Pentafluorophenyl 4-nitrobenzenesulfonate as a peptide coupling reagent

Article abstract of DOI:10.1081/SCC-100000180

Protected dipeptides were obtained in good yield by coupling Boc- or Fmoc-protected amino acids with amino acid esters in the presence of pentafluorophenyl 4-nitrobenzenesulfonate (PFNB) as peptide coupling agent and 1-hydroxybenzotriazole as catalyst.

Full text of DOI:10.1081/SCC-100000180

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PENTAFLUOROPHENYL 4-NITROBENZENESULFONATE
AS A PEPTIDE COUPLING REAGENT
Khanitha Pudhom a & Tirayut Vilaivan b
a Organic Synthesis Research Unit, Department of Chemistry, Faculty of Science,
Chulalongkorn University, Phayathai Road, Patumwan, Bangkok, 10330, Thailand
b Organic Synthesis Research Unit, Department of Chemistry, Faculty of Science,
Chulalongkorn University, Phayathai Road, Patumwan, Bangkok, 10330, Thailand
Version of record first published: 09 Nov 2006.
To cite this article: Khanitha Pudhom & Tirayut Vilaivan (2001): PENTAFLUOROPHENYL 4-NITROBENZENESULFONATE AS A
PEPTIDE COUPLING REAGENT, Synthetic Communications: An International Journal for Rapid Communication of Synthetic
Organic Chemistry, 31:1, 61-70
To link to this article: http://dx.doi.org/10.1081/SCC-100000180
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SYNTHETIC COMMUNICATIONS, 31(1), 61–70 (2001)
PENTAFLUOROPHENYL
4-NITROBENZENESULFONATE AS A
PEPTIDE COUPLING REAGENT
Khanitha Pudhom and Tirayut Vilaivan∗
Organic Synthesis Research Unit, Department of Chemistry,
Faculty of Science, Chulalongkorn University, Phayathai
Road, Patumwan, Bangkok 10330, Thailand
ABSTRACT
Protected dipeptides were obtained in good yield by coupling
Boc- or Fmoc-protected amino acids with amino acid esters in the
presence of pentafluorophenyl 4-nitrobenzenesulfonate (PFNB) as
peptide coupling agent and 1-hydroxybenzotriazole as catalyst.
Peptide bond formation between an N-protected amino acid and an amino
acid ester is perhaps the most crucial step in peptide synthesis. Among many pep-
tide coupling reagents known to date (1–3), sulfonate esters of 1-hydroxybenzo-
triazoles have been reported to be efficient reagents for peptide and oligonucleotide
synthesis (4–9), and for sulfonylation of amines (10). The more stable and crys-
talline sulfonate esters of strongly acidic phenols were reported to be unreactive in
peptide coupling reactions (4–6), although they have been successfully used as sul-
fonyl transfer agents to phenols and amines (11). We have recently shown that aryl
esters of N-protected amino acids can be prepared in good yield by reactions of the
amino acids with sulfonate esters of phenols bearing electron-withdrawing groups
∗ To whom correspondence should be addressed.
61
Copyright ꢀ 2001 by Marcel Dekker, Inc.
www.dekker.com
C

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PUDHOM AND VILAIVAN
such as p-nitrophenol and pentafluorophenol in the presence of an organic base and
a catalytic amount of HOBt (12). Since these aryl esters of amino acids are good
acylating agents, it is therefore of interest to investigate the possibility of using aryl
sulfonates to assist peptide bond formation without prior isolation of the aryl esters.
Boc-glycine reacted slowly and incompletely with pentafluorophenyl 4-
nitrobenzenesulfonate (PFNB, 1) (12) in the presence of triethylamine in DMF, but
the addition of 0.1 eq of HOBt at room temperature caused rapid disappearance of
the starting materials. After stirring for 15 min at room temperature, an equivalent
of glycine ethyl ester hydrochloride was added, followed by another equivalent
of triethylamine, and the reaction mixture was stirred for another 60 min. Sim-
ple aqueous work-up afforded Boc-glycylglycine ethyl ester in 90% yield. Under
similar conditions, many other Boc- and Fmoc-dipeptides were obtained in good
yield and purity after simple aqueous work-up followed by chromatography, if
1
necessary (Table 1). All products gave the expected H NMR spectra and satis-
factory elemental analysis (C, H, N). It was found also that pre-activation of the
amino acid was not necessary and all components may be added simultaneously to
give similar yields of the same products. Interestingly, no N-nitrobenzenesulfonyl
amino acid ester was observed as a side product, although the amino group is
expected to be a better nucleophile than the carboxylate group.
