Expansion of phosphane treasure box for staudinger peptide ligation

Article abstract of DOI:10.1021/acs.joc.0c01319

A smooth traceless ligation strategy using an airstable phosphane probe (2-(diphenylphosphanyl)phenyl)methanol as a C-terminus activator has been demonstrated at simple and sterically hindered amino acid junctions (Gly, Ala, Trp, Glu). This Staudinger pep

Full text of DOI:10.1021/acs.joc.0c01319

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Article
Expansion of Phosphane Treasure Box for Staudinger Peptide Ligation
Kiran Bajaj, Girinath G. Pillai, Rajeev Sakhuja, and Dalip Kumar
J. Org. Chem., Just Accepted Manuscript • DOI: 10.1021/acs.joc.0c01319 • Publication Date (Web): 04 Sep 2020
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The Journal of Organic Chemistry
Expansion of Phosphane Treasure Box for Staudinger Peptide Ligation
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Kiran Bajaj,a* Girinath G. Pillai,b Rajeev Sakhuja,a Dalip Kumara
aDepartment of Chemistry, Birla Institute of Technology and Science, Pilani 333031, Rajasthan, India
bChemistry Division, Nyro Research India, Kochi, Kerala, India
E-mail: kbajaj26@gmail.com
For Table of Contents Only
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Staudinger
Peptide Ligation
Trp
Glu
Ala
Gly
Peptide
P
Peptide
N3
HO
O
O
Peptide
OH
O
P
O
Peptide
Peptide
N
H
75-95% yields
ABSTRACT:
A
smooth traceless ligation strategy using an air-stable phosphane probe, (2-
(diphenylphosphanyl)phenyl)methanol as a C-terminus activator has been demonstrated at simple and sterically-
hindered amino acid junctions (Gly, Ala, Trp, Glu). This Staudinger Peptide Ligation proceeds via formation of a
7-membered transition state to afford di-, tetra- and pentapeptides in 78-95% yields. The experimental results of
ligation at Gly junction and regioselective ligation at Glu junction were theoretically studied by computational
calculations. These findings established the versatile behavior of our synthesized phoshphane probe for
Staudinger Peptide Ligation towards synthesizing peptides and proteins of choice.
Introduction
Total chemical synthesis of proteins has always been a challenge owing to vast complexities and structure
diversities present in them. Immense advancements have been witnessed during the past decades in protein
syntheses; of which, Native Chemical Ligation (NCL) has been much appreciated and categorically explored
under various aspect to convene the requirements of ligation at various amino acid sites.1 Consequently,
Aldehyde Capture Ligation2 (ACL) and α-Ketoacid-Hydroxylamine3 (KAHA) amide-forming ligation strategies
have been developed, which expanded the chemists toolbox for peptide synthesis and its bioconjugation, albeit
with some inherent limitations. Another pioneer approach introduced by Bertozzi’s group4 was Staudinger
Peptide Ligation (SPL) that utilizes the bioorthogonality and chemoselectivity of Staudinger reaction. In a typical
SPL, C-side of one of the peptide fragment gets activated with specially designed phosphinyl group to capture the
azido peptidic fragment to furnish aza-ylide, which on intramolecular nucleophilic attack on the neighbouring
acyl group produces amidophosphonium salt via the formation of cyclic iminophosphorane tetrahedral
intermediate. Finally, hydrolysis of amidophosphonium salt affords a stable amide bond between two peptide
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fragments. Further, Raine, Marx, Mamat and Lam groups have expanded the scope of SPL using different
phosphinols and phosphinothiols to prepare peptides, ranging in yields from very low-to-high (Scheme 1i-iv).5
Notably, most of these strategies worked predominantly at Gly junction. However to utilize SPL at non-glycyl
junctions, specific functionalized phosphane probes have been used to afford peptide bond in good yields
(Scheme 1iv).5e Recently, during the natural product synthesis of Yaku’amide B, Inque et. al prepared an enamide
dipeptide fragment in 76% yield by performing SPL between 2-(bis(4-(trifluoromethyl)phenyl)phosphanyl)
phenolic ester of protected alanine and alkenyl azide in THF/H2O for 7 days (Scheme 1v).6 Strikingly, in the past
decade, the application of SPL has been expanded towards in vitro and in vivo biological and chemical labeling of
proteins, glycans, biotin, nucleic acids, fatty acids, lipids and several organic fluorophores.7 However, this
expedition has been achieved at the cost of earlier reported phosphane probes, probably due to their stability or
limitation to ligate bulky amino acids at ligation junction.
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O
O
Staudinger Peptde Ligation
N3
N
H
M
Previous works
Gly-Gly; 95%
Phe-Gly; 92%
Gly-Ser(Bn); 92%
Ala-Ala; 36%
2001
2002
2006
Org. Lett.
