Synthesis and enzymatic evaluation of phosphoramidon and its β anomer: Anomerization of α-L-rhamnose triacetate upon phosphitylation

Article abstract of DOI:10.1016/j.bmc.2013.07.052

A novel and efficient strategy for the synthesis of phosphoramidon and its β anomer has been developed by manipulating the anomerization of α-l-rhamnose triacetate upon phosphitylation. The experimental results suggest that proton transfer, bond rotation, and N atom are the key factors for the anomerization. The determined K_i and K_d values establish that phosphoramidon prepared by this method possesses excellent biological activity, and indicate that the contacts of rhamnose moiety with the enzyme have limited contribution to the binding.

Full text of DOI:10.1016/j.bmc.2013.07.052

Bioorganic & Medicinal Chemistry 21 (2013) 6778–6787
Contents lists available at ScienceDirect
Bioorganic & Medicinal Chemistry
journal homepage: www.elsevier.com/locate/bmc
Synthesis and enzymatic evaluation of phosphoramidon
and its b anomer: Anomerization of
a-L-rhamnose
triacetate upon phosphitylation
⇑
Qi Sun , Qingkun Yang, Shanshan Gong, Quanlei Fu, Qiang Xiao
Jiangxi Key Laboratory of Organic Chemistry, Jiangxi Science and Technology Normal University, 605 Fenglin Avenue, Nanchang, Jiangxi 330013, PR China
a r t i c l e i n f o
a b s t r a c t
Article history:
A novel and efficient strategy for the synthesis of phosphoramidon and its b anomer has been developed
Received 13 June 2013
Revised 30 July 2013
Accepted 31 July 2013
Available online 8 August 2013
by manipulating the anomerization of a-L-rhamnose triacetate upon phosphitylation. The experimental
results suggest that proton transfer, bond rotation, and N atom are the key factors for the anomerization.
The determined Ki and Kd values establish that phosphoramidon prepared by this method possesses
excellent biological activity, and indicate that the contacts of rhamnose moiety with the enzyme have
limited contribution to the binding.
Keywords:
Ó 2013 Elsevier Ltd. All rights reserved.
Phosphoramidon
Phosphoramidite
Phosphoramidate
Anomerization
Glycopeptide
1. Introduction
H-phosphonate monoester,7 and di(p-methoxy-benzyl)-N,N-dii-
sopropylphosphoramidite.8 However, all these methods afforded
Phosphoramidon (1), a naturally occurring glycopeptide first
isolated from a strain of Streptomyces tanashiensis by Umezawa
and co-workers in 1972, has a unique chemical structure featuring
their target molecules in only low to moderate yields with compli-
cated purification procedures. We report herein a novel and efficient
route for the total synthesis of phosphoramidon and its b anomer by
a phosphoramidate linkage between
a
-
L
-rhamnose and
L
-leucine-
manipulating the anomerization of
phosphitylation.
a-L-rhamnose triacetate upon
L-tryptophan. As a microbial metabolite, phosphoramidon exhibits
potent inhibitory activity against thermolysin, a zinc endopepti-
dase isolated from Bacillus thermoproteolyticus (Ki = 32 nM).1 It is
also identified as an inhibitor of endothelin converting enzyme
2. Results and discussion
(ECE) (IC50 = 0.69 lM), a zinc-containing enzyme responsible for
2.1. Synthesis of phosphoramidon and its b anomer
activation of endothelin (ET-1). As a potent vasoconstrictor, ET-1
causes a variety of pathophysiological conditions, such as hyper-
tension, stroke, and cardiac failure.2 Phosphoramidon has been
extensively utilized as a reference inhibitor for investigation of
ECE-related diseases and development of novel therapeutic agents
with better metabolic stability and ECE specificity.3 Meanwhile,
phosphoramidon inhibits other metalloproteases, such as neutral
endopeptidase (NEP)4 and bacterial elastases,5 rendering this natu-
ral compound a valuable tool in biological and medicinal research.
Despite its versatile biological activities, only a few methods have
been reported for the chemical synthesis of phosphoramidon and its
analogues. These approaches involved different P(III) and P(V) inter-
To overcome the problems of known methods, our synthetic
strategy employs three building blocks, including
triacetate (2), benzyl-N,N-diisopropylchlorophosphoramidite (3),
and NH2- -leucine- -tryptophan benzyl ester (4), all of which can
a-L-rhamnose
L
L
be efficiently prepared from inexpensive starting materials and
contain easily removable protecting groups. As shown in Scheme 1,
peracetylation of
L-rhamnose (5) followed by selective deprotec-
tion of anomeric acetyl group afforded 2 as pure
a
anomer. Starting
from PCl3 (7), phosphitylating reagent 3 was obtained in high yield
and purity based on a modified procedure in our work.9 EDC cou-
pling of N-Boc-L-leucine (9) and L-tryptophan-OBn (10) gave dipep-
tide 11. Removal of Boc with TFA and simple alkaline workup
afforded 4 in almost quantitative yield.
mediates, such as L
-rhamnose-1-phosphate,1b dichlorophosphate,6
The construction of the phosphoramidate linkage was achieved
efficiently via three consecutive steps, including phosphitylation of
⇑
Corresponding author. Tel.: +86 791 83805183; fax: +86 791 83826894.
E-mail address: sunqi96@tsinghua.org.cn (Q. Sun).
0968-0896/$ - see front matter Ó 2013 Elsevier Ltd. All rights reserved.
http://dx.doi.org/10.1016/j.bmc.2013.07.052

Q. Sun et al. / Bioorg. Med. Chem. 21 (2013) 6778–6787
6779
Scheme 1. Design and synthesis of the building blocks for phosphoramidon (1). Reagents and conditions: (a) Ac2O, NaOAc, reflux, 4 h; (b) 1,2-diamino ethane, HOAc, THF, rt,
24 h (76% over 2 steps); (c) BnOH, CH2Cl2, 0 °C to rt, 6 h; (d) (iPr)2NH, Et3N, Et2O, 0 °C to rt, 12 h (82% over 2 steps); (e) EDC, HOBt, CH2Cl2, rt, 12 h (89%); (f) 20% TFA, CH2Cl2, rt,
2 h (99%).
the anomeric OH of 2 with 3, hydrolysis of phosphoramidite inter-
mediate 12 with an acidic catalyst, and oxidative coupling of the
resulting H-phosphonate diester 13 with 4 (Scheme 2). However,
it was found that if the reaction was performed with DBU as base
and diethyl ether as solvent at 0 °C in low concentration (condition
mixture of
was phosphitylated under condition b. The
separable on silica gel chromatography, which afforded 14a and
14b in 16% and 53% yield ( /b = 1:3.3), respectively.
a
and b anomers (14a and 14b) was obtained, if 2
a
/b mixture was readily
a
Final deprotection of 14a and 14b was conducted by catalytic
hydrogenation and deacetylation with NaOMe in a one pot manner
(Scheme 2). Due to the fact that phosphoramidate is susceptible to
solvolysis at both low and high pH, two benzyl esters were ratio-
nally introduced in our design and avoided harsh deprotection
conditions used in previous methods.6,7b,11 Hydrogenation of 14a
and 14b with 5% Pd/C in anhydrous MeOH removed the benzyl
groups in 3 h. Then, a solution of NaOMe in MeOH was added in
drops to reach a final concentration of 0.05 M and pH 9.0. The
deacetylation completed in 3 h with only trace amounts of byprod-
a),
a anomer of 12 was the sole product. In contrast, if the reaction
was conducted with Et3N as base and CH2Cl2 as solvent at 35 °C in
high concentration (condition b), the b anomer in turn became the
major product (a
/b = 1:3.6).10 After salt and extra base were re-
moved, 1H-tetrazole-catalyzed hydrolysis of 12 prepared under
either condition a or b afforded the corresponding batch of H-phos-
phonate diester 13. Oxidative coupling of 13 with dipeptide 4 using
CCl4 and Et3N gave phosphoramidate 14. For the reaction based on
condition a, 14a was isolated in 72% yield (
a only). Otherwise, a
Scheme 2. Synthesis of phosphoramidon (1) and its b anomer (15). Reagents and conditions: (a) 3, DBU, Et2O, 0 °C, 30 min, [2] = 0.057 M; (b) 3, Et3N, CH2Cl2, 35 °C, 30 min,
[2] = 0.46 M; (c) 1H-tetrazole, H2O, CH3CN, rt, 30 min; (d) 4, CCl4, Et3N, CH3CN, 0 °C to rt, 30 min [72% for 14a ( only, condition a)/16% for 14a and 53% for 14b ( /b = 1:3.3,
condition b) over 3 steps]; (e) H2, 5% Pd/C, MeOH, rt, 3 h; (f) NaOMe, MeOH, rt, 3 h (92% for 1/93% for 15 over 2 steps).
a
a

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Q. Sun et al. / Bioorg. Med. Chem. 21 (2013) 6778–6787
Table 2
ucts. It was found that when the final concentration of NaOMe ex-
ceeded 0.1 M, more byproducts appeared on TLC. After neutraliza-
tion with diluted HCl in MeOH, crude 1 and 15 were easily purified
by size-exclusion gel chromatography and recrystallization respec-
tively in high yields. The purity of 1 and 15 determined by NMR
and reverse-phase HPLC analysis was over 98%.
