manipulating these building
blocks. Seebach7 and Hunter8 have previously elaborated amino
acids such as 4 and 5 into peptides containing up to twenty amino
60 acid residues,11 using solution phase peptide coupling reactions.
However it would be desirable to be able to employ these
building blocks within the manifold of solid phase peptide
synthesis, given the operational simplicity and the rapidity of this
technique.12 Challenges associated with solid phase peptide
65 synthesis include strongly basic peptide coupling conditions, and
the standard technique of employing a large reagent excess in
each peptide coupling step; both of these issues could prove
problematic with the nonꢀcommercial amino acids 4 and 5.
Accordingly, the aim of this study was to investigate whether it is
There are several synthetic methods for incorporating amino
acids such as 1−5 into peptides and proteins. Fluorinated
This journal is © The Royal Society of Chemistry [year]
[journal], [year], [vol], 00–00 | 1

Organic & Biomolecular Chemistry
Page 2 of 8
View Online
50 acidꢀstable linkage in case small amounts of HF continued to be
formed. When PyBOP and Nꢀmethylmorpholine were employed
as the peptide coupling reagents (both equimolar relative to 9), a
moderate yield of the desired peptide 14 was eventually obtained,
along with a smaller quantity of the undesired elimination
55 product 13 (Table 1, entry 2).
Due to a temporary exhaustion of the available stock of Fmocꢀ
protected amino acid 9, further SPPS investigations were
performed using the building block 10. In the next experiment
(Table 1, entry 3), a single coupling of 10 was employed, with 1.2
60 equivalents of 10 and an equimolar quantity of HOBt and DIC
(ie. no base). Gratifyingly, a moderate yield of the desired peptide
15 was obtained (Table 1, entry 3). Finally, the yield of 15 was
able to be increased to 83% by employing 1.5 equivalents of 10
(Table 1, entry 4). The absence of base in the key coupling step
65 resulted in complete suppression of the undesired HF elimination
pathway, and standard peptide coupling conditions (HBTU,
diisopropylethylamine, DMF) were able to be used for all other
coupling steps in the sequence. This suggests that the fluorinated
amino acid is sensitive to HF elimination only when converted
70 into the corresponding active ester; in constrast, once
incorporated into a peptide the difluoro motif is stable even under
basic conditions.
The optimised SPPS conditions (Table 1, entry 4) were then
employed to generate a small library of fluorinated peptides
75 (Figure 2). Overall yields ranged from moderate to good,
although in most cases it was necessary purify the target peptide
by preparative reverseꢀphase HPLC in order to remove minor
impurities such as the ArgꢀAspꢀPhe tripeptide (ie. lacking the
fluorinated amino acid).
Scheme 1 Synthesis of Fmocꢀprotected building blocks.
possible to efficiently manipulate backboneꢀfluorinated amino
acids such as 4 and 5 using Fmocꢀstrategy solid phase peptide
synthesis.12
5
Results and discussion
The first task was to assemble the required collection of Fmocꢀ
protected fluorinated amino acids. This was accomplished by
10 treatment of the appropriate unprotected amino acids 5, 6 and 8
with Nꢀ(9ꢀfluorenylmethoxycarbonyloxy)succinimide (Fmocꢀ
OSu) under standard conditions13 (Scheme 1). The protected
amino acids 7, 9 and 10 were obtained in good yields, and were
fully characterised according to the standard suite of analytical
15 techniques. The NMR spectra14 of 7, 9 and 10 revealed the
presence of rotamers at 300 K, the signals of which were
observed to coalesce at elevated temperature (~330 K).
With building blocks 7, 9 and 10 (and their enantiomers) in
hand, attention was next turned toward their use in solid phase
20 peptide synthesis. The peptides targeted in this study were
analogues of the tetrapeptide sequence RGDF (arginineꢀglycineꢀ
aspartateꢀphenylalanine), where the glycine residue was to be
replaced with various fluorinated amino acids. These peptide
targets would allow the efficiency of peptide coupling at both the
25 Cꢀ and Nꢀtermini of the fluorinated amino acids to be
investigated, and would also allow the compatibility with some
sidechain protecting groups to be determined.
80
Peptides 14–22 are analogues of the RGD tripeptide, a
sequence that is commonly found in extracellular matrix proteins
and which is recognised by cellꢀsurface receptors known as
integrins.16 There is considerable interest in creating RGD
analogues that are selective for individual integrin receptor
85 subtypes, because such molecules have a variety of potential
therapeutic applications;17 αVβ3 integrin is a target of particular
interest because this receptor has a proꢀangiogenic function and is
upregulated in solid tumours.18 It has previously been shown that
αVβ3 integrin selectivity is exhibited by synthetic peptides in
Initial experiments were performed with the fluorinated amino
acid 9 (Table 1, entry 1). 2ꢀChlorotrityl chloride resin loaded with
30 aspartateꢀphenylalanine dipeptide (11) was treated for 2 h with a
solution of 9 (1 equivalent relative to resin loading) and HBTU (1
equivalent) in
a minimal volume of dimethylformamide
containing 0.4 M diisopropylethylamine, and this coupling step
was then repeated with 0.5 equivalents of 9/HBTU for 2h. A
35 standard sequence of deprotection, peptide coupling and cleavage
steps followed; a double coupling of arginine was employed to
compensate for any reduced nucleophilicity of the amino group
of the fluorinated amino acid. This reaction sequence (Table 1,
entry 1) eventually delivered the undesired product 13 which had
40 suffered HF elimination (alkene geometry of 13 confirmed by
3JHF = 34.5 Hz).15 The undesired elimination reaction likely
occurred during HBTU/diisopropylethylamine activation of 9
prior to its addition to the resin; a dramatic colour change of the
solution from colourless to dark orange was observed at this step.
45 The presence of excess base was thought to be responsible for an
efficient E2 elimination, therefore alternative conditions were
investigated in which only one equivalent of base was employed
(Table 1, entry 2). For this second experiment it was also decided
to switch to Wang resin as the solid support (12), to offer a more
90
Fig. 2 Peptides prepared by SPPS in this study. Yields refer to purified
materials and are based on the initial resin loading of Fmocꢀ
phenylalanine.
