An Allyl Protection and Improved Purification Strategy Enables the Synthesis of Functionalized Phosphonamidate Peptides

Article abstract of DOI:10.1055/s-0036-1588393

For modern biophysical methods such as isothermal titration calorimetry, high purity of the inhibitor of interest is indispensable. Herein, we describe a procedure for the synthesis and purification of functionalized phosphonamidate peptides that is able to generate inhibitors for the metalloprotease thermolysin for use in biophysical experiments. The method utilizes an allyl ester/alloc protection strategy and takes advantage of a fast and effective solid-phase extraction (SPE) purification step. Applying this strategy, we were able to synthesize a series of highly polar inhibitors featuring amino- and hydroxy-functionalized side chains in excellent purity.

Full text of DOI:10.1055/s-0036-1588393

SYNTHESIS
0
0
3
9
-
7
8
8
1
1
4
3
7
-
2
1
0
X
© Georg Thieme Verlag Stuttgart · New York
2017, 49, A–J
paper
A
J. Cramer, G. Klebe
Paper
Synthesis
An Allyl Protection and Improved Purification Strategy Enables
the Synthesis of Functionalized Phosphonamidate Peptides
Jonathan Cramer
Gerhard Klebe*
Universität Marburg, Institut für Pharmazeutische Chemie,
Marbacher Weg 6, 35032 Marburg, Germany
klebe@staff.uni-marburg.de
Received: 04.11.2016
Accepted after revision: 13.12.2016
Published online: 12.01.2017
works on the surface of the protein–ligand complex and
their influence on binding thermodynamics.12 In this con-
text, a high purity of the inhibitors used for the measure-
ments is of vital importance. In isothermal titration calo-
rimetry experiments, the enthalpic term is strongly depen-
dent on the applied inhibitor concentration. Inaccuracies in
the latter property can easily lead to false data interpreta-
tion when studied samples are not sufficiently pure.13
Herein, we report a modified strategy for the synthesis
and purification of Cbz-protected phosphonamidates (Fig-
ure 1), utilizing allyl ester/alloc protection and an efficient
solid-phase extraction (SPE) protocol that allows the facile
introduction of a variety of functional groups at the P1′-
and P2′-positions of a peptidomimetic inhibitor. For a pro-
spective biophysical study, we envisaged the introduction
of polar functional groups to the basic scaffold of thermoly-
sin inhibitors, while simultaneously adhering to the N-ter-
minal Cbz-protecting group, which has to be seen as an in-
tegral part of the inhibitor scaffold. The most commonly
employed strategy in the synthesis of phosphonamidate
peptides utilizes alkyl or benzyl esters as protecting groups
for the phosphonamidate and C-terminal carboxylic acid
functions. The deprotection is consequently accomplished
by hydrolytic cleavage,12b treatment with TMSBr,14 NaI11a or
hydrogenolysis,4 respectively (Scheme 1). These strategies
combine the advantage of mild reaction conditions with
easily removable reagents, thus avoiding the formation of
major side products. For the solid-phase synthesis of aryl
phosphonamidate peptides by a Staudinger reaction, the
DOI: 10.1055/s-0036-1588393; Art ID: ss-2016-t0779-op
Abstract For modern biophysical methods such as isothermal titration
calorimetry, high purity of the inhibitor of interest is indispensable.
Herein, we describe a procedure for the synthesis and purification of
functionalized phosphonamidate peptides that is able to generate in-
hibitors for the metalloprotease thermolysin for use in biophysical ex-
periments. The method utilizes an allyl ester/alloc protection strategy
and takes advantage of a fast and effective solid-phase extraction (SPE)
purification step. Applying this strategy, we were able to synthesize a
series of highly polar inhibitors featuring amino- and hydroxy-function-
alized side chains in excellent purity.
Key words thermolysin, metalloprotease, inhibitor synthesis, phos-
phonamidate, solid-phase extraction, allyl/alloc protection
The introduction of phosphonamidate moieties in pep-
tidic structures has repeatedly yielded highly potent transi-
tion-state analogous inhibitors for a broad range of pro-
teases such as thermolysin (TLN),1 HIV protease,2 carboxy-
peptidase A,3 angiotensin-converting enzyme,4 leucine
aminopeptidase,5 endothelin-converting enzyme6 and hu-
man collagenase.7 In the form of phosphonamidate ester
conjugates, the phosphonamidate group has also been in-
corporated in haptens designed for the generation of cata-
lytic antibodies8 and utilized for the bio-orthogonal modifi-
cation of proteins.9 This scope of applications highlights the
applicability of phosphonamidates in biochemical and bio-
medical research. While phosphonamidate esters are suffi-
ciently stable under physiological conditions,9,10 the inher-
ent instability of free phosphonamidates has been deemed
as a major disadvantage and has prevented the broad appli-
cation of these compounds.11
O
P
R1
O
H
H
N
N
Cbz
N
OH
HO
H
O
R2
In our group, P(V)-based peptidomimetic inhibitors of
thermolysin have proven to be valuable tool compounds to
study the formation of contiguously connected water net-
Figure 1 Basic scaffold of phosphonamidate-type inhibitors of ther-
molysin
© Georg Thieme Verlag Stuttgart · New York — Synthesis 2017, 49, A–J

B
J. Cramer, G. Klebe
Paper
Synthesis
necessity of protecting groups can even be eliminated.15
The purification of the delicate deprotected phosphonami-
dates is usually achieved by employing ion exchange chro-
matography or preparative RP-HPLC. However, in various
cases, the samples are subsequently used in biochemical as-
says without any final purification step.5
The synthesis of phosphonic acid diallyl ester 2 as the
precursor of the basic building block for the TLN inhibitor
series was accomplished by a one-pot reaction of benzyl
carbamate (1) and paraformaldehyde in glacial acetic acid
followed by an Arbuzov reaction with triallyl phosphite.
The resulting diallyl ester 2 was then selectively trans-
previous work
O
H
a
N
P
Cbz
OH
OAll
OR1
OH
R3
R3
3
O
O
TMSBr,
O
NH
R2
O
NH
R2
LiOH, NaI
or H2, Pd/C
O
P
O
P
O
P
H
H
H
c
N
N
N
Cbz
N
PG
N
H
PG
N
H
AllO
H
O
HO
R1
R
O
H
Boc
N
R1 = alkyl, benzyl
N
H
OAll
O
R
b
or
this work:
R
H
N
Boc
N
R
H
OAll
OLi
O
R
R
O
O
Pd(PPh3)4,
HNEt2,
then LiOH
O
NH
X
O
NH
X
O
O
H
O
P
H
H
N
N
P
N
H
N
H
OAll
Cbz
N
Cbz
N
N
AllO
H
LiO
H
Alloc
O
n
n
NH
Alloc
O
Alloc
X = O, NH
X = OH, NH2
n = 2 (4, 65%), 3 (5, 89%),
4 (6, 81%)
n = 1 (7, 72%), 2 (8, 56%),
3 (9, 92%), 4 (10, 67%)
Scheme 1 Synthetic strategies for the deprotection of phosphonami-
date protease inhibitors
O
H
Alloc
N
N
OAll
Due to the incompatibility of the desired carboxybenzyl
phosphonopeptides to hydrogenolytic side-chain deprotec-
tion and the unsatisfactory range of hydrolytically labile
protecting groups for functional groups such as amines, we
investigated different strategies as alternatives to estab-
lished protocols. Taking into consideration the orthogonali-
ty and ease of purification after deprotection, an allyl es-
ter/alloc strategy was chosen for side-chain and backbone
protection. Through the use of diethylamine as a nucleo-
philic scavenger in the palladium-catalyzed deallylation re-
action, all reagents and side products, except for the cata-
lyst, are volatile and can be easily removed under reduced
pressure. The considerable difference in polarity between
the palladium catalyst and the deprotected phosphonopep-
tides allows for facile separation of the two species by liq-
uid–liquid extraction or reversed-phase chromatography.
N
O
O
12, 97%
11, 96%
O
O
H
H
N
N
OAll
OAll
O
O
13, 94%
14, 86%
Alloc
Alloc
HN
O
O
O
H
H
N
N
OAll
OAll
O
O
15, 92%
16, 65%
O
P
O
P
H
H
a, b
c
N
Cbz NH2
N
O
Cbz
OH
Cbz
OAll
OAll
OAll
O
1
2
3
OAll
O
Scheme 2 Reagents and conditions: (a) (CHO)n, AcOH, 50 °C, 4 h; (b)
P(OAll)3, neat, 110 °C, overnight (64%, two steps); (c) NaOH, H2O, 1 h,
81%.