Effect of temperature on anomerization of 12a
Entry Solvent/
Base
a/b ratio (20 °C)
a
/b ratio (0 °C)
a/b ratio (ꢁ20 °C)
1
2
Et2O/DBU
THF/DBU
4.1:1
3.0:1
a
4.3:1
only
—
a
only
a
All the reactions listed in Table 2 were performed with [2] = 0.057 M.
2.2. Mechanistic study and control of the anomerization of
rhamnose triacetate upon phosphitylation
a-L-
To validate the proposed mechanism, more experiments con-
cerning the key factors, the acidic proton and bond rotation, were
conducted. Based on the hypothesis that more basic reaction con-
dition should reduce the proton transfer and formation of b ano-
mer, the reaction was tested in different solvents with Et3N or
DBU as base. As expected (Table 1), the stronger basicity of DBU
As mentioned above, the experimental results demonstrated
the efficacy of combining phosphoramidite chemistry12 and Ather-
ton-Todd reaction13 for the construction of phosphoramidate at the
anomeric position of glycosyl substrates. However, to our surprise,
when 2 (pure
a anomer) was used in our initial attempt (Et3N, CH2-
significantly increased the ratio of
a anomer. Meanwhile, solvents
Cl2, 20 °C, [2] = 0.057 M, condition c in Table 1), 14a and 14b were
isolated in nearly equal amount, which was an interesting phe-
nomenon that has never been mentioned in literature.9,14 13C
NMR spectra of crude 12 and 13 were obtained, and the data
that could serve as Lewis bases, such as THF and Et2O, also lowered
the portion of b anomer by quenching the acidic proton. The pos-
sibility of using weak organic bases, such as pyridine, lutidine,
and N-methylimidazole, to favor the formation of 12b in CH2Cl2
was also explored. But the reaction was sluggish in the absence
of a strong base.
showed that the a/b ratios were almost the same as 14a/14b. Fur-
thermore, no b anomer was observed on 13C NMR spectrum, when
2 was treated with 1.0 equiv of HCl, Et3N, or Et3NꢀHCl respectively
in CH2Cl2 after 12 h. These results suggested that the anomeriza-
tion happened during the process of phosphitylation.
In the following research, the effect of reaction temperature on
the a/b ratio of 12 was investigated. The reaction was performed at
lower temperatures to slow down the rotation of C1ꢁC2 bond,
thereby decreasing the portion of b anomer. The data in Table 2
showed that when the reaction with Et2O as solvent and DBU as
As illustrated in Scheme 3, we postulated that upon the substi-
tution of chloride with anomeric OH group, if the resulting acidic
proton was neutralized by the base, the
a anomeric configuration
base was lowered to 0 °C,
a anomer of 12 was obtained as the only
remained untouched (12a, path A). However, the acidic proton
may also be quickly transferred to the adjacent oxygen atom on
C5, concerted with attack of nitrogen atom on phosphoramidite
at C1, to cause pyranose ring opening (17). Rotation of C1–C2 bond
(18) followed by attack of OH on C1 and reclosure of the ring in-
verted the anomeric configuration (12b, path B). Although the
bond rotation may involve multiple factors, the steric repulsion be-
tween the N-isopropyl groups and acetyl group on C2 could be a
possible driving force.
product (Scheme 2, condition a). The reaction with THF/DBU exhib-
ited a similar trend, but required ꢁ20 °C to completely eliminate
the generation of b anomer.
In contrast, the reaction of 2 and 3 in CH2Cl2 with Et3N as base
was performed at 35 °C to promote the formation of b anomer.
Meanwhile, it was found that the concentration of 2 also played
a key role in the anomerization. The data in Table 3 showed that
both higher temperature and concentration favored the formation
of b anomer. The lowest a/b ratio (1:3.6) was obtained, when the
reaction was conducted at 35 °C with [2] = 0.46 M (Scheme 2, con-
dition b).
Table 1
Effects of solvent and base on anomerization of 12a
2.3. Assignment of the anomeric configurations
Entry
Solvent
a
/b ratio (Et3N as base)
a/b ratio (DBU as base)
In analysis of 1H NMR data of 1 and 15, we found that the 3JH1,H2
and 3JH1,P values of 1 (0–1 and 7.6 Hz), which were used for assign-
ment of the
1
2
3
4
CH2Cl2
CH3CN
THF
1.1:1 (condition c)
1.5:1
2.1:1
3.0:1
4.1:1
1.3:1
N.R.
N.R.
a
anomeric configuration of 1 in previous reports,1a,6–8
Et2O
were only of slight difference to those of 15 (0–1 and 8.4 Hz) due to
a
the axial OH at C2 position (UH1,H2 = 60°) of both 1 and 15. Accord-
All the reactions listed in Table 1 were performed at 20 °C with [2] = 0.057 M.
Scheme 3. A proposed mechanism for the anomerization of a-L-rhamnosyl moiety upon phosphitylation.

Q. Sun et al. / Bioorg. Med. Chem. 21 (2013) 6778–6787
6781
Table 3
2.4. Synthesis of
with control of anomeric configuration
a- and b-L-rhamnosyl-1-phosphoramidates
Effects of temperature and concentration of 2 on anomerization of 12a
Entry
[2] (M)
a
/b ratio (20 °C)
a/b ratio (35 °C)
Our finding that anomerization of 2 upon reacting with chloro-
phosphoramidite 3 could be manipulated by using different reac-
1
2
3
0.057
0.23
1:0.9
1:1.4
—
1:1.0
1:2.1
1:3.6
0.46b
tion conditions provided a novel approach for constructing
a and
b phosphoramidate linkage at the anomeric position of rhamnose
with control of stereochemistry. To prove this point, seven
a
All the reactions listed in Table 3 were performed with CH2Cl2/Et3N.
The maximum concentration for phosphitylation reaction of 2 in CH2Cl2 at
b
35 °C.
a-2,3,4-O-triacetyl-L-rhamnosyl-1-phosphoramidates (19a–25a)
and their b anomers (19b–25b) were prepared according to this
1
new method in good yields. Moreover, the JC1,H1 values of these
pairs provided valuable references for determination of the ano-
meric configurations of
tives (Table 4).
L-rhamnosyl-1-phosphoramidate deriva-
2.5. Enzymatic evaluation of phosphoramidon and its b anomer
To examine the biological activity of 1 and its b anomer 15 pre-
pared by our new method, the inhibitory effects of these two com-
pounds on thermolysin were investigated according to literature
methods. The inhibitor constants (Ki) of 1 and 15 at pH 7.5 were
determined based on the Henderson plots of inhibition of a sub-
strate cleavage (Fig. 2).1b,1c,16 The dissociation constants of the EI
complexes (Kd) were calculated from the fluorometric titration
curves by non-linear least-square method (Fig. 3).17
The Ki and Kd values of 1 obtained in our experiments were
(3.0 0.1) ꢂ 10ꢁ8 M and (3.2 0.6) ꢂ 10ꢁ8 M respectively, and
compared well with those reported previously.1c,1d It was deter-
mined that b anomer 15 also exhibited potent inhibitory activity
against thermolysin with a Ki of (2.8 0.8) ꢂ 10ꢁ7 M and Kd of
(2.1 0.5) ꢂ 10ꢁ7 M, which were about one order of magnitude
Figure 1. 2D NOSEY NMR spectrum of 15.
ing to literature precedents, measuring one-bond CꢁH coupling
constants is a reliable method to distinguish anomeric pairs in
higher than those of 1, indicating that altering the a configuration
1
pyranoses.15 The JC1,H1 values of these two compounds (170 Hz
of 1 to b only moderately decreased the binding affinity of 15 to
thermolysin. This result confirmed that N-phosphoryl dipeptide
moiety in 1 and 15 is the primary structural feature for effective
inhibition, and the interactions between rhamnose moiety and S1
subsite of thermolysin make only a relatively small contribution
to the binding.1c,1e,18
for 1 and 159 Hz for 15) matched well with the typical values of
- and b-glycosyl anomers (170 Hz for a and 160 Hz for b). In
addition, two-dimensional NMR experiments unambiguously
confirmed the b configuration of 15 by detection of a nuclear Over-
hauser effect (NOE) between H1 and H3/H5 (Fig. 1).
a
Table 4
1JC1,
Values of 14a,b, 19a,b–25a,b
H1
Compounds
1JC1,H1 of
a
anomer (Hz)
1JC1,H1 of b anomer (Hz)
14a/14b
19a/19b
20a/20b
21a/21b
22a/22b
23a/23b
24a/24b
25a/25b
176
176
171
176
176
177
175
175
162
164
161
163
161
161
162
161

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Q. Sun et al. / Bioorg. Med. Chem. 21 (2013) 6778–6787
Figure 2. Henderson plots of 1 (A) and 15 (B) inhibition towards the cleavage of 3-(2-furylacryloyl)-glycyl-L-leucine amide (FAGLA) by thermolysin in pH 7.5 Tris buffer at
25 °C. [E]0 = 4.0 ꢂ 10ꢁ8 M, [A]1 = 5.0 ꢂ 10ꢁ4 M, [A]2 = 4.0 ꢂ 10ꢁ4 M, [A]3 = 3.0 ꢂ 10ꢁ4 M, [A]4 = 2.0 ꢂ 10ꢁ4 M, [A]5 = 1.0 ꢂ 10ꢁ4 M, [I]1 = 1.0 ꢂ 10ꢁ8 M, [I]2 = 2.0 ꢂ 10ꢁ8 M,
[I]3 = 4.0 ꢂ 10ꢁ8 M, [I]4 = 6.0 ꢂ 10ꢁ8 M, [I]5 = 8.0 ꢂ 10ꢁ8 M. It Indicates total concentration of inhibitor. vi and v0 Indicate velocity in the presence and in the absence of the
inhibitor, respectively. The increasing slopes with increasing concentrations of A indicate that the inhibition is in a competitive manner.
