HOBt·DCHA-mediated synthesis of sterically hindered peptides employing Fmoc-amino acid chlorides in both solution-phase and solid phase methods

Article abstract of DOI:10.1080/00397910802219536

The synthesis of peptides employing Fmoc-amino acid chlorides in presence of HOBt·DCHA salt in solution as well as by the solid-phase methods is described. The coupling was found to be complete in 30 min and free from racemization. The synthesis of β-casomorphin by solid-phase protocol employing Fmoc-amino acid chloride/HOBt·DCHA in DMF-CH2Cl2 has also been outlined. The final peptide was obtained in 80% yield and was fully characterized. Copyright Taylor & Francis Group, LLC.

Full text of DOI:10.1080/00397910802219536

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HOBt·DCHA-Mediated Synthesis
of Sterically Hindered Peptides
employing Fmoc-Amino Acid
Chlorides in Both Solution-
Phase and Solid Phase Methods
Vommina V. Sureshbabu a , Naremaddepalli S.
Sudarshan a & G. Chenna Krishna a
a Department of Studies in Chemistry , Central
College Campus, Bangalore University , Bangalore,
India
Published online: 09 Sep 2008.
To cite this article: Vommina V. Sureshbabu , Naremaddepalli S. Sudarshan &
G. Chenna Krishna (2008) HOBt·DCHA-Mediated Synthesis of Sterically Hindered
Peptides employing Fmoc-Amino Acid Chlorides in Both Solution-Phase and
Solid Phase Methods, Synthetic Communications: An International Journal for
Rapid Communication of Synthetic Organic Chemistry, 38:15, 2625-2637, DOI:
10.1080/00397910802219536
To link to this article: http://dx.doi.org/10.1080/00397910802219536
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Synthetic Communications1, 38: 2625–2637, 2008
Copyright # Taylor & Francis Group, LLC
ISSN: 0039-7911 print/1532-2432 online
DOI: 10.1080/00397910802219536
HOBtꢀDCHA-Mediated Synthesis of Sterically
Hindered Peptides employing Fmoc-Amino
Acid Chlorides in Both Solution-Phase and
Solid Phase Methods
Vommina V. Sureshbabu, Naremaddepalli S. Sudarshan,
and G. Chenna Krishna
Department of Studies in Chemistry, Central College Campus,
Bangalore University, Bangalore, India
Abstract: The synthesis of peptides employing Fmoc-amino acid chlorides
in presence of HOBtꢀDCHA salt in solution as well as by the solid-phase methods
is described. The coupling was found to be complete in 30 min and free from
racemization. The synthesis of b-casomorphin by solid-phase protocol employing
Fmoc-amino acid chloride=HOBtꢀDCHA in DMF–CH2Cl2 has also been
outlined. The final peptide was obtained in 80% yield and was fully characterized.
Keywords: Fmoc-amino acid chloride, fmoc-amino acid-OBt ester, HOBtꢀDCHA
INTRODUCTION
One of the many contributions of Louis Carpino, University of Massa-
chusetts, is the discovery of shelf-stable Fmoc-amino acid chlorides,[1,2]
which have been demonstrated to be rapid, efficient, acylating agents
useful in peptide chemistry. Because of the high degree of activation asso-
ciated with them, the acylation using acid chlorides[3,4] makes coupling of
very hindered or weakly nucleophlic systems possible, whereas the use of
other methods gives inferior results. Those Fmoc-amino acid chlorides
bearing a tert-butyl type side chain functional group such as Asp(tBu),
Received March 18, 2007
Address correspondence to Vommina V. Sureshbabu, Department of Studies in
Chemistry, Central College Campus, Bangalore University, Dr. B. R. Ambedkar
Veedhi, Bangalore 560 001, India. E-mail:hariccb@rediffmail.com
2625