2
| Journal Name, [year], [vol], 00–00
This journal is © The Royal Society of Chemistry [year]

Page 3 of 8
Organic & Biomolecular Chemistry
View Online
Table 1 Investigation of different SPPS protocols.a
Entry Starting material
Reaction conditions
Product (yield)
(a) 9 (1+0.5 eq), HBTU (1+0.5 eq), DIPEA (0.4 M), DMF
(b) Piperidine (10%), DMF
1
(c) FmocꢀArg(Pbf) (3+3 eq), HBTU (2.9+2.9 eq), DIPEA (0.4 M), DMF
(d) Piperidine (10%), DMF
(e) TFA (95%), TIS (2.5%), H2O
(a) 9 (1+0.5 eq), PyBOP (1+0.5 eq), NMM (1+0.5 eq), DMF
(b) Piperidine (10%), DMF
2
(c) FmocꢀArg(Pbf) (3+3 eq), PyBOP (3+3 eq), NMM (3+3 eq), DMF
(d) Piperidine (10%), DMF
(e) TFA (95%), TIS (2.5%), H2O
(a) 10 (1.2 eq), HOBt (1.2 eq), DIC (1.2 eq), DMF
(b) Piperidine (10%), DMF
3
4
(c) FmocꢀArg(Pbf) (3+3 eq), HBTU (2.9+2.9 eq), DIPEA (6+6 eq), DMF
(d) Piperidine (10%), DMF
(e) TFA (95%), TIS (2.5%), H2O
(a) 10 (1.5 eq), HOBt (1.5 eq), DIC (1.5 eq), DMF
(b) Piperidine (10%), DMF
(c) FmocꢀArg(Pbf) (3+3 eq), HBTU (2.9+2.9 eq), DIPEA (6+6 eq), DMF
(d) Piperidine (10%), DMF
(e) TFA (95%), TIS (2.5%), H2O
a Abbreviations: Solid black circle = 2ꢀchlorotrityl chloride resin; shaded grey circle = Wang resin; HBTU = OꢀbenzotriazoleꢀN,N,N’,N’ꢀ
tetramethyluronium hexafluorophosphate; DIPEA = diisopropylethylamine; DMF = dimethylformamide; FmocꢀArg(Pbf) = NαꢀFmocꢀNωꢀ(2,2,4,6,7ꢀ
pentamethyldihydrobenzofuranꢀ5ꢀsulfonyl)ꢀ ꢀarginine; TFA = trifluoroacetic acid; TIS = triisopropylsilane; PyBOP = (benzotriazolꢀ1ꢀ
yloxy)tripyrrolidinophosphonium hexafluorophosphate; NMM = Nꢀmethylmorpholine; HOBt = hydroxybenzotriazole; DIC = diisopropylcarbodiimide.
5
L
which the RGD sequence is constrained into
a “bent”
10 conformation.19 Since the fluorinated amino acids employed in
this study are known to adopt uniqe and wellꢀdefined
conformations,7,8 it became of interest to investigate whether any
of the peptides 14–22 exhibited integrin binding, and/or αVβ3
integrin selectivity. Accordingly, cell adhesion assays were
15 performed with αVβ3, αVβ6, αVβ5 and α5β1 integrins.20
Disappointingly however, only the control peptide 20 was found
to exhibit any detectable integrin activity (at αVβ3 and αVβ5).14
The lack of activity of the fluorinated peptides 14–19 could be
attributed to the altered length of the backboneꢀhomologated
20 amino acid residues;21 in the future, it may be of interest to
investigate peptide analogues in which one or more methylene
groups are removed from the arginine sidechain in order to
restore the natural distance between the reactive groups of the
arginine and aspartate sidechains which provide key binding
25 interactions at integrin receptors.17 Indeed, work is currently
underway in our laboratories towards the incorporation of
backboneꢀfluorinated amino acids such as 9 and 10 into a variety
of peptide systems, including a second generation of fluorinated
RGD peptide analogues, using the solid phase synthesis methods
30 developed here.
Conclusions
Protocols have been developed that allow backboneꢀfluorinated
amino acids to be efficiently incorporated into peptides using
Fmocꢀstrategy SPPS. Fluorinated peptides are obtained in
35 moderate to good overall yields (suffering only minimal HF
elimination), using only a slight excess of the nonꢀcommercial
fluorinated amino acid. The peptides created in this study did not
possess significant biological activity in the context of integrin
receptor ligands; nevertheless, the synthetic methodology
40 described herein should expedite the future creation of a variety
of fluorinated peptides for other applications in biotechnology
and medicine.
Experimental section
Solvents, reagents and instrumentation
45 Water was obtained from a Millipore filtration system. N,Nꢀ
Dimethylformamide was purchased in peptide synthesis grade
and stored over 4Å molecular sieves. All other reagents and
solvents were purchased in the highest available quality and used
as received. Eluting solvents for chromatography are reported as
50 volume/volume mixtures. Where indicated, NMR signals were
This journal is © The Royal Society of Chemistry [year]
Journal Name, [year], [vol], 00–00 | 3

Organic & Biomolecular Chemistry
Page 4 of 8
View Online
After the last Fmoc deprotection, the resin was washed with DMF
PRECAUTION: due to the possibility that small amounts of HF 55 (3 × 1 min) and DCM (3 × 1 min). The resin was agitated with a
assigned using information from COSY experiments. SAFETY
could be formed as a sideꢀproduct in these experiments,
appropriate personal protective equipment was employed
(including a tube of calcium gluconate gel kept close at hand).
solution of 95 : 2.5 : 2.5 TFA/TIS/H2O for 2h. The resin was
drained and washed with TFA (2 × 1 min). The combined
cleavage solutions were concentrated in vacuo. The residue was
dissolved in water (~20 mL per mmol of peptide) and this
60 solution was washed four times with an equal volume of diethyl
ether. The aqueous layer was freezeꢀdried to afford the crude
peptide, which was purified if necessary by reverseꢀphase HPLC
eluting with 0→30% acetonitrile/water (containing 0.1% v/v
TFA) over 40 min and monitoring at 254 nm.
5
General procedure A: synthesis of Fmoc-protected
fluorinated amino acids
A solution of FmocꢀOꢀsuccinimide (1 mmol) in 1,4ꢀdioxane
(7.5 mL) was added to a solution of the appropriate fluorinated
10 amino acid (1 mmol) and sodium carbonate (3 mmol) in water
(7.5 mL) at 0 °C. The mixture was stirred at room temperature
overnight, then acidified with dilute hydrochloric acid. The
mixture was concentrated onto silica, and the crude product was
purified by flash chromatography eluting with 90 : 8 : 2
15 chloroform/ methanol/ acetic acid.
65 (R)-3-((((9H-Fluoren-9-yl)methoxy)carbonyl)amino)-2-
fluoropropanoic acid (7)
The title compound was prepared from (R)ꢀ3ꢀaminoꢀ2ꢀ
fluoropropanoic acid hydrochloride (6)22 according to General
Procedure A on 1.40 mmol scale. The product was obtained as a
70 white solid (0.445 g, 96% yield); m.p. 138–139 °C; [α]D –3.3
(c 0.94, MeOH); IR (neat) vmax (cm–1) 3305, 3062, 1718, 1535,
General procedure B: SPPS: preparation of resin
Solid phase peptide synthesis was conducted manually in a sinterꢀ
fitted polypropylene syringe. Wang resin (100–200 mesh) preꢀ
loaded with Fmocꢀphenylalanine (0.65 mmol/g resin loading) was
20 agitated in DCM for 1h, then drained and washed with DMF (3 ×
1 min).