17, 74%
Scheme 3 Reagents and conditions: (a) SOCl2, CH2Cl2, 0 °C to r.t., 3 h;
(b) 4 M HCl in dioxane, r.t., 1 h; (c) DIPEA, CH2Cl2, 40 °C, overnight.
© Georg Thieme Verlag Stuttgart · New York — Synthesis 2017, 49, A–J

C
J. Cramer, G. Klebe
Paper
Synthesis
formed into the monoallyl ester 3 by hydrolysis with aque-
ous NaOH (Scheme 2). Phosphonamidate coupling was
achieved by reacting the corresponding phosphonochlori-
date with the free amino group of a peptidic building block
(Scheme 3). The required phosphonochloridate was gener-
ated from 3 by activation with SOCl2. As suggested by
Hirschmann et al., the phosphonochloridate was treated
with DIPEA prior to the addition of the coupling partner to
generate an intermediate phosphonylammonium species
with an increased reactivity toward nucleophiles.16 Deal-
lylation of the protected phosphonamidates was achieved
by a palladium-catalyzed allyl transfer reaction using di-
ethylamine as the nucleophile. This method generated the
fully deprotected inhibitor as a diethylammonium salt. Af-
ter removal of the volatile side products, most hydrophobic
contaminants were separated by a liquid–liquid extraction
step. Analysis of the raw reaction products by qNMR indi-
cated high conversion along with a purity of 70–80%, with
excess diethylamine and catalyst residue as the major con-
taminants.
Table 1 Synthesis of Peptidic Phosphonamidate Inhibitors
1. Pd(PPh3)4, Et2NH, CH2Cl2, r.t., 3 h
2. aqueous workup with LiOH
O
P
O
P
H
N
H
N
Cbz
Cbz
AllO
LiO
Entry
Reactant
Product
Isolated yielda
Purity (qNMR)
O
O
H
H
N
N
18: 73%
19: 63%
20: 66%
18: 95%
19: 97%
20: 99%
N
H
OAll
N
OLi
NH2
1
H
O
O
n
n
NH
Alloc
n = 2 (4), 3 (5), 4 (6)
n = 2 (18), 3 (19), 4 (20)
21: 78%
22: 72%
23: 72%
24: 75%
21: 99%
22: 99%
23: 96%
24: 96%
H
H
H
N
N
N
NH2
n
2
N
Alloc
N
n
H
H
O
O
n = 1 (7), 2 (8), 3 (9), 4 (10)
n = 1 (21), 2 (22), 3 (23), 4 (24)
Alloc
N
NH
3
4
5
6
66%
59%
75%
72%
97%
97%
97%
96%
N
N
N
H
N
H
O
O
11
12
13
25
O
O
O
O
O
H
N
H
N
N
H
OAll
OAll
N
H
OLi
OLi
26
27
O
H
N
H
N
N
H
N
H
O
O
O
O
O
O
H
N
H
N
N
H
OLi
N
H
OAll
28
14
© Georg Thieme Verlag Stuttgart · New York — Synthesis 2017, 49, A–J

D
J. Cramer, G. Klebe
Paper
Synthesis
Entry
Reactant
Product
Isolated yielda
Purity (qNMR)
Alloc
HN
H2N
O
O
O
H
O
N
H
N
H
OLi
OLi
OLi
N
7
79%
96%
N
H
OAll
O
O
O
O
29
15
Alloc
O
HO
O
H
N
H
N
N
H
8
9
77%
77%
97%
97%
N
H
OAll
O
30
16
17
O
O
O
N
H
OAll
N
H
O
31
For compounds containing a primary amino group, the
above procedure led to the formation of non-stoichiometric
mixtures of the inherently zwitterionic substances with di-
ethylamine. Due to the rather close similarity of the pKa val-
ues of diethylamine and the primary amino group, no con-
sistent salt formation could be achieved. Additionally, the
diethylammonium salts proved to be highly hygroscopic.
Incubation of the raw reaction product with aqueous LiOH
solution prior to liquid–liquid extraction led to the forma-
tion of uniform lithium salts with greatly reduced hygro-
scopicity. By using a semi-preparative HPLC setup for the
purification of functionalized phosphonopeptides, we fre-
quently failed to achieve sufficient separation efficiencies,
had trouble with detection and overall poor sample recov-
ery. This might be attributed to the fact that the lability of
the reaction product does not allow for the addition of an
acidic modifier such as TFA to the mobile phase, which, in
consequence, severely deteriorates the peak shape. Addi-
tionally, there are reports that some phosphonamidates
seem to show a tendency for decomposition during chro-
matographic purification, even without the addition of
acidic modifiers.17 Regularly, we also observed hydrolysis in
preparative HPLC runs. As an alternative purification meth-
od, we investigated the use of solid-phase extraction (SPE).
While predominantly used as a technique for sample
preparation in analytical setups,18 SPE can be seen as a
small-scale chromatography system that retains high sam-
ple concentration (narrow bands) and reduces exposure of
the analyte to mobile and stationary phases to a minimum.
While the separation efficiency is comparatively low, this
technique has been successfully used for preparative purifi-
cation of synthetic peptides, modified sugars and anthocy-
anidin-glucosides from plant material.19
SPE cartridges with various phases and surface modifi-
cations are readily available from commercial suppliers, a
number of which were tested for the purification of phos-
phonamidate peptides. The eluted fractions were analyzed
by HPLC. Amino- and cyano-modified silica gel phases and
polymeric DPA-6S resin, however, did not show any reten-
tion of the desired compounds or contaminants in re-
versed-phase mode. The use of a zwitterionic ammonium–
sulfonic acid phase in HILIC mode was also investigated.
This technique enabled the efficient separation of the de-
sired products from apolar reaction side products. Unfortu-
nately, direct application of alkaline samples to the column
led to hydrolysis of the surface modification. When the
sample pH was adjusted, separation of the ionic product
from buffer substances proved impossible. Efficient purifi-
cation could be achieved with a C18-modified silica gel
phase. Here, the contaminants showed a strong retention,
while the desired phosphonopeptides could be easily elut-
ed with water or water/acetonitrile mixtures. By using
these SPE phases, we were able to obtain the desired inhib-
itors with high purity. Table 1 shows a variety of phospho-
nopeptides prepared following the reported protocol. The
scope of the procedure includes inhibitors featuring polar
and basic functional groups. Additionally, the use of a palla-
dium-catalyzed deprotection reaction grants access to
compounds featuring a hydrolytically labile ester group,
which have been challenging to prepare by traditional
methods.20
In conclusion, the novel SPE protocol enables a fast and
efficient purification of the inhibitors while simultaneously
reducing the amount of compound degradation in aqueous
solvents. The purity of the isolated products is excellent
© Georg Thieme Verlag Stuttgart · New York — Synthesis 2017, 49, A–J

E
J. Cramer, G. Klebe
Paper
Synthesis
and allows their use in biophysical assays such as isother-
mal titration calorimetry, a method that relies heavily on
the accurate knowledge of ligand concentrations.
1H NMR (400 MHz, CDCl3): δ = 3.50–3.70 (m, 2 H), 4.45–4.57 (m, 2 H),
5.10 (s, 2 H), 5.22 (dd, J = 10.4, 0.9 Hz, 1 H), 5.28–5.37 (m, 1 H), 5.82–
5.96 (m, 1 H), 6.85 (br s, 1 H), 7.27–7.38 (m, 5 H).
13C NMR (101 MHz, CDCl3): δ = 66.9, 67.0, 67.5, 118.7, 128.3, 128.4,
128.7, 132.5, 132.5, 136.2, 156.5.
31P NMR (162 MHz, CDCl3): δ = 25.2.
MS (ESI–): m/z [M – H]– calcd for C12H15NO5P: 284.23; found: 284.10.
Melting points were determined with a Krüss KSP1N melting point
meter. 1H, 13C and 31P NMR spectra were recorded on a JEOL ECX-400
or JEOL ECA-500 instrument. All chemical shift values are reported in
ppm relative to the non-deuterated solvent signal. An external stan-
dard was used for 31P NMR spectra (85% H3PO4) and 13C NMR spectra
in D2O (trimethylsilyl propanoic acid). Quantitative NMR spectra
were recorded using recommended instrument parameters for quan-
titative analysis (see the Supporting Information). Relaxation delay
was set to 50–60 s to ensure complete relaxation between pulses. Di-
methyl terephthalate (Sigma-Aldrich, TraceCERT 99.99%) was used as
an internal standard for quantification. All qNMR spectra were re-
corded in deuterated methanol. ESI-MS spectra were recorded on an
Applied Biosystems Q-Trap 2000 system. For high-resolution ESI-MS,
an LTQ-FT Ultra mass spectrometer (Thermo Fischer Scientific) was
used. For HPLC chromatography, a Shimadzu LC-20 system equipped
with a diode array detector was used. Analytic separations were car-
ried out with an MN Nucleodur 100-5 C18 ec 4.6 × 250 mm column
using a water/acetonitrile gradient with the addition of 0.1% TFA.