Although HOBt is a well-known acylation catalyst in peptide synthesis (13),
catalysis of sulfonation reactions is much less common (14). We propose that
HOBt catalyzes this reaction by first reacting with pentafluorophenyl
Table 1. Protected Dipeptides Obtained from Coupling of Protected Amino Acids Em-
ploying Pentafluorophenyl 4-Nitrobenzenesulfonate (PFNB) as Coupling Agent
N-Protected
Amino Acid
Amino Acid
Ester
Yield
(%)b
Dipeptide Producta
Boc-Gly-Gly-OEt
Boc-Gly-L-Ala-OMe
Boc-Gly-Sar-OEt
Boc-L-Leu-Gly-OEt
Boc-L-Leu-L-Ala-OMe
Fmoc-Gly-Gly-OEt
Fmoc-L-Val-Gly-OEt
Fmoc-L-Val-L-Ala-OMe
Fmoc-L-Val-Sar-OEt
Fmoc-L-Phe-Gly-OEt
Fmoc-L-Lys(Boc)-Sar-OEt
Fmoc-L-Ser(tBu)-L-Leu-OMe
Fmoc-L-Trp(Boc)-Gly-OEt
Boc-Gly-OH
Boc-Gly-OH
Boc-Gly-OH
H-Gly-OEt
H-L-Ala-OMe
H-Sar-OEt
H-Gly-OEt
H-L-Ala-OMe
H-Gly-OEt
H-Gly-OEt
H-L-Ala-OMe
H-Sar-OEt
H-Gly-OEt
H-Sar-OEt
H-L-Leu-OMe
H-Gly-OEt
90
82
92
95
84
96
97
91
95
94
92
96
94
Boc-L-Leu-OH
Boc-L-Leu-OH
Fmoc-Gly-OH
Fmoc-L-Val-OH
Fmoc-L-Val-OH
Fmoc-L-Val-OH
Fmoc-L-Phe-OH
Fmoc-L-Lys(Boc)-OH
Fmoc-L-Ser(tBu)-OH
Fmoc-L-Trp(Boc)-OH
aAll products gave clean 1H NMR spectra.
bYield after purification by passing through a short silica gel column.

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63
Scheme 1.
4-nitrobenzenesulfonate to give benzotriazol-1-yl 4-nitrobenzensulfonate (2) as
the reactive intermediate. Benzotriazolyl sulfonates are known to react with car-
boxylate ions to give mixed sulfonic-carboxylic anhydrides (4–6), which could
undergo a nucleophilic attack by the amino component either directly or after
a reaction with the pentafluorophenoxide ion generated in the first step to form
pentafluorophenyl esters (Scheme 1). Formation of the aryl ester by an alternative
SNAr pathway also might be considered possible, due to the presence of electron-
withdrawing groups on the aromatic ring of the phenol. Indeed, it was previously
noted that 4-nitrophenyl 4-nitrobenzenesulfonate reacted with morpholine under
harsh conditions and in the absence of catalysts to give both 4-nitrophenyl mor-
pholine and 4-nitrobenzenesulfonylmorpholine (15). However, we have evidence
against this mechanism, since 4-nitrophenyl 4-nitrobenzensulfonate and similar
aryl 4-nitrobenzenesulfonates reacted with morpholine in the presence of HOBt to
give only 4-nitrobenzenesulfonylmorpholine, resulting from nucleophilic attack at
thesulfonylgroupandnotarylmorpholines, whichwouldbetheSNArproduct. Fur-
thermore, even though the product from sulfonylation of morpholine has no ability
to react further with nucleophiles, the rate of reaction increased substantially in the
presence of HOBt (a reaction between 4-nitrophenyl 4-nitrobenzenesulfonate and
morpholine was completed in 30 min in the presence of 0.1 eq HOBt, and less than
10% completed after 120 min without HOBt). This suggests that, in the PFNB-
mediated peptide bond formation, HOBt catalyzes the nucleophilic attack of the
sulfonate ester rather than subsequent acylation steps. The putative mixed sulfonic-
carboxylic anhydrides or benzotriazol-l-yl 4-nitrobenzenesulfonate intermediates
have not yet been successfully isolated, probably due to their high reactivities.