1
1450, 1259, 1156, 1104; H NMR (300 MHz, MeOD) δ 7.80–
7.59 (m, 4H, ArH), 7.42–7.26 (m, 4H, ArH), 5.00 (ddd, J = 49.1,
6.6, 3.5 Hz, 1H, αꢀCHF), 4.31 (d, J = 6.8 Hz, 2H, Fmoc CH2),
75 4.16 (t, J = 6.8 Hz, 1H, Fmoc CH), 3.71 (ddd, J = 24.5, 14.7, 3.3
Hz, 1H, βꢀCHH), 3.58 (ddd, J = 22.2, 14.7, 6.6 Hz, βꢀCHH);
13C {1H} NMR (75 MHz, MeOD) δ 171.5 (d, J = 23.4 Hz,
CO2H), 158.8, 145.2, 142.5, 128.7, 128.1, 126.2, 120.9, 88.9 (d,
J = 184.4 Hz, αꢀCHF), 68.0, 48.3, 43.7 (d, J = 21.5 Hz, βꢀCH2);
80 19F NMR (282 MHz, MeOD) δ –197.1 (ddd, J = 47.7, 23.9,
23.9 Hz, 1F); 19F {1H} NMR (282 MHz, MeOD) δ –197.1 (s,
1F); MS (ESI, +ve) m/z 352 (MNa+, 52%); HRMS (ESI, +ve)
C18H16NO4FNa+ requires m/z 352.0956, found 352.0954.
General procedure C: SPPS: Fmoc deprotection
The resin was washed with DMF (3 × 1 min). The resin was
agitated with a solution of 10% piperidine in DMF (2 × 3 min),
25 then washed with DMF (3 × 1 min), DCM (3 × 1 min) and DMF
(3 × 1 min). The deprotection solutions were combined and
diluted 100ꢀfold with 10% piperidine in DMF, and the
absorbance of the diluted solution was measured at 301 nm
against 10% piperidine in DMF as reference. The resin loading
30 was determined by calculating the concentration of the
piperidineꢀfulvene adduct (ε = 7800 M–1cm–1) in the deprotection
solution.
(S)-3-((((9H-Fluoren-9-yl)methoxy)carbonyl)amino)-2-
85 fluoropropanoic acid (ent-7)
The title compound was prepared from (S)ꢀ3ꢀaminoꢀ2ꢀ
fluoropropanoic acid hydrochloride (entꢀ6)22 according to
General Procedure A on 1.34 mmol scale. The product was
obtained as a white solid (0.200 g, 45% yield); m.p. 127–132 °C;
90 [α]D +2.3 (c 1.54, MeOH); IR data, 1H NMR data, 13C {1H}
NMR data, 19F NMR data, 19F {1H} NMR data and MS data
identical to that of 7 above; HRMS (ESI, +ve) C18H16NO4FNa+
requires m/z 352.0956, found 352.0953.
General procedure D: SPPS: peptide coupling (commercial
amino acids)
35 The resin was washed with DMF (3 × 1 min). A solution was
prepared of the appropriate Fmocꢀprotected amino acid (3 equiv.
relative to resin loading) and HBTU (2.9 equiv. relative to resin
loading) in minimal DMF. DIPEA (6 equiv. relative to resin
loading) was added to this solution, and the mixture was
40 immediately added to the resin and agitated for 1.5 h. The resin
was drained and washed with DMF (3 × 1 min), DCM (3 ×
1 min) and DMF (3 × 1 min).
(2R,3S)-4-((((9H-Fluoren-9-yl)methoxy)carbonyl)amino)-2,3-
95 difluorobutanoic acid (9)
The title compound was prepared from (2R,3S)ꢀ4ꢀaminoꢀ2,3ꢀ
difluorobutanoic acid hydrochloride (8)9 according to General
Procedure A on 0.18 mmol scale. The product was obtained as a
white solid (0.059 g, 92% yield); m.p. 171–174 °C; [α]D –4.5
100 (c 1.21, MeOH); IR (neat) vmax (cm–1) 3439, 1714, 1540, 1455,
General procedure E: SPPS: peptide coupling (fluorinated
amino acids)
45 The resin was washed with DMF (3 × 1 min). A solution was
prepared of the appropriate Fmocꢀprotected amino acid
(1.5 equiv. relative to resin loading), HOBt (1.5 equiv. relative to
resin loading) and DIC (1.5 equiv. relative to resin loading) in
minimal DMF. This solution was stirred for 20 min, then added
50 to the resin and agitated overnight. The resin was drained and
washed with DMF (3 × 1 min), DCM (3 × 1 min) and DMF (3 ×
1 min).
1
1275; H NMR (300 MHz, MeOD) δ 7.79–7.75 (m, 2H, ArH),
7.65–7.61 (m, 2H, ArH), 7.40–7.26 (m, 4H, ArH), 5.31–4.81 (m,
2H, αꢀCHF + βꢀCHF), 4.34 (d, J = 6.7 Hz, 2H, Fmoc CH2), 4.18
(t, J = 6.7 Hz, 1H, Fmoc CH); 13C {1H} NMR (100 MHz, MeOD,
105 330 K) δ 168.4 (m, CO2H), 158.0, 144.5, 141.8, 127.9, 127.3,
125.3, 120.1, 91.4 (dd, J = 180.3, 21.3 Hz, CHF), 88.7 (dd, J =
195.4, 23.8 Hz, CHF), 67.2, 47.8, 40.8 (dd, J = 26.6, 5.7 Hz,
γꢀCH2); 19F NMR (282 MHz, MeOD) δ –199.7 (m, 1F, βꢀCHF),
–204.3 (m, 1F, αꢀCHF); 19F {1H} NMR (282 MHz, MeOD) δ
General procedure F: SPPS: cleavage of peptide from resin
4
| Journal Name, [year], [vol], 00–00
This journal is © The Royal Society of Chemistry [year]

Page 5 of 8
Organic & Biomolecular Chemistry
View Online
–199.7 (d, J = 13.3 Hz, 1F, βꢀCHF), –204.3 (d, J = 13.3 Hz, 1F,
αꢀCHF); MS (ESI, +ve) m/z 384 (MNa+, 100%); HRMS (ESI,
+ve) C19H17F2NO4Na+ requires m/z 384.1018, found 384.1013.
3.12 (m, 1H, Arg δꢀCH2), 2.95 (dd, J = 14.0, 8.6 Hz, 1H, Phe βꢀ
CHH), 2.85 (dd, J = 17.1, 6.1 Hz, 1H, Asp βꢀCHH), 2.69 (dd, J =
17.1, 8.0 Hz, 1H, Asp βꢀCHH), 1.86 (m, 2H, Arg βꢀCH2), 1.56
(m, 2H, Arg γꢀCH2); 19F NMR (282 MHz, D2O) δ –76.0 (s, 3F,
60 TFA), –129.3 (d, J = 34.5 Hz, 1F, αꢀF); 19F {1H} NMR
(282 MHz, D2O) δ –76.0 (s, 3F, TFA), –129.3 (s, 1F, αꢀF); MS
(ESI, +ve) m/z 538 (MH+, 100%).
(2S,3R)-4-((((9H-Fluoren-9-yl)methoxy)carbonyl)amino)-2,3-
difluorobutanoic acid (ent-9)
5
The title compound was prepared from (2S,3R)ꢀ4ꢀaminoꢀ2,3ꢀ
difluorobutanoic acid hydrochloride (entꢀ8)9 according to General
Procedure A on 0.36 mmol scale. The product was obtained as a
L
L
-Arginyl-[(2R,3S)-4-amino-2,3-difluorobutanoyl]-L-aspartyl-
-phenylalanine, TFA salt (14)
white solid (0.061 g, 46% yield); m.p. 172–176 °C; [α]D +3.7
1
10 (c 1.08, MeOH); IR data, H NMR data, 13C {1H} NMR data, 65 The title compound was prepared on 25 ꢀmol scale according to
19F NMR data, 19F {1H} NMR data and MS data identical to that
of 9 above; HRMS C19H17F2NO4Na+ requires m/z 384.1018,
found 384.1022.