J. Org. Chem.
J. Org. Chem.
, 9
, 4993
, 9824
(i)
M
=
S
PPh2
(ii)
M
=
S
2PPh2
Gly-Gly; 39%
2009
Methods Enzymol.
, 25
Gly-Phe; 36% (X = O)
Ala-Phe; 32% (X = O)
Gly-Leu; 6% (X = O)
Gly-Gly; 15% (X = S)
Phe-Gly; 35% (X = S)
2003
2000
2001
Tetrahedron Lett.
Org. Lett.
, 4515
, 1939
, 9
(iii)
(iv)
M
X
=
Org. Lett.
PPh2
S
M
=
Ala-Ala; 82% (R = 4-OMe)
Phe-Ala; 84% (R = 4-OMe)
2006
J. Org. Chem.
J. Am.Chem.Soc.
, 9824
2007
, 11421
P
Ala-Gly; 59% (R = 4-CH2CH2NMe2)
Gly-Gly; 55% (R = 3-CH2CH2NMe2)
R
R
R = 4-OMe, 3-CH2CH2NMe2, 4-CH2CH2NMe2
OMe
2020
Angew. Chem. Int. Ed.
, 4564
(v)
M
=
O
O
H
N
NHBn
P
Boc
N
H
O
CF3
CF3
After 7 days, 76%
Present work
* Successful LIgation at Gly, Ala, Trp, Glu Sites
* Ligation via 7-membered transition state
* Synthesis of di-, tetra- & pentapeptides
* Good to excellent yields (78-95%)
Ph
Ph
P
(vi)
M
=
O
* Supported by Computational Studies
Scheme 1. Previous and present works on phosphane probes for Staudinger Peptide Ligation
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1
The Journal of Organic Chemistry
In view of the above discussion, we herein disclose the synthesis, application and computational studies
of a new phosphane probe, (2-(diphenylphosphanyl)phenyl)methanol towards SPL on a variety of amino acid
junctions to afford di-, tetra- and pentapeptides in good-to-excellent yields under ambient conditions at glycyl and
non-glycyl junctions (Scheme 1vi).
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Results & Discussion
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At the outset of the proposed work, the synthesis of phosphane probe, (2-
(diphenylphosphanyl)phenyl)methanol (2) was achieved in high yield by reduction of commercially available 2-
(diphenylphosphanyl)benzoic acid (1) with lithium aluminum hydride in dry THF at room temperature (Scheme
2). To demonstrate the efficacy of phosphane probe 2 towards peptide synthesis via traceless SPL, different N-
protected amino acids (3a-e) and peptides (3f-i) were activated with (2-(diphenylphosphanyl)phenyl)methanol (2)
using DCC/DMAP in dichloromethane to furnish their corresponding phosphinyl benzyl esters (4a-i) in 68-78%
yields (Scheme 2).
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R
O
H
PG
N
N
OH
H
O
R'
n
HO
LiAlH4 (3.0 equiv)
THF, r.t., 2 h
HOOC
R
O
P
3a-i
H
P
P
PG
N
N
O
DCC (1.0 equiv)
DMAP (1.0 equiv)
DCM, r.t., 12-14 h
H
O
R'
n
4
2
, 80%
1
Cbz
O
Cbz
N
N
NH
H
H
O
P
O
P
4b, 70%
Boc
O
N
4a, 68%
H
O
P
O
O
R
4c, 72%
Boc
O
O
P
H
N
N
Cbz
H
O
P
N
O
H
O
O
H
P
4f, 70%
Cbz
N
4d: R = Bn, 78%
4e: R = H, 68%
N
O
H
O
O
O
O
H
O
P
4g, 70%
Cbz
N
O
H
N
N
H
Boc
N
N
O
H
O
O
P
H
O
4h, 70%
4i, 71%
Scheme 2. Synthesis of N-protected amino acyl/peptidyl phosphinyl benzyl esters
As a model reaction, the coupling of N-Cbz protected glycyl phosphinyl benzyl ester (4a) with 2-azido-N-(p-
tolyl)acetamide (5a, α-N3-Phe-p-tol) was carried out under various reaction conditions to obtain the desired
ligated native dipeptide 6aa (Table 1). Interestingly, the reaction between the two model substrates (4a & 5a) in
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THF at room temperature for 12 h afforded N-Cbz-Gly-Phe-p-tol (6aa) in 50% yield (Table 1, entry 1). The
formation of 6aa could be explained by the formation of iminophosphorane (I) that undergoes intramolecular
nucleophilic attack on ester carbonyl of phosphane unit to generate amido phosphonium salt (III) via a seven-
membered intermediate (II). Thereafter, III upon hydrolysis produces native peptide bond and the oxidized
phosphinyl alcohol 2a' as a by-product that could be reused after reduction. The use of THF-H2O (3:1) in place of
THF further declined the yield of 6aa to 32% due to hydrolysis of iminophosphorane intermediate (I) that to the
oxidized ester (4a') and reduced C-protected phenylalanyl amide (5a') (Table 1, entry 2). Solvent and temperature
screening studies suggested DMF to be an ideal solvent for the desired transformation, producing 6aa in 70%
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yield at 40 C (Table 1, entries 3-6). Gratifyingly, the use of pyridine or triethylamine as a base drastically
improved the yield of 6aa to 82-90% in DMF (Table 1, entries 7-8). It is worth mentioning that after the addition
of triethylamine, the reaction mixture acquires a pH between 7-8 that is an optimal pH range for peptide ligation.
