Synthesis of sterically hindered peptide analogs using diphenyl phosphite as the coupling reagent

Article abstract of DOI:10.1016/j.bioorg.2005.05.003

Some model sterically hindered peptide analogs were synthesized with various yields using diphenyl phosphite as the coupling reagent under mild conditions. The experimental procedure is straightforward and the products are easily isolated. This method may be convenient and efficient for the synthesis of hindered peptides.

Full text of DOI:10.1016/j.bioorg.2005.05.003

Bioorganic Chemistry 33 (2005) 386–392
www.elsevier.com/locate/bioorg
Synthesis of sterically hindered peptide analogs
using diphenyl phosphite as the coupling reagent
Ting Yang a, Changxue Lin a, Hua Fu a,¤, Yuyang Jiang a,b,
Yufen Zhao a
a Key Laboratory of Bioorganic Phosphorus Chemistry and Chemical Biology, Ministry of Education,
Department of Chemistry, Tsinghua University, Beijing 100084, PR China
b Key Laboratory of Chemical Biology, Guangdong Province, Graduate School of Shenzhen,
Tsinghua University, Shenzhen 518057, PR China
Received 20 March 2005
Available online 11 July 2005
Abstract
Some model sterically hindered peptide analogs were synthesized with various yields using
diphenyl phosphite as the coupling reagent under mild conditions. The experimental procedure
is straightforward and the products are easily isolated. This method may be convenient and
eYcient for the synthesis of hindered peptides.
2005 Elsevier Inc. All rights reserved.
Keywords: Hindered peptide; Coupling reagent; Diphenyl phosphite; Secondary amine
1. Introduction
Peptides exhibit a wide variety of biological activities [1,2]. Some have also
served as drugs or lead compounds in drug development and others have proven
useful in studies directed toward the elucidation of biochemical pathways [3,4].
Hence, it is important to develop eYcient approaches for the synthesis of peptides.
*
Corresponding author. Fax: +86 10 62781695.
E-mail address: fuhua@mail.tsinghua.edu.cn (H. Fu).
0045-2068/$ - see front matter 2005 Elsevier Inc. All rights reserved.
doi:10.1016/j.bioorg.2005.05.003

T. Yang et al. / Bioorganic Chemistry 33 (2005) 386–392
387
In recent years, peptide coupling reactions have signiWcantly advanced due to the
development of eYcient coupling reagents [5]. Coupling reagents responsible for
the formation of azide, mixed anhydride, and acid halide intermediates have gained
substantial popularity in peptide coupling reactions [6]. Other kinds of coupling
reagents have also been developed, such as phosphonium reagents [7], uronium
reagents [8], immonium reagents [9], carbodiimide reagents [10], imidazolium
reagents [11], organophosphorus reagents [12], and acid halogenating reagents [13].
However, peptides isolated from fungi, bacteria, marine sponges, and other lower
animal forms, are usually N-methylated and exhibit a variety of biological activi-
ties. The chain elongations of these sterically hindered peptides are not readily
accomplished using ordinary coupling reagents [14]. The development of eYcient
coupling reagents for hindered peptides has attracted much attention. Preparation
of several simple model peptides was studied using diphenyl phosphite (DPP) and
triphenyl phosphite as coupling reagents in the presence of tertiary amines in the
1970s [15,16]. No further progress has not been reported over the past 30 years.
Here, we have explored the utility of this reagent (DPP) for the one-pot conversion
of acids and sterically hindered amines to amides.
2. Materials and methods
1
Pyridine was dried over CaH2 and freshly distilled. H NMR and 13C NMR
spectra were recorded on a JOEL JNM-ECA 300 spectrometer using tetramethylsi-
lane as the internal standard. High resolution mass spectra were recorded on a Bru-
ker APEX-II fourier transform ion cyclotron resonance (FT-ICR) MS instrument
equipped with 4.7 T super-conduction magnet and an analytical electrospray
source.
2.1. General procedure for synthesis of sterically hindered peptides
The acid (1 mmol) and amine (1 mmol) were dissolved in 5 mL of anhydrous
pyridine. Subsequently, diphenyl phosphate (2 mmol) in 3 mL of dry pyridine was
added dropwise to the solution at 0 °C. The reaction temperature was warmed to
room temperature and the stirring was lasted about 1.5 h. The solvent was removed
under reduced pressure and puriWcation via silica gel column chromatography pro-
vided the corresponding amides using petroleum:ethyl acetate (ratio from 7:1 to
1:3) as eluent.
2.2. Phenyl-piperidin-1-yl-methanone (1)
1H NMR (300 MHz, CDCl3): ꢀ 7.37 (s, 5H, aromatic), 3.69 (br, 2H, N–CH2), 3.32
(br, 2H, N–CH2), 1.51–1.65 (m, 6H, CH2CH2CH2). 13C NMR (75 MHz, CDCl3): ꢀ
170.00 (CO), 136.12, 129.05, 128.07, 126.46 (–Ph), 48.46, 42.81 (2£ N–CH2), 26.18,
25.36, 24.23 (CH2CH2CH2). Positive ion HR-ICRESI-MS: [M+H]+ m/z Cacld
190.1232. Found 190.1241.