General Procedures B–F, employing a double coupling of Fmocꢀ
arginine. The product was obtained as a sticky white solid
(7.3 mg, 46% overall yield based on initial resin loading);
analytical HPLC (0→25% acetonitrile/water (containing 0.1%
70 v/v formic acid) over 30 min) retention time = 8.8 min, >95%
purity; [α]D –16.4 (c 0.21, H2O); IR (neat) vmax (cm–1) 3398,
(2S,3S)-4-((((9H-Fluoren-9-yl)methoxy)carbonyl)amino)-2,3-
15 difluorobutanoic acid (10)
1
The title compound was prepared from (2S,3S)ꢀ4ꢀaminoꢀ2,3ꢀ
difluorobutanoic acid hydrochloride (5)9 according to General
Procedure A on 0.32 mmol scale. The product was obtained as a
white solid (0.107 g, 92% yield); m.p. 142–148 °C; [α]D –16.0 (c
20 0.094, MeOH); IR (neat) vmax (cm–1) 3320, 3065, 2949, 1705,
2927, 1675, 1543, 1398, 1204, 1137; H NMR (300 MHz, D2O,
COSY) δ 7.41–7.26 (m, 5H, Ph), 5.30 (ddd, J = 47.4, 18.1,
2.3 Hz, 1H, αꢀCHF), 5.02 (ddddd, J = 46.8, 22.3, 8.0, 3.1, 2.3 Hz,
75 1H, βꢀCHF), 4.82 (m, 1H, Asp αꢀH), 4.71 (m, 1H, Phe αꢀH), 4.05
(t, J = 6.4 Hz, 1H, Arg αꢀH), 3.68 (ddd, J = 29.3, 15.0, 3.1 Hz,
1H, CHFCHH), 3.59 (m, 1H, CHFCHH), 3.29 (dd, J = 14.2,
5.4 Hz, 1H, Phe βꢀCHH), 3.24 (t, J = 6.6 Hz, 2H, Arg δꢀCH2),
3.06 (dd, J = 14.0, 8.9 Hz, 1H, Phe βꢀCHH), 2.91 (dd, J = 17.1,
80 5.2 Hz, 1H, Asp βꢀCHH), 2.77 (dd, J = 17.1, 8.7 Hz, 1H, Asp βꢀ
CHH), 1.95 (m, 2H, Arg βꢀCH2), 1.66 (m, 2H, Arg γꢀCH2);
13C {1H} NMR (75 MHz, D2O) δ 172.5, 171.6, 168.8, 167.4,
165.1 (dd, J = 21.4, 8.4 Hz, CHFCO), 154.5, 134.3, 126.9, 126.3,
124.8, 87.7 (dd, J = 201.7, 21.8 Hz, CHF), 87.1 (dd, J = 192.7,
85 22.7 Hz, CHF), 51.9, 50.5, 47.3, 37.9, 36.1 (dd, J = 22.7, 10.0 Hz,
CHFCH2), 34.3, 32.9, 25.6, 21.1; 19F NMR (282 MHz, D2O) δ
–76.0 (s, 3F, TFA), –198.7 (m, 1F, βꢀCHF), –202.1 (ddd, J =
47.6, 22.1, 13.3 Hz, 1F, αꢀCHF); 19F NMR (282 MHz, D2O) δ
–76.0 (s, 3F, TFA), –198.7 (d, J = 13.3 Hz, 1F, βꢀCHF), –202.1
90 (d, J = 13.3 Hz, 1F, αꢀCHF); MS (ESI, +ve) m/z 558 (MH+,
100%); HRMS (ESI, +ve) C23H34F2N7O7+ requires m/z 558.2482,
found 558.2486.
1
1538, 1429, 1265, 1126; H NMR (300 MHz, MeOD) δ 7.78–
7.74 (m, 2H, ArH), 7.64–7.60 (m, 2H, ArH), 7.39–7.25 (m, 4H,
ArH), 5.22–4.82 (m, 2H, αꢀCHF + βꢀCHF); 4.37 (d, J = 6.7 Hz,
2H, Fmoc CH2), 4.17 (t, J = 6.7 Hz, 1H, Fmoc CH), 3.59–3.44
25 (m, 2H, γꢀCHH + γꢀCHH); 13C {1H} NMR (75 MHz, MeOD) δ
170.5 (dd, J = 26.0, 6.1 Hz, CO2H), 159.3, 145.6, 143.0, 129.2,
128.6, 126.6, 121.4, 91.9 (dd, J = 179.3, 18.5 Hz, CHF), 88.9 (dd,
J = 182.1, 15.7 Hz, CHF), 68.3, 48.9, 42.1 (dd, J = 25.0, 4.6 Hz,
γꢀCH2); 19F NMR (282 MHz, MeOD) δ –204.1 (m, 1F, βꢀCHF),
30 –211.8 (m, 1F, αꢀCHF); 19F {1H} NMR (282 MHz, MeOD) δ
–204.1 (d, J = 8.7 Hz, 1F, βꢀCHF), –211.8 (d, J = 8.7 Hz, 1F, αꢀ
CHF); MS (ESI, +ve) m/z 384 (MNa+, 100%); HRMS
C19H17F2NO4Na+ requires m/z 384.1018, found 384.1018.
(2R,3R)-4-((((9H-Fluoren-9-yl)methoxy)carbonyl)amino)-2,3-
35 difluorobutanoic acid (ent-10)
The title compound was prepared from (2R,3R)ꢀ4ꢀaminoꢀ2,3ꢀ
difluorobutanoic acid hydrochloride (entꢀ5)9 according to General
Procedure A on 0.10 mmol scale. The product was obtained as a
white solid (0.032 g, 86% yield); m.p. 137–140 °C; [α]D +16.9 (c
40 0.172, MeOH); IR data, 1H NMR data, 13C {1H} NMR data,
19F NMR data, 19F {1H} NMR data and MS data identical to that
of 10 above; HRMS C19H17F2NO4Na+ requires m/z 384.1018,
found 384.1023.