In order to diminish the premature hydrolysis of imino-phosphorane I, experiments were performed in pure
solvents (THF, 1-4-dioxane, DMF), water was added after 12 h of the reaction, and the reaction mixture was
stirred for another 30 minutes before the isolation of desired ligated product. This was done to retard the
hydrolysis of I before its intramolecular acyl migration to give native peptide in high yield.
Table 1. Selective Optimizationa of reaction parameters for the synthesis of ligated product 6aa
O
H
Cbz
N
p-Tol
Cbz
O
N
N
H
N
H
H
H
O
O
P
N
N
N
N
p-Tol
6aa
O
5a
4a
HO
O
H2O
-N2
O
P
Ph Ph
Cbz
O
2a'
N
H
O
P
Ph
Ph
O
4a'
Cbz
O
N
O
NH2
H
NEt3
Ph
Ph
P
O
PPh2 O
N
H2O
O
-NEt3
NEt3
O
P
N
O
Cbz
p-Tol
Ph
p-Tol NH
Ph
N
N
N
H
H
p-Tol NH
5a'
O
NH
Cbz
O
NH
I
p-Tol
III
II
Entry
1c
2
3c
3c
4
5c
6c
7c
8c
Solvent
THF
Temperature
25 oC
Additive
6aa (%)b
50
-
-
-
-
-
-
-
THF-H2O (3:1)
Dioxane
DMF
DMF-H2O (3:1)
DMF
25 oC
32
52
58
36
70
68
82
90
25 oC
25 oC
25 oC
40 oC
DMF
DMF
DMF
60 oC
40 oC
Pyridine
Et3N
40 oC
aReaction conditions: 4a (1.0 equiv), 5a (1.1 equiv), pyridine/Et3N (1.0 equiv) in solvent (5 mL) at the specified
temperature for 12 h; bIsolated Yields; cWater (0.5 mL) was added after 12 h and stirred the reaction contents for
30 min. at the same temperature.
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The Journal of Organic Chemistry
The pathway for this reaction was further obtained by employment of reaction modeling and energy profile
(Figure 1). Single-point energy calculations were carried out at the BP86 /def2-TZVP level of theory to get more
accurate results. Transition state reaction path sampling computations were performed to confirm the feasibility
of transition states connected to the appropriate reactants and products. To measure the conformer pre-
organization of the transition state (I) for the nucleophilic attack, the distance [d(N-C)] between the N and the C
atoms was calculated, and was found to be 3.60 Å. This further confirmed the viability of acyl migration via 7-
membered transition state. We also calculated the frequencies and energies of all structure configurations with
thermochemical parameters (Enthalpy: ∆H, Temperature x Entropy: T∆S, Gibbs free energy: ∆G) to ascertain the
spontaneity of the reaction, which agreed well with the experimental results (Table 2). The role of triethylamine
in the above transformation could be envisioned in stabilizing intermediate II that further accelerates the acyl
migration to afford the ligated product in high yields, which also got support from the computed energies of I-III
(Table 2).
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Figure 1. Energy profile representation referring to the change in activation energy and enthalpy change for SPL
at Gly junction
Table 2. Thermodynamic parameters at BP86/TZVP level of theory at 298.15 K and 1 atm
Structure no.