While these conditions led to hydrolysis of the analyte, the purity of
the sample, regarding other contaminants, can be determined never-
theless. The absence of hydrolytic cleavage products in the sample
was confirmed by 1H and 31P NMR. For SPE purification, Thermosci-
entific HyperSep C18 500 mg 3 mL cartridges were used.
Phosphonamidate Coupling; General Procedure
SOCl2 (4.0 equiv) was dissolved in dry CH2Cl2 under an argon atmo-
sphere and cooled to 0 °C. A solution of benzyl {[(allyloxy)(hy-
droxy)phosphoryl]methyl}carbamate (3) (2.0 equiv) was added to the
solution over 30 min. The cooling bath was removed and the reaction
was stirred at r.t. for 3 h. All volatile components were removed under
reduced pressure. In a second flask, the Boc-protected peptidic build-
ing block (1.0 equiv) (syntheses detailed in the Supporting Informa-
tion) was treated with HCl (4 M in dioxane, 4.0 equiv) at r.t. for 1 h
and all volatile components were subsequently removed under re-
duced pressure. The activated phosphonic acid was dissolved in dry
CH2Cl2. DIPEA (3.0 equiv) and the deprotected peptide were added
consecutively to the solution, after which the mixture was heated to
40 °C and stirred overnight. The solution was diluted with EtOAc and
washed with 5% citric acid (3 × 10 mL), 1 M HCl (10 mL) and 1 M
NaOH (3 × 10 mL). The separated organic phase was washed with
brine and dried over MgSO4. The product was obtained as a mixture
of diastereomers and used in the next step without further purifica-
tion.
Compound 4
Benzyl {[Bis(allyloxy)phosphoryl]methyl}carbamate (2)
Prepared from peptide S3 (178 mg, 0.39 mmol), HCl (0.5 mL, 4 M
solution in dioxane, 2.00 mmol), 3 (222 mg, 0.78 mmol), SOCl2 (186
mg, 1.56 mmol), and DIPEA (151 mg, 1.17 mmol). The product was
obtained as a yellow oil (158 mg, 0.25 mmol, 65%).
1H NMR (400 MHz, CDCl3): δ = 0.84–1.03 (m, 6 H), 1.41–1.54 (m, 1 H),
1.57–1.92 (m, 3 H), 1.99–2.22 (m, 1 H), 2.99–3.16 (m, 1 H), 3.32–3.48
(m, 1 H), 3.51–3.99 (m, 4 H), 4.41–4.76 (m, 6 H), 5.03–5.42 (m, 8 H),
5.75–5.99 (m, 3 H), 7.25–7.40 (m, 5 H).
13C NMR (101 MHz, CDCl3): δ = 21.8, 22.0, 23.0, 23.1, 24.61, 24.64,
32.2, 37.1, 44.0, 50.0, 53.9, 65.6, 65.68, 65.73, 65.8, 66.36, 66.43,
67.37, 67.41, 117.78, 117.83, 118.1, 118.4, 119.3, 119.4, 128.2, 128.32,
128.34, 128.37, 128.65, 128.67, 131.46, 131.52, 132.95, 133.00, 136.3,
136.4, 156.6, 156.7, 171.9, 172.1, 174.28, 174.32.
Benzyl carbamate (1) (756 mg, 5.00 mmol, 1.0 equiv) and paraformal-
dehyde (180 mg, 6.00 mmol, 1.2 equiv) were suspended in AcOH and
heated to 50 °C for 4 h. The solvent was removed under reduced pres-
sure and triallyl phosphite (2.02 g, 10.00 mmol, 2.0 equiv) was added
to the residue. The mixture was heated at 110 °C for 14 h. All volatile
components were removed under reduced pressure and the crude
product was purified by silica gel chromatography (cyclohexane/EtOAc,
1:1). The product was obtained as a colorless oil (1.042 g, 3.20 mmol,
64%).
1H NMR (400 MHz, CDCl3): δ = 3.66 (dd, J = 11.2, 6.0 Hz, 2 H), 4.55 (dd,
J = 7.6, 6.5 Hz, 4 H), 5.05 (s, 1 H), 5.11 (s, 2 H), 5.24 (dd, J = 10.4, 1.1 Hz,
2 H), 5.34 (dd, J = 17.1, 1.2 Hz, 2 H), 5.84–5.99 (m, 2 H), 7.28–7.41 (m,
5 H).
31P NMR (162 MHz, CDCl3): δ = 27.6, 28.2.
13C NMR (101 MHz, CDCl3): δ = 36.2, 37.8, 67.0, 67.1, 67.4, 118.7,
MS (ESI+): m/z [M + H]+ calcd for C29H44N4O9P: 623.66; found: 623.26.
118.7, 128.3, 128.4, 128.7, 132.6, 132.6, 136.2, 156.2.
31P NMR (162 MHz, CDCl3): δ = 24.0.
Compound 5
MS (ESI): m/z [M + H]+ calcd for C15H21NO5P: 326.31; found: 326.08.
Prepared from peptide S8 (235 mg, 0.50 mmol), HCl (0.5 mL, 4 M
solution in dioxane, 2.00 mmol), 3 (285 mg, 1.00 mmol), SOCl2 (238
mg, 2.00 mmol), and DIPEA (194 mg, 1.50 mmol). The product was
obtained as a yellow oil (283 mg, 0.44 mmol, 89%).
1H NMR (400 MHz, CDCl3): δ = 0.81–1.02 (m, 6 H), 1.38–1.95 (m, 7 H),
3.02–3.29 (m, 2 H), 3.41–4.00 (m, 4 H), 4.36–4.68 (m, 6 H), 5.00–5.43
(m, 8 H), 5.79–6.04 (m, 3 H), 7.25–7.43 (m, 6 H).
13C NMR (101 MHz, CDCl3): δ = 21.9, 22.0, 23.0, 23.0, 24.56, 24.59,
25.9, 26.1, 29.0, 38.0, 40.4, 43.88, 43.94, 52.2, 53.8, 65.6, 66.1, 66.2,
67.3, 67.4, 117.8, 118.1, 118.3, 119.1, 119.2, 128.2, 128.3, 128.6,
131.55, 131.58, 132.87, 132.94, 133.0, 133.1, 136.3, 156.7, 156.8,
171.8, 172.1, 173.9, 174.0.
Benzyl {[(Allyloxy)(hydroxy)phosphoryl]methyl}carbamate (3)
An aqueous solution of NaOH (80 mg, 2.00 mmol, 3.5 equiv) was add-
ed to compound 2 (189 mg, 0.58 mmol, 1.0 equiv) and the mixture
was shaken vigorously until the solution cleared. After stirring for 1 h
at r.t., the mixture was washed with EtOAc (3 × 15 mL) and the organ-
ic phase was discarded. The aqueous phase was acidified to pH 1 and
extracted with EtOAc (3 × 15 mL). The combined organic phases were
washed with saturated NaCl solution and dried over MgSO4. The
crude product was washed with hot MTBE to give the product as a
colorless solid (135 mg, 0.47 mmol, 81%); mp 91–92 °C.
© Georg Thieme Verlag Stuttgart · New York — Synthesis 2017, 49, A–J

F
J. Cramer, G. Klebe
Paper
Synthesis
31P NMR (162 MHz, CDCl3): δ = 27.5, 28.4.
MS (ESI+): m/z [M + H]+ calcd for C30H46N4O9P: 637.69; found: 637.51.
13C NMR (101 MHz, CDCl3): δ = 21.8, 22.0, 23.0, 23.1, 24.7, 29.7, 36.2,
36.3, 37.7, 37.8, 39.4, 39.6, 43.8, 53.4, 53.6, 65.5, 65.7, 67.3, 117.8,
118.1, 118.3, 128.3, 128.4, 128.7, 132.8, 133.0, 136.3, 156.9, 174.2,
174.5.
31P NMR (162 MHz, CDCl3): δ = 27.9, 28.2.
MS (ESI): m/z [M + H]+ calcd for C25H40N4O7P: 539.59; found: 539.51.