L
L
-Arginyl-[(2S,3S)-4-amino-2,3-difluorobutanoyl]-L-aspartyl-
-phenylalanine, TFA salt (15)
95 The title compound was prepared on 25 ꢀmol scale according to
General Procedures B–F, employing a double coupling of Fmocꢀ
arginine. The product was obtained as a sticky white solid (13.8
mg, 83% overall yield based on initial resin loading); analytical
HPLC (0→25% acetonitrile/water (containing 0.1% v/v formic
100 acid) over 30 min) retention time = 8.7 min, >90% purity; [α]D
–11.6 (c 0.28, H2O); IR (neat) vmax (cm–1) 3195, 2925, 2856,
1667, 1538, 1397, 1201, 1134; 1H NMR (300 MHz, D2O, COSY)
δ 7.41–7.24 (m, 5H, Ph), 5.22 (ddd, J = 46.3, 29.6, 1.3 Hz, 1H, αꢀ
CHF), 5.04 (ddddd, J = 46.2, 27.2, 8.1, 3.6, 1.3 Hz, 1H, βꢀCHF),
105 4.81 (m, 1H, Asp αꢀH), 4.68 (m, 1H, Phe αꢀH), 4.07 (t, J =
6.5 Hz, 1H, Arg αꢀH), 3.86 (ddd, J = 28.9, 14.7, 3.6 Hz, 1H,
CHFCHH), 3.63 (ddd, J = 14.7, 14.7, 8.2 Hz, 1H, CHFCHH),
3.23 (dd, J = 14.1, 5.2 Hz, 1H, Phe βꢀCHH), 3.19 (t, J = 6.8 Hz,
2H, Arg δꢀCH2), 3.06 (dd, J = 14.1, 8.2 Hz, 1H, Phe βꢀCHH),
110 2.92 (dd, J = 17.0, 5.7 Hz, 1H, Asp βꢀCHH), 2.80 (dd, J = 17.0,
8.2 Hz, 1H, Asp βꢀCHH), 1.95 (m, 2H, Arg βꢀCH2), 1.66 (m, 2H,
Arg γꢀCH2); 13C {1H} NMR (75 MHz, D2O) δ 174.9, 174.3,
L
-Arginyl-[(Z)-4-amino-2-fluorobut-2-enoyl]-
L-aspartyl-L-
45 phenylalanine, TFA salt (13)
The title compound was prepared on 25 ꢀmol scale according to
the procedure described in Table 1, entry 1. The product was
obtained as a fluffy white solid (11.2 mg, 67% overall yield based
on initial resin loading); analytical HPLC (0→25%
50 acetonitrile/water (containing 0.1% v/v formic acid) over 30 min)
retention time = 7.3 min, >90% purity; 1H NMR (300 MHz, D2O)
δ 7.31–7.15 (m, 5H, Ph), 5.97 (dt, J = 34.5, 7.0 Hz, 1H, GABA βꢀ
H), 4.70–4.58 (m, 2H, Asp αꢀH + Phe αꢀH), 4.14 (ddd, J = 16.1,
7.0, 1.6 Hz, 1H, GABA γꢀCHH), 4.00–3.92 (m, 2H, GABA γꢀ
55 CHH + Arg αꢀH), 3.19 (dd, J = 14.0, 5.4 Hz, 1H, Phe βꢀCHH),
This journal is © The Royal Society of Chemistry [year]
Journal Name, [year], [vol], 00–00 | 5

Organic & Biomolecular Chemistry
Page 6 of 8
View Online
171.6, 170.3, 168.5 (dd, J = 21.3, 2.9 Hz, CHFCO), 157.1, 136.8,
129.7, 129.1, 127.5, 89.8 (dd, J = 181.3, 20.7 Hz, CHF), 89.7 (dd,
(m, 2H, Arg γꢀCH2); 13C {1H} NMR (75 MHz, D2O) δ 175.5,
174.4, 171.5, 170.3, 168.4 (dd, J = 21.5, 3.6 Hz, CHFCO), 157.1,
J = 193.8, 22.8 Hz, CHF), 54.6, 53.3, 50.2, 40.7, 37.0, 35.4, 28.3, 60 137.1, 129.7, 129.0, 127.4, 90.1 (dd, J = 181.2, 18.0 Hz, CHF),
23.8; 19F NMR (282 MHz, D2O) δ –76.0 (s, 3F, TFA), –203.3 (m,
1F, βꢀCHF), –209.6 (ddd, J = 46.2, 26.8, 10.4 Hz, 1F, αꢀCHF);
19F {1H} NMR (282 MHz, D2O) δ –76.0 (s, 3F, TFA), –203.3 (d,
J = 10.4 Hz, 1F, βꢀCHF), –209.6 (d, J = 10.4 Hz, 1F, αꢀCHF);
89.6 (dd, J = 194.5, 19.0 Hz, CHF), 54.9, 53.3, 50.2, 40.7, 39.5
(dd, J = 22.6, 6.6 Hz, CHFCH2), 37.1, 35.8, 28.3, 23.8; 19F NMR
(282 MHz, D2O) δ –73.5 (s, 3F, TFA), –201.1 (m, 1F, βꢀCHF),
–207.0 (ddd, J = 46.1, 26.9, 10.7 Hz, 1F, αꢀCHF); 19F NMR
5
MS (ESI, +ve) m/z 558 (MH+, 100%); HRMS (ESI, +ve) 65 (282 MHz, D2O) δ –73.5 (s, 3F, TFA), –201.1 (d, J = 10.7 Hz,
C23H34F2N7O7+ requires m/z 558.2482, found 558.2481.
1F, βꢀCHF), –207.0 (d, J = 10.7 Hz, 1F, αꢀCHF); MS (ESI, +ve)
+
m/z 558 (MH+, 100%); HRMS (ESI, +ve) C23H34F2N7O7
10
L
L
-Arginyl-[(2S,3R)-4-amino-2,3-difluorobutanoyl]-L-aspartyl-
-phenylalanine, TFA salt (16)
requires m/z 558.2482, found 558.2491.