Dipole
G
H
TS
4a + 5a
I
0.00 (459.17)
+6.78
0.00 (968.48)
+0.32
0.00 (156.22)
-6.47
2.29+6.45
6.75
II
-24.53
-32.57
-8.04
14.05
III
6aa
2a′
+11.88
-34.59
-42.91
+5.11
-43.38
-57.60
-6.76
-8.78
-14.70
11.97
2.24
3.43
All energy values are in kcal/mol and normalised to factor of 10. Dipole is in debye units
We next explored the coupling of glycyl phosphinyl benzyl ester (4a) with several other α-azido amides
(substrates: 5b-g) derived from hydrophobic, aromatic, and basic α-amino acids (Gly, Ala, Leu, Val, Tyr, Lys)
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under our optimized conditions (Scheme 3). Previous findings have revealed that the rate and efficacy of ligation
with reported earlier phoshane ligands is controlled by bulkiness of migrating group at the phosphane site.5f In
striking contrast, using our phosphane probe (2), different α-azido amides varying in bulkiness and functionalities
in their side chains smoothly underwent acyl migration on glycyl phosphinyl ester (4a) to produce their respective
native dipeptides (products: 6ab-6ag) in 80-95% yields. Formation of 6ac-6ag in excellent yields rules out a
deeper role of sterics and electronics of the side chain of azido peptidic fragment in the SPL process using
phosphane probe 2. The elimination of steric factor could be well understood by visualizing the bulky effect of
side chain in α-azido amide (for example, phenyl moiety in 5a) that does not impose any steric interference
during the intramolecular C-N bond formation via 7-membered transition state from iminophosphorane
intermediate (I). “[Insert link to here]”
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Ph2P
R2
R1
O
R1
H
H
N
Et3N (1 equiv)
N
Pg
Pg
O
N3
N
H
N
H
N
(i) DMF, 40oC, 12h
(ii) H2O, 40oC, 0.5h
H
O
O
R2
O
6
4a-e
5a-g
O
O
O
H
H
N
H
Cbz
N
Boc
Cbz
N
N
H
N
N
H
N
H
N
N
H
H
H
O
O
O
6ab, 95%
Gly
Ala
6bc, 80%
OH
6af, 92%
O
H
O
O
Bn
Cbz
Cbz
N
O
N
N
H
H
H
O
Cbz
N
O
H
N
N
H
Boc
N
H
O
N
H
N
H
6ac, 95%
O
6db, 90%
O
H
6ag, 88%
HN
N
Cbz
N
H
N
Glu
O
O
H
Bn
O
O
O
H
N
Boc
6ad, 80%
N
H
N
H
Trp
6dc, 88%
O
H
O
OH
Cbz
N
N
N
HN
Cbz
H
H
O
O
H
O
Boc
N
H
N
N
H
N
6ae, 80%
H
N
N
O
H
H
O
6eb, 73%
6cb, 80%
Scheme 3. Scope of N-protected amino acyl phosphinyl benzyl esters & α-azido amides
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The Journal of Organic Chemistry
To further expand the ligation at other amino acid junctions using phosphane probe (2), the coupling of
phosphinyl benzyl esters of protected Ala, Trp and Glu (substrates: 4b-e) with α-azido amides (substrates: 5b-c)
was attempted under optimized conditions. Delightfully, the dipeptides fragments, Ala-Ala (6bc), Trp-Gly (6cb),
Glu-Gly (6db, 6eb) and Glu-Ala (6dc) were furnished in 73-90% yields, thereby establishing the generality of
phosphane probe 2 at non-glycyl junctions as well (Scheme 3). We have also investigated the utility of phosphane
probe 2 with phosphinyl benzyl ester of unprotected Glu (4e) that underwent smooth transformation to the desired
dipeptide 6eb in 73% yield (Scheme 3). This further generalize the scope of SPL via phosphane probe 2 at
unprotected Glu junction. In the process of SPL, the configurations at the chiral centres of the two coupling
partners were retained after ligation. This was evident by the formation of high diastereomeric excess observed in
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the H NMR spectra (>97% de) of peptides (6bc & 6dc). To further ascertain this, N-Cbz-L-Ala-DL-Ala-p-tol
was synthesized using standard EDC coupling strategy, and its 1H NMR was compared with that of N-Cbz-L-Ala-
L-Ala-p-tol (6bc) prepared by SPL (SI). The comparative spectral analysis clearly confirms the absence of other
diasteromer in 6bc, thus eliminating the possibility of epimerization following described SPL.
Interestingly the ligation at glutamic acid junction (Compounds 6db and 6dc) exhibited regioselective amino acyl
migration at glutamyl junction that was further studied theoretically (Figure 2). Formation of 6db via SPL can be
explained by regioselective migration of amino acyl group in intermediate imino phosphorane (4d5b) at C-side of
phosphinyl benzyl ester, which is closest migrating centre as compared to its side chain benzyl ester, furnishing
native dipeptide (6db) in 90% yield, rather than its isopeptidyl phosphonate ester (6db′). This indicated
differential accessibility of intramolecular attack of the nitrogen on the two available carbonyl electrophilic sites
in iminophospharane intermediate (4d5b), preferring seven-membered (A1-7m) over eleven-membered transition
state (B1-11m) (Figure 2).