Figure 3. Fluorometric titration curves of [thermolysin-1] (A) and [thermolysin-15] (B) complexs in pH 7.5 Tris buffer at 25 °C.
DF (relative) represents the relative increment
of the fluorescence against the total fluorescence intensity of the enzyme and inhibitor at a concentration equal to that of the enzyme,
DF (relative) = DF/(FE + FI,eq).
[E]0 = 1.15 ꢂ 10ꢁ6 M, kex = 280 nM, kem = 360 nM. Kd values were calculated by the non-linear least-squares method.
assays reveal that the samples synthesized by this new method
have excellent biological activity. Additionally, the slightly lowered
inhibitory activity of 15 against thermolysin indicates that the con-
tacts of sugar moiety with the enzyme have limited contribution to
the free energy of binding.
3. Conclusions
In summary, we developed a novel and efficient route for the
total synthesis of phosphoramidon (1) and its b anomer (15). Com-
paring to the few known methods, this new route features easily
accessible building blocks, efficient installation of phosphorami-
date at anomeric position of a-L-rhamnose triacetate (2) with con-
4. Experimental
4.1. Chemistry
trol of stereochemistry, fast removal of all protecting groups under
mild conditions, and simple purification procedures. The mecha-
nism of anomerization of 2 in the reaction with chlorophosph-
oramidite 3 was proposed to involve two steps, including the
acidic proton transfer induced N atom attack at C1 and pyranose
ring opening, and subsequent bond rotation and reclosure of the
ring. The definite assignment of the anomeric configurations of 1
4.1.1. General
Chemical reagents and solvents were obtained from Acros,
Aldrich, Alfa Aesar, and Beijing Chemical Works. Commercial grade
reagents were used without further purification unless otherwise
noted. Anhydrous solvents were obtained through standard labo-
ratory protocols. Reactions were monitored by analytical thin-
layer chromatography on plates coated with 0.25 mm silica gel
60 F254 (Qingdao Haiyang Chemicals, China). TLC plates were visu-
alized by UV irradiation (254 nM) or stained with 20% sulfuric acid
in ethanol. Flash column chromatography employed silica gel (par-
1
and 15 was achieved by measuring JC1,H1 and NOE effect of H1
with H3/H5. Our finding that anomerization of a-L-rhamnose triac-
etate upon phosphitylation could be manipulated by using differ-
ent reaction conditions offered a novel and efficient approach for
constructing
of anomeric configuration. The JC1,H1 values of a series of novel
-rhamnosyl-1-phosphoramidates (19a,b–25a,b) prepared by this
a- and b-glycosyl-1-phosphoramidate with control
1
L
ticle size 32–63 lM, Qingdao Haiyang Chemicals, China). Size-
new method provide useful information for identification of the
anomeric configurations of related phosphorus-containing glyco-
sides. The Ki and Kd values of 1 and 15 determined by enzymatic
exclusion chromatography employed Sephadex LH-20 gel. NMR
spectra were obtained with a Bruker AV-400 instrument with
chemical shifts reported in parts per million (ppm, d) referenced

Q. Sun et al. / Bioorg. Med. Chem. 21 (2013) 6778–6787
6783
to CDCl3 or D2O. IR spectra were recorded on a Bruker Vertex-70
spectrometer. HPLC traces were recorded on an Agilent 1200
instrument equipped with an Eclipse XDB-C18 analytical column
(20 mL ꢂ 2), dried with anhydrous Na2SO4, and concentrated in va-
cuo. Flash column chromatography on silica gel (petroleum ether/
ethyl acetate 5:1) afforded 11 (456 mg, 90%) as a white solid; mp:
119–120 °C; 1H NMR (400 MHz, CDCl3): d 8.02 (s, 1H), 7.51 (d,
J = 7.7 Hz, 1H), 7.34–7.32 (m, 4H), 7.21–7.16 (m, 3H), 7.09 (t,
J = 7.4 Hz, 1H), 6.86 (s, 1H), 6.53 (d, 1H), 5.05 (s, 2 H), 4.94 (dd,
J1 = 5.4 Hz, J2 = 12.7 Hz, 1H), 4.79 (d, J = 7.6 Hz, 1H), 4.07 (br, 1H),
3.32 (t, J = 3.7 Hz, 2H), 1.62–1.54 (m, 2H), 1.40 (s, 10H), 0.86 (d,
J = 5.9 Hz, 6H) ppm; 13C NMR (100 MHz, CDCl3): d 172.1, 171.4,
155.5, 136.0, 135.1, 128.5, 128.4, 127.6, 123.0, 122.2, 119.6,
118.6, 111.2, 109.7, 79.9, 67.2, 53.1, 52.9, 41.4, 28.2, 27.6, 24.6,
22.8, 21.8 ppm; IR (KBr): vmax 3377, 3335, 3284, 2961, 1720,
(4.6 ꢂ 150 mm, 5
lM; Agilent Technologies). Low-resolution and
high-resolution mass spectra were obtained with a Bruker amaZon
SL mass spectrometer and a Bruker Dalton micrOTOF-Q II spec-
trometer respectively at JXSTNU analysis center and reported as
m/z.
4.1.2. Synthesis of phosphoramidion, b anomer, and
intermediates
4.1.2.1. 2,3,4-Tri-O-acetyl-6-deoxy-
slurry of sodium acetate (6.4 g, 78 mmol) in acetic anhydride
(150 mL, 1.58 mol) was added -rhamnose (20.0 g, 122 mmol) in
a-L-mannopyranose (2). To
1665, 1521, 1456, 1364, 1275, 1166, 1047, 1020, 744, 688 cmꢁ1
;
HRMS (ESI+): m/z calcd for C29H38N3O5 [M+H]+ 508.2806; found
508.2815.
L
four portions over 30 min. The reaction was refluxed for 2 h, and
stirred at 20 °C for 2 h. The solution was poured into ice water
(400 mL) and extracted with ethyl acetate (250 mL ꢂ 2). The com-
bined organic phase was washed with saturated NaHCO3 aqueous
solution (300 mL), and dried over anhydrous Na2SO4. Concentra-
tion in vacuo afforded 6 as yellow syrup (38.1 g, 94%). To a solution
of ethylenediamine (10.7 mL, 160 mmol) in THF (200 mL) was
added acetic acid (9.2 mL, 160 mmol). The reaction was stirred
for 30 min. A solution of 6 (38.1 g, 114 mmol) in THF (50 mL)
was added, and the reaction was stirred overnight. After the sol-
vent was removed in vacuo, the residue was dissolved in ethyl ace-
tate (400 mL), washed with deionized H2O (150 mL), saturated
NaHCO3 aqueous solution (150 mL), and 5% HCl aqueous solution
(150 mL), dried with anhydrous Na2SO4, and concentrated in va-
cuo. Flash column chromatography on silica gel (petroleum
ether/ethyl acetate 3:1) afforded 2 (24.1 g, 74%) as a white solid,
mp: 94–95 °C; 1H NMR (400 MHz, CDCl3): d 5.29–5.25 (m, 1H),
5.15 (d, J = 2.0 Hz, 1H), 5.07 (t, J = 9.9 Hz, 1H), 4.15–4.08 (m, 1H),
3.37 (br, 1H), 2.15 (s, 3H), 2.05 (s, 3H), 1.99 (s, 3H), 1.82–1.77 (m,
1H), 1.21 (d, J = 6.2 Hz, 3H) ppm; 13C NMR (100 MHz, CDCl3): d
170.3, 170.1, 92.0, 71.1, 70.2, 68.7, 66.3, 20.9, 20.8, 20.7, 17.4
ppm; IR (KBr): vmax 3435, 2988, 2910, 1732, 1445, 1381, 1222,
1053, 835, 785, 692, 578 cmꢁ1; LRMS (ESI+): m/z calcd for
4.1.2.4. L-Leucine-L-tryptophan benzyl ester (4). Compound 11
(507 mg, 1 mmol) was dissolved in a solution of trifluoroacetic acid
in CH2Cl2 (20% v/v, 20 mL) and stirred at 20 °C for 2 h. After the sol-
vent was removed in vacuo, the residue was dissolved in CH2Cl2
(30 mL), washed with saturated NaHCO3 aqueous solution
(20 mL ꢂ 2), dried with anhydrous Na2SO4, and concentrated in va-
cuo to afford 4 (405 mg, 99%) as a white solid. Due to the concern of
potential cyclization of dipeptide,7b crude 4 was directly applied to
the next step without further purification.