L
-Argininyl-[(S)-3-amino-2-fluoropropanoyl]-
L-aspartyl-L-
The title compound was prepared on 25 ꢀmol scale according to
General Procedures B–F, employing a double coupling of Fmocꢀ
arginine. The product was obtained as a sticky white solid
15 (6.0 mg, 36% overall yield based on initial resin loading);
analytical HPLC (0→100% acetonitrile/water (containing 0.1%
v/v formic acid) over 10 min) retention time = 7.3 min, >90%
70 phenylalanine, TFA salt (18)
The title compound was prepared on 50 ꢀmol scale according to
General Procedures B–F, employing a double coupling of Fmocꢀ
arginine. The product was obtained as a sticky white solid
(22.9 mg, 72% overall yield based on initial resin loading);
purity; [α]D +8.5 (c 0.20, H2O); IR (neat) vmax (cm–1) 3358, 2959, 75 analytical HPLC (0→100% acetonitrile/water (containing 0.1%
2926, 2874, 1684, 1527, 1464, 1407, 1205, 1139; 1H NMR
20 (300 MHz, D2O, COSY) δ 7.41–7.26 (m, 5H, Ph), 5.29 (ddd, J =
47.2, 18.8, 2.3 Hz, 1H, αꢀCHF), 5.02 (ddddd, J = 47.2, 22.1, 8.5,
3.0, 2.3 Hz, 1H, βꢀCHF), 4.82 (m, 1H, Asp αꢀH), 4.65 (m, 1H,
v/v formic acid) over 10 min) retention time = 7.3 min, >95%
purity; [α]D +4.0 (c 0.33, H2O); IR (neat) vmax (cm–1) 3274, 2926,
1
2855, 1655, 1534, 1431, 1400, 1186, 1133; H NMR (300 MHz,
D2O, COSY) δ 7.42–7.27 (m, 5H, Ph), 5.09 (dt, J = 47.5, 4.6 Hz,
Phe αꢀH), 4.04 (t, J = 6.1 Hz, 1H, Arg αꢀH), 3.73 (ddd, J = 17.3, 80 1H, CHF), 4.76–4.64 (m, 2H, Asp αꢀH + Phe αꢀH), 4.00 (t, J =
14.9, 8.5 Hz, 1H, CHFCHH), 3.52 (ddd, J = 30.5, 14.9, 3.0 Hz,
25 1H, CHFCHH), 3.25 (dd, J = 14.0, 5.2 Hz, 1H, Phe βꢀCHH), 3.20
(t, J = 6.7 Hz, 2H, Arg δꢀCH2), 3.06 (dd, J = 14.0, 8.5 Hz, 1H,
Phe βꢀCHH), 2.92 (dd, J = 17.1, 5.6 Hz, 1H, Asp βꢀCHH), 2.80
6.4 Hz, 1H, Arg αꢀH), 3.71 (dd, J = 24.8, 4.3 Hz, 2H, CHFCH2),
3.27 (dd, J = 14.0, 5.1 Hz, 1H, Phe βꢀCHH), 3.21 (m, 2H, Arg δꢀ
CH2), 3.06 (dd, J = 14.0, 8.9 Hz, 1H, Phe βꢀCHH), 2.90 (dd, J =
17.0, 5.8 Hz, 1H, Asp βꢀCHH), 2.78 (dd, J = 17.0, 7.9 Hz, 1H,
(dd, J = 17.1, 8.4 Hz, 1H, Asp βꢀCHH), 1.92 (m, 2H, Arg βꢀ 85 Asp βꢀCHH), 1.89 (m, 2H, Arg βꢀCH2), 1.64 (m, 2H, Arg γꢀ
CH2), 1.64 (m, 2H, Arg γꢀCH2); 13C {1H} NMR (75 MHz, D2O) δ
30 175.5, 174.5, 171.6, 170.1, 167.8 (m, CHFCO), 157.1, 137.1,
129.7, 129.1, 127.5, 55.1, 53.3, 50.3, 40.7, 40.5 (dd, J = 9.1,
1.9 Hz, CHFCH2), 37.1, 35.7, 28.3, 23.8 [2 × CHF signals
CH2); 13C {1H} NMR (75 MHz, D2O) δ 175.2, 174.3, 171.9,
170.2, 169.8 (d, J = 21.8 Hz, CHFCO), 157.2, 137.0, 129.7,
129.1, 127.5, 89.3 (d, J = 186.9 Hz, CHF), 54.7, 53.4, 50.1, 41.3
(d, J = 21.1 Hz, CHFCH2), 40.7, 36.9, 35.6, 28.3, 23.9; 19F NMR
overlapping or obscured]; 19F NMR (282 MHz, D2O) δ –76.0 (s, 90 (282 MHz, D2O) δ –76.0 (s, 3F, TFA), –195.9 (dt, J = 48.7,
3F, TFA), –198.2 (m, 1F, βꢀCHF), –201.7 (m, 1F, αꢀCHF);
35 19F NMR (282 MHz, D2O) δ –76.0 (s, 3F, TFA), –198.2 (d, J =
12.5 Hz, 1F, βꢀCHF), –201.7 (d, J = 12.5 Hz, 1F, αꢀCHF); MS
(ESI, +ve) m/z 558 (MH+, 100%); HRMS (ESI, +ve)
C23H34F2N7O7+ requires m/z 558.2482, found 558.2474.
25.0 Hz, 1F, CHF); 19F {1H} NMR (282 MHz, D2O) δ –76.0 (s,
3F, TFA), –195.9 (s, 1F, CHF); MS (ESI, +ve) m/z 526 (MH+,
+
100%); HRMS (ESI, +ve) C22H33FN7O7 requires m/z 526.2420,
found 526.2424.
95
L
-Argininyl-[(R)-3-amino-2-fluoropropanoyl]-
L-aspartyl-L-
phenylalanine, TFA salt (19)
L
L
-Arginyl-[(2R,3R)-4-amino-2,3-difluorobutanoyl]-L-aspartyl-
-phenylalanine, TFA salt (17)
40
The title compound was prepared on 50 ꢀmol scale according to
General Procedures B–F, employing a double coupling of Fmocꢀ
arginine. The product was obtained as a sticky white solid
The title compound was prepared on 25 ꢀmol scale according to
General Procedures B–F, employing a double coupling of Fmocꢀ
arginine. The product was obtained as a sticky white solid 100 (17.3 mg, 54% overall yield based on initial resin loading);
(7.9 mg, 47% overall yield based on initial resin loading);
45 analytical HPLC (0→100% acetonitrile/water (containing 0.1%
analytical HPLC (0→100% acetonitrile/water (containing 0.1%
v/v formic acid) over 10 min) retention time = 7.3 min, >95%
purity; [α]D –16.2 (c 0.26, H2O); IR (neat) vmax (cm–1) 3215,
v/v formic acid) over 10 min) retention time = 7.2 min, >95%
purity; [α]D –13.3 (c 0.26, H2O); IR (neat) vmax (cm–1) 3206,
2926, 2855, 1655, 1533, 1429, 1186, 1134; H NMR (300 MHz,
1
1
1651, 1556, 1531, 1415, 1178, 1129; H NMR (300 MHz, D2O, 105 D2O, COSY) δ 7.42–7.27 (m, 5H, Ph), 5.10 (dt, J = 47.1, 4.1 Hz,
COSY) δ 7.41–7.25 (m, 5H, Ph), 5.19 (ddd, J = 46.2, 29.3,
50 1.3 Hz, 1H, αꢀCHF), 5.03 (ddddd, J = 46.5, 26.9, 8.5, 3.4, 1.3 Hz,
1H, βꢀCHF), 4.81 (m, 1H, Asp αꢀH), 4.68 (m, 1H, Phe αꢀH), 4.06
(t, J = 6.4 Hz, 1H, Arg αꢀH), 3.87 (ddd, J = 15.2, 14.7, 8.5 Hz,
1H, CHF), 4.76–4.70 (m, 2H, Asp αꢀH + Phe αꢀH), 4.02 (t, J =
6.4 Hz, 1H, Arg αꢀH), 3.83–3.72 (m, 2H, CHFCH2), 3.30 (dd, J =
14.1, 5.2 Hz, 1H, Phe βꢀCHH), 3.23 (m, 2H, Arg δꢀCH2), 3.06
(dd, J = 14.1, 9.0 Hz, 1H, Phe βꢀCHH), 2.88 (dd, J = 17.2,
1H, CHFCHH), 3.65 (ddd, J = 29.8, 14.7, 3.4 Hz, 1H, 110 5.1 Hz, 1H, Asp βꢀCHH), 2.75 (dd, J = 17.2, 8.8 Hz, 1H, Asp βꢀ
CHFCHH), 3.27 (dd, J = 14.2, 5.4 Hz, 1H, Phe βꢀCHH), 3.22 (t,
55 J = 6.8 Hz, 2H, Arg δꢀCH2), 3.05 (dd, J = 14.0, 8.6 Hz, 1H, Phe
βꢀCHH), 2.91 (dd, J = 16.9, 5.4 Hz, 1H, Asp βꢀCHH), 2.76 (dd, J
= 16.9, 8.5 Hz, 1H, Asp βꢀCHH), 1.95 (m, 2H, Arg βꢀCH2), 1.66
CHH), 1.92 (m, 2H, Arg βꢀCH2), 1.65 (m, 2H, Arg γꢀCH2);
13C {1H} NMR (50 MHz, D2O) δ 175.0, 174.3, 171.8, 170.2,
170.0 (d, J = 22.0 Hz, CHFCO), 157.2, 137.0, 129.7, 129.1,
127.5, 89.4 (d, J = 188.7 Hz, CHF), 54.4, 53.3, 50.0, 40.7, 40.6
6
| Journal Name, [year], [vol], 00–00
This journal is © The Royal Society of Chemistry [year]

Page 7 of 8
Organic & Biomolecular Chemistry
View Online
(d, J = 31.0 Hz, CHFCH2), 36.8, 35.5, 28.3, 23.9; 19F NMR
(282 MHz, D2O) δ –76.0 (s, 3F, TFA), –196.4 (ddd, J = 47.5,
26.4, 23.7 Hz, 1F, CHF); 19F {1H} NMR (282 MHz, D2O) δ
5H, Arg δꢀCH2 + GABA γꢀCH2 + Phe βꢀCHH), 3.03 (dd, J =
14.7, 9.4 Hz, 1H, Phe βꢀCHH), 2.81 (dd, J = 16.8, 5.4 Hz, 1H,
Asp βꢀCHH), 2.68 (dd, J = 16.8, 8.3 Hz, Asp βꢀCHH), 2.24 (t, J
–76.0 (s, 3F, TFA), –196.4 (s, 1F, CHF); MS (ESI, +ve) m/z 526 60 = 7.3 Hz, 2H, GABA αꢀCH2), 1.91 (m, 2H, Arg βꢀCH2), 1.74 (m,
+
5
(MH+, 100%); HRMS (ESI, +ve) C22H33FN7O7 requires m/z
526.2420, found 526.2423.