Figure 2. Energy profile representation referring to the change in activation energy and enthalpy change for SPL
at Glu junction for regioselective amino acyl migration via SPL
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This can be further rationalized by computationally calculating the distance between two migrating
centres, (N to C) in transition states A1-7m and B1-11m for the possibility of corresponding amide bond
formations (Figure 3).
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B1-11m
A1-7m
Figure 3. Theoretically calculated bond distance between migrating sites for the two possible transition states
A1-7m [3.4 Å] and B1-11m [4.8 Å] for SPL at Glu junction from a pre-organised conformational analysis
The established computational procedure consists of complete conformational search, optimization of the
conformer to conform the proximity function and transition state search studies as validated in previous work.8 To
measure the conformer pre-organization for the nucleophilic attack, the distance represented as d(N-C) between
the N atom and the C atom is considered as the proximity function.9 The pre-organized conformers of A1-7m and
B1-11m resulted 3.40 Å distance in 7-membered and 4.80 Å in 11-membered transition states, respectively. All
thermochemical parameters (Enthalpy: H, Temperature x Entropy: TS, Gibbs free energy: G) were also
calculated along with transition state reaction path sampling computations to verify the spontaneity of the reaction
towards the formation of 6db (Table 3).
Table 3. Thermodynamic parameters at BP86/TZVP level of theory at 298.15 K and 1 atm
Structure no.
4d + 5b
4d5b
Dipole
3.25+3.06
7.82
G
0.00 (882.95)
-5.84
H
0.00 (1003.27)
-7.42
TS
0.00 (120.32)
-1.57
A1-7m
B1-11m
A2-7m
B2-11m
6db
-5.73
-4.90
-20.30
-16.42
-45.72
-24.10
-7.23
-7.10
-18.42
-19.88
-50.13
-26.42
-1.50
-2.20
-1.88
-3.46
-4.41
-2.31
9.2
7.25
9.82
5.75
2.75
2.16
6db′
All energy values are in kcal/mol and normalised to factor of 10. Dipole in debye units
Next, the versatility of phosphane probe 2 was extended towards the synthesis of short peptides in solution phase
by performing Staudinger Peptide Ligation on different peptidyl phosphinyl benzyl esters as illustrated in Scheme
4. Contentedly, dipeptidyl phosphinyl benzyl esters [Cbz-Ile-Ala-OCH2C6H4PPh2 (4f), Cbz-Val-Glu(OMe)-
OCH2C6H4PPh2 (4g)] and tripeptidyl phosphinyl benzyl ester [Cbz-Ile-Gly-Gly-OCH2C6H4PPh2 (4h)] reacted
comfortably with α-azido amide/ α-azido dipeptidyl esters [N3-Phe-NH-p-Tol (5a), N3-Trp-Ph-OEt (5h), N3-Gly-
Phe-OEt (5i)] to furnish corresponding tetrapeptides (6fh, 6gi, 6ha) and pentapeptide (6hh) in 79-85% yields
(Scheme 4).The successful syntheses of these short peptides in high yields resulting in the formation of new
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peptide bonds at Ala-Trp, Glu-Gly, Gly-Phe and Gly-Trp junctions established the generality of phosphane 2 in
1
2
the Staudinger Peptide Ligation approach.
3
4
5
6
7
R1
O
R1
O
R
O
R
O
Et3N (1 equiv)
H
H
H
H
Cbz
N
N
R''
Cbz
N
N
R''
N
O
N3
X
N
N
X
(i) DMF, 40 oC, 12 h
(ii) H2O, 40 oC ,0.5 h
H
H
H
R2
O
O
R'
m
R2
O
O
R'
m
Ph2P
n
8
n
5e-g
9
6
4f-h
10
11
12
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O
O
H
N
H
N
NH
Cbz
N
N
OEt
O
O
H
N
H
H
H
O
O
Cbz
N
N
N
OEt
H
H
O
O
O
OMe
6gi 80%
6fh, 79%
O
O
O
O
H
H
H
H
Cbz
N
N
OEt
Cbz
N
N
N
N
H
N
N
N
H
N
H
H
H
H
O
O
O
O
O
NH
6ha, 85%
6hh, 82%
Scheme 4. Syntheses of short peptides by SPL
Finally, the scope of optimized SPL was applied towards peptide coupling using unprotected phosphinyl
benzyl ester. For this purpose, Boc-Gly-Gly-OCH2C6H4PPh2 (4i) was deprotected using HCl/dioxane at room
temperature to afford corresponding crude Cl-NH3 -Gly-Gly-OCH2C6H4PPh2 (4i′), which on coupling with α-N3-
+
o
Trp-Phe-OEt (5h) using 2 equivalent of triethyl amine in DMF at 40 C for 12 h produced corresponding
unprotected tetrapeptidyl ester (6i′h) in 40% yield (Scheme 5).