Compound 6
Prepared from peptide S9 (242 mg, 0.50 mmol), HCl (0.5 mL, 4 M
solution in dioxane, 2.00 mmol), 3 (285 mg, 1.00 mmol), SOCl2 (238
mg, 2.00 mmol), and DIPEA (194 mg, 1.50 mmol). The product was
obtained as a yellow oil (262 mg, 0.40 mmol, 81%).
Compound 10
1H NMR (400 MHz, CDCl3): δ = 0.83–1.06 (m, 6 H), 1.28–2.11 (m, 9 H),
3.02–3.25 (m, 2 H), 3.40–3.96 (m, 4 H), 4.40–4.76 (m, 6 H), 5.02–5.45
(m, 8 H), 5.76–6.03 (m, 3 H), 6.91–7.04 (m, 1 H), 7.26–7.49 (m, 5 H).
13C NMR (101 MHz, CDCl3): δ = 21.8, 22.0, 22.3, 23.0, 23.1, 24.5, 29.3,
31.3, 38.0, 38.2, 39.4, 39.6, 40.2, 40.3, 43.7, 52.2, 52.4, 53.8, 65.6,
66.08, 66.13, 67.3, 117.7, 118.1, 118.4, 119.08, 119.12, 128.2, 128.3,
128.6, 131.6, 132.9, 133.0, 133.1, 136.3, 156.7, 172.0, 174.0.
Prepared from peptide S17 (272 mg, 0.70 mmol), HCl (1.0 mL, 4 M
solution in dioxane, 4.00 mmol), 3 (399 mg, 1.40 mmol), SOCl2 (333
mg, 2.80 mmol), and DIPEA (271 mg, 2.10 mmol). The product was
obtained as a brown solid (258 mg, 0.47 mmol, 67%); mp 112–113 °C.
1H NMR (400 MHz, CDCl3): δ = 0.85–1.05 (m, 6 H), 1.41–1.79 (m, 7 H),
3.11–3.29 (m, 4 H), 3.50–3.93 (m, 3 H), 4.39–4.64 (m, 4 H), 5.01–5.43
(m, 7 H), 5.81–6.05 (m, 2 H), 7.10–7.19 (m, 1 H), 7.29–7.49 (m, 5 H).
31P NMR (162 MHz, CDCl3): δ = 27.6, 28.3.
MS (ESI+): m/z [M + H]+ calcd for C31H48N4O9P: 651.72; found: 651.42.
13C NMR (101 MHz, CDCl3): δ = 21.9, 22.1, 23.0, 23.1, 24.7, 26.5, 26.6,
27.3, 38.0, 38.2, 39.1, 40.6, 43.7, 53.3, 53.6, 65.4, 65.6, 67.3, 117.8,
118.1, 118.3, 128.3, 128.4, 128.7, 132.9, 133.1, 136.3, 156.6, 156.8,
173.8, 174.2.
31P NMR (162 MHz, CDCl3): δ = 27.8, 28.2.
MS (ESI): m/z [M + NH4]+ calcd for C26H45N5O7P: 570.31; found:
Compound 7
Prepared from peptide S11 (197 mg, 0.57 mmol), HCl (0.5 mL, 4 M
solution in dioxane, 2.00 mmol), 3 (328 mg, 1.15 mmol), SOCl2 (271
mg, 2.28 mmol), and DIPEA (221 mg, 1.71 mmol). The product was
obtained as a brown solid (185 mg, 0.36 mmol, 72%); mp 118–119 °C.
570.44.
1H NMR (400 MHz, CDCl3): δ = 0.61–1.01 (m, 6 H), 1.33–1.86 (m, 3 H),
3.40–3.77 (m, 2 H), 3.73–3.98 (m, 1 H), 4.25–4.68 (m, 6 H), 4.94–5.46
(m, 6 H), 5.67–5.95 (m, 2 H), 7.19–7.47 (m, 5 H).
13C NMR (101 MHz, CDCl3): δ = 22.0, 22.1, 22.95, 23.01, 24.6, 24.7,
43.7, 46.4, 53.1, 53.4, 65.2, 65.9, 67.3, 117.9, 118.0, 128.26, 128.33,
128.6, 132.6, 132.9, 133.0, 136.36, 136.41, 156.8, 175.2, 175.4.
Compound 11
Prepared from peptide S19 (192 mg, 0.50 mmol), HCl (0.5 mL, 4 M
solution in dioxane, 2.00 mmol), 3 (285 mg, 1.00 mmol), SOCl2 (238
mg, 2.00 mmol), and DIPEA (194 mg, 1.50 mmol). The product was
obtained as a brown oil (264 mg, 0.48 mmol, 96%).
1H NMR (400 MHz, CDCl3): δ = 0.79–1.04 (m, 6 H), 1.22–1.56 (m, 2 H),
1.72–1.95 (m, 1 H), 3.22–3.81 (m, 10 H), 4.23–4.37 (m, 1 H), 4.40–4.68
(m, 4 H), 5.00–5.42 (m, 6 H), 5.78–6.05 (m, 2 H), 7.19–7.44 (m, 5 H).
13C NMR (101 MHz, CDCl3): δ = 21.4, 21.7, 23.4, 23.5, 24.4, 24.6, 38.3,
39.3, 42.1, 42.2, 43.6, 43.9, 45.3, 49.1, 49.6, 53.5, 65.0, 65.2, 66.5, 67.2,
67.3, 117.9, 118.0, 128.2, 128.3, 128.59, 128.64, 132.7, 132.9, 133.0,
136.4, 154.9, 155.0, 156.4, 172.6, 173.2.
31P NMR (162 MHz, CDCl3): δ = 27.6, 28.1.
MS (ESI+): m/z [M + H]+ calcd for C23H36N4O7P: 511.54; found: 511.48.
Compound 8
Prepared from peptide S15 (179 mg, 0.50 mmol), HCl (0.5 mL, 4 M
solution in dioxane, 2.00 mmol), 3 (285 mg, 1.00 mmol), SOCl2 (238
mg, 2.00 mmol), and DIPEA (194 mg, 1.50 mmol). The product was
obtained as a colorless solid (146 mg, 0.28 mmol, 56%); mp 148–
149 °C.
31P NMR (162 MHz, CDCl3): δ = 27.4, 28.0.
MS (ESI+): m/z [M + NH4]+ calcd for C26H43N5O7P: 568.63; found:
568.44.
1H NMR (400 MHz, CDCl3): δ = 0.82–0.98 (m, 6 H), 1.37–1.51 (m, 1 H),
1.56–1.95 (m, 2 H), 3.22–3.88 (m, 7 H), 4.40–4.60 (m, 4 H), 5.03–5.42
(m, 6 H), 5.64–6.00 (m, 4 H), 6.98–7.11 (m, 1 H), 7.27–7.44 (m, 5 H).
13C NMR (101 MHz, CDCl3): δ = 21.8, 22.1, 23.0, 23.1, 24.7, 38.0, 38.1,
39.86, 39.92, 40.7, 40.9, 43.7, 53.6, 53.8, 65.6, 65.8, 67.4, 67.5, 118.0,
118.3, 118.4, 128.3, 128.4, 128.7, 132.8, 133.0, 136.2, 156.9, 174.6.
Compound 12
Prepared from peptide S21 (164 mg, 0.50 mmol), HCl (0.5 mL, 4 M
solution in dioxane, 2.00 mmol), 3 (285 mg, 1.00 mmol), SOCl2 (238
mg, 2.00 mmol), and DIPEA (194 mg, 1.50 mmol). The product was
obtained as a brown oil (240 mg, 0.48 mmol, 97%).
1H NMR (400 MHz, CDCl3): δ = 0.78–0.98 (m, 6 H), 1.54–1.75 (m, 3 H),
3.32–3.52 (m, 2 H), 3.70–3.97 (m, 2 H), 4.29–4.43 (m, 2 H), 4.47–4.71
(m, 3 H), 4.98–5.45 (m, 6 H), 5.75–6.03 (m, 2 H), 7.23–7.46 (m, 5 H).
31P NMR (162 MHz, CDCl3): δ = 27.8, 28.4.
MS (ESI): m/z [M + H]+ calcd for C24H38N4O7P: 525.56; found: 525.26.
13C NMR (101 MHz, CDCl3): δ = 21.8, 22.8, 24.8, 37.0, 38.4, 40.6, 51.4,
66.1, 66.8, 116.8, 118.8, 128.0, 128.1, 128.5, 131.7, 134.4, 134.5, 136.8,
156.8, 156.9, 166.9, 172.9.