2H, GABA βꢀCH2), 1.63 (m, 2H, Arg γꢀCH2); 13C {1H} NMR
(75 MHz, D2O) δ 178.4, 177.4, 176.9, 174.9, 172.2, 159.7, 139.3,
132.2, 131.6, 130.1, 56.8, 55.9, 52.8, 43.2, 41.7, 39.4, 38.2, 35.6,
30.9, 27.6, 26.5; MS (ESI, +ve) m/z 522 (MH+, 100%); HRMS
65 (ESI, +ve) C23H35N7O7+ requires m/z 22.2671, found 522.2675.
L
-Argininyl-glycinyl-
L-aspartyl-L-phenylalanine, TFA salt
(20)
The title compound was prepared on 0.1 mmol scale according to
Cell adhesion assay
10 General Procedures B–D & F. The product was obtained as a
sticky white solid (0.053 g, 92% overall yield based on initial
resin loading); analytical HPLC retention time = 7.0 min
(0→25% acetonitrile/water (containing 0.1% v/v formic acid)
over 30 min), >95% purity; [α]D –4.7 (c 0.53, H2O); IR (neat)
Reagents and methods were utilised mostly as described.20 The
following cell lines were used, with ligands in brackets: K562ꢀ
α5β1
(Fibronectin),
K562ꢀαvβ3
(LAPꢀβ1),
K562ꢀαvβ5
70 (Vitronectin), K562ꢀαvβ6 (LAPꢀβ1). The divalent cation used to
facilitate adhesion was 2 mM MgCl2. Adhesion was quantified by
cell labelling with the fluorescent dye BCECFꢀAM (Life
Technologies), where cell suspensions at 6×106 cells/mL were
incubated with 0.66 ꢀL/mL of 30 mM BCECFꢀAM at 37 °C for
75 10 minutes, before dispensing into the assay plate. At the assay
conclusion cells that adhered were lysed using 50 ꢁl/well of 0.5%
Triton Xꢀ100 in H2O to release fluorescence. Fluorescence
intensity was detected using an Envision® plate reader (Perkin
Elmer). For active antagonists in the assay, data were fitted to a
80 4 parameter logistic equation for IC50 determinations.
15 vmax (cm–1) 3203, 1645, 1531, 1416, 1026; H NMR (300 MHz,
1
D2O, COSY) δ 7.40–7.26 (m, 5H, Ph), 4.71–4.60 (m, 2H, Asp αꢀ
H + Phe αꢀH), 4.11 (t, J = 6.4 Hz, 1H, Arg αꢀH), 4.04 (d, J =
16.7 Hz, 1H, Gly αꢀCHH), 3.92 (d, J = 16.7 Hz, 1H, Gly αꢀ
CHH), 3.28–3.20 (m, 3H, Arg δꢀCH2 + Phe βꢀCHH), 3.04 (dd, J
20 = 13.8, 8.3 Hz, 1H, Phe βꢀCHH), 2.82 (dd, J = 16.9, 5.5 Hz, 1H,
Asp βꢀCHH), 2.69 (dd, J = 16.9, 8.1 Hz, 1H, Asp βꢀCHH), 1.97
(m, 2H, Arg βꢀCH2), 1.72 (m, 2H, Arg γꢀCH2); 13C {1H} NMR
(75 MHz, D2O) δ 175.7, 174.7, 172.0, 170.9, 170.5, 157.2, 137.2,
129.7, 129.1, 127.5, 55.1, 53.2, 50.6, 42.7, 40.8, 37.2, 36.0, 28.3,
25 23.8; MS (ESI, +ve) m/z 494 (MH+, 100%); HRMS (ESI, +ve)
C21H32N7O7+ requires m/z 494.2358, found 494.2360.
Acknowledgements
L
-Arginyl-β-alanyl-L-aspartyl-L-phenylalanine, TFA salt (21)
This work was funded by a University of Sydney Postdoctoral
Research Fellowship and a UNSW startꢀup grant awarded to LH.
The authors thank Ms Irene AravadinosꢀBelerhas for the
85 synthesis of nonꢀfluorinated control peptides.