2626
V. V. Sureshbabu, N. S. Sudarshan, and G. C. Krishna
Cys(tBu), Lys(Boc), Ser(tBu), Thr(tBu), Trp(Boc), Tyr(tBu) and
Asp(Pmc) can also be generated using in situ activation via bis(trichlor-
ophenyl)carbonate. The acid chloride coupling mediated by an organic
tertiary amine (pyridine, diisopropylethylamine (DIEA)), an essential
component of such a reaction protocol, is known to result in the forma-
tion of oxazol-5(4H)-one,[5] leading to stereomutation and=or premature
deblocking of Fmoc-group, due to the prolongation of the course of the
reaction (usually required for the incorporation of hindered amino acids).
This has been circumvented by the use of co-coupling agents such as sil-
ver cyanide[6,7] potassium salts of 1-hydroxybenzotriazole (KOBt)[8,9]
and 1-hydroxy-7-aza-benzotriazole (KOAt),[10,11] t-butyldimethylsilyloxy
benzotriazole (TBDMS) derivatives of HOBt (TBMS-OBt) and HOAt
(TBDMS-OAt),[12,13]
and
zinc
dust.[14,15]
The
use
of
KOBt=KOAt=TBDMS-OBt or TBDMS-OAt converts acid chloride to
its benzotriazole=azabenzotriazole ester, which acts as an acylating agent,
whereas in the presence of zinc dust–mediated coupling, the liberated
HCl is directly abstracted. There are few limitations in the use of some
of these reagents. The formation of KCl as a side product avoids the
use of KOBt or KOAt in solid phase. The zinc dust–mediated coupling
is limited to acylation reactions in solution phase only. TBDMS deriva-
tives cannot be prepared and stored for long periods and need to be
freshly prepared every time. Although the use of these co-coupling agents
is satisfactory in solution phase, their utility in solid phase is not a
straightforward protocol.
This article demonstrates the efficient synthesis of peptides
employing Fmoc-amino acid chlorides mediated by HOBtꢀDCHA salt.
Employing the present protocol, the synthesis of H2N-Val-Pro-Gly-
Val-Gly-OH (VPGVG)[16] in solution phase and b-casomorphin (H-T
yr-Pro-Phe-Pro-Gly-OH) in solid phase have been accomplished.
1-Hydroxybenzotriazole (HOBt), introduced by Konig and Geiger,[17]
is well known to suppress racemization in carbodiimide-mediated cou-
plings. Further, the other popular coupling agents 1-[bis(dimethylamino)-
methylene]-1H-benzotriazolium hexaflur ophosphate-3-oxide (HBTU)[18]
and 2-(1H-benzotriazole-1-yl)1,1,3,3-tetramethylluroniumtetrafluorobo-
rate (TBTU)[19] also possess HOBt as part of their structure. Our interest
in developing a simple non–Schotten–Baumann[20] protocol for acid
chloride coupling has led to explore the use of HOBtꢀDCHA salt as a
co-coupling agent in base-free conditions. Katritzky et al. have employed
the benzotriazole amide derivatives of N-protected amino acids for the
acylation of amino acids in an aqueous medium in the presence of triethyl
amine.[21] In contrast to this, the present synthesis uses the rapidly and in
situ generated –OBt ester as acylating agent in a completely base-free
organic medium. The Bt-amide derivatives of amino acids are prepared

HOBtꢀDCHA-Mediated Peptide Synthesis
2627
through a two-step protocol comprising activation of benzotriazoles with
thionyl chloride followed by treatment of N-protected amino acids with
activated Bt. However, the Fmoc-acid chlorides are prepared easily in a
single step by treating the Fmoc-amino acids directly with thionyl chlor-
ide. Also, preparation of the HOBT–DCHA reagent is simple, involving
the addition of an equimolar quantity of dicyclohexylamine to anhydrous
HOBt in tetrahydrofuran (THF) solution. Upon standing, the salt sepa-
rates out, which can be crystallized and stored for a long time.
RESULTS AND DISCUSSION
In a typical reaction, the mixture of Fmoc-amino acid chloride (1 equiva-
lent) and HOBtꢀDCHA (2.2 equivalents) in THF was initially stirred for
about 5 min to generate–OBt ester (which is evidenced from IR absorption
spectrum by the disappearance of the band at 1786cmꢁ1 pertaining to acid
chloride and the appearance of the band at 1820cmꢁ1 pertaining to –OBt
ester formation), and then amino acid methyl ester salt was added and stir-
red. As monitored by thin layer chromatography (TLC), high pressure
liquid chromatography (HPLC), and infrared spectrum (IR) analysis
(Scheme 1), the coupling was found to be complete in about 30min.
The reaction mixture was filtered to remove the precipitated
DCHAꢀHCl salt, and after simple workup, the peptide was isolated.
The efficacy of HOBtꢀDCHA-mediated coupling is further demonstrated
by the synthesis of dipeptides containing Caa-dialkylglycines[22,23] (H2N-
CR1R2-COOH) with linear substitutents [a-aminoisobutric acid (Aib),
diethylglycine (Deg), dipropylglycine (Dpg), and dibutylglycine (Dbg)]
and cyclic substitutents [1-aminocyclopentane-1-carboxylic acid (Ac5 c),
and 1-aminocyclo hexane-1-carboxylic acid (Ac6c), and 1-aminocyclo-
heptane-1-carboxylic acid (Ac7c)]. The duration of such couplings
required about 2 h, and the resulting peptides were obtained in 65–72%
yield (Table 1). Further, the coupling of Fmoc-amino acid–chloride
mediated using HOBtꢀDCHA, and using an aqueous NaHCO3 solution
of amino acid, resulted in Fmoc-peptide acid with moderate yield. On
the other hand, the use of bis-N,O-trimethylsilyl amino acid[24] gave good
yields with analytically pure compounds (Scheme 2).
Scheme 1. Synthesis of Na-Fmoc-protected peptide esters.