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T. Yang et al. / Bioorganic Chemistry 33 (2005) 386–392
2.3. Morpholin-4-yl-phenyl-methanone (2)
1H NMR (300 MHz, CDCl3): ꢀ 7.40 (s, 5H, aromatic), 3.47–3.67 (m, 8H,
N(C2H4)2O). 13C NMR (75 MHz, CDCl3):
ꢀ 170.04 (Ph–CO), 135.00,
129.53, 128.22, 126.76 (–Ph), 66.51 (2 £ O–CH2), 47.86, 42.28 (2 £ N–CH2).
Positive ion HR-ICRESI-MS: [M+H]+ m/z Cacld 192.1025. Found
192.1032.
2.4. N-Methyl-N-phenyl-benzamide (3)
1H NMR (300MHz, CDCl3): ꢀ 7.01–3.31 (m, 10H, aromatic), 3.49 (s, 3H, CH3).
13C NMR (75 MHz, CDCl3): ꢀ 170.59 (CO), 144.71, 135.72, 129.46, 129.00, 128.55,
127.58, 126.74, 126.37 (2£Ph), 38.27 (CH3). Positive ion HR-ICRESI-MS: [M+H]+
m/z Cacld 212.1075. Found 212.1092.
2.5. 1-Piperidin-1-yl-ethanone (4)
1H NMR (300MHz, CDCl3): ꢀ 3.57 (t, 2H, N–CH2, 2J D6.87 Hz), 3.39 (t, 2H, N–
CH2, 2J D6.87 Hz), 2.12 (s, 3H, CH3CO), 1.51–1.67 (m, 6H, CH2CH2CH2). 13C NMR
(75MHz, CDCl3): ꢀ 156.45 (CO), 47.52 (N–CH2), 42.82 (N–CH2), 26.26, 25.38, 24.23
(CH2CH2CH2), 21.16 (CH3CO). Positive ion HR-ICRESI-MS: [M+H]+ m/z Cacld
128.1075. Found 128.1089.
2.6. 2-(9H-Fluoren-9-yl)-N-(2-morpholin-4-yl-2-oxo-ethyl)-acetamide (5)
1H NMR (300 MHz, CDCl3): ꢀ 7.26–7.74 (m, 8H, Ar of Fmoc), 5.99 (br, s,
2
1H, NH of Gly), 4.37 (d, 2H, CH2 of Fmoc, J D 7.23 Hz), 4.21 (t, 1H, CH of
Fmoc, 2J D 6.87 Hz), 3.98 (d, 2H, CH2 of Gly, 2J D 4.47 Hz), 3.32–3.61 (m,
8H, N(C2H4)2O). 13C NMR (75 MHz, CDCl3):
ꢀ 166.51 (CO of Gly),
156.06 (CO of Fmoc), 143.64, 141.03, 127.49, 126.85, 124.93, 119.76 (Ar
mof Fmoc), 66.86 (CH2 of Fmoc), 66.41, 66.02 (2 £ O–CH2 of morpholinyl),
46.87 (CH of Fmoc), 44.50, 42.29 (2 £ N–CH2 of morpholinyl), 42.01 (Gly-
CH2). Positive ion HR-ICRESI-MS: [M+H]+ m/z Cacld 367.1658. Found
367.1665.
2.7. 2-(9H-Fluoren-9-yl)-N-[(methyl-phenyl-carbamoyl)-methyl]-acetamide (6)
1H NMR (300 MHz, CDCl3): ꢀ 7.15–7.73 (m, 13H, Ar of Fmoc and Ph), 5.85 (br
s, 1H, Gly-NH), 4.31 (d, 2H, CH2 of Fmoc,2J D 7.23 Hz), 4.18 (t, 1H, of CH
2
of Fmoc, J D 6.87 Hz), 3.71 (s, 2H, CH2 of Gly), 3.27 (s, 3H, N–CH3). 13C NMR
(75 MHz, CDCl3): ꢀ 168.13 (CO of Gly), 156.01 (CO of Fmoc), 143.74,
141.69, 141.06, 130.01, 128.45, 127.48, 126.99, 126.87, 124.99, 119.76 (Ar of Fmoc
and Ph), 66.86 (CH2 of Fmoc), 46.95 (CH of Fmoc), 43.19 (CH2 of Gly), 37.33 (N–
CH3). Positive ion HR-ICRESI-MS: [M+H]+ m/z Cacld 387.1609. Found
387.1622.