31P NMR (162 MHz, CDCl3): δ = 19.3.
MS (ESI+): m/z [M + NH4]+ calcd for C23H38N4O7P: 513.55; found:
Compound 9
Prepared from peptide S16 (334 mg, 0.90 mmol), HCl (1.0 mL, 4 M
solution in dioxane, 4.00 mmol), 3 (513 mg, 1.80 mmol), SOCl2 (428
mg, 3.60 mmol), and DIPEA (349 mg, 2.70 mmol). The product was
obtained as a yellow solid (446 mg, 0.83 mmol, 92%); mp 100–101 °C.
513.41.
1H NMR (400 MHz, CDCl3): δ = 0.80–1.03 (m, 6 H), 1.39–2.00 (m, 5 H),
3.07–3.32 (m, 4 H), 3.37–3.92 (m, 2 H), 4.39–4.64 (m, 4 H), 5.03–5.46
(m, 6 H), 5.77–5.98 (m, 2 H), 7.28–7.42 (m, 5 H).
© Georg Thieme Verlag Stuttgart · New York — Synthesis 2017, 49, A–J

G
J. Cramer, G. Klebe
Paper
Synthesis
Compound 13
Compound 16
Prepared from peptide S22 (171 mg, 0.50 mmol), HCl (0.5 mL, 4 M
solution in dioxane, 2.00 mmol), 3 (285 mg, 1.00 mmol), SOCl2 (238
mg, 2.00 mmol), and DIPEA (194 mg, 1.50 mmol). The product was
obtained as a brown oil (239 mg, 0.47 mmol, 94%).
Prepared from peptide S26 (221 mg, 0.50 mmol), HCl (0.5 mL, 4 M
solution in dioxane, 2.00 mmol), 3 (285 mg, 1.00 mmol), SOCl2 (238
mg, 2.00 mmol), and DIPEA (194 mg, 1.50 mmol). The product was
obtained as a brown oil (199 mg, 0.33 mmol, 65%).
1H NMR (400 MHz, CDCl3): δ = 0.86–1.04 (m, 6 H), 1.23–1.49 (m, 3 H),
1.53–1.82 (m, 3 H), 3.45–3.81 (m, 2 H), 3.84–4.05 (m, 1 H), 4.44–4.73
(m, 5 H), 5.09 (d, J = 10.8 Hz, 2 H), 5.19–5.47 (m, 4 H), 5.71 (dd, J =
28.8, 23.4 Hz, 1 H), 5.80–6.01 (m, 2 H), 7.28–7.44 (m, 5 H).
13C NMR (101 MHz, CDCl3): δ = 21.4, 21.6, 21.9, 23.0, 25.0, 37.7, 38.1,
39.1, 39.5, 41.09, 41.13, 50.6, 50.9, 51.0, 51.1, 65.25, 65.31, 65.37,
65.43, 66.0, 67.3, 118.2, 118.3, 118.9, 128.2, 128.26, 128.31, 128.6,
131.7, 132.80, 132.85, 132.90, 136.4, 156.7, 156.8, 172.7, 173.0, 173.7,
173.9.
1H NMR (500 MHz, CDCl3): δ = 0.85–1.03 (m, 6 H), 1.57–1.80 (m, 3 H),
3.58–3.79 (m, 2 H), 4.11–4.46 (m, 3 H), 4.49–4.70 (m, 7 H), 5.04–5.18
(m, 2 H), 5.18–5.40 (m, 6 H), 5.81–5.97 (m, 3 H), 7.28–7.41 (m, 5 H).
13C NMR (101 MHz, CDCl3): δ = 21.8, 23.0, 24.9, 40.9, 41.1, 51.2, 51.3,
54.2, 54.2, 65.6, 66.1, 66.2, 67.5, 67.5, 69.0, 118.5, 118.9, 119.0, 119.4,
119.4, 128.3, 128.4, 128.7, 131.4, 131.7, 132.7, 132.7, 136.3, 154.6,
156.8, 169.6, 172.5.
31P NMR (162 MHz, CDCl3): δ = 27.1, 27.7.
MS (ESI+): m/z [M + NH4]+ calcd for C28H44N4O10P: 627.65; found:
31P NMR (162 MHz, CDCl3): δ = 27.4, 27.6.
MS (ESI+): m/z [M + NH4]+ calcd for C24H40N4O7P: 527.58; found:
627.33.
527.43.
Compound 17
Prepared from peptide S28 (172 mg, 0.50 mmol), HCl (0.5 mL, 4 M
solution in dioxane, 2.00 mmol), 3 (285 mg, 1.00 mmol), SOCl2 (238
mg, 2.00 mmol), and DIPEA (194 mg, 1.50 mmol). The product was
obtained as a yellow oil (189 mg, 0.37 mmol, 74%).
1H NMR (400 MHz, CDCl3): δ = 0.86–1.04 (m, 6 H), 1.42–1.64 (m, 4 H),
1.65–1.91 (m, 2 H), 2.92–3.17 (m, 1 H), 3.38–3.85 (m, 2 H), 3.98–4.17
(m, 1 H), 4.39–4.73 (m, 4 H), 5.01–5.39 (m, 7 H), 5.76–6.02 (m, 2 H),
7.28–7.44 (m, 5 H).
13C NMR (101 MHz, CDCl3): δ = 16.87, 16.90, 21.3, 21.5, 23.0, 23.1,
24.5, 24.6, 37.9, 38.1, 39.3, 39.6, 43.17, 43.23, 43.58, 43.64, 52.2, 52.3,
65.0, 65.1, 65.2, 65.3, 66.0, 66.1, 67.20, 67.24, 69.3, 69.4, 117.8, 118.0,
119.03, 119.06, 128.1, 128.2, 128.3, 128.57, 128.59, 131.38, 131.42,
132.9, 133.0, 136.3, 136.4, 156.4, 170.2, 174.0, 174.5.
Compound 14
Prepared from peptide S23 (185 mg, 0.50 mmol), HCl (0.5 mL, 4 M
solution in dioxane, 2.00 mmol), 3 (285 mg, 1.00 mmol), SOCl2 (238
mg, 2.00 mmol), and DIPEA (194 mg, 1.50 mmol). The product was
obtained as a brown oil (231 mg, 0.43 mmol, 86%).
1H NMR (400 MHz, CDCl3): δ = 0.82–1.01 (m, 9 H), 1.01–1.15 (m, 3 H),
1.59–1.83 (m, 3 H), 2.04–2.43 (m, 1 H), 3.48–3.84 (m, 4 H), 4.43–4.69
(m, 4 H), 5.05–5.15 (m, 2 H), 5.17–5.39 (m, 4 H), 5.79–6.01 (m, 2 H),
7.27–7.42 (m, 5 H).
13C NMR (101 MHz, CDCl3): δ = 17.5, 17.6, 19.3, 19.4, 21.6, 21.7, 22.9,
23.0, 24.9, 24.9, 32.1, 32.2, 37.8, 38.1, 39.3, 39.5, 40.9, 41.0, 50.9, 51.1,
60.0, 60.6, 65.2, 65.3, 65.4, 65.97, 66.01, 67.17, 67.23, 117.9, 118.0,
118.85, 118.88, 128.1, 128.2, 128.55, 128.57, 131.7, 132.80, 132.87,
132.90, 133.0, 136.4, 156.55, 156.60, 156.7, 156.8, 172.6, 172.9, 173.0,
173.1.
31P NMR (162 MHz, CDCl3): δ = 26.6, 27.5.
MS (ESI+): m/z [M + Na]+ calcd for C24H35N2NaO8P: 533.51; found:
533.34.
31P NMR (162 MHz, CDCl3): δ = 27.9, 28.3.
MS (ESI+): m/z [M + NH4]+ calcd for C26H44N4O7P: 555.63; found:
555.37.
Phosphonamidate Deprotection; General Procedure
The protected phosphonamidate (1.0 equiv) was dissolved in CH2Cl2
and diethylamine (20.0 equiv) and Pd(PPh3)4 (10 mol%) were consec-
utively added to the solution. After stirring at r.t. for 3 h, all volatile
components were removed under reduced pressure. The residue was
taken up in CH2Cl2 and the solution was washed with H2O containing
LiOH (2.2 equiv) (3 × 2 mL). The combined aqueous phases were ex-
tracted with CH2Cl2 and lyophilized. The crude product was purified
using a reversed-phase SPE cartridge (500 mg, C18). The SPE column
was conditioned with MeCN (1 mL) followed by H2O (5 mL). The sam-
ple was dissolved in H2O (1 mL), slowly passed through the column
and eluted using H2O or H2O/MeCN mixtures. The eluted fractions
were analyzed by HPLC. All fractions containing the desired product
in sufficiently pure form were combined and lyophilized. The purity
of the products was analyzed by quantitative NMR spectroscopy.