2628
V. V. Sureshbabu, N. S. Sudarshan, and G. C. Krishna
Table 1. List of peptides made by the HOBtꢀDCHA method
Yield (%) Mp (ꢂC) [a]D
(c1.0, DMF)
25
SI. no.
Peptide
1
2
3
4
5
6
7
8
9
Fmoc-Phg-Phe-OMe
Fmoc-D-Phg-Phe-OMe
Fmoc-Leu-Ala-OMe
Fmoc-Dpg-Dbg-OMe
Fmoc-Aib-Aib-OMe
Fmoc-Ac6c-Ac6c-OMe
Fmoc-Deg-Leu-OMe
Fmoc-Ac7c-Ac5c-OMe
Fmoc-D-Phg-Phe-OH
Fmoc-Tyr(Bzl)-Pro-OH
Fmoc-Asp(OBn)-Ala-OH
Fmoc-Ser(OBn)-Val-OH
86
87
88
72
72
70
70
71
82
76
80
78
194–196
194–194
160–162
gum
70–71
164–165
55–57
207–209
175–177
112–114
148–149
120–122
þ 24.0
ꢁ23.4
ꢁ28.0
—
—
—
—
—
þ 43.6
þ 24.6
þ 38.6
ꢁ28.4
10
11
12
The 1H NMR analysis of Carpino’s diastereomeric dipeptides Fmoc-
L or D-Phg-Phe-OMe[2] prepared by employing the present protocol
indicated that the C-methylene doublets of phenylalanine and methyl
ester singlets are distinct. Thus the coupling is free from racemization.
Further, their HPLC analysis [Rt-values: L,L-isomer (13.19) and D,L-iso-
mer (14.26)] also confirmed the same. Employing the present protocol for
coupling and tris (2-aminoethyl) amine (TAEA) for deprotection of the
Fmoc-group, the pentapeptide Fmoc-VPGVG synthesis has been
accomplished. The purity of the final peptide Fmoc-VPGVG was satis-
1
factory and was fully characterized by H NMR and mass spectra.
The yield was 72%.
Although Fmoc-amino acid fluorides[25–27] have been used with
or without any organic base successfully as coupling agents in SPPS,
the use of Fmoc-amino acid chlorides invariably requires an equimolar
quantity of a base. Carpino et al. intially employed a 1:1 mixture of HOBt
and pyridine [or diisopropylethylamine (DIEA) or N-methylmorpholine
(NMM)] in the coupling. The addition of a base is well known to promote
Scheme 2. Synthesis of Na-Fmoc-protected peptide acids.

HOBtꢀDCHA-Mediated Peptide Synthesis
2629
one or more side reactions in Fmoc chemistry. The use of already known
co-coupling agents (KOBt, KOAt, etc.) is not satisfactory in SPPS.
In the present studies, HOBtꢀDCHA can be used in SPPS also. This
is mainly feasible because of two reasons: 1) HOBtꢀDCHA dissolves in
DMF (or NMP) : CH2Cl2 (2 : 8) and converts Fmoc-amino acid chloride
to its–OBt ester, and 2) the side product DCHAꢀHCl salt can be easily
and completely removed, after coupling, by washing with a 1:1 mixture
of CHCl3 and CH2Cl2. The solid-phase synthesis of b-casomorphin using
Wang resin as solid support and Fmoc-amino acid chlorides (3 equiv.)
and HOBtꢀDCHA (3 equiv.) in DMF:CH2Cl2 (2:8) for coupling starting
from Fmoc-Gly-O-CH2-C6H4-O-resin was carried out. The crude pep-
tide, on HPLC analysis, was found to be about 91% pure. It was further
purified by RP-HPLC and found to be more than 99% pure. A simple
coupling for 30 min was found to result in the completion of the coupling.
Fmoc group was deprotected using 20% piperidine. After the removal of
Fmoc group from Fmoc-Tyr(Bzl)-Pro-Phe-Pro-Gly-OCH2-C6H4-O-
resin, the peptide was cleaved by using TFA=H2O=thioanisole=phenol
mixtures at room temperature for 1.5 h and subjected to catalytic hydro-
genation using 10% Pd=C=methanol=hydrogen gas to remove the Bzl
group from the peptide, which was purified.
CONCLUSION
HOBtꢀDCHA is a stable and crystalline solid. Its preparation, isolation,
and purification is very simple. It has been now demonstrated that the
coupling of Fmoc-amino acid chlorides can be accomplished employing
HOBtꢀDCHA under non–Schotten–Baumann conditions. The acylation
was efficient, resulting in good yields. The scaling of coupling up to
10 mmol has not posed any practical difficulties. Thus, it has now been
demonstrated that HOBtꢀDCHA finds utility in a solid-phase method
for the synthesis of peptides employing Fmoc-amino acid chlorides under
non–Schotten–Baumann conditions.
EXPERIMENTAL
The melting points were determined by the capillary method and are
uncorrected. Specific rotations were recorded on a Rudolf Research
Autopol IV automatic polarimeter. IR spectra were recorded on a
Nicolet model impact 400D FT-IR spectrometer (KBr pellets,
1
3 cmꢁ1 resolution) and H NMR spectra on a Bruker 400-MHz instru-
ment. Unless otherwise mentioned, all amino acids used have
L-configuration. Analytical HPLC was performed on a Waters LC