Racemization of the N-terminal amino acid during coupling reactions also
was studied using two model couplings between an N-acyl-protected amino acid
(N-Bz-L-Phe-OH) and a urethane protected amino acid (Boc-L-Phe-OH) with H-
L-Leu-OMe. Control reactions between N-BZ-DL-Phe-OH or N-Boc-DL-Phe-OH
1
and H-L-Leu-OMe gave crude products that showed clearly distinguishable H-
NMR signals, due to each diastereomeric dipeptide product in equal amounts.

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PUDHOM AND VILAIVAN
Table 2. Racemization Studies
Test System
Reagenta
Ratio of L/L:D/L Isomer
N-Boc-L-Phe-OH
+ H-L-Leu-OMe
DCC + HOBt · H2O 0.1 eq
HBTU
>98:2
>98:2
>98:2
(1) + HOBt · H2O 0.1 eq
N-Bz-L-Phe-OH
+ H-L-Leu-OMe
DCC + HOBt · H2O 0.1 eq
65:35
68:32
50:50
50:50
HBTU
(1) + HOBt · H2O 0.1 eq
(1) + HOBt · H2O 2 eq
aAll reactions were carried out in DMF unless otherwise indicated.
Therefore the ratio of products containing L-Phe and D-Phe could be measured
1
directly from integration of H-NMR spectra. The results revealed that no sig-
nificant racemization took place when Boc-L-Phe-OH, but not N-BZ-L-Phe-OH,
was employed as the carboxy component (Table 2), which is consistent with other
known sulfonyl-based peptide coupling agents (6,7), except in one case where
intromolecular reaction is possible (16). Changing the solvent (DMF, MeCN) and
the base (Et3N, DIEA), and increasing the amount of HOBt up to 2 eq did not yield
any improvements, indicating that racemization of the mixed carboxylic-sulfonic
anhydride of the N-acylamino acid takes place much faster than its trapping by
the auxiliary nucleophile. HBTU and DCC in the presence of HOBt gave a some-
what lower degree of racemization under similar conditions, while those of DCC
alone gave very impure product in poor yield. The use of (1) as peptide coupling
reagent should therefore be limited to coupling of urethane-protected amino acid
in a stepwise fashion and should not be used for fragment coupling.
In conclusion, pentafluorophenyl 4-nitrobenzensulfonate (1), which is a sta-
ble and crystalline reagent, may be used as an efficient peptide coupling agent in the
presence of a tertiary organic base and a catalytic amount of 1-hydroxybenzotri-
azole. The speed of the reaction and simplicity of product purification make this
reagent an alternative to established peptide coupling agents, especially when
urethane-protected amino acids are employed as the carboxyl component where
racemization would not be a problem. The possibility of using (1) in solid phase
peptide synthesis and detailed mechanistic aspects of the coupling reaction are
currently under investigation.
EXPERIMENTAL
General
Melting points were recorded on a Fisher-John melting point apparatus and
are quoted uncorrected. Specific rotations were measured on a Perkin-Elmer 341

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65
polarimeter and [α]D-values are given in units of 10−1 deg · cm2 · g−1. IR spec-
tra were recorded on a Nicolet Model Impact 410 Fourier Transform Infrared
Spectrometer using KBr disk. Elemental Analyses were performed on a Perkin
Elmer Elemental Analyzer 2400 CHNS/O at the Research Equipment Centre,
1
Chulalongkorn University. Routine H NMR spectra were obtained on a Bruker
ACF 200 (Chulalongkorn University, Bangkok) operating at 200 MHz (1H) and
50.28 MHz (13C). High field NMR experiments were performed on a JEOL
JNM500 at at the Research Equipment Centre, Chulalongkorn University. Chem-
ical shifts are reported in parts per million (ppm, δ) downfield relative to the inter-
nal standard tetramethylsilane. Unless otherwise noted, all chemicals and solvents
were obtained from commercial suppliers (Aldrich, Fluka, and Merck) and were
used as received. Reactions were performed under an atmosphere of dry nitrogen.