T. Yang et al. / Bioorganic Chemistry 33 (2005) 386–392
389
2.8. 2-(9H-Fluoren-9-yl)-N-[2-methyl-1-(morpholine-4-carbonyl)-propyl]-acetamide (7)
1H NMR (300 MHz, CDCl3): ꢀ 7.17–7.76 (m, 8H, Ar of Fmoc), 5.78 (br, d, 1H, NH
of Val, 2J D 8.94Hz), 4.18–4.50 (m, 4H, CH of Val, CH and CH2 of Fmoc), 3.48–3.72
(m, 8H, N(C2H4)2O), 1.92–2.03 (m, CH of Val), 0.97 (d, 3H, CH3 of Val, 2J D6.87 Hz),
2
0.93 (d, 3H, CH3 of Val, J D6.87 Hz). 13C NMR (75 MHz, CDCl3): ꢀ 170.46 (Val–
CO), 156.40 (CO of Fmoc), 143.67, 141.16, 127.59, 126.95, 124.99, 119.86 (Ar of
Fmoc), 66.99 (CH2 of Fmoc), 66.67, 66.51 (2 £ O–CH2 of morpholinyl), 55.10 (CH of
Val), 47.05 (CH of Fmoc), 46.23, 42.34 (2£N–CH2 of morpholinyl), 31.43 (CH of
Val), 19.47, 17.27 (2£ CH3 of Val). Positive ion HR-ICRESI-MS: [M+H]+ m/z Cacld
409.2127. Found 409.2141.
2.9. 2-(2-9H-Fluoren-9-yl-acetylamino)-3,N-dimethyl-N-phenyl-butyramide (8)
1H NMR (300MHz, CDCl3): ꢀ 7.21–7.73 (m, 13H, Ar of Fmoc and Ph), 5.67 (br,
d, 1H, NH of Val, 2J D9.27 Hz), 4.17–4.41 (m, 4H, CH of Val, CH and CH2 of Fmoc),
3.27 (s, 3H, N–CH3), 1.81–1.91 (m, 1H, CH of Val), 0.83 (d, 3H, CH3 of Val,
2
2J D6.87 Hz), 0.74 (d, 3H, CH3 of Val, J D6.87 Hz). 13C NMR (75MHz, CDCl3): ꢀ
171.85 (CO of Val), 155.87 (CO of Fmoc), 143.69, 142.51, 141.23, 129.58, 128.00,
127.43, 127.28, 126.80, 124.98, 119.72 (Ar of Fmoc and Ph), 66.63 (CH2 of Fmoc),
55.93 (CH of Val), 46.98 (CH of Fmoc), 37.51 (N–CH3), 31.39 (CH of Val), 19.32,
17.15 (2£CH3 of Val). Positive ion HR-ICRESI-MS: [M+H]+ m/z Cacld 429.2178.
Found 429.2188.
2.10. 2-(9H-Fluoren-9-yl)-N-[1-(morpholine-4-carbonyl)-2-phenyl-propyl]-
acetamide (9)
1H NMR (300MHz, CDCl3): ꢀ 7.26–7.84 (m, 13H, Ar of Fmoc and Phe), 6.04–6.10
(m, 1H, NH of Phe), 4.92–5.02 (m, 1H, CH of Phe), 4.38–4.51 (m, 2H, CH2 of Fmoc),
2
4.26 (t, 1H, CH of Fmoc, J D6.87Hz), 2.89–3.66 (m, 10H, CH2 of N(C2H4)2O and
Phe). 13C NMR (75 MHz, CDCl3): ꢀ 169.92 (CO of Phe), 155.68 (CO of Fmoc),
143.66, 141.15, 135.83, 129.44, 128.54, 127.60, 127.14, 126.94, 124.98, 119.87 (Ar of
Fmoc and Phe), 67.00 (Fmoc-CH2), 66.23, 65.83 (2£ O–CH2 of morpholinyl), 51.16
(CH of Phe), 46.97 (CH of Fmoc), 45.87, 42.17 (2 £ N–CH2 of morpholinyl), 40.12
(CH2 of Phe). Positive ion HR-ICRESI-MS: [M+H]+ m/z Cacld 457.2127. Found
457.2136.
2.11. 2-(2-9H-Fluoren-9-yl-acetylamino)-N-methyl-3,N-diphenyl-butyramide (10)
1H NMR (300MHz, CDCl3): ꢀ 6.82–7.75 (m, 18H, Ar of Fmoc, Phe and Ph), 5.65
(br, s, 1H, NH of Phe), 4.60 (m, 1H, CH of Phe), 4.15–4.36 (m, 3H, CH and CH2 of
Fmoc), 3.22 (s, 3H, N–CH3), 2.72–2.97 (m, 2H, CH2 of Phe). 13C NMR (75 MHz,
CDCl3): ꢀ 171.53 (CO of Phe), 155.39 (CO of Fmoc), 143.82, 142.33, 141.21, 136.28,
129.69, 129.33, 128.38, 127.60, 127.23, 126.98, 126.80, 125.17, 125.10, 119.90 (Ar of
Fmoc, Phe and Ph), 66.86 (CH2 of Fmoc), 52.69 (CH of Phe), 47.06 (CH of Fmoc),