2630
V. V. Sureshbabu, N. S. Sudarshan, and G. C. Krishna
3000 system equipped with Waters 484 tunable absorbance UV detec-
tor and millipore 745 data module. Water RP 48-deltapack column
(3.9 mm ꢃ 300 mm, 15 m spherical) was used for analysis. Thin-layer
chromatography (TLC) analysis was carried out using the precoated
silica gel G254 plates using 1) RfA; CHCl3–methanol–acetic acid
40:2:1; 2) RfB; ethyl acetate–nhexane (35:65). Deprotonation of Fmoc
group used TAEA, and mass spectra were obtained using a Kratos
PCKompact SEQ V1.2.2 spectrometer.
Synthesis of HOBtꢀDCHA Salt (2)
Dicyclohexylamine (DCHA) (0.238 mL, 1.2 mmol) was added to a sus-
pension of anhydrous HOBt (0.135 g, 1 mmol) in CH2Cl2 (5 mL) at rt
and stirred for 1–2 h. Hexane (2 mL) was added and left for recrystalliza-
tion. The resulting HOBtꢀDCHA crystals were filtered, washed with 10%
CH2Cl2 in hexane (10 mL), and dried thoroughly to obtain 0.284 g (90%)
ꢂ
1
of the title compound,[28] mp 137–139 C; H NMR (CDCl3): d 1.0–1.7
(12H, m), 1.7–2.1 (8H, m), 3.05 (2H, t), 7.25 (2H, m), 7.6–7.8 (2H,
m);13C NMR (CDCl3):d 24.8, 25.0, 29.3, 52.8, 53.0, 111.3, 118.4, 123.4,
127.7, 143.3; FAB (M þ 1)þ : m=z 317.4.
General Procedure for the Synthesis of Na-Protected Peptide Esters (4)
To a previously stirred solution of Fmoc-amino acid chloride
(1 mmol) and HOBtꢀDCHA (0.695 g, 2.2 mmol) in dry THF (5 mL),
a suspension of amino acid ester hydrochloride salt (1 mmol) in
THF (5 mL) was added, and the reaction mixture was stirred. The
completion of the reaction was monitored by TLC. The reaction mix-
ture was filtered and washed with organic solvent (THF, 10 mL) to
remove DCHAꢀHCl as a white precipitate. The filtrate was concen-
trated, and CH2Cl2 (15 mL) was added. It was washed with 20%
sodium bisulphate (10 mL ꢃ 3), to remove excess DCHA, water
(10 mL ꢃ 2), 5% HCl (10 mL ꢃ 3), and water, then dried over anhy-
drous Na2SO4. The solvent was removed by evaporation under
reduced pressure and product was crystallized using CH2Cl2–hexane
(2:8) to yield the peptide.
Data
Fmoc-Phg-Phe-OMe: 1H NMR (CDCl3): d 2.91 (d, 2H), 3.10 (t, 1H), 3.62
(s, 3H), 4.21 (t, 1H), 4.53 (d, 2H), 5.20 (d, 1H), 6.23 (d, 1H), 6.91 (1H, d),