The title compound was prepared on 0.1 mmol scale according to
General Procedures B–D & F. The product was obtained as a
30 sticky white solid (0.053 g, 94% overall yield based on initial
resin loading); analytical HPLC (0→25% acetonitrile/water
(containing 0.1% v/v formic acid) over 30 min) retention time =
11.2 min, >95% purity; [α]D –5.6 (c 0.53, H2O); IR (neat) vmax
Notes and references
a School of Chemistry, The University of New South Wales, Sydney NSW
2052, Australia. Fax: +612 9385 6141; Tel: +612 9385 4474; E-mail:
l.hunter@unsw.edu.au
(cm–1) 3207, 1651, 1531, 1416, 1183, 1044; H NMR (400 MHz,
1
35 D2O, COSY) δ 7.29–7.14 (m, 5H, Ph), 4.57–4.52 (m, 2H, Asp αꢀ
H + Phe αꢀH), 3.85 (t, J = 6.2 Hz, 1H, Arg αꢀH), 3.38 (m, 2H, βꢀ
Ala βꢀCH2), 3.15 (dd, J = 13.9, 4.8 Hz, 1H, Phe βꢀCHH), 3.09 (t,
J = 6.2 Hz, 2H, Arg δꢀCH2), 2.94 (dd, J = 13.9, 8.7 Hz, 1H, Phe
βꢀCHH), 2.71 (dd, J = 17.0, 5.0 Hz, 1H, Asp βꢀCHH), 2.59 (dd, J
40 = 17.0, 8.5 Hz, Asp βꢀCHH), 2.38 (t, J = 5.8 Hz, 2H, Gly αꢀCH2),
1.78 (m, 2H, Arg βꢀCH2), 1.51 (m, 2H, Arg γꢀCH2);
13C {1H} NMR (100 MHz, D2O) δ 175.1, 174.1, 173.4, 171.9,
169.3, 156.6, 136.5, 129.2, 128.6, 127.0, 54.3, 52.7, 50.0, 40.2,
36.6, 35.8, 35.3, 34.5, 27.8, 23.4; MS (ESI, +ve) m/z 508 (MH+,
90 b School of Chemistry, The University of Sydney, NSW 2006, Australia.
c Department of Screening and Compound Profiling, GlaxoSmithKline
Research & Development, Stevenage SG1 2NY, United Kingdom.
† Electronic Supplementary Information (ESI) available: NMR spectra,
LCMS traces and cell adhesion assay data. See DOI: 10.1039/b000000x/
95
1
2
3
4
5
6
7
8
(a) B. C. Buer and E. N. G. Marsh, Protein Sci., 2012, 21, 453–462;
(b) C. Jäckel and B. Koksch, Eur. J. Org. Chem., 2005, 4483–4503.
S. K. Holmgren, K. M. Taylor, L. E. Bretscher and R. T. Raines,
Nature, 1998, 392, 666–667.
S. K. Holmgren, L. E. Bretscher, K. M. Taylor and R. T. Raines,
Chem. Biol., 1999, 6, 63–70.
J. L. KitevskiꢀLeBlanc and R. S. Prosser, Prog. Nucl. Magn. Reson.
Spectrosc., 2012, 62, 1–33.
M. Schüler, D. O’Hagan and A. M. Z. Slawin, Chem. Commun.,
2005, 4324–4326.
(a) D. O’Hagan, Chem. Soc. Rev., 2008, 37, 308–319; (b) L. Hunter,
Beilstein J. Org. Chem., 2010, 6, No. 38, doi:10.3762/bjoc.6.38.
R. I. Mathad, F. Gessier, D. Seebach and B. Jaun, Helv. Chim. Acta,
2005, 88, 266–280.
(a) L. Hunter, K. A. Jolliffe, M. J. T. Jordan, P. Jensen and R. B.
Macquart, Chem. Eur. J., 2011, 17, 2340–2343; (b) Z. Wang and L.
Hunter, J. Fluorine Chem., 2012, in press.
(a) I. Yamamoto, M. J. T. Jordan, N. Gavande, M. R. Doddareddy,
M. Chebib and L. Hunter, Chem. Commun., 2012, 48, 829–831; (b)
L. Hunter, Chim. Oggi, 2012, 30, 20–22.
100
105
110
115
+
45 100%); HRMS (ESI, +ve) C22H34N7O7 requires m/z 508.2514,
found 508.2513.
L
-Arginyl-GABA-L-aspartyl-L-phenylalanine, TFA salt (22)
The title compound was prepared on 0.1 mmol scale according to
General Procedures B–D & F. The product was obtained as a
50 sticky white solid (0.059 g, 94% overall yield based on initial
resin loading); analytical HPLC (0→25% acetonitrile/water
(containing 0.1% v/v formic acid) over 30 min) retention time =
11.0 min, >95% purity; [α]D –7.9 (c 0.59, H2O); IR (neat) vmax
(cm–1) 3212, 1651, 1556, 1416, 1180, 1128; H NMR (300 MHz,
1
9
55 D2O, COSY) δ 7.38–7.23 (m, 5H, Ph), 4.70–4.63 (m, 2H, Asp αꢀ
H + Phe αꢀH), 3.98 (t, J = 6.4 Hz, 1H, Arg αꢀH), 3.27–3.14 (m,
This journal is © The Royal Society of Chemistry [year]
Journal Name, [year], [vol], 00–00 | 7

Organic & Biomolecular Chemistry
Page 8 of 8
View Online
10 (a) O. M. Rennert and H. S. Anker, Biochemistry, 1963, 2, 471–476;
(b) Y. Tang, G. Ghirlanda, W. A. Petka, T. Nakajima, W. F. DeGrado
and D. A. Tirrell, Angew. Chem. Int. Ed., 2001, 40, 1494–1496.
11 R. I. Mathad, B. Jaun, O. Flögel, J. Gardiner, M. Löweneck, J. D. C.
Codée, P. H. Seeberger and D. Seebach, Helv. Chim. Acta, 2007, 90,
2251–2273.
5
12 W. C. Chan and P. D. White, Fmoc Solid Phase Peptide Synthesis: A
Practical Approach, 2000, Oxford.
13 J. W. Perich, Int. J. Peptide Protein Res., 1994, 44, 288–294.
10 14 See Electronic Supplementary Information for full details.
15 W.R. Dolbier, Guide to Fluorine NMR for Organic Chemists, 2009,
Wiley.
16 (a) R. O. Hynes, Cell, 2002, 110, 673–687; (b) Y. Takada, X. Ye and
S. Simon, Genome Biol., 2007, 8, No. 215, doi:10.1186/gbꢀ2007ꢀ8ꢀ5ꢀ
15
20
25
30
35
215.
17 (a) D. Heckmann and H. Kessler, Methods Enzymol., 2007, 426, 463–
503; (b) G. Casiraghi, G. Rassu, L. Auzzas, P. Burreddu, E. Gaetani,
L. Battistini, F. Zanardi, C. Curti, G. Nicastro, L. Belvisi, I. Motto,
M. Castorina, G. Giannini and C. Pisano, J. Med. Chem., 2005, 48,
7675–7687.
18 R. Rathinam and S. K. Alahari, Cancer Metastasis Rev., 2010, 29,
223–237.
19 M. A. Dechantsreiter, E. Planker, B. Mathä, E. Lohof, G. Hölzemann,
A. Jonczyk, S. L. Goodman and H. Kessler, J. Med. Chem., 1999, 42,
3033–3040.
20 S. B. Ludbrook, S. T. Barry, C. J. Delves and C. M. T. Horgan,
Biochem. J., 2003, 369, 311–318.
21 βꢀAmino acids have previously been shown to be tolerated in place
of glycine in bioactive RGD peptide analogues; see P. E. Thompson,
D. L. Steer, M. I. Aguilar and M. T. W. Hearn, Bioorg. Med. Chem.
Lett., 1998, 8, 2699–2704.
22 D. Gani, P. B. Hitchcock and D. W. Young, J. Chem. Soc., Perkin 1,
1985, 1363–1372.
8
| Journal Name, [year], [vol], 00–00
This journal is © The Royal Society of Chemistry [year]