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T. Yang et al. / Bioorganic Chemistry 33 (2005) 386–392
39.64 (CH2 of Phe), 37.54 (N–CH3). Positive ion HR-ICRESI-MS: [M+H]+ m/z
Cacld 477.2178. Found 477.2192.
3. Results and discussion
The synthetic route for sterically hindered peptide analogs is summarized in
Scheme 1. The reaction of N-[(9H-Xuoren-9-yl-methoxy)carbonyl]-glycine (Fmoc-
Gly) and N-methyl phenylamine (entry 6 in Table 1) was carried out in the presence
of DPP. Accordingly, an equivalent of Fmoc-Gly and N-methyl phenylamine were
dissolved in dry pyridine, and then two equivalents of DPP in anhydrous pyridine
were added dropwise to the solution at 0°C. The solution was warmed to room tem-
perature. The purpose for the addition of excess DPP was to remove the trace
amount of water in the solution. About 1.5 h later, TLC showed that the starting
materials were quantitatively transferred into Fmoc-Gly-N(Me)-Ph, and the target
product Fmoc-Gly-N(Me)-Ph was obtained in excellent yield (96%) after isolation
using silica gel column chromatography. Encouraged by the promising result, we suc-
cessfully synthesized a series of amides through reaction of various acids and second-
ary amines in the presence of DPP. Their coupling also gave yields ranging from 65
to 95% as shown in Table 1. The structures of the target products were determined by
1H, 13C NMR spectroscopy and ESI-MS.
Compared with other kinds of coupling reagents, DPP has Wve advantages. The
method provides reasonably high yields for the synthesis of the hindered peptides
and the experimental procedure results in a high regioselectivity. Reaction of the acid
with DPP in pyridine could lead to a phosphonic-carboxylic mixed anhydride inter-
mediates and the nucleophilic attack of the amino group could occur on the carbonyl
rather than on the phosphorus in the anhydrides to give an amidate. Also, the excess
coupling reagent (diphenyl phosphite, 31P NMR 2.0ppm) and its byproduct (phenyl
phosphite, 31P NMR 1.6ppm) are easily removed during puriWcation. Fourth, the
coupling reaction can be carried out by a one-pot procedure. For some organophos-
phorus reagents such as diphenylphosphorochloridate (DPP-Cl), the carboxylic acid
was reacted Wrst and the amine cannot be added to the resulting solution until DPP-
Cl is used up. Finally, the coupling reagent DPP is inexpensive, and the coupling of
hindered peptides occurs fairly rapid.
In conclusion, some sterically hindered peptide analogs were synthesized using
diphenyl phosphite as the coupling reagent. The method is straightforward, occurs
Scheme 1. Synthetic route for amides using diphenyl phosphite as the coupling agent.

T. Yang et al. / Bioorganic Chemistry 33 (2005) 386–392
391
Table 1
Reaction yields of acids and amines in the coupling of diphenyl phosphite
Entry
1
R1-COOH
R2-NH-R3
Yield (%)
65
2
95
3
4
65
92
CH3COOH
Fmoc-Gly
5
93
6
7
Fmoc-Gly
Fmoc-Val
96
93
8
9
Fmoc-Val
Fmoc-Phe
92
88
10
Fmoc-Phe
90
under mild conditions, and the desired products are readily puriWed. Hence, it may
Wnd use for synthesis of hindered peptides on a large scale.
Acknowledgments
This work was supported by the Excellent Dissertation Foundation of the Chinese
Ministry of Education (No. 200222), the Excellent Young Teacher Program of MOE,

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T. Yang et al. / Bioorganic Chemistry 33 (2005) 386–392
P.R.C., and the National Natural Science Foundation of China (Grant No.
20472042).
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