HOBtꢀDCHA-Mediated Peptide Synthesis
2631
7.30–7.90 (m, 18H);13C NMR (CDCl3): d 38.2, 47.3, 52.5, 54.4, 66.8, 67.8,
120.1, 124.1, 126.3, 126.5, 127.2, 127.5, 128.2, 128.7, 130.9, 133.4, 135.8,
141.5, 144.0, 155.6, 167.5, 171.9; ES MS: m=z 535.3 [M þ H]þ , Anal.
calcd. for C33H30N2O5: C, 74.14; H, 5.66;N, 5.24. Found: C, 74.04; H,
5.61; N, 5.28%.
1
Fmoc-(D)Phg-Phe-OMe: H NMR (CDCl3): d 2.81 (d, 2H), 3.10 (t,
1H), 3.62 (s, 3H), 4.20 (t, 1H), 4.51 (d, 2H), 5.21 (d, 1H), 6.22 (d, 1H),
6.90 (d, 1H), 7.28–7.90 (m, 18H); 13C NMR (CDCl3): d38.3, 47.3, 52.3,
54.4, 66.8, 67.8, 120.0, 124.0, 126.2, 126.5, 127.2, 127.5, 128.2, 128.7,
130.9, 133.4, 135.8, 141.5, 144.0, 155.6, 167.5, 171.9; ES MS: m=z 535.3
[M þ H]þ, Anal. calcd. for C33H30N2O5: C, 74.14;H, 5.66;N, 5.24.
Found: C, 74;02; H, 5.60; N, 5.30%.
1
Fmoc-Leu-Ala-OMe: H NMR (CDCl3): d 0.97 (d, 6H), 1.62 (m,
3H), 3.75 (s, 3H), 4.25 (t, 1H), 4.41 (t, 1H), 4.53 (d, 2H), 5.20 (m, 1H),
6.33 (m, 1H), 7.27–7.80 (m, 8H); 13C NMR (CDCl3): d 17.4, 22.4, 22.9,
38.3, 46.5, 47.5, 52.3, 52.9, 66.8, 120.0, 125.1, 127.1, 141.5, 144.0,
155.6, 172.0, 173.1; ES MS: m=z 439.4 [M þ H]þ , Anal. calcd. for
C25H30N2O5: C, 68.47; H, 6.90; N, 6.39. Found; C, 68.41; H, 6.70; N,
6.21%.
1
Fmoc-Dpg-Dbg-Ome: H NMR (CDCl3): d 0.81–2.02 (m, 40H), 3.60
(s, 3H), 4.11 (d, 1H), 4.20 (t, 2H), 6.51 (s, 1H), 6.92 (s, 1H), 7.20–7.81 (m,
8H);[13]C NMR (CDCl3): d 13.3, 14.0, 18.9, 22.3, 24.7, 37.6, 40.9, 47.4,
52.3, 61.2, 66.7, 66.9, 120.1, 124.0, 127.1, 127.2, 141.4, 144.0, 155.3,
170.2, 175.1; ES MS: m=z 565.6 [M þ H]þ , Anal. Calcd. for
C34H48N2O5: C, 72.31; H, 8.57; N, 4.96. Found:C, 72.10;H, 8.49;N,
4.90%.
1
Fmoc-Aib-Aib-Ome: H NMR (CDCl3): d 1.00–1.21 (m, 12H), 3.80
(s, 3H), 4.11 (d, 1H), 4.31 (t, 2H), 6.92 (s, 1H), 7.42 (s, 1H), 7.20–7.80
(m, 8H); [13]C NMR (CDCl3): d 25.2, 26.9, 47.4, 52.1, 55.9, 60.7, 66.9,
120.0, 124.0, 127.0, 127.6, 141.5, 144.1, 155.3, 171.2, 175.3; ES MS:
m=z 425.5 [M þ H]þ , Anal. calcd. for C24H28N2O5: C, 67.91; H, 6.65;
N, 6.60. Found: C, 67.90; H, 6.54; N, 6.50%.
Fmoc-Ac6c-Ac6c-Ome: 1H NMR (CDCl3): d 1.11–2.30 (m, 20H),
3.71 (s, 3H), 4.20 (d, 1H), 4.52 (t, 2H), 6.50 (s, 1H), 7.01 (s, 1H), 7.20–
7.80 (m, 8H);[13] C NMR (CDCl3): d 20.6, 24.1, 25.5, 33.3, 38.8, 47.5,
52.3, 60.8, 68.5, 66.8, 120.1, 124.1, 126.3, 127.2, 141.5, 144.7, 155.0,
170.1, 174.5; ES MS: m=z 505.5 [M þ H]þ , Anal. calcd. for
C30H36N2O5: C, 71.41;H, 7.19;N, 5.55. Found: C, 71.01; H, 7.08; N,
5.56%.
1
Fmoc-Deg-Leu-OMe: H NMR (CDCl3): d 0.81–2.02 (m, 18H), 3.70
(s, 3H), 4.10 (d, 1H), 4.20 (d, 1H), 4.41 (t, 2H), 4.60 (d, 1H), 6.81 (s,
1H), 7.20–7.80 (m, 8H);13 C NMR (CDCl3): d 7.1, 23.0, 24.0, 35.8,
41.2, 47.3, 48.8, 52.3, 66.9, 70.0, 119.9, 124.0, 126.2, 127.3, 141.5,