4.1.2.5. Benzyl(5S,8S)-8-(1H-indol-3-ylmethyl)-5-(2-methylpro-
pyl)-6-oxo-1-phenyl-3-{[(2S,3R,4R,5R,6S)-3,4,5-tris(acetyloxy)-
6-methyltetrahydro-2H-pyran-2-yl]oxy}-2-oxa-4,7-diaza-3-
phosphanonan-9-oate-3-oxide (14a). General method for the
synthesis of
a-L-rhamnosyl-1-phosphoramidates: To a solution of
2 (580 mg, 2 mmol) in anhydrous diethyl ether (25 mL) at 0 °C
were added DBU (0.50 mL, 3.4 mmol) and 3 (821 mg, 3.0 mmol)
in anhydrous diethyl ether (10 mL) dropwise under an atmosphere
of argon. The reaction was stirred for 30 min at 0 °C. The precipi-
tated salt was removed by filtration, and the filtrate was concen-
trated in vacuo to afford crude 12. To a solution of 12 in CH3CN
(10 mL) was added 1H-tetrazole (280 mg, 4 mmol) and deionized
H2O (0.1 mL, 5.6 mmol). The reaction was stirred for 30 min. After
the solvent was removed in vacuo, the residue was dissolve in CH2-
Cl2 (50 mL), and washed with 2% HCl aqueous solution (30 mL ꢂ 2)
and deionized H2O (30 mL ꢂ 2). The combined organic phase was
dried with anhydrous Na2SO4 and concentrated in vacuo to afford
crude 13 as yellow syrup. To a solution of 4 (326 mg, 0.8 mmol) in
CH3CN (5 mL) was added TEA (0.21 mL, 1.5 mmol), CCl4 (0.49 mL,
5 mmol) and a solution of crude 12 (444 mg, 1 mmol) in CH3CN
(5 mL) at 0 °C. The reaction was stirred for 30 min at 20 °C. The
reaction was concentrated in vacuo and ethyl acetate (5 mL) was
added to the residue. The precipitation was removed by filtration,
and the filtrate was concentrated in vacuo. Flash column chroma-
tography on silica gel (petroleum ether/ethyl acetate 2:1 to 1:1)
afforded 14a (490 mg, 72%) as a white solid, mp: 63–65 °C; 1H
NMR (400 MHz, CDCl3): d 8.77, 8.40 (s, 1H), 7.49 (d, J = 7.8 Hz,
1H), 7.32–7.22 (m, 10H), 7.14 (dd, J1 = 6.8 Hz, J2 = 11.9 Hz, 1H),
7.08–6.92 (m, 4H), 5.61, 5.33 (d, J = 6.4 Hz, 1H), 5.33 (s, 1H),
5.27–5.23 (m, 1H), 5.10–4.88 (m, 6H), 4.02–3.94 (m, 1H), 3.79–
3.74 (m, 2H), 3.33–3.29 (m, 2H), 2.22 (s, 1H), 2.10–1.97 (m, 9H),
1.71–1.53 (m, 2H), 1.39–1.34 (m, 1H), 1.17, 1.08 (d, J = 6.0 Hz,
3H), 0.89–0.82 (m, 6H) ppm; 13C NMR (100 MHz, CDCl3): d 172.5,
171.5, 169.8, 136.1, 135.7, 135.1, 128.5, 127.5, 123.3, 121.9,
119.3, 118.4, 111.3, 109.4, 94.5, 70.2, 69.2, 68.7, 68.4, 68.2, 67.1,
54.1, 52.7, 43.4, 27.4, 24.2, 22.7, 21.6, 20.6, 17.1 ppm; 31P NMR
(161 MHz, CDCl3): d 6.42, 5.46 ppm; IR (KBr): vmax 3410, 3064,
2962, 1757, 1678, 1517, 1458, 1381, 1222, 1170, 1039, 960, 746,
C
12H19O8 [M+H]+ 291.1; found 291.1.
4.1.2.2. Benzyl-N,N-diisopropylchlorophosphoramidite (3). To a
cooled solution of PCl3 (59.2 mL, 682 mmol) in CH2Cl2 (200 mL)
at 0 °C was added a solution of benzyl alcohol (14.2 mL, 136 mmol)
in CH2Cl2 (50 mL) dropwise over 3 h under an atmosphere of argon.
The reaction was stirred at 20 °C for 2 h. Concentration in vacuo
afforded 8 as light yellow oil (28.5 g, 99%). To a solution of 8
(28.5 g, 134 mmol) in ether (120 mL) was added a solution of diiso-
propylamine (19.3 mL, 136 mmol) and triethylamine (19.0 mL,
136 mmol) in ether (50 mL) dropwise under an atmosphere of ar-
gon at ꢁ20 °C over 3 h. The reaction was stirred overnight at 20 °C.
The triethylammonium chloride was removed by filtration, and the
filtrate was concentrated in vacuo. Vacuum distillation (90 °C, 8
mm Hg) of the residue oil afforded 3 (28.4 g, 82%) as colorless
oil; 1H NMR (400 MHz, CDCl3): d 7.37 (s, 4H), 7.33–7.32 (m, 5H),
4.99–4.91 (m, 2H), 3.90–3.84 (m, 2H), 1.35 (d, J = 6.0 Hz, 6H),
1.27 (d, J = 5.6 Hz, 6H) ppm; 13C NMR (100 MHz, CDCl3): d 137.5,
128.5, 127.9, 127.3, 67.6, 46.1, 46.0, 24.1, 23.3 ppm; 31P NMR
(161 MHz, CDCl3): d 181.57 ppm.
4.1.2.3. N-(tert-Butoxycarbonyl)-L-leucyl-L-tryptophan benzyl
ester (11). To a solution of 9 (231 mg, 1 mmol) in CH2Cl2 (20 mL)
at 0 °C was added EDCꢀHCl (201 mg, 1.05 mmol) and HOBt (148
mg, 1.1 mmol) under an atmosphere of argon. The reaction was
stirred for 30 min. 10 (293 mg, 1 mmol) in CH2Cl2 (5 mL) was
added dropwise and stirred overnight at 20 °C. The reaction solu-
tion was washed with saturated NaHCO3 aqueous solution
696, 603, 495 cmꢁ1; HRMS (ESI+): m/z calcd for C43H53N3O13
P
[M+H]+ 850.3311; found 850.3297.

6784
Q. Sun et al. / Bioorg. Med. Chem. 21 (2013) 6778–6787
4.1.2.6.
Methyl(2S)-2-{[(benzyloxy){[(2S,3R,4R,5R,6S)-3,4,5-
4.98–4.93 (m, 1H), 4.89–4.68 (m, 1H), 4.28–4.05 (m, 1H), 4.01–
3.95 (m, 1H), 3.71, 3.66 (s, 3H), 3.36, 3.28 (t, J = 10.6 Hz, 1H),
3.05, 2.94 (t, J = 5.1 Hz, 2H), 2.14, 2.13 (s, 3H), 2.04, 2.03 (s, 3H),
1.98 (s, 3H), 1.19, 1.12 (d, J = 6.2 Hz, 3H) ppm; 13C NMR
(400 MHz, CDCl3): d 172.6, 169.8, 169.6, 135.7, 129.4, 128.6,
128.4, 127.8, 127.7, 127.1, 94.5, 70.3, 69.2, 68.4, 68.3, 68.2, 55.6,
55.2, 40.3, 20.6, 17.1 ppm; 31P NMR (161 MHz, CDCl3): d 5.44,
4.36 ppm; IR (KBr): vmax 3469, 3049, 2952, 1749, 1517, 1441,
1371, 1220, 1052, 960, 744, 700, 593, 502 cmꢁ1; HRMS (ESI+):
m/z calcd for C29H37NO12P [M+H]+ 622.2048; found 622.2035.