O
H
NH
O
H
Boc
N
Cl
HCl/Dioxane
r,t, 1 h
N
O
N
O
H
H3N
O
H
O
+
N
Ph2P
O
N3
OEt
Ph2P
4i
O
4i'
5h
Et3N (2 equiv)
(i) DMF, 40 oC, 12 h
(ii) H2O, 40 oC , 0.5 h
NH
O
H
O
H
N
N
H2N
N
OEt
H
O
O
6i'h, 40%
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Scheme 5. Synthesis of unprotected tetrapeptidyl ester by SPL
In summary, an efficient and broadly
1
2
tolerable
phosphane
probe,
(2-
3
4
5
6
7
8
9
(diphenylphosphaneyl)phenyl)methanol 2 is synthesized and categorically investigated as a C-terminus activator
for various amino acids/peptide junctions (Gly, Ala, Trp, Glu), achieving SPL via their coupling with different α-
azido amides/esters derived from hydrophobic, aromatic, and basic α-amino acids (Gly, Ala, Leu, Val, Tyr, Lys).
Native peptides are produced in high yields by employing the described traceless SPL. Quantum chemical
calculations were done to understand the reaction mechanism and energy profiles of compounds/transition states
with most stable conformers. A regioselective SPL was observed at Glu junction via 7-membered transition state
over 11-membered transition state, which was computationally verified. The conducted studies expanded the
realm of phoshphane treasure box for the synthesis of proteins via Staudinger Peptide Ligation at non-glycyl sites
also. Thus, a synthetic protocol based on SPL using phosphane probe 2 and appropriate bioactive azides could
open comfortable doors for peptide syntheses and drug discovery.
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Experimental Section
General computational methods. To generate complete conformational MMX forcefield in PCMODEL v9.3
software was used.10 To obtain global minima conformer, molecular restructuring was considered to prioritise
cyclization property. For pre-optimization process, MOPAC 2006 was used with PM3 parameterization.11
Density functional theory (DFT) calculations with BP86 hybrid functional and def2-TZVP basis set which is
employed in ORCA v4.2.0 ab initio software was used for geometry optimizations.12 TmoleX client version from
COSMOlogic GmbH was used for output data analysis as well as to generate different representations.13 Full
GMMX conformational search of the transition state was carried out with a parameter of 10,000 conformers,
which were subsequently pruned, filtered and ranked in the ascending order of the b(N-C) proximity function.8
Transition state search analysis. 3D geometry structures were optimized using ORCA 4.2.1 software. Geometry
optimizations were carried out at BP86 (GGA)/def2-TZVP level of theory with RI approximation, starting from
transition structures (TS).14 Transition states were related to minima (IS) through intermediates. Minima was
obtained from the saddle point optimization via potential energy scan by using large triple-zeta valence basis set
def2-TZVP was justified to minimize errors.15 Vibration frequencies studies were performed at the same level of
theory to confirm the convergence criteria with respect to transition states including any second-order or third-
order saddle point. This step is mandatory to eliminate the small imaginary frequencies and re-optimize again for
the complete geometry convergence. Solvation effects were considered in the calculation using conductor-like
polarizable continuum model (CPCM).16 The solvent reaction field is described by polarization charges on the
surface of the cavity to elucidate the thermodynamic parameters (H, TS, G) from frequency calculations at
the temperature 298.15 K.
General experimental methods. All chemicals were obtained from commercial suppliers and used without
further purification. Solvents were distilled following standard procedure before usage. All reactions were carried
out in oven-dried glassware with magnetic stirring. Purification of reaction products was carried out by flash
column chromatography using silica gel 60 (100-120 mesh). TLC visualization was accompanied by UV light,
KMnO4 solution and phosphomolybidic acid stain. Nuclear Magnetic Resonance spectra were recorded on Bruker
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1
13
400 spectrometer. All H NMR (400 MHz) and C NMR (101 MHZ) spectra were recorded either in CDCl3 or
DMSO-d6 with TMS as standard in δ units, parts per million (ppm). All coupling constants J were reported in Hz.
The following abbreviations were used to describe peak splitting patterns when appropriate: s = singlet, d =
doublet, t = triplet, dd = doublet of doublet, m = multiplet and brs = broad singlet. High resolution mass
spectrometry (HRMS) was performed with an Agilent 6210 instrument using time-of-flight (TOF-MS) with
electro spray ionization (ESI). Melting points were determined on a capillary point apparatus equipped with a
digital thermometer and are uncorrected.