2632
V. V. Sureshbabu, N. S. Sudarshan, and G. C. Krishna
144.4, 155.0, 171.8, 172.1; ES MS: m=z 481.4 [M þ H]þ , Anal. calcd. for
C28H36 N2O5: C, 69.98; H, 7.55; N, 5.83. Found: C, 69.90;H, 7.51; N,
5.84%.
Fmoc-Ac7c-Ac5c-OMe: 1H NMR (CDCl3): d 1.51–2.00 (m, 20H),
4.11 (s, 3H), 4.31 (t, 2H), 4.50 (d, 1H), 5.01 (s, 1H), 6.50 (s, 1H), 7.20–
8.01 (m, 8H);[13]C NMR (CDCl3): d 21.6, 23.4, 25.9, 32.7, 39.6, 47.4,
52.3, 63.9, 65.3, 66.9, 120.0, 124.1, 126.2, 127.3, 141.4, 144.7, 155.3,
170.1, 175.1; ES MS: m=z 505.5 [M þ H]þ , Anal. calcd. for
C30H36N2O5: C, 71.41; H, 7.19; N, 5.55. Found: C, 71.32; H, 7.16; N,
5.60%.
General Procedure for the Synthesis of Na-Protected Peptide Acids (6)
To a stirred solution of Fmoc-amino acid chloride (1 mmol) and
HOBtꢀDCHA (0.695 g, 2.2 mmol) in dry THF (5 mL), a TMS activated
amino acid (1.2 mmol) was added. Trimethylsilyl chloride (TMS-Cl,
1.2 mmol) and triethylamine (TEA, 2.5 mmol) were added to a stirred
suspension of amino acid (1 mmol) in DCM (10 mL) and refluxed for
2–4 h. The completion of the reaction was monitored by TLC. The reac-
tion mixture was concentrated to remove THF, and the residue was dis-
solved in sodium carbonate solution (10%, 15 mL). The aqueous phase
was washed with ether (2 ꢃ 10 mL) and acidified using dil. HCl. The com-
pound was extracted in ethyl acetate (2 ꢃ 10 mL). The organic phase was
washed with water (2 ꢃ 10 mL) and brine (10 mL) and dried over anhy-
drous sodium sulphate. It was concentrated under vacuum to get the
desired compound as a white solid in good yield.
Data
Fmoc-(D)Phg-Phe-OH: 1H NMR (DMSO): d 2.90 (d, 2H), 3.70 (m, 2H),
4.25 (m, 3H), 4.90 (d, 1H), 5.31 (d, 1H), 7.10–7.50 (m, 14H), 7.55 (d, 2H),
7.80 (d, 2H), 12.01 (br, 1H); 13C NMR (DMSO): d 36.6, 47.4, 49.4, 66.4,
74.9, 120.0, 124.0, 125.0, 126.3, 126.4, 126.8, 127.3, 128.4, 129.6, 130.9,
133.6, 140.7, 141.5, 144.5, 155.3, 166.5, 172.7; ES MS: m=z 521.3
[M þ H]þ , Anal. calcd. for C32H28N2O5: C, 73.83; H, 5.42; N, 5.38.
Found: C, 73.79; H, 5.43; N, 5.40%.
1
Fmoc-Tyr(Bn)-Pro-OH: H NMR (DMSO): d 2.12 (m, 4H), 3.00 (d,
2H), 3.51 (m, 2H), 3.82 (t, 1H), 4.20 (d, 2H), 4.40 (t, 1H), 5.80 (br, 1H),
7.20–7.90 (m, 17H), 12.5 (br, 1H); 13C NMR (DMSO): d 20.6, 29.1, 36.7,
47.4, 52.0, 58.4, 60.1, 69.4, 74.4, 118.9, 120.0, 124.2, 127.0, 127.2, 127.6,
128.2, 129.9, 130.6, 136.8, 141.5, 144.7, 153.4, 157.5, 163.6, 175.4; ES MS:

HOBtꢀDCHA-Mediated Peptide Synthesis
2633
m=z 577.4 [M þ H]þ , Anal. calcd. for C35H32N2O6: C, 72.90; H, 5.59;
N, 4.86. Found: C, 72.82; H, 5.60; N, 4.88%.
1
Fmoc-Asp(OBn)-Ala-OH: H NMR (DMSO): d 1.21 (d, 3H), 2.60
(d, 2H), 3.61–3.72 (m, 2H), 4.20 (t, 1H), 4.31 (m, 2H), 4.80 (s, 2H),
5.30 (d, 1H), 5.70 (br, 1H), 7.10–7.52 (m, 9H), 7.61 (d, 2H), 7.80 (d,
2H), 8.00 (d, 1H), 12.4 (br, 1H); 13C NMR (DMSO): d 17.6, 39.3, 44.9,
47.2, 54.8, 63.8, 66.5, 120.0, 124.0, 126.2, 127.2, 128.1, 128.6, 141.5,
142.8, 144.7, 155.4, 169.9, 171.0, 175.9; ES MS: m=z 517.4 [M þ H]þ ,
Anal. calcd. for C29H28N2O7: C, 67.43; H, 5.46; N, 5.42. Found:
C, 67.40; H, 5.47; N, 5.45%.
Fmoc-Ser(OBn)-Val-OH: 1H NMR (DMSO): d 0.91 (t, 6H), 1.81 (m,
1H), 3.40–3.72 (m, 4H), 4.10 (t, 1H), 4.32 (d, 2H), 4.91 (s, 2H), 5.20 (d,
1H), 5.83 (br, 1H), 7.18–7.54 (m, 9H), 7.62 (d, 2H), 7.85 (d, 2H), 8.00
(d, 1H), 12.6 (br, 1H); 13C NMR (DMSO): d 18.9, 30.2, 47.4, 52.3,
58.1, 66.5, 69.7, 73.0, 120.0, 124.0, 126.5, 127.2, 127.9, 128.0, 128.3,
137.6, 141.5, 144.7, 155.0, 170.9, 128.3, 137.6, 141.5, 144.7, 155.0,
170.9, 174.8; ES MS: m=z 517.5 [M þ H]þ , Anal. calcd. for
C30H32N2O6: C, 69.75; H, 6.24; N, 5.42. Found: C, 69.62; H, 6.21; N,
5.39%.
Synthesis of Fmoc-Val-Pro-Gly-Val-Gly-OBzl
Fmoc-Val-Gly-Obn (I)
To H-Gly-OBn (1.65 g, 10 mmol) in THF (10 mL), a mixture of Fmoc-
Val-Cl (3.6 g, 10 mmol) and HOBtꢀDCHA (3.5 g, 11 mmol) in THF
(20 mL) was added and stirred untill the completion of reaction. The
workup of the reaction mixture followed by column purification gave
the peptide (I) as a white crystalline solid (4.3 g, 90%); mp 182–184 ꢂC;
ꢂ
1
[a ]25D þ 32.2 (c 1.0, CHCl3); RfA, 0.51; RfB, 0.65; H NMR (CDCl3):
d 0.90–1.01 (d, J ¼ 6.4 Hz, 6H), 1.29 (m, 1H), 3.81 (m, 1H), 4.20–4.40
(m, 4H), 5.00 (s, 2H), 6.70 (m, 1H), 7.20–7.43 (m, 9H), 7.58 (d,
J ¼ 6.9 Hz, 2H), 7.76 (d, J ¼ 6.8 Hz, 2H); 13C NMR (CDCl3): d 18.6,
29.1, 40.4, 47.3, 55.1, 67.4, 69.5, 119.9, 124.1, 126.4, 126.8, 127.2,
127.4, 128.7, 133.5, 141.5, 143.4, 155.2, 169.4, 172.4; ES MS: m=z 509.3
[M þ Na]þ , Anal. calcd. for C29H30N2O5: C, 71.59; H, 6.21; N, 5.76,
Found: C, 71.55; H, 6.19; N, 5.73%.
Fmoc-Gly-Val-Gly-OBn (II)
Fmoc-Gly-Cl (2.7 g, 8.8 mmol) and HOBtꢀDCHA (3.1 g, 9.7 mmol) in
THF (20 mL), were added to a solution of free peptide H-Val-Gly-OBn