tris(acetyloxy)-6-methyltetrahydro-2H-pyran-2-yl]oxy}phosphoryl]
amino}propanoate (19a). Starting from -alanine methyl ester
L
hydrochloride (112 mg, 0.8 mmol), 19a was synthesized accord-
ing to the procedure described for 14a. Flash column chromatog-
raphy afforded 19a (306 mg, 70%) as colorless syrup; 1H NMR
(400 MHz, CDCl3): d 7.39–7.31 (m, 5H), 5.58, 5.52 (d, J = 7.0 Hz,
1H), 5.29–5.23 (m, 2H), 5.09–5.04 (m, 3H), 4.07–3.90 (m, 2H),
3.72, 3.69 (s, 3H), 3.50 (dd, J1 = 10.2 Hz, J2 = 13.7 Hz, 1H), 2.13,
2.12 (s, 3H), 2.03 (s, 3H), 1.97 (s, 3H), 1.40, 1.32 (d, J = 7.1 Hz,
3H), 1.20, 1.14 (d, J = 6.2 Hz, 3H) ppm; 13C NMR (100 MHz,
CDCl3): d 173.8, 169.6, 135.7, 128.5, 128.4, 127.9, 127.7, 94.5,
70.3, 69.3, 68.7, 68.4, 68.3, 52.4, 50.0, 20.6, 17.1 ppm; 31P NMR
(161 MHz, CDCl3): d 5.14, 4.23 ppm; IR (KBr): vmax 3629, 3211,
4.1.2.10. Methyl(2S)-2-{[(benzyloxy){[(2S,3R,4R,5R,6S)-3,4,5-tris
(acetyloxy)-6-methyltetrahydro-2H-pyran-2-yl]oxy}phosphoryl]
amino}-3-(1H-indol-3-yl)propanoate (23a). Starting from L-tryp-
2592, 1747, 1446, 1377, 1222, 1020, 750, 595 cmꢁ1
(ESI+): m/z calcd for
546.1723.
;
HRMS
tophan methyl ester hydrochloride (204 mg, 0.8 mmol), 23a was
synthesized according to the procedure described for 14a. Flash
column chromatography afforded 23a (360 mg, 68%) as colorless
C
23H33NO12 [M+H]+ 546.1735; found
syrup; 1H NMR (400 MHz, CDCl3):
d 8.58 (s, 1H), 7.51 (d,
4.1.2.7.
tris(acetyloxy)-6-methyltetrahydro-2H-pyran-2-yl]oxy}phosphoryl]
amino}-4-methylpentanoate (20a). Starting from -leucine
Methyl(2S)-2-{[(benzyloxy){[(2S,3R,4R,5R,6S)-3,4,5-
J = 7.6 Hz, 1H), 7.33–7.25 (m, 6H), 7.16–7.04 (m, 3H), 5.55 (d,
J = 6.6 Hz, 1H), 5.26–5.19 (m, 2H), 5.07–4.95 (m, 2H), 4.91–4.86
(m, 1H), 4.28–4.24 (m, 1H), 3.93–3.88 (m, 1H), 3.60 (s, 3H), 3.52
(t, J = 10.7 Hz, 1H), 3.24 (d, J = 5.4 Hz, 2H), 2.12 (s, 3H), 2.03 (s,
3H), 1.98 (s, 3H), 1.07 (d, J = 6.2 Hz, 3H) ppm; 13C NMR
(100 MHz, CDCl3): d 173.0, 169.9, 169.8, 169.7, 136.1, 135.9,
128.5, 128.3, 127.6, 127.5, 123.4, 121.9, 119.4, 118.4, 111.3,
109.3, 94.5, 70.3, 69.3, 68.5, 68.3, 68.2, 54.8, 52.3, 29.9, 20.7,
20.6, 17.1 ppm; 31P NMR (161 MHz, CDCl3): d 5.69 ppm; IR
(KBr): vmax 3293, 2932, 1749, 1434, 1368, 1215, 1151, 1035, 970,
L
methyl ester hydrochloride (145 mg, 0.8 mmol), 20a was synthe-
sized according to the procedure described for 14a. Flash column
chromatography afforded 20a (325 mg, 69%) as colorless syrup;
1H NMR (400 MHz, CDCl3): d 7.38–7.30 (m, 5H), 5.57, 5.50 (d,
J = 6.8 Hz, 1H), 5.28–5.21 (m, 2H), 5.10–5.01 (m, 3H), 4.05–3.96
(m, 1H), 3.92–3.78 (m, 1H), 3.70, 3.66 (s, 3H), 3.43–3.34 (m, 1H),
2.11 (s, 3H), 2.02, 2.01 (s, 3H), 1.95 (s, 3H), 1.75–1.56 (m, 1H),
1.58–1.38 (m, 2H), 1.20, 1.11 (d, J = 6.2 Hz, 3H), 0.91 (t, J = 6.7 Hz,
3 H), 0.86 (t, J = 5.8 Hz, 3H) ppm; 13C NMR (100 MHz, CDCl3): d
174.0, 169.7, 169.5, 135.8, 128.5, 128.4, 127.9, 127.7, 94.6, 70.3,
69.2, 68.7, 68.4, 68.1, 52.9, 52.2, 43.6, 24.3, 22.5, 21.8, 20.6, 20.5,
17.2 ppm; 31P NMR (161 MHz, CDCl3): d 6.12, 4.90 ppm; IR
(KBr): vmax 3647, 3496, 3215, 2956, 1747, 1373, 1218, 1052, 954,
744, 682, 600 cmꢁ1; HRMS (ESI+): m/z calcd for C31H38N2O12
P
[M+H]+ 661.2157; found 661.2142.
4.1.2.11. (2S,3R,4R,5R,6S)-2-{[(Benzyloxy)(dodecylamino)phos-
phoryl]oxy}-6-methyltetrahydro-2H-pyran-3,4,5-triyltriacetate
(24a). Starting from dodecylamine (177 mg, 0.8 mmol), 24a was
synthesized according to the procedure described for 14a. Flash
column chromatography afforded 24a (352 mg, 70%) as colorless
syrup; 1H NMR (400 MHz, CDCl3): d 7.40–7.30 (m, 5H), 5.57, 5.53
(d, J = 7.2 Hz, 1H), 5.31–5.25 (m, 2H), 5.10–5.05 (m, 3H), 4.07–
3.96 (m, 1H), 2.94–2.84 (m, 2H), 2.75–2.70 (m, 1H), 2.14 (s,
3H), 2.04, 2.03 (s, 3H), 1.98 (s, 3H), 1.45–1.41 (m, 2H), 1.23–
1.13 (m, 21H), 0.86 (t, J = 6.4 Hz, 3H) ppm; 13C NMR (100 MHz,
CDCl3): d 169.8, 136.0, 128.5, 128.4, 127.8, 127.7, 94.3, 70.4,
69.4, 68.4, 68.3, 68.1, 41.4, 31.8, 31.6, 29.6, 29.3, 29.2, 26.5,
22.6, 20.7, 17.3, 14.1 ppm; 31P NMR (161 MHz, CDCl3): d 6.93,
6.12 ppm; IR (KBr): vmax 3475, 3255, 2925, 2858, 1753, 1462,
739, 603, 499 cmꢁ1; HRMS (ESI+): m/z calcd for C26H39NO12
P
[M+H]+ 588.2204; found 588.2217.
4.1.2.8.
tris(acetyloxy)-6-methyltetrahydro-2H-pyran-2-yl]oxy}phosphoryl]
amino}pentanedioate (21a). Starting from -glutamic dimethyl
Dimethyl(2S)-2-{[(benzyloxy){[(2S,3R,4R,5R,6S)-3,4,5-
L
ester hydrochloride (212 mg, 0.8 mmol), 21a was synthesized
according to the procedure described for 14a. Flash column
chromatography afforded 21a (311 mg, 63%) as colorless syrup;
1H NMR (400 MHz, CDCl3): d 7.40–7.31 (m, 5H), 5.57, 5.51 (d,
J = 6.8 Hz, 1H), 5.28–5.20 (m, 2H), 5.10–5.03 (m, 3H), 4.02–
3.86 (m, 2H), 3.73, 3.70 (s, 3H), 3.63, 3.62 (s, 3H), 3.58–3.53
(m, 1H), 2.44–2.35 (m, 2H), 2.12 (s, 3H), 2.03 (d, J = 3.4 Hz,
4H), 1.96 (s, 3H), 1.93–1.85 (m, 1H), 1.20, 1.12 (d, J = 6.2 Hz,
3H) ppm; 13C NMR (100 MHz, CDCl3): d 172.8, 169.7, 169.5,
135.7, 128.5, 128.4, 127.9, 94.6, 70.3, 69.2, 68.8, 68.5, 68.4,
68.2, 53.6, 52.5, 51.6, 29.6, 29.3, 20.6, 17.1 ppm; 31P NMR
1371, 1220, 1153, 2049, 960, 740, 597, 507 cmꢁ1
;
HRMS
(ESI+): m/z calcd for
C31H51NO10P
[M+H]+ 628.3245; found
628.3258.