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Synthesis of (2-(diphenylphosphanyl)phenyl)methanol (2). To a solution of 2-(diphenylphosphaneyl)benzoic
acid (1) (1.0 g, 3.26 mmol) in dry THF (30 mL), lithium aluminium hydride (LAH) (0.370 g, 9.804 mmol) was
o
added at 0 C in three portions. The resultant reaction mixture was stirred at room temperature for 2 h till the
completion of reaction that was monitored by TLC. Upon completion, THF of reaction mixture was evaporated to
dryness under reduced pressure and ethyl acetate (10 mL) was added. The organic layer was washed with brine (3
x 10 mL), dried over anhydrous magnesium sulfate, filtered and dried under reduced pressure to give 2 as yellow
oil (0.760 g). The spectroscopic data are in accordance with literature.17
General procedure for the synthesis of phosphinyl benzyl esters (4a-i). To a solution of N-protected amino
acid/peptide (3a-i) (1.0 equiv) in dry DCM (10 mL), DCC (1.0 equiv) and DMAP (1.0 equiv) were added at room
temperature. After 10 min. of stirring, a solution of (2-(diphenylphosphanyl)phenyl)methanol (2) (1.0 equiv) in
DCM (5 mL) was added, and the reaction mixture was stirred for 12-14 h. The completion of the reaction was
monitored by TLC. Upon completion, the reaction mixture was filtered through celite and the filtrate was
subjected to flash column chromatography to afford phosphinyl benzyl esters (4a-i).
2-(Diphenylphosphanyl)benzyl ((benzyloxy)carbonyl)glycinate (Cbz-Gly-OC6H4PPh2) (4a). Cbz-Gly-OH
(0.500 g, 2.39 mmol), PPh2C6H4CH2OH (0.700 g, 2.39 mmol), DCC (0.490 g, 2.39 mmol), DMAP (0.290 g, 2.39
mmol) were used following the above general procedure; Purification of this crude product by flash column
chromatography (silica gel, 0–10% ethyl acetate/hexanes), Rf = 0.31 (silica gel, EtOAc/Hexanes, 9:1); Colorless
oil, Yield 68% (0.780 g); 1H NMR (400 MHz, Chloroform-d) δ 7.54 – 7.46 (m, 1H), 7.42 – 7.29 (m, 14H), 7.27 –
7.21 (m, 3H), 7.03 – 6.98 (m, 1H), 5.47 (s, 2H), 5.12 (s, 2H), 4.96 (t, J = 5.6 Hz, 1H), 3.51 (d, J = 5.6 Hz, 2H);
13C{1H} NMR (101 MHz, Chloroform-d) δ 169.6, 156.3, 139.7, 139.6 (d, JCP = 25.1 Hz), 137.3, 137.1, 136.5 (d,
JCP = 9.5 Hz), 134.3, 134.2, 134.0, 130.2 (d, JCP = 5.2 Hz), 129.3, 129.1, 129.1, 128.9, 128.8, 128.8, 128.7, 128.7,
128.6, 128.4, 128.3, 128.3, 128.2, 127.1, 67.2, 66.5, 66.4 (d, J = 21.4 Hz), 42.5; 31P{1H} NMR (162 MHz,
Chloroform-d) δ -16.1; HRMS (ESI) m/z calcd for C29H26NO4PNa [M+Na]+ 506.1492, found 506.1488.
2-(Diphenylphosphanyl)benzyl ((benzyloxy)carbonyl)-L-alaninate (Cbz-L-Ala-OC6H4PPh2) (4b). Cbz-L-
Ala-OH (0.200 g, 0.89 mmol), PPh2C6H4CH2OH (0.260 g, 0.89 mmol), DCC (0.180 g, 0.89 mmol), DMAP
(0.110 g, 0.89 mmol) were used following the above general procedure; Purification of this crude product by
flash column chromatography (silica gel, 0–20% ethyl acetate/hexanes), Rf = 0.52 (silica gel, EtOAc/Hexanes,
8:2); Colorless oil, Yield 70% (0.310 g); 1H NMR (400 MHz, Chloroform-d) δ 7.40 – 7.32 (m, 1H), 7.28 – 7.20
(m, 15H), 7.18 – 7.11 (m, 2H), 6.90 – 6.83 (m, 1H), 5.31 – 5.12 (m, 1H), 5.09 (dd, J = 12.4, 4.4 Hz, 2H), 5.02 (s,
2H), 4.36 (p, J = 7.6 Hz, 1H), 1.34 (d, J = 7.2 Hz, 3H); 13C{1H} NMR (101 MHz, Chloroform-d) δ 171.4, 154.4,
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138.5 (d, JCP = 24.9 Hz), 135.3 (d, JCP = 9.6 Hz), 134.3, 133.1, 133.0, 132.9, 132.7 (d, JCP = 8.7 Hz), 128.5 (d, JCP
= 5.4 Hz), 128.2, 127.9, 127.8, 127.8, 127.6, 127.6, 127.5, 127.5, 127.4, 127.1 (d, JCP = 1.4 Hz), 127.0, 66.1, 64.9
(d, JCP = 23.0 Hz), 48.6 (d, JCP = 16.3 Hz), 17.5 (d, JCP = 26.2 Hz); 31P{1H} NMR (162 MHz, Chloroform-d) δ -
16.5; HRMS (ESI) m/z calcd for C30H29NO4P [M+H]+ 498.1829, found 498.1821.