Compound 15
Prepared from peptide S24 (221 mg, 0.50 mmol), HCl (0.5 mL, 4 M
solution in dioxane, 2.00 mmol), 3 (285 mg, 1.00 mmol), SOCl2 (238
mg, 2.00 mmol), and DIPEA (194 mg, 1.50 mmol). The product was
obtained as a brown oil (280 mg, 0.46 mmol, 92%).
1H NMR (400 MHz, CDCl3): δ = 0.83–1.01 (m, 6 H), 1.58–1.77 (m, 3 H),
3.37–3.74 (m, 3 H), 4.10 (m, 2 H), 4.37–4.70 (m, 7 H), 5.02–5.37 (m, 8
H), 5.81–5.98 (m, 3 H), 7.27–7.41 (m, 5 H).
13C NMR (101 MHz, CDCl3): δ = 21.6, 23.0, 24.9, 38.1, 39.6, 40.4, 45.2,
45.4, 51.3, 51.3, 54.8, 55.4, 65.2, 65.3, 65.5, 65.5, 65.97, 66.03, 66.1,
67.2, 67.3, 117.8, 118.2, 118.3, 118.9, 128.16, 128.18, 128.3, 128.55,
128.58, 131.7, 132.8, 136.3, 136.4, 156.7, 157.5, 171.3, 171.5, 172.6,
172.7.
Compound 18
31P NMR (162 MHz, CDCl3): δ = 27.6.
MS (ESI+): m/z [M + NH4]+ calcd for C28H45N5O9P: 626.67; found:
Compound 4 (93 mg, 0.15 mmol) was deprotected using Pd(PPh3)4
(17 mg, 0.02 mmol), Et2NH (219 mg, 3.00 mmol), and LiOH (14 mg,
0.33 mmol). The product was obtained as a colorless solid (57 mg,
95% purity by qNMR, 0.11 mmol, 73%); decomposition above 200 °C.
626.38.
1H NMR (400 MHz, D2O): δ = 0.82–0.94 (m, 6 H), 1.35–1.50 (m, 2 H),
1.56–1.68 (m, 1 H), 1.85–1.94 (m, 1 H), 1.99–2.07 (m, 1 H), 2.78–2.89
(m, 2 H), 3.21–3.35 (m, 2 H), 3.62–3.67 (m, 1 H), 4.16 (dd, J = 8.4, 5.0
Hz, 1 H), 5.05–5.19 (m, 2 H), 7.34–7.43 (m, 5 H).
© Georg Thieme Verlag Stuttgart · New York — Synthesis 2017, 49, A–J

H
J. Cramer, G. Klebe
Paper
Synthesis
13C NMR (101 MHz, D2O): δ = 21.2, 22.2, 24.0, 36.8, 39.7 (d, JC–P = 135.7
Hz), 43.21, 43.27, 52.5, 54.0, 67.1, 127.8, 128.4, 128.8, 136.4, 158.2,
177.4, 177.7.
1H NMR (400 MHz, D2O): δ = 0.74–0.94 (m, 6 H), 1.33–1.50 (m, 2 H),
1.52–1.71 (m, 1 H), 2.69–2.90 (m, 2 H), 3.12–3.43 (m, 4 H), 3.53–3.73
(m, 1 H), 4.98–5.27 (m, 2 H), 7.29–7.55 (m, 5 H).
31P NMR (162 MHz, D2O): δ = 18.2.
13C NMR (101 MHz, D2O): δ = 21.1, 22.3, 24.1, 38.8, 39.42 (d, JC–P
=
137.9 Hz), 39.46, 42.9, 54.4, 67.1, 127.8, 128.4, 128.8, 136.4, 158.2,
179.0.
31P NMR (162 MHz, D2O): δ = 18.4.
HRMS (ESI): m/z [M + H]+ calcd for C19H32N4O7P: 459.2003; found:
459.2003.
HRMS (ESI): m/z [M + Na]+ calcd for C17H29N4O5PNa: 423.1768; found:
Compound 19
423.1766.
Compound 5 (96 mg, 0.15 mmol) was deprotected using Pd(PPh3)4
(17 mg, 0.02 mmol), Et2NH (219 mg, 3.00 mmol), and LiOH (14 mg,
0.33 mmol). The product was obtained as a colorless solid (51 mg,
97% purity by qNMR, 0.10 mmol, 63%); decomposition above 200 °C.
Compound 23
Compound 9 (81 mg, 0.15 mmol) was deprotected using Pd(PPh3)4
(17 mg, 0.02 mmol), Et2NH (219 mg, 3.00 mmol), and LiOH (13 mg,
0.30 mmol). The product was obtained as a colorless solid (47 mg,
96% purity by qNMR, 0.11 mmol, 72%); decomposition above 200 °C.
1H NMR (400 MHz, D2O): δ = 0.80–1.09 (m, 6 H), 1.44–1.98 (m, 7 H),
2.73–3.00 (m, 2 H), 3.19–3.57 (m, 2 H), 3.67–3.87 (m, 1 H), 4.15–4.33
(m, 1 H), 5.06–5.42 (m, 2 H), 7.38–7.75 (m, 5 H).
13C NMR (101 MHz, D2O): δ = 21.2, 22.2, 24.0, 25.0, 28.8, 39.5, 39.7 (d,
JC–P = 136.1 Hz), 43.2, 54.0, 54.4, 67.1, 127.8, 128.4, 128.8, 138.0,
158.3, 177.4, 178.3.
1H NMR (400 MHz, D2O): δ = 0.65–0.98 (m, 6 H), 1.29–1.52 (m, 2 H),
1.51–1.73 (m, 3 H), 2.55–2.76 (m, 2 H), 3.01–3.38 (m, 4 H), 3.46–3.67
(m, 1 H), 4.98–5.22 (m, 2 H), 7.24–7.53 (m, 5 H).
31P NMR (202 MHz, D2O): δ = 18.2.
HRMS (ESI): m/z [M + H]+ calcd for C20H34N4O7P: 473.2160; found:
13C NMR (101 MHz, D2O): δ = 21.1, 22.2, 24.0, 29.3, 36.4, 37.5, 40.0 (d,
JC–P = 136.2 Hz), 43.2, 54.2, 67.1, 127.8, 128.4, 128.8, 136.4, 158.3,
178.4.
473.2161.
31P NMR (162 MHz, D2O): δ = 18.1.
HRMS (ESI): m/z [M + Na]+ calcd for C18H31N4O5PNa: 437.1924; found:
Compound 20
437.1923.
Compound 6 (100 mg, 0.15 mmol) was deprotected using Pd(PPh3)4
(17 mg, 0.02 mmol), Et2NH (219 mg, 3.00 mmol), and LiOH (14 mg,
0.33 mmol). The product was obtained as a colorless solid (50 mg,
99% purity by qNMR, 0.10 mmol, 66%); decomposition above 200 °C.
Compound 24
Compound 10 (50 mg, 0.09 mmol) was deprotected using Pd(PPh3)4
(10 mg, 0.01 mmol), Et2NH (132 mg, 1.80 mmol), and LiOH (8 mg,
0.18 mmol). The product was obtained as a colorless solid (30 mg,
96% purity by qNMR, 0.07 mmol, 75%); decomposition above 200 °C.
1H NMR (400 MHz, D2O): δ = 0.85–1.01 (m, 6 H), 1.38–1.61 (m, 6 H),
1.61–1.76 (m, 1 H), 2.61–2.73 (m, 2 H), 3.15–3.25 (m, 2 H), 3.26–3.47
(m, 2 H), 3.61–3.76 (m, 1 H), 5.19 (s, 2 H), 7.38–7.60 (m, 5 H).
13C NMR (101 MHz, D2O): δ = 21.2, 22.2, 24.0, 25.7, 27.6, 38.8, 39.0,
39.7 (d, JC–P = 136.4 Hz), 43.3, 54.1, 67.1, 127.8, 128.4, 128.8, 136.4,
158.3, 178.0.
1H NMR (500 MHz, D2O): δ = 0.91–1.02 (m, 6 H), 1.33–1.53 (m, 5 H),
1.73–1.84 (m, 4 H), 2.72–2.81 (m, 2 H), 3.30–3.38 (m, 2 H), 3.74–3.76
(m, 1 H), 4.22 (dd, J = 7.2, 5.3 Hz, 1 H), 5.17–5.23 (m, 2 H), 7.47–7.54
(m, 5 H).