4.1.2.12. (2S,3R,4R,5R,6S)-2-{[(Benzyloxy)(morpholin-4-yl)phos-
phoryl]oxy}-6-methyltetrahydro-2H-pyran-3,4,5-triyltriacetate
(25a). Starting from morpholine (70 mg, 0.8 mmol), 25a was syn-
thesized according to the procedure described for 14a. Flash col-
umn chromatography afforded 25a (297 mg, 70%) as colorless
syrup; 1H NMR (400 MHz, CDCl3): d 7.39–7.33 (m, 5H), 5.56, 5.52
(d, J = 7.0 Hz, 1H), 5.32–5.19 (m, 2H), 5.11–5.03 (m, 3H), 4.04–
3.87 (m, 1H), 3.60–3.53 (m, 4H), 3.14–3.05 (m, 4H), 2.13, 2.12 (s,
3H), 2.03, 2.02 (s, 3H), 1.97 (s, 3H), 1.23, 1.13 (d, J = 6.2 Hz, 3H)
ppm; 13C NMR (400 MHz, CDCl3): d 169.9, 169.7, 169.6, 135.7,
128.6, 128.0, 127.8, 94.3, 70.2, 69.3, 68.7, 68.5, 68.3, 66.7, 44.4,
20.6, 17.2 ppm; 31P NMR (161 MHz, CDCl3): d 6.12, 5.26 ppm; IR
(KBr): vmax 3642, 3483, 2976, 2863, 1749, 1447, 1373, 1218,
1060, 958, 704, 606, 482 cmꢁ1; HRMS (ESI+): m/z calcd for
C23H33NO11P [M+H]+ 530.1786; found 530.1772.
(161 MHz, CDCl3):
d 5.95, 4.96 ppm; IR (KBr): vmax 3649,
2954, 1741, 1452, 1373, 1220, 1046, 947, 750, 595, 502 cmꢁ1
;
HRMS (ESI+): m/z calcd for
C26H37NO14P
[M+H]+ 618.1946;
found 618.1958.
4.1.2.9.
tris(acetyloxy)-6-methyltetrahydro-2H-pyran-2-yl]oxy}phosphoryl]
amino}-3-phenylpropanoate (22a). Starting from -phenylalanine
Methyl(2S)-2-{[(benzyloxy){[(2S,3R,4R,5R,6S)-3,4,5-
L
methyl ester hydrochloride (133 mg, 0.8 mmol), 22a was synthe-
sized according to the procedure described for 14a. Flash column
chromatography afforded 22a (333 mg, 67%) as colorless syrup;
1H NMR (400 MHz, CDCl3): d 7.35–7.11 (m, 10H), 5.50, 5.42
(d, J = 6.7 Hz, 1H), 5.26–5.19 (m, 2H), 5.05 (t, J = 10.0 Hz, 1H),

Q. Sun et al. / Bioorg. Med. Chem. 21 (2013) 6778–6787
6785
4.1.2.13.
Benzyl(5S,8S)-8-(1H-indol-3-ylmethyl)-5-(2-methyl-
4.1.2.16. Dimethyl (2S)-2-{[(benzyloxy){[(2R,3R,4R,5R,6S)-3,4,
5-tris(acetyloxy)-6-methyltetrahydro-2H-pyran-2-yl]oxy}phos-
phoryl]amino}pentanedioate (21b). Starting from L-glutamic di-
propyl)-6-oxo-1-phenyl-3-{[(2R,3R,4R,5R,6S)-3,4,5-tris(acetyl-
oxy)-6-methyltetrahydro-2H-pyran-2-yl]oxy}-2-oxa-4,7-diaza-
3-phosphanonan-9-oate-3-oxide (14b). General method for the
methyl hydrochloride (212 mg, 0.8 mmol), 21b was synthesized
according to the procedure described for 14b. Flash column chro-
matography afforded 21b (237 mg, 48%) as colorless syrup; 1H
NMR (400 MHz, CDCl3): d 7.34–7.25 (m, 5H), 5.46–5.37 (m, 2H),
5.08–4.92 (m, 4H), 3.90–3.81 (m, 1H), 3.68 (s, 3H), 3.63 (d,
J = 7.6 Hz, 2H), 3.59 (s, 3H), 2.43–2.30 (m, 2H), 2.13 (s, 3H), 2.07
(s, 1H), 2.02 (s, 3H), 1.95 (s, 3H), 1.90–1.79 (m, 1H), 1.25, 1.23 (d,
J = 6.1 Hz, 3H) ppm; 13C NMR (100 MHz, CDCl3): d 172.9, 170.2,
169.8, 135.8, 128.5, 128.4, 127.7, 93.5, 71.1, 70.5, 70.0, 69.0, 68.6,
53.3, 52.5, 51.6, 29.4, 29.1, 20.6, 17.2 ppm; 31P NMR (161 MHz,
CDCl3): d 5.92 ppm; IR (KBr): vmax 3647, 3467, 2952, 1741, 1467,
1436, 1373, 1220, 1056, 928, 744, 595, 505 cmꢁ1; HRMS (ESI+):
m/z calcd for C26H37NO14P [M+H]+ 618.1946; found 618.1933.
synthesis of b-L-rhamnosyl-1-phosphoramidates: To a solution
of 2 (580 mg, 2.0 mmol) in anhydrous CH2Cl2 (3.0 mL) at 35 °C
was added Et3N (0.47 mL, 3.4 mmol) and a solution of 3 (821
mg, 3.0 mmol) in CH2Cl2 (1.4 mL) dropwise under an atmosphere
of dry argon. The reaction was stirred for 30 min at 35 °C. The pre-
cipitated salt was removed by filtration, and the filtrate was con-
centrated in vacuo to afford crude 12. The acid-catalyzed
hydrolysis and oxidative coupling were performed according to
the procedures described for 14a. Flash column chromatography
(petroleum ether/ethyl acetate 3:1 to 2:1) afforded 14b (360
mg, 53%) as a white solid; mp: 64–66 °C; 1H NMR (400 MHz,
CDCl3): d 8.71, 8.55 (s, 1H), 7.49, 7.47 (d, J = 7.8 Hz, 1H), 7.32–
7.19 (m, 10H), 7.14–6.90 (m, 4H), 5.49–5.31 (m, 2H), 5.06 (s,
1H), 5.05–4.83 (m, 6H), 3.76–3.64 (m, 2H), 3.55–3.36 (m, 3H),
2.33 (s, 1H), 2.10, 2.06 (s, 3H), 2.03 (s, 3H), 1.99, 1.96 (s, 3H),
1.70–1.49 (m, 2H), 1.38–1.29 (m, 1H), 1.21, 1.14 (d, J = 6.1 Hz,
3H), 0.82–0.76 (m, 6H) ppm; 13C NMR (100 MHz, CDCl3): d
172.6, 171.6, 170.3, 169.9, 136.1, 135.8, 135.3, 128.4, 127.7,
123.4, 121.9, 119.4, 118.5, 111.4, 109.5, 93.5, 71.1, 70.6, 70.2,
69.2, 68.6, 67.1, 53.9, 52.9, 43.3, 27.5, 24.2, 22.7, 21.8, 20.6, 17.2
ppm; 31P NMR (161 MHz, CDCl3) d 6.49 ppm; IR (KBr): vmax
3413, 3066, 2953, 1752, 1661, 1520, 1460, 1368, 1215, 1046,
925, 739, 598, 496 cmꢁ1; HRMS (ESI+): m/z calcd for C43H53N3O13-
P [M+H]+ 850.3311; found 850.3325.
4.1.2.17.
tris(acetyloxy)-6-methyltetrahydro-2H-pyran-2-yl]oxy}phosphoryl]
amino}-3-phenylpropanoate (22b). Starting from -phenylalaine
Methyl(2S)-2-{[(benzyloxy){[(2R,3R,4R,5R,6S)-3,4,5-
L
methyl ester hydrochloride (133 mg, 0.8 mmol), 22b was synthe-
sized according to the procedure described for 14b. Flash column
chromatography afforded 22b (224 mg, 45%) as colorless syrup;
1H NMR (400 MHz, CDCl3): d 7.35–7.08 (m, 10H), 5.49–5.18 (m,
2H), 5.06–4.78 (m, 4H), 4.26–4.13 (m, 1H), 3.68, 3.62 (s, 3H),
3.54–3.38 (m, 2H), 3.09–2.90 (m, 2H), 2.13 (d, J = 6.7 Hz, 3H),
2.05 (s, 3H), 1.98 (s, 3H), 1.23 (d, J = 6.1 Hz, 3H) ppm; 13C NMR
(100 MHz, CDCl3): d 172.4, 170.3, 170.1, 169.8, 135.8, 129.5,
129.4, 128.5, 128.3, 127.8, 127.7, 127.1, 93.4, 71.2, 70.5, 70.1,
69.1, 68.4, 55.2, 52.3, 40.3, 20.7, 17.2 ppm; 31P NMR (161 MHz,
CDCl3): d 5.62 ppm; IR (KBr): vmax 3479, 2979, 1749, 1446, 1373,
1220, 1029, 869, 747, 691, 600, 490 cmꢁ1; HRMS (ESI+): m/z calcd
for C29H37NO12P [M+H]+ 622.2048; found 622.2061.