Pentafluorophenyl 4-nitrobenzenesulfonate (1) was prepared from pentaflu-
orophenol and 4-nitrobenzenesulfonyl chloride in pyridine (12) (83% yield) as a
yellow crystalline solid after recrystallization from ethyl acetate-hexane. It can
be stored at room temperature for years without deterioration. m.p. 108◦–109◦C;
Anal. calcd. C: 39.0, H: 1.1, N: 3.8 found : C: 38.9, H: 1.4, N: 3.7%; IR νmax
(KBr)/cm−1 3117, 1610, 1537, 1400, 1351, 1200, 999. δH (CDCl3, 200 MHz) 8.20
(2H, d, J = 7.5 Hz), 8.45 (2H, d, J = 7.5 Hz).
General Procedure for Peptide Coupling Using (1)
as Coupling Reagent
A solution of N-protected amino acid (0.3 mmol), pentafluorophenyl 4-
nitrobenzenesulfonate (0.3 mmol), and HOBt · H2O (0.03 mmol) in DMF (3 mL)
was added to triethylamine (0.3 mmol) when using N-Boc protected amino acid
or DIEA (0.3 mmol) when using N-Fmoc protected amino acid as the carboxyl
component with stirring. After stirring for 15 min at room temperature, a solution
of an amino acid methyl or ethyl ester hydrochloride (0.3 mmol) and triethylamine
or DIEA (0.3 mmol) was added to the reaction mixture. The reaction mixture was
allowed to react for 1 h at room temperature and diluted with dichloromethane.
This solution was washed with 5% HCl, 5% NaHCO3, H2O, and brine and then
dried over MgSO4. The dried solution was evaporated under reduced pressure and
the residue was purified by flash column chromatography (SiO2, CH2Cl2:Hexane)
to give analytically pure dipeptide.
In an alternative modification, the reaction was performed and worked up
identically except that all reactants were added simultaneously at the beginning.
N-tert-Butoxycarbonylglycylglycine ethyl ester (Boc-Gly-Gly-OEt)
Colorless oil (70.0 mg, 90% yield). Anal. Calcd. for C11H20O5N2: C, 50.8;
H, 7.7; N, 10.8%. Found: C, 50.8; H, 7.7; N, 10.7%. IR νmax (neat)/cm−1 3330,

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PUDHOM AND VILAIVAN
2981, 2936, 1682, 1531, 1455, 1371, 1251, 1205, 1171, 1029, 947, 864. 1H NMR
(CDCl3) δH 1.26 (3H, t, ethyl CH3), 1.42 (9H, s, Boc CH3 (x3)), 3.81 (2H, d, Glyl
CH2), 4.01 (2H, d, Gly2 CH2), 4.18 (2H, q, ethyl CH2), 5.30 (1H, br t, NH), 6.78
(1H, br t, NH).
N-tert-Butoxycarbonylglycyl-L-alanine methyl ester (Boc-Gly-L-Ala-OMe)
Colorless oil (67.4 mg, 82% yield), [α]2D0 + 10.7 (c = 1.00, CHCl3). Anal.
Calcd. for C11H20O5N2: C, 50.8; H, 7.7; N, 10.8%. Found: C, 50.7; H, 7.8; N,
10.5%. IR νmax (neat)/cm−1 3320, 2980, 2938, 1744, 1674, 1528, 1456, 1369,
1280, 1249, 1217, 1168, 1055. 1H NMR (CDCl3) δH 1.40 (3H, d, Ala CH3), 1.44
(9H, s, Boc CH3 (x3)), 3.72 (3H, s, OCH3), 3.80 (2H, d, Gly CH2), 4.57 (1H, m,
Ala CαH), 5.24 (1H, br m, Gly NH), 6.75 (1H, br d, Ala NH).