13C NMR (101 MHz, D2O): δ = 21.3, 21.8, 22.2, 24.0, 26.4, 31.1, 39.2,
39.7 (d, JC–P = 136.0 Hz), 43.2, 54.0, 54.6, 67.1, 127.7, 128.3, 128.8,
136.5, 158.3, 177.2, 178.5.
31P NMR (202 MHz, D2O): δ = 18.2.
HRMS (ESI): m/z [M + H]+ calcd for C21H36N4O7P: 487.2316; found:
487.2315.
31P NMR (162 MHz, D2O): δ = 17.9.
HRMS (ESI): m/z [M + Na]+ calcd for C19H33N4O5PNa: 451.2081; found:
451.2084.
Compound 21
Compound 7 (77 mg, 0.15 mmol) was deprotected using Pd(PPh3)4
(17 mg, 0.02 mmol), Et2NH (219 mg, 3.00 mmol), and LiOH (14 mg,
0.33 mmol). The product was obtained as a colorless solid (39 mg,
99% purity by qNMR, 0.12 mmol, 78%); decomposition above 200 °C.
1H NMR (400 MHz, D2O): δ = 0.75–0.90 (m, 6 H), 1.31–1.47 (m, 2 H),
1.55–1.68 (m, 1 H), 3.17–3.34 (m, 2 H), 3.51–3.61 (m, 1 H), 3.92 (s, 2
H), 5.01–5.16 (m, 2 H), 7.29–7.49 (m, 5 H).
Compound 25
Compound 11 (44 mg, 0.08 mmol) was deprotected using Pd(PPh3)4
(9 mg, 0.01 mmol), Et2NH (117 mg, 1.60 mmol), and LiOH (8 mg, 0.18
mmol). The product was obtained as a colorless solid (24 mg, 97% pu-
rity by qNMR, 0.05 mmol, 66%); decomposition above 200 °C.
1H NMR (400 MHz, D2O): δ = 0.81–0.91 (m, 6 H), 1.22–1.42 (m, 2 H),
1.57–1.76 (m, 1 H), 2.61–2.93 (m, 4 H), 3.07–3.24 (m, 2 H), 3.27–3.63
(m, 4 H), 4.06–4.18 (m, 1 H), 5.03–5.14 (m, 2 H), 7.31–7.46 (m, 5 H).
13C NMR (101 MHz, D2O): δ = 21.0, 22.3, 24.0, 39.7 (d, JC–P = 137.0 Hz),
43.0, 47.9, 54.1, 67.1, 127.7, 128.4, 128.8, 136.4, 158.3, 178.5.
31P NMR (162 MHz, D2O): δ = 17.9.
HRMS (ESI): m/z [M + Na]+ calcd for C16H27N4O5PNa: 409.1611; found:
13C NMR (101 MHz, D2O): δ = 21.2, 22.5, 24.0, 39.7 (d, JC–P = 135.2 Hz),
42.6, 43.5, 44.1, 44.5, 46.0, 49.2, 67.1, 127.9, 128.4, 128.8, 136.4,
158.1, 175.4.
409.1614.
31P NMR (162 MHz, D2O): δ = 18.0.
HRMS (ESI): m/z [M + Na]+ calcd for C19H31N4O5PNa: 449.1924; found:
Compound 22
449.1922.
Compound 8 (146 mg, 0.28 mmol) was deprotected using Pd(PPh3)4
(32 mg, 0.03 mmol), Et2NH (404 mg, 5.60 mmol), and LiOH (26 mg,
0.62 mmol). The product was obtained as a colorless solid (82 mg,
99% purity by qNMR, 0.09 mmol, 72%); decomposition above 200 °C.
© Georg Thieme Verlag Stuttgart · New York — Synthesis 2017, 49, A–J

I
J. Cramer, G. Klebe
Paper
Synthesis
Compound 26
HRMS (ESI): m/z [M + Na]+ calcd for C18H29N4O7PNa: 467.1666; found:
467.1665.
Compound 12 (74 mg, 0.15 mmol) was deprotected using Pd(PPh3)4
(17 mg, 0.02 mmol), Et2NH (219 mg, 3.00 mmol), and LiOH (14 mg,
0.33 mmol). The product was obtained as a colorless solid (39 mg,
97% purity by qNMR, 0.09 mmol, 59%); decomposition above 200 °C.
Compound 30
Compound 16 (91 mg, 0.15 mmol) was deprotected using Pd(PPh3)4
(17 mg, 0.02 mmol), Et2NH (219 mg, 3.00 mmol), and LiOH (14 mg,
0.33 mmol). The product was obtained as a colorless solid (55 mg,
97% purity by qNMR, 0.11 mmol, 77%); decomposition above 200 °C.
1H NMR (500 MHz, D2O): δ = 0.91–1.04 (m, 6 H), 1.63–1.75 (m, 3 H),
3.35–3.46 (m, 2 H), 3.51–3.63 (m, 2 H), 4.26–4.35 (m, 1 H), 5.17–5.28
(m, 2 H), 7.44–7.58 (m, 5 H).
1H NMR (400 MHz, D2O): δ = 0.70–0.92 (m, 6 H), 1.48–1.68 (m, 3 H),
3.15–3.42 (m, 2 H), 3.55–3.80 (m, 3 H), 4.10–4.22 (m, 1 H), 5.01–5.20
(m, 2 H), 7.23–7.49 (m, 5 H).
13C NMR (101 MHz, D2O): δ = 20.9, 22.4, 24.6, 39.5 (d, JC–P = 136.7 Hz),
40.7, 54.1, 56.8, 63.8, 67.1, 127.8, 128.4, 128.8, 136.5, 158.2, 174.0,
179.9.
13C NMR (101 MHz, D2O): δ = 20.9, 22.4, 24.6, 38.4, 39.2 (d, JC–P = 135.2
Hz), 44.3, 53.7, 67.1, 127.9, 128.4, 128.8, 136.5, 158.2, 174.2, 179.9.
31P NMR (202 MHz, D2O): δ = 20.2.
HRMS (ESI): m/z [M + Na]+ calcd for C17H26N3O7PNa: 438.1401; found:
438.1400.
31P NMR (162 MHz, D2O): δ = 18.9.
HRMS (ESI): m/z [M + Na]+ calcd for C18H28N3O8PNa: 468.1506; found:
Compound 27
Compound 13 (76 mg, 0.15 mmol) was deprotected using Pd(PPh3)4
(17 mg, 0.02 mmol), Et2NH (219 mg, 3.00 mmol), and LiOH (14 mg,
0.33 mmol). The product was obtained as a colorless solid (50 mg,
97% purity by qNMR, 0.11 mmol, 75%); decomposition above 200 °C.
468.1504.
Compound 31
1H NMR (500 MHz, D2O): δ = 0.93 (d, J = 6.2 Hz, 3 H), 0.98 (d, J = 6.1
Hz, 3 H), 1.33 (d, J = 7.1 Hz, 3 H), 1.61–1.73 (m, 3 H), 3.20–3.44 (m, 2
H), 3.77–3.83 (m, 1 H), 4.24–4.28 (m, 1 H), 5.16–5.25 (m, 2 H), 7.46–
7.57 (m, 5 H).
Compound 17 (77 mg, 0.15 mmol) was deprotected using Pd(PPh3)4
(17 mg, 0.02 mmol), Et2NH (219 mg, 3.00 mmol), and LiOH (14 mg,
0.33 mmol). The product was obtained as a colorless solid (53 mg,
97% purity by qNMR, 0.12 mmol, 77%); decomposition above 200 °C.
1H NMR (400 MHz, D2O): δ = 0.81–0.98 (m, 6 H), 1.33–1.47 (m, 4 H),
1.53–1.82 (m, 2 H), 3.08–3.43 (m, 2 H), 3.74–3.91 (m, 1 H), 4.81–4.85
(m, 1 H), 5.05–5.24 (m, 2 H), 7.20–7.58 (m, 5 H).
13C NMR (101 MHz, D2O): δ = 20.3, 20.9, 22.5, 24.6, 39.4 (d, JC–P = 136.1
Hz), 40.8, 50.9, 53.8, 67.1, 127.8, 128.4, 128.8, 136.5, 158.3, 177.1,
179.9.
13C NMR (101 MHz, D2O): δ = 16.8, 21.0, 22.4, 23.9, 39.7 (d, JC–P = 136.1
Hz), 42.8, 52.8, 67.1, 72.7, 127.8, 128.4, 128.8, 136.4, 158.3, 177.3,
178.6.