2634
V. V. Sureshbabu, N. S. Sudarshan, and G. C. Krishna
(2.3 g, 8.8 mmol), obtained by the deprotection of I (4.3 g, 9 mmol) using
TAEA[4] (45 mL) in THF (10 mL). Workup of the reaction mixture fol-
lowed by column purification gave the peptide (II) as a white crystalline
solid (4.4 g, 88%) ; mp 176–179 ꢂC; [a][25]D þ 30.4ꢂ(c 1.0, CHCl3); 1H
NMR (CDCl3): d 0.90–1.01 (d, J ¼ 6.3 Hz, 6H), 1.30 (m, 1H), 3.67 (m,
4H), 3.93 (m, 1H), 4.20–4.45 (m, 5H), 5.01 (s, 2H), 6.20 (d, 1H), 7.20–
7.81 (m, 13H), 13C NMR (CDCl3): d 18.6, 29.1, 40.4, 46.2, 47.1, 57.5,
67.4, 69.5, 119.9, 124.1, 126.2, 126.9, 127.2, 127.4, 128.7, 133.2, 141.5,
143.4, 157.1, 165.4, 169.4, 173.4; ES MS: m=z 566.2 [M þ Na]þ , Anal.
calcd. for C31H33N3O6: C, 68.49; H, 6.12; N, 7.73, Found: C, 68.55; H,
6.19; N, 5.79%.
Fmoc-Pro-Gly-Val-Gly-OBn (III)
Fmoc-Pro-Cl (2.6 g, 8.0 mmol) and HOBtꢀDCHA (2.78 g, 8.8 mmol) in
THF (20 mL) were added to a solution of the free peptide H-Gly-Val-
Gly-OBn (2.2 g, 8.0 mmol), obtained by the deprotection of the peptide
(II) (4.4 g, 8.1 mmol) using tris(2-aminoethyl)amine (TAEA) (40 mL) in
THF (10 mL). Workup of the reaction mixture followed by column puri-
ficationꢂgave the title prꢂoduct (III) as a crystalline solid (4.6 g, 78%) mp
1
158–60 C; [a]25D þ 24.2 (c 1.0, CHCl3); H NMR (CDCl3): d 0.90–1.01
(d, J ¼ 6.7 Hz, 6H), 1.29 (m, 1H), 1.73–2.24 (m, 6H), 3.67 (m, 4H),
3.93 (m, 1H), 4.11 (t, 1H), 4.20–4.45 (m, 5H), 5.00 (s, 2H), 5.10 (d,
1H), 6.20 (d, 1H), 7.20–7.81 (m, 13H); 13C NMR (CDCl3): d 18.6, 26.8,
28.4, 29.4, 40.2, 43.6, 46.9, 47.3, 57.5, 59.4, 67.4, 69.6, 119.9, 124.1, 126.4,
127.0, 127.2, 127.4, 128.9, 133.3, 141.5, 143.7, 165.5, 167.8, 167.9, 169.4,
170.2; ES MS: m=z 663.7 [M þ Na]þ , Anal. calcd. for C36H40N4O7: C,
67.48; H, 6.29; N, 8.74, Found: C, 67.20; H, 6.29; N, 8.73%.
Fmoc-Val-Pro-Gly-Val-Gly-OBn (IV)
Fmoc-Val-Cl (2.3 g, 7.0 mmol) and HOBtꢀDCHA (2.43 g, 7.7 mmol) in
THF (20 mL)were added to a solution of the free peptide H-Pro-Gly-
Val-Gly-OBn (2.91 g, 7.0 mmol), obtained by the deprotection of the pep-
tide (III) (4.6 g, 7.1 mmol) using TAEA (35 mL) in THF (10 mL).
Workup of the reaction mixture followed by column purification gave
the title product (IV) as a crystalline solid (4.6 g, 67%); mp 98–100 ꢂC;
[a]25D–18.7 ꢂ (c 1.0, CHCl3); H NMR (CDCl3): d 0.89–1.10 (m, 12H),
1
1.28 (m, 2H), 1.73–2.24 (m, 6H), 3.65 (d, 1H), 3.95 (m, 1H), 4.02 (d,
2H), 4.13 (d, 2H), 4.18 (m, 3H), 4.20–4.40 (m, 2H), 4.45 (t, 1H), 5.01
(s, 2H), 5.10 (m, 2H), 6.01 (m, 1H), 7.20–7.84 (m, 13H); 13C NMR
(CDCl3): d 18.6, 18.8, 27.0, 27.3, 30.1, 30.2, 40.1, 42.4, 47.1, 48.4, 54.6,