N-tert-Butoxycarbonylglycylsarcosine ethyl ester (Boc-Gly-Sar-OEt)
Colorless oil (75.6 mg, 92% yield). Anal. Calcd. for C12H22O5N2: C, 52.5;
H, 8.1; N, 10.2% Found: C, 52.7; H, 8.1; N, 10.2%. IR νmax (neat)/cm−1 3422,
2980, 2937, 1715, 1664, 1488, 1412, 1370, 1204, 1172, 1052. 1H NMR (CDCl3)
δH 1.22 (3H, 2xt, CH3 rotamers), 1.40 (9H, s, Boc CH3 (x3)), 2.95, 2.99 (3H, 2xs,
Sar CH3 rotamers), 3.84, 3.99 (2H, 2xd, Gly CH2 rotamers), 3.95, 4.08 (2H, 2xs,
Sar CH2 rotamers), 4.14 (2H, q, ethyl CH2), 5.42 (1H, br m, NH)
N-tert-Butoxycarbonyl-L-leucylglycine ethyl ester (Boc-L-Leu-Gly-OEt)
White solid (89.6 mg, 95% yield), m.p. 76◦–78◦C, [α]D20 −20.0 (c = 1.02,
CHCl3). Anal. Calcd. for C15H28O5N2: C, 56.9; H, 8.9; N, 8.8%. Found: C, 56.9;
H, 8.8; N, 8.9%. IR νmax (KBr)/cm−1 3325, 2959, 2871, 1757, 1664, 1541, 1468,
1392, 1370, 1304, 1250, 1198, 1174, 1100, 1044, 1024. 1H NMR (CDCl3) δH 0.92
(6H, dd, isopropyl CH3 (x2)), 1.28 (3H, t, ethyl CH3), 1.41 (9H, s, Boc CH3 (x3)),
1.55–1.90 (3H, br m, Leu CH, CH2), 4.00 (2H, d, Gly CH2), 4.15 (1H, m, Leu
CαH), 4.19 (2H, q, ethyl CH2), 4.96 (1H, br d, Leu NH), 6.70 (1H, br m, Gly NH).
N-tert-Butoxycarbonyl-L-leucyl-L-alanine methyl ester (Boc-L-Leu-L-Ala-OMe)
White solid (79.6 mg, 84% yield), m.p. 97◦–99◦C, [α]D20 −31.7 (c = 1.00,
CHCl3). Anal. Calcd. for C15H28O5N2: C, 56.9; H, 8.9; N, 8.8%. Found: C, 56.8;
H, 8.7; N, 8.7%. IR νmax (KBr)/cm−1 3313, 2961, 1756, 1687, 1655, 1531, 1457,

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1392, 1368, 1293, 1250, 1206, 1168, 1050, 1025. 1H NMR (CDCl3) δH 0.95 (6H,
dd, isopropyl CH3 (x2)), 1.39 (3H, d, Ala CH3), 1.44 (9H, s, Boc CH3 (x3)), 1.65
(3H, br m, Leu CH, CH2), 3.73 (3H, s, OCH3), 4.08 (1H, m, Leu CαH), 4.55 (1H,
m, Ala CαH), 4.87 (1H, br d, Leu NH), 6.60 (1H, br d, Ala NH).
N-9-Fluorenylmethoxycarbonylglycylglycine ethyl ester (Fmoc-Gly-Gly-OEt)
White solid (110.0 mg, 96% yield), m.p. 115◦–116◦C. Anal. Calcd. for
C21H22O5N2: C, 66.0; H, 5.8; N, 7.3%. Found: C, 65.9; H, 5.8; N, 7.3%. IR νmax
(KBr)/cm−1 3426, 3068, 2982, 1718, 1674, 1536, 1451, 1403, 1252, 1215, 1050.