31P NMR (202 MHz, D2O): δ = 18.9.
HRMS (ESI): m/z [M + Na]+ calcd for C18H28N3O7PNa: 452.1557; found:
452.1555.
31P NMR (202 MHz, D2O): δ = 18.3.
Compound 28
HRMS (ESI): m/z [M + Na]+ calcd for C18H27N2O8PNa: 453.1397; found:
453.1395.
Compound 14 (81 mg, 0.15 mmol) was deprotected using Pd(PPh3)4
(17 mg, 0.02 mmol), Et2NH (219 mg, 3.00 mmol), and LiOH (14 mg,
0.33 mmol). The product was obtained as a colorless solid (52 mg,
96% purity by qNMR, 0.11 mmol, 72%); decomposition above 200 °C.
Acknowledgment
1H NMR (400 MHz, D2O): δ = 0.71–0.95 (m, 12 H), 1.46–1.63 (m, 3 H),
1.91–2.05 (m, 1 H), 3.10–3.34 (m, 2 H), 3.39–3.47 (m, 1 H), 4.12–4.23
(m, 1 H), 5.02–5.20 (m, 2 H), 7.32–7.47 (m, 5 H).
This work was funded by the European Research Council (ERC) of the
European Union (grant 268145-DrugProfilBind).
13C NMR (101 MHz, D2O): δ = 16.8, 18.8, 20.9, 22.4, 24.6, 31.5, 39.3 (d,
JC–P = 136.8 Hz), 40.9, 53.9, 60.8, 67.1, 127.8, 128.4, 128.8, 136.5,
158.3, 176.2, 179.8.
Supporting Information
Supporting information for this article is available online at
31P NMR (162 MHz, D2O): δ = 18.5.
HRMS (ESI): m/z [M + Na]+ calcd for C20H32N3O7PNa: 480.187; found:
http://dx.doi.org/10.1055/s-0036-1588393.
S
u
p
p
ortiInfogrmoaitn
S
u
p
p
o
nrtogI
f
rmoaitn
480.1868.
References
Compound 29
(1) Bartlett, P. A.; Marlowe, C. K. Biochemistry 1983, 22, 4618.
(2) McLeod, D. A.; Brinkworth, R. I.; Ashley, J. A.; Janda, K. D.;
Wirsching, P. Bioorg. Med. Chem. Lett. 1991, 1, 653.
(3) Jacobsen, N. E.; Bartlett, P. A. J. Am. Chem. Soc. 1981, 103, 654.
(4) Elliott, R. L.; Marks, N.; Berg, M. J.; Portoghese, P. S. J. Med. Chem.
1985, 28, 1208.
(5) Mucha, A.; Kunert, A.; Grembecka, J.; Pawelczak, M.; Kafarski, P.
Eur. J. Med. Chem. 2006, 41, 768.
(6) Bertenshaw, S. R.; Rogers, R. S.; Stern, M. K.; Norman, B. H.;
Moore, W. M.; Jerome, G. M.; Branson, L. M.; McDonald, J. F.;
McMahon, E. G.; Palomo, M. A. J. Med. Chem. 1993, 36, 173.
Compound 15 (91 mg, 0.15 mmol) was deprotected using Pd(PPh3)4
(17 mg, 0.02 mmol), Et2NH (219 mg, 3.00 mmol), and LiOH (14 mg,
0.33 mmol). The product was obtained as a colorless solid (56 mg,
96% purity by qNMR, 0.12 mmol, 79%); decomposition above 200 °C.
1H NMR (500 MHz, D2O): δ = 0.89–1.04 (m, 6 H), 1.61–1.75 (m, 3 H),
2.84–3.08 (m, 2 H), 3.28–3.52 (m, 2 H), 3.78–3.90 (m, 1 H), 4.20–4.29
(m, 1 H), 5.13–5.34 (m, 2 H), 7.39–7.64 (m, 5 H).
13C NMR (101 MHz, D2O): δ = 20.8, 22.5, 24.7, 39.5 (d, JC–P = 134.7 Hz),
40.5, 44.4, 54.1, 56.0, 67.1, 127.8, 128.4, 128.8, 136.5, 158.2, 173.9,
179.8.
31P NMR (202 MHz, D2O): δ = 18.7.
© Georg Thieme Verlag Stuttgart · New York — Synthesis 2017, 49, A–J

J
J. Cramer, G. Klebe
Paper
Synthesis
(7) Mookhtiar, K. A.; Marlowe, C. K.; Bartlett, P. A.; van Wart, H. E.
Biochemistry 1987, 26, 1962.
(8) Hua, T. D.; Lamaty, F.; Souriau, C.; Rolland-Fulcrand, V.; Lazaro,
R.; Viallefont, P.; Lefranc, M. P.; Weill, M. Amino Acids 1996, 10,
167.
(9) Vallee, M. R. J.; Majkut, P.; Wilkening, I.; Weise, C.; Muller, G.;
Hackenberger, C. P. R. Org. Lett. 2011, 13, 5440.
(10) (a) Vallee, M. R. J.; Artner, L. M.; Dernedde, J.; Hackenberger, C.
P. R. Angew. Chem. Int. Ed. 2013, 52, 9504. (b) Vallee, M. R. J.;
Majkut, P.; Krause, D.; Gerrits, M.; Hackenberger, C. P. R. Chem.
Eur. J. 2015, 21, 970.
(13) (a) Krimmer, S. G.; Klebe, G. J. Comput. Aided Mol. Des. 2015, 29,
867. (b) Gruner, S.; Neeb, M.; Barandun, L. J.; Sielaff, F.; Hohn, C.;
Kojima, S.; Steinmetzer, T.; Diederich, F.; Klebe, G. Biochim. Bio-
phys. Acta 2014, 1840, 2843.
(14) Kishore Kumar, G. D.; Saenz, D.; Lokesh, G. L.; Natarajan, A. Tet-
rahedron Lett. 2006, 47, 6281.
(15) Wilkening, I.; del Signore, G.; Hackenberger, C. P. R. Chem.
Commun. 2011, 47, 349.
(16) Hirschmann, R.; Yager, K. M.; Taylor, C. M.; Witherington, J.;
Sprengeler, P. A.; Phillips, B. W.; Moore, W.; Smith, A. B. III.
J. Am. Chem. Soc. 1997, 119, 8177.
(11) (a) Medina, P.; de Ingrassia, L. S.; Mulliez, M. E. J. Org. Chem.
2003, 68, 8424. (b) Mucha, A.; Grembecka, J.; Cierpicki, T.;
Kafarski, P. Eur. J. Org. Chem. 2003, 4797. (c) Christianson, D. W.;
Lipscomb, W. N. J. Am. Chem. Soc. 1986, 108, 545.
(12) (a) Biela, A.; Sielaff, F.; Terwesten, F.; Heine, A.; Steinmetzer, T.;
Klebe, G. J. Med. Chem. 2012, 55, 6094. (b) Krimmer, S. G.; Betz,
M.; Heine, A.; Klebe, G. ChemMedChem 2014, 9, 833. (c) Biela, A.;
Nasief, N. N.; Betz, M.; Heine, A.; Hangauer, D.; Klebe, G. Angew.
Chem. 2013, 125, 1868. (d) Biela, A.; Betz, M.; Heine, A.; Klebe,
G. ChemMedChem 2012, 7, 1423. (e) Biela, A.; Nasief, N. N.; Betz,
M.; Heine, A.; Hangauer, D.; Klebe, G. Angew. Chem. Int. Ed.
2013, 52, 1822.
(17) Blackburn, G. M.; Wentworth, P. US Patent 5807688, 1998.
(18) Hennion, M.-C. J. Chromatogr. A 1999, 856, 3.
(19) (a) Kamysz, W.; Okroj, M.; Lempicka, E.; Ossowski, T.; Lukasiak,
J. Acta Chromatogr. 2004, 14, 180. (b) Nilsson, U. J.; Fournier, E.
J.; Hindsgaul, O. Bioorg. Med. Chem. 1998, 6, 1563. (c) Asada, T.;
Koi, Y.; Arakawa, R.; Zhu, F.; Sadaoka, M.; Tamura, H. J. Chro-
matogr. A 2014, 1351, 21.
(20) Morgan, B.; Scholtz, J. M.; Ballinger, M. D.; Zipkin, I. D.; Bartlett,
P. A. J. Am. Chem. Soc. 1991, 113, 297.
© Georg Thieme Verlag Stuttgart · New York — Synthesis 2017, 49, A–J