4.1.2.14.
tris(acetyloxy)-6-methyltetrahydro-2H-pyran-2-yl]oxy}phosphoryl]
amino}propanoate (19b). Starting from -alanine methyl ester
Methyl(2S)-2-{[(benzyloxy){[(2R,3R,4R,5R,6S)-3,4,5-
L
hydrochloride (112 mg, 0.8 mmol), 19b was synthesized accord-
ing to the procedure described for 14b. Flash column chromatog-
raphy afforded 19b (209 mg, 48%) as colorless syrup; 1H NMR
(400 MHz, CDCl3): d 7.39–7.26 (m, 5H), 5.58, 5.52 (d, J = 6.8 Hz,
1H), 5.27–5.23 (m, 2H), 5.09–5.03 (m, 3H), 4.0–3.90 (m, 2H),
3.72, 3.69 (s, 3H), 3.54 (t, J = 9.6 Hz, 1H), 2.12 (s, 3H), 2.03 (s,
3H), 1.96 (s, 3H), 1.39, 1.32 (d, J = 7.2 Hz, 3H), 1.19, 1.14 (d,
J = 6.2 Hz, 3H) ppm; 13C NMR (100 MHz, CDCl3): d 172.9, 169.2,
169.0, 168.7, 134.9, 127.5, 126.8, 126.7, 92.4, 70.1, 69.5, 69.0,
68.1, 67.5, 51.4, 48.7, 19.7, 19.5, 16.2 ppm; 31P NMR (161MHz,
CDCl3): d 5.53 ppm; IR (KBr): vmax 3344, 2985, 1747, 1444,
1373, 1218, 1056, 739, 592, 505 cmꢁ1; HRMS (ESI+): m/z calcd
for C23H33NO12P [M+H]+ 546.1735; found 546.1749.
4.1.2.18.
tris(acetyloxy)-6-methyltetrahydro-2H-pyran-2-yl]oxy}phosphoryl]
amino}-3-(1H-indol-3-yl)propanoate (23b). Starting from -tryp-
Methyl(2S)-2-{[(benzyloxy){[(2R,3R,4R,5R,6S)-3,4,5-
L
tophan methyl ester hydrochloride (204 mg, 0.8 mmol), 23b was
synthesized according to the procedure described for 14b. Flash
column chromatography afforded 23b (248 mg, 47%) as colorless
syrup; 1H NMR (400 MHz, CDCl3): d 8.62, 8.47 (s, 1H), 7.56, 7.53
(d, J = 7.8 Hz, 1H), 7.38–7.26 (m, 6H), 7.19–6.99 (m, 3H), 5.50–
4.82 (m, 6H), 4.32–4.26 (m, 1H), 3.72–3.06 (m, 7H), 2.12, 2.10 (s,
3H), 2.06, 2.05 (s, 3H), 2.01, 1.99 (s, 3H), 1.16 (d, J = 6.1 Hz, 3H)
ppm; 13C NMR (100 MHz, CDCl3): d 173.1, 170.4, 170.1, 169.8,
136.0, 135.9, 128.5, 128.3, 127.8, 127.2, 123.5, 122.0, 119.5,
118.7, 111.3, 109.7, 93.5, 71.1, 70.9, 70.7, 70.5, 70.0, 69.0, 68.3,
54.5, 52.4, 30.2, 20.7, 20.6, 17.2 ppm; 31P NMR (161 MHz, CDCl3):
d 6.38, 5.87 ppm; IR (KBr): vmax 3348, 2947, 1753, 1460, 1379,
1232, 1055, 927, 875, 740, 592 cmꢁ1; HRMS (ESI+): m/z calcd for
4.1.2.15.
tris(acetyloxy)-6-methyltetrahydro-2H-pyran-2-yl]oxy}phosphoryl]
amino}-4-methylpentanoate (20b). Starting from -leucine
Methyl(2S)-2-{[(benzyloxy){[(2R,3R,4R,5R,6S)-3,4,5-
C
31H38N2O12P [M+H]+ 661.2157; found 661.2148.
L
methyl ester hydrochloride (145 mg, 0.8 mmol), 20b was synthe-
sized according to the procedure described for 14b. Flash column
chromatography afforded 20b (230 mg, 49%) as colorless syrup;
1H NMR (400 MHz, CDCl3): d 7.37–7.31 (m, 5H), 5.58, 5.50 (d,
J = 6.84, 1H), 5.27–5.22 (m, 2H), 5.09–5.03 (m, 3H), 4.04–3.97 (m,
1H), 3.92–3.80 (m, 1H), 3.72, 3.68 (s, 3H), 3.26 (dd, J1 = 10.4 Hz,
J2 = 17.9 Hz, 1H), 2.13 (s, 3H), 2.03 (s, 3 H), 1.97 (s, 3H), 1.76–
1.67 (m, 1H), 1.56–1.38 (m, 2H), 1.21, 1.12 (d, J = 6.1 Hz, 3H),
0.91–0.85 (m, 6H) ppm; 13C NMR (100 MHz, CDCl3): d 174.0,
169.8, 169.7, 169.6, 135.8, 128.5, 128.4, 127.9, 127.7, 94.6, 70.3,
69.2, 68.7, 68.4, 68.1, 52.9, 52.2, 43.7, 24.4, 22.6, 21.8, 20.7, 20.5,
17.5 ppm; 31P NMR (161 MHz, CDCl3): d 6.34 ppm; IR (KBr): vmax
3631, 3492, 3211, 1747, 1460, 1373, 1218, 1055, 957, 743, 607,
4.1.2.19. (2R,3R,4R,5R,6S)-2-{[(Benzyloxy)(dodecylamino)phos-
phoryl]oxy}-6-methyltetrahydro-2H-pyran-3,4,5-triyltriacetate
(24b). Starting from dodecylamine (177 mg, 0.8 mmol), 24b was
synthesized according to the procedure described for 14b. Flash
column chromatography afforded 24b (226 mg, 45%) as colorless
syrup; 1H NMR (400 MHz, CDCl3); d 7.35–7.25 (m, 5H), 5.46 (d,
J = 14.4 Hz, 1H), 5.40 (t, J = 8.2 Hz, 1H), 5.06–5.01 (m, 3H), 4.99–
4.92 (m, 1H), 3.61–3.56 (m, 1H), 2.88–2.81 (m, 3H), 2.13, 2.10 (s,
3H), 2.01 (s, 3H), 1.95 (s, 3H), 1.39 (d, J = 5.6 Hz, 2H), 1.25–1.20
(m, 21H), 0.84 (t, J = 6.6 Hz, 3H) ppm; 13C NMR (100 MHz, CDCl3):
d 170.1, 169.9, 169.6, 136.0, 128.4, 128.3, 128.1, 127.7, 127.6, 93.4,
71.0, 70.5, 70.1, 69.2, 68.2, 41.2, 31.7, 31.4, 29.4, 29.1, 26.4, 22.5,
20.5, 17.3, 13.9 ppm; 31P NMR (161 MHz, CDCl3): d 8.33 ppm; IR
(KBr): vmax 3479, 3240, 2927, 2856, 1755, 1460, 1373, 1220,
460 cmꢁ1
; HRMS (ESI+): m/z calcd for C26H39NO12P
[M+H]+
588.2204; found 588.2213.

6786
Q. Sun et al. / Bioorg. Med. Chem. 21 (2013) 6778–6787
1163, 1049, 970, 732, 599, 493 cmꢁ1; HRMS (ESI+): m/z calcd for
31H51NO10P [M+H]+ 628.3245; found 628.3257.
3282, 2956, 1662, 1579, 1519, 1404, 1236, 1099, 1051, 931,
883, 837, 742, 686, 587, 526, 491 cmꢁ1; HRMS (ESIꢁ): m/z calcd
for C23H33N3O10P [MꢁH]ꢁ 542.1909; found 542.1916.
C
4.1.2.20. (2R,3R,4R,5R,6S)-2-{[(Benzyloxy)(morpholin-4-yl)phos-
phoryl]oxy}-6-methyltetrahydro-2H-pyran-3,4,5-triyltriacetate
(25b). Starting from morpholine (0.07 mL, 0.8 mmol), 25b was
synthesized according to the procedure described for 14b. Flash
column chromatography afforded 25b (203 mg, 48%) as colorless
syrup; 1H NMR (400 MHz, CDCl3): d 7.37–7.32 (m, 5H), 5.50,
5.49 (d, J = 11.6 Hz, 1H), 5.44, 5.38 (d, J = 8.4 Hz, 1H), 5.11–4.92
(m, 4H), 3.64–3.61 (m, 1H), 3.58–3.52 (m, 4H), 3.09–3.02 (m, 4H),
2.16, 2.13 (s, 3H), 2.05 (s, 3H), 1.98 (s, 3H), 1.28 (t, J = 6.0 Hz, 3H)
ppm; 13C NMR (400 MHz, CDCl3): d 170.0, 169.9, 169.7, 135.8,
128.5, 127.9, 93.5, 71.2, 70.6, 70.1, 69.2, 68.5, 66.6, 44.4, 20.6,
17.4 ppm; 31P NMR (161 MHz, CDCl3): d 6.30, 5.77 ppm; IR (KBr):
vmax 3473, 2978, 2863, 2922, 1749, 1642, 1469, 1373, 1218, 1157,
4.2. Enzymatic assays
4.2.1. Determination of the inhibitor constants (Ki) of 1 and 15
The Ki values were determined in a 50 mM Tris–HCl, 10 mM
CaCl2 buffer (pH 7.5) with FA-Gly-Leu-NH2 (FAGLA, Bachem) as a
substrate by using an Agilent 8453 UV–vis spectrophotometer in
triplicate. Henderson plots were employed for the calculation of
Ki values.