1H NMR (CDCl3) δH 1.27 (3H, t, ethyl CH3), 3.92 (2H, d, Gly1 CH2), 4.02 (2H,
d, Gly2 CH2), 4.18 (3H, m, ethyl CH2, Fmoc aliphatic CH), 4.41 (2H, d, Fmoc
CH2), 5.62 (1H, br t, NH), 6.62 (1H, br m, NH), 7.35 (4H, m, Fmoc aromatic CH),
7.58 (2H, d, Fmoc aromatic CH), 7.74 (2H, d, Fmoc aromatic CH).
N-9-Fluorenylmethoxycarbonyl-L-valylglycine ethyl ester
(Fmoc-L-Val-Gly-OEt)
White solid (122.8 mg, 97% yield), m.p. 197◦–198◦C, [α]D20 −18.0 (c =
1.00, CHCl3). Anal. Calcd. for C24H28O5N2: C, 67.9; H, 6.6; N, 6.6%. Found: C,
67.9; H, 6.5; N, 6.7%. IR νmax (KBr)/cm−1 3440, 3069, 2966, 1705, 1661, 1535,
1451, 1296, 1214, 1111, 1030. 1H NMR (CDCl3) δH 0.95 (6H, dd, isopropyl CH3
(x2)), 1.27 (3H, t, ethyl CH3), 2.16 (1H, m, Val CH), 4.04 (3H, dd, Gly CH2, Val
CαH), 4.20 (3H, m, ethyl CH2, Fmoc aliphatic CH), 4.42 (2H, m, Fmoc CH2),
5.38 (1H, d, Val NH), 6.47 (1H, br t, Gly NH), 7.34 (4H, m, Fmoc aromatic CH),
7.59 (2H, d, Fmoc aromatic CH), 7.76 (2H, d, Fmoc aromatic CH).
N-9-Fluorenylmethoxycarbonyl-L-valyl-L-alanine methyl ester
(Fmoc-L-Val-L-Ala-OMe)
White solid (116.3 mg, 91% yield), m.p. 208◦–209◦C, [α]D20 −18.3 (c =
1.02, CHCl3). Anal. Calcd. for C24H28O5N2: C, 67.9; H, 6.6; N, 6.6%. Found: C,
67.9; H, 6.6; N, 6.5%. IR νmax (KBr)/cm−1 3432, 3067, 2962, 1704, 1661, 1532,
1452, 1336, 1295, 1226, 1151. 1H NMR (CDCl3) δH 0.98 (6H, dd, isopropyl CH3
(x2)), 1.40 (3H, d, Ala CH3), 2.11 (1H, m, Val CH), 3.72 (3H, s, OCH3), 4.04 (1H,
m, Val CαH), 4.23 (1H, m, Fmoc aliphatic CH), 4.40 (2H, m, Fmoc CH2), 4.59
(1H, m, Ala CαH), 5.48 (1H, d, Val NH), 6.47 (1H, d, Ala NH), 7.32 (4H, m, Fmoc
aromatic CH), 7.57 (2H, d, Fmoc aromatic CH), 7.76 (2H, d, Fmoc aromatic CH).

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PUDHOM AND VILAIVAN
N-9-Fluorenylmethoxycarbonyl-L-valylsarcosine ethyl ester
(Fmoc-L-Val-Sar-OEt)
Colorless oil (117.8 mg, 95% yield), [α]2D0 −14.5 (c = 1.03, CHCl3). Anal.
Calcd. for C25H30O5N2: C, 68.5; H, 6.9; N, 6.4%. Found: C, 68.4; H, 6.8; N, 6.3%.
IR νmax (neat)/cm−1 3431, 3314, 3065, 2967, 1718, 1640, 1529, 1479, 1452, 1374,
1206, 1111, 1093. 1H NMR (CDCl3) δH 1.00 (6H, dd, isopropyl CH3 (x2), 1.26
(3H, t, ethyl CH3), 2.07 (1H, m, Val CH), 3.18 (3H, s, Sar CH3), 3.71, 3.80 (1H,
2xs, Sar CH2 rotamers), 4.05–4.64 (8H, m, ethyl CH2, Sar CH2 rotamers, Val CαH,
Fmoc CH2, Fmoc aliphatic CH), 5.57 (1H, d, NH), 7.35 (4H, m, Fmoc aromatic
CH), 7.59 (2H, d, Fmoc aromatic CH), 7.74 (2H, d, Fmoc aromatic CH).