HOBtꢀDCHA-Mediated Peptide Synthesis
2635
55.9, 58.6, 67.4, 69.4, 119.9, 124.1, 126.2, 127.0, 127.3, 127.5, 128.7, 133.5,
141.5, 143.4, 155.2, 165.4, 167.8, 167.9, 168.4, 170.4; ES MS: m=z 763.7
[M þ Na]þ , Anal. calcd. for C41H49N5O8: C, 66.56; H, 6.68; N, 9.47,
Found: C, 66.47; H, 6.57; N, 9.40%.
Synthesis of b-Casomorphin (H-Tyr-Pro-Phe-Pro-Gly-OH)
The peptide was synthesized using manual Fmoc-chemistry with a
solid-phase synthesis strategy on Wang resin (substitution 0.6 mmol=g)
employing Fmoc-Tyr(OBzl)-COCl (0.92 mg, 1.8 mmol, 3 eq), Fmoc-
Pro-COCl (0.64 mg, 1.8 mmol,
3 eq), Fmoc-Phe-COCl (0.72 mg,
1.8 mmol, 3 eq), Fmoc-Pro-COCl (0.64 mg, 1.8 mmol, 3 eq), and
HOBtꢀDCHA (0.63 mg, 1.98 mmol, 3.1 eq) in DMF–CH2Cl2 (10 mL,
1:1). The synthesis was carried out using 1 g of Fmoc-Gly-Wang resin.
A single coupling for 30 min was sufficient for the completion of each
coupling. The DCHAꢀHCl formed as a white precipitate was removed
by washings using CHCl3–CH2Cl2 (1:1). After deprotection of Fmoc
group using 20% pipyridine in DMF, the peptide was cleaved using tri-
fluroacetic acid (TFA) (10 mL)= H2O (0.5 mL)=thioanisole (0.5 mL)=
phenol (0.75 g) mixture at room temperature for 1 h. The crude peptide
was subjected to catalytic hydrogenation using 10% Pd=C (50 mg)=
methanol (10 mL)=hydrogen gas to remove the benzyl group and
was purified by RP-HPLC to obtain the final peptide (275 mg,
ꢂ
ꢂ
1
79%). Mp 151–153 C; [a][25]D–48.5 (c 0.5, DMF); H NMR (CDCl3):
d 1.10–2.10 (m, 14H), 2.31 (t, 2H), 2.90 (t, 2H), 3.11 (t, 1H), 5.20 (br,
1H), 5.40 (br, 1H), 5.71 (br, 1H), 7.20–8.10 (m, 9H), 12.0–13.0 (1H,
COOH); 13C NMR (CDCl3): d 27.3, 29.0, 33.9, 35.4, 36.1, 50.6, 51.3,
51.8, 54.3, 58.1, 58.8, 127.1, 128.9, 129.4, 129.5, 130.1, 130.2, 140.1,
155.4, 166.0, 168.6, 169.4, 170.3, 177.2; ES MS: m=z 602.65 [M þ Na]þ,
, Anal. calcd. for C30H37N5O7: C, 62.16; H, 6.43; N, 12.08, Found: C,
62.12;H, 6.40; N, 12.00%; R, 8.45 [(Waters C-18 deltapak column
(3.9 ꢃ 300 mm, 15m); using eluent acetonitrile 0.1% trifluoroacetic acid
(TFA) and water (65:35; isocratic, flow rater 1 mL=min, monitoring at
220 nm).
ACKNOWLEDGMENTS
We are grateful to the Department of Science and Technology,
Government of India, for financial support. We thank Professor Fred
Naider, City of University of New York (CUNY), New York, for useful
discussions and S. I. F, and M. B. U. of Indian Institute of Science (IISc),
Bangalore, for providing NMR and mass facilities.

2636
V. V. Sureshbabu, N. S. Sudarshan, and G. C. Krishna
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