20 L), and thermolysin (Sigma, 40 nM, 10
25 °C for 15 min in cuvette (2 mL).
A mixture of buffer (970 lL), inhibitor (0–80 nM,
l
l
L) was incubated at
a
A solution of FAGLA
(0.1–0.5 mM, 1.0 mL) in Tris buffer pH 7.5 was added into the cuv-
ette. The absorbance decrease upon cleavage of FAGLA by thermol-
ysin was recorded at 340 nm wavelength for 5 min. The
concentration of thermolysin was determined from the molar
1060, 956, 704, 607, 489 cmꢁ1
;
HRMS (ESI+): m/z calcd for
C
23H33NO11P [M+H]+ 530.1786; found 530.1796.
extinction coefficient e278 = 66,300. The Ki values of 1 and 15 were
4.1.2.21.
(2S)-2-({(2S)-2-[(Hydroxy{[(2S,3R,4R,5R,6S)-3,4,5-tri-
determined according to Eq. 1. When E0 was fixed, dose-response
hydroxy-6-methyltetrahydro-2H-pyran-2-yl]oxy}phosphoryl)
amino]-4-methylpentanoyl}amino)-3-(1H-indol-3-yl)propio-
nate, disodium salt (1, phosphoramidon). To a solution of 14a
(85 mg, 0.1 mmol) in anhydrous MeOH (20 mL) was added 5%
Pd/C (15 mg). The reaction was stirred under an atmosphere of
hydrogen for 3 h at 20 °C. Then, NaOMe (32 mg, 0.6 mmol) in
anhydrous MeOH (6 mL) was added to the solution dropwise at
0 °C and stirred for 3 h at 20 °C. The pH value of the reaction
solution was adjusted to 7.5 by adding a solution of aqueous
HCl in MeOH (0.12 M). Removal of Pd/C by filtration and concen-
tration in vacuo afforded crude 1 as a white solid. Size-exclusion
chromatography (Sephadex LH-20, deionized H2O, 2.5 ꢂ 70 cm)
afforded 1 (54 mg, 92%) as disodium salt, a white solid; mp:
measurements were repeated at increasing concentrations of At.
The slopes of plots of It/(1ꢁvi/v0) against v0/vi increases for this
competitive binding. Replots of the slopes against At were linear
for competitive binding, and the extrapolated intercepts on the
ordinate yielded the Ki values.
It=ð1 ꢁ vi
=
v0Þ ¼ Et þ Ki½ðAt þ KaÞ=Kaꢃv0
=vi
ð1Þ
4.2.2. Determination of the dissociation constants (Kd) of the E-
1 and E-15 complexes
The fluorescence spectra of thermolysin (E), 1, 15, and their
binding complexes at the same molar concentration were re-
corded with a Hitachi F-4600 fluorescence spectrophotometer
with kex = 280 nm. To a solution of thermolysin (1.15 ꢂ 10ꢁ6 M,
176–179 °C [Commercial phosphoramidon (Sigma) from
a
20
microbial source, mp: 173–178 °C]; [
a
]
D = ꢁ17.86° (c = 1.0,
D = ꢁ15.7° (H2O) for di-dicyclohexylammonium
1H NMR (400 MHz, D2O): d 7.68 (d, J = 7.8 Hz, 1H), 7.50
980 lL) in Tris buffer pH 7.5 in a cuvette was added a solution
20
H2O) [Lit. [
a]
of the inhibitor (1.15 ꢂ 10ꢁ4 M, 2
lL) in Tris buffer pH 7.5. The
mixed solution was incubated at 25 °C for 15 min. The percentage
salt ]1a
;
(d, J = 8.0 Hz, 1H), 7.26–7.15 (m, 3H), 5.26 (d, J = 7.8 Hz, 1H),
4.54 (t, J = 6.0 Hz, 1H), 3.85–3.77 (m, 3H), 3.49–3.38 (m, 3H),
3.20, 3.16 (d, J = 7.6 Hz, 1H), 1.53 (br, 1H), 1.27 (d, J = 5.8 Hz,
3H), 1.17–1.11 (m, 1H), 0.95–0.88 (m, 1H), 0.83 (d, J = 6.5 Hz,
3H), 0.79 (d, J = 6.5 Hz, 3H) ppm; 13C NMR (100 MHz, D2O): d
178.5, 177.1, 136.3, 127.3, 124.5, 121.9, 119.3, 119.0, 111.9,
110.4, 95.5, 72.4, 71.3, 70.0, 69.6, 55.8, 54.9, 43.2, 27.9, 24.0,
22.6, 21.2, 17.1 ppm; 31P NMR (161 MHz, D2O): d 4.06 ppm; IR
(KBr): vmax 3508, 3425, 3383, 3278, 2952, 1166, 1579, 1516,
1402, 1234, 1101, 1047, 835, 746, 586, 526, 497 cmꢁ1; HRMS
(ESIꢁ): m/z calcd for C23H33N3O10P [MꢁH]ꢁ 542.1909; found
542.1921.
of the increment of fluorescence intensity relative to the total
fluorescence intensity (
titration was repeated by adding a solution of the inhibitor
(2 L) until the total fluorescence intensity remained constant.
The values of f and Kd were determined as adjustable parame-
DF/(FE + FI)) was recorded at 360 nm. The
l
D
ters by the nonlinear least-squares method according to Eqs.
(2)–(4) in triplicate.
qffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffi
1
2
2
D
F ¼ ð½Eꢃ0 þ ½Iꢃ0 þ Kd ꢁ ð½Eꢃ0 þ ½Iꢃ0 þ KdÞ ꢁ 4½Eꢃ0½Iꢃ0Þ
D
f
ð2Þ
D
D
F ¼ FEI ꢁ FE ꢁ FI
ð3Þ
ð4Þ
4.1.2.22. (2S)-2-({(2S)-2-[(Hydroxy{[(2R,3R,4R,5R,6S)-3,4,5-tri-
hydroxy-6-methyltetrahydro-2H-pyran-2-yl]oxy}phosphoryl)
amino]-4-methylpentanoyl}amino)-3-(1H-indol-3-yl)propio-
nate, disodium salt (15). Starting from 14b (85 mg, 0.1 mmol),
compound 15 was synthesized according to the procedure de-
scribed for 1. Recrystallization from ethyl acetate/MeOH/H2O
Fmax
¼ Df½Eꢃ0
Acknowledgments
We thank the National Natural Science Foundation of China
(No. 21262014), Natural Science Foundation of Jiangxi Province
(No. 20114BAB203008), Project of the Science Funds of Jiangxi
Education Office (No. GJJ12589), Key Project of Chinese Ministry
of Education (No. 212092), and Scientific Research Foundation of
Chinese Ministry of Human Resources and Social Security for Re-
turned Chinese Scholars for financial support.
afforded 15 (55 mg, 93%) as disodium salt, a white solid; mp:
20
204–206 °C; [
a
]
D = ꢁ4.88° (c = 1.0, H2O); 1H NMR (400 MHz,
D2O): d 7.72 (d, J = 7.2 Hz, 1H), 7.52 (d, J = 7.6Hz, 1H), 7.25–
7.19 (m, 3H), 4.99 (d, J = 8.0 Hz, 1H), 4.56 (s, 1H), 3.88 (s, 1H),
3.59–3.38 (m, 5H), 3.23–3.18 (m, 1H), 1.54 (s, 1H), 1.30 (s, 3H),
1.20 (s, 1H), 1.00 (s, 1H), 0.83, 0.79 (d, J = 5.2 Hz, 6H) ppm; 13C
NMR (100 MHz, D2O):
d 178.4, 176.9, 136.2, 127.7, 124.4,
Supplementary data
121.8, 119.2, 119.0, 111.9, 110.3, 94.9, 72.6, 72.5, 72.0, 71.5,
55.8, 54.8, 43.3, 27.8, 23.9, 22.5, 21.4, 17.0 ppm; 31P NMR
(161 MHz, D2O): d 4.90 ppm; IR (KBr): vmax 3504, 3417, 3381,
Supplementary data associated with this article can be found, in
the online version, at http://dx.doi.org/10.1016/j.bmc.2013.07.052.

Q. Sun et al. / Bioorg. Med. Chem. 21 (2013) 6778–6787
6787
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the a a/b
/b anomer ratios of 12 were estimated from 13C NMR of C1 atom. The
ratios estimated by 13C NMR method were close to the values determined by
isolated yields of 14a and 14b (within 10% difference). For examples of
estimation of diastereomeric ratios by 13C NMR, see: (a) Helder, R.; Arends, R.;