N-9-Fluorenylmethoxycarbonyl-L-phenylalanylglycine ethyl ester
(Fmoc-L-Phe-Gly-OEt)
White solid (129.2 mg, 94% yield), m.p. 185◦–187◦C, [α]D20 −14.2 (c =
1.02, CHCl3). Anal. Calcd. for C28H28O5N2: C, 71.2; H, 6.0; N, 5.9%. Found: C,
71.1; H, 6.0; N, 5.8%. IR νmax (KBr)/cm−1 3468, 3304, 1738, 1695, 1654, 1541,
1262, 1210, 1038. 1H NMR (CDCl3) δH 1.26 (3H, t, ethyl CH3), 3.10 (2H, br d, Phe
CH2), 4.20 (3H, m, ethyl CH2, Fmoc aliphatic CH), 4.42 (3H, br, m, Phe aliphatic
CαH, Fmoc CH2), 5.32 (1H, br m, Gly NH), 6.30 (1H, br m, Phe NH), 7.10–7.47
(9H, m, Phe aromatic CH, Fmoc aromatic CH), 7.52 (2H, m, Fmoc aromatic CH),
7.76 (2H, d, Fmoc aromatic CH).
N-9-Fluorenylmethoxycarbonyl-N-ε-tert-butoxycarbonyl-L-lysylsarcosine
ethyl ester (Fmoc-L-Lys(Boc)-Sar-OEt)
Colorless oil (160.5 mg, 92% yield), [α]2D0 +0.5 (c = 1.02, CHCl3). Anal.
Calcd. for C31H43O7N3: C, 65.4; H, 7.6; N, 7.4%. Found: C, 65.6; H, 7.5; N, 7.4%.
IR νmax (neat)/cm−1 3431, 3066, 2977, 2938, 1711, 1649, 1517, 1453, 1409, 1252,
1173. 1H NMR (CDCl3) δH 1.26 (3H, 2xt, ethyl CH3 rotamers), 1.41 (9H, s, Boc
CH3 (x3)), 1.48–1.95 (8H, br m, Lys CH2), 2.96, 3.14 (3H, 2xs, Sar CH3 rotamers),
3.78, 3.86 (1H, 2xs, Sar CH2 rotamers), 4.20 (4H, m, ethyl CH2, Sar CH2 rotamers,
Fmoc aliphatic CH), 4.35 (2H, d, Fmoc CH2), 4.73 (1H, m, Lys, CαH), 5.74 (1H,
d, Lys NH), 7.35 (4H, m, Fmoc aromatic CH), 7.59 (2H, d, Fmoc aromatic CH),
7.75 (2H, d, Fmoc aromatic CH).
N-9-Fluorenylmethoxycarbonyl-(O-tert-butyl)-L-seryl-L-leucine methyl ester
(Fmoc-L-Ser(tBu)-L-Leu-OMe)
White solid (146.7 mg, 96% yield), m.p. 105◦–106◦C, [α]D20 +22.5 (c =
1.01, CHCl3). Anal. Calcd. for C29H28O6N2: C, 68.2; H, 7.5; N, 5.5%. Found: C,

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PEPTIDE COUPLING REAGENT
69
68.4; H, 7.4; N, 5.4%. IR νmax (KBr)/cm−1 3427, 3249, 3064, 3036, 2962, 2873,
1
1756, 1727, 1661, 1508, 1451, 1361, 1281, 1224, 1200, 1153, 1096, 1066. H
NMR (CDCl3) δH 0.93 (6H, d, isopropyl CH3 (x2)), 1.22 (9H, s, Boc CH3 (x3)),
1.44–1.80 (3H, br m, Leu CH, CH2), 3.38 (1H, t, Ser CH2), 3.71 (3H, s, OCH3),
3.81 (1H, dd, Ser CH2), 4.24 (2H, m, Leu CαH, Fmoc aliphatic CH), 4.40 (2H, d,
Fmoc CH2), 4.60 (1H, br m, Ser CαH), 5.76 (1H, br m, NH), 7.35 (4H, m, Fmoc
aromatic CH), 7.58 (2H, d, Fmoc aromatic CH), 7.74 (2H, d, Fmoc aromatic CH).
N-9-Fluorenylmethoxycarbonyl-Nin-tert-butoxycarbonyltryptophanylglycine
ethyl ester (Fmoc-Trp(Boc)-Gly-OEt)
20
White solid (172.1 mg, 94% yield), m.p. 80◦–81◦C, [α]D −10.3 (c = 1.11,
CHCl3). Anal. Calcd. for C35H37O7N3: C, 68.7; H, 6.1; N, 6.9%. Found: C, 68.9;
H, 6.3; N, 7.0%. IR νmax (KBr)/cm−1 3329, 3066, 2980, 2936, 1733, 1672, 1531,
1
1478, 1454, 1374, 1336, 1309, 1257, 1160, 1088, 1027, 939. H NMR (CDCl3)
δH 1.23 (3H, t, ethyl CH3), 1.64 (9H, s, Boc, CH3 (x3)), 3.20 (2H, br d, Trp CH2),
3.90 (2H, d, Gly CH2), 4.16 (3H, m, ethyl CH2, Fmoc aliphatic CH), 4.38 (2H, d,
Fmoc CH2), 4.54 (1H, m, Trp CαH), 5.50 (1H, br m, Trp NH), 6.26 (1H, br m, Gly
NH), 7.17–7.65 (10H, m, Fmoc aromatic CH, Trp indole CH), 7.75 (2H, d, Fmoc
aromatic CH), 8.11 (1H, d, Trp indole CH).
ACKNOWLEDGMENTS
We acknowledge financial support from the Department of Chemistry and
Rachadaphisek Somphot Endowment (grants for development of new faculty staff)
from Chulalongkorn University. We also thank the Research Equipment Centre,
Chulalongkorn University, for microanalysis service.
REFERENCES AND NOTES
1. Bodanszky, M. Peptide Chemistry, A Practical Textbook; Springer-Verlag:
Berlin, 1993.
2. Atherton, E.; Sheppard, R.C. Solid Phase Peptide Synthesis, A Practical
Approach; IRL Press: Oxford, 1989.
3. Fields, G.B.; Noble R.L. Int. J. Peptide Protein Res. 1990, 35, 161.
4. Itoh, M.; Nojima, H.; Notani, J.; Hagiwara, D.; Takai, K. Tetrahedron Lett.
1974, 35, 3089.
5. Itoh, M.; Hagiwara, D.; Notani, J. Synthysis 1975, 456.
6. Itoh, M.; Nojima, H.; Notani, J.; Hagiwara, D.; Takai, K. Bull. Chem. Soc.
Jpn. 1978, 51, 3320.

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PUDHOM AND VILAIVAN
7. Devadas, B.; Kundu, B.; Srivastava, A.; Mathur, K.B. Tetrahedron Lett. 1993,
34, 6455.
8. Reese, C.B.; Pei-Zhou, Z. J. Chem. Soc., Perkin Trans. I 1993, 2291.
9. Furukawa, M.; Hokama, N.; Okawara, T. Synthesis 1983, 42.
10. Kim, S.Y.; Sung, N.-D.; Choi J.-K.; Kim, S.S. Tetrahedron Lett. 1999, 117.
11. Neuvile, L.; Bigot, A.; Dau, M.E.T.H.; Zhu, J. J. Org. Chem. 1999, 64, 7638.
12. Pudhom, K.; Vilaivan, T. Tetrahedron Lett. 1999, 40, 5939.
13. Konig, W.; Geiger, R. Ber. 1987, 788.
14. Fujii, T.; Sakakibara, S. Bull. Chem. Soc. Jpn. 1974, 47, 3146.
15. Kotsuki, H.; Kobayashi, S.; Suenaga, H.; Nishizawa, H. Synthesis 1990,
1145.
16. Cabaret, D.; Wakselman, M. Tetrahedron Lett. 1994, 35, 9613.
Received in the U.K. January 31, 2000

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