1
2
3
4
5
6
2-(Diphenylphosphanyl)benzyl (tert-butoxycarbonyl)-L-tryptophanate (Boc-L-Trp-OC6H4PPh2) (4c). Boc-
L-Trp-OH (0.200 g, 0.66 mmol), PPh2C6H4CH2OH (0.190 g, 0.66 mmol), DCC (0.140 g, 0.66 mmol), DMAP
(0.080 g, 0.66 mmol) were used following the above general procedure; Purification of this crude product by
flash column chromatography (silica gel, 0–20% ethyl acetate/hexanes), Rf = 0.30 (silica gel, EtOAc/Hexanes,
8:2); Pale yellow solid, Yield 72% (0.270 g), mp 165-168 oC; 1H NMR (400 MHz, Chloroform-d) δ 8.12 (s, 1H),
7.39 (d, J = 8.0 Hz, 1H), 7.26 – 7.19 (m, 10H), 7.17 – 7.10 (m, 4H), 7.06 (t, J = 8.0 Hz, 1H), 6.98 (t, J = 8.0 Hz,
1H), 6.87 (dd, J = 7.2, 4.0 Hz, 1H), 6.71 (d, J = 2.4 Hz, 1H), 5.34 (d, J = 12.4 Hz, 1H), 5.27 (d, J = 12.8 Hz, 1H),
4.86 (d, J = 8.4 Hz, 1H), 4.36 – 4.26 (m, 1H), 3.00 (dd, J = 14.8, 5.4 Hz, 1H), 2.82 (dd, J = 14.8, 5.5 Hz, 1H),
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13
1.32 (s, 9H); C{1H} NMR (101 MHz, Chloroform-d) δ 171.9, 155.3, 139.8 (d, JCP = 24.9 Hz), 136.8 (d, JCP
=
16.0 Hz), 136.5, 136.4 (d, JCP = 9.7 Hz), 136.2, 134.1, 134.1, 134.0 (d, JCP = 8.9 Hz), 133.8, 129.7, 129.6, 129.3,
129.1, 129.0, 128.8, 128.8, 128.7, 128.7, 127.9, 123.0, 122.1, 119.6, 119.0, 111.3, 110.2, 79.8, 65.8 (d, JCP = 23.8
Hz), 60.6, 54.5, 28.7, 27.7; 31P{1H} NMR (162 MHz, Chloroform-d) δ -16.5 HRMS (ESI) m/z calcd for
C35H36N2O4P [M+H]+ 579.2407, found 579.2399.
5-Benzyl
1-(2-(diphenylphosphanyl)benzyl)
(tert-butoxycarbonyl)-L-glutamate
(Boc-L-Glu(OBn)-
C6H4PPh2) (4d). Boc-L-Glu(OBn)-OH (0.300 g, 0.89 mmol), PPh2C6H4CH2OH (0.260 g, 0.89 mmol), DCC
(0.180 g, 0.89 mmol), DMAP (0.110 g, 0.89 mmol) were used following the above general procedure;
Purification of this crude product by flash column chromatography (silica gel, 0–10% ethyl acetate/hexanes), Rf =
0.53 (silica gel, EtOAc/Hexanes, 8:2); Colorless oil, Yield 78% (0.420 g); 1H NMR (400 MHz, Chloroform-d) δ
7.37 – 7.33 (m, 1H), 7.29 – 7.18 (m, 12H), 7.17 – 7.09 (m, 5H), 6.88 – 6.84 (m, 1H), 5.37 (d, J = 12.4 Hz, 1H),
5.29 (d, J = 12.4 Hz, 1H), 4.99 (s, 2H), 4.80 (t, J = 7.2 Hz, 1H), 3.98 – 3.93 (m, 1H), 2.19 (t, J = 8.0 Hz, 2H), 1.91
– 1.77 (m, 2H), 1.32 (s, 9H); 13C{1H} NMR (101 MHz, Chloroform-d) δ 172.6, 171.7, 155.4, 139.56 (d, JCP
=
24.9 Hz), 136.9 (d, JCP = 16.3 Hz), 136.4 (d, JCP = 9.7 Hz), 135.9, 134.2, 134.1, 134.0, 133.9 (d, JCP = 8.2 Hz),
129.8 (d, JCP = 5.1 Hz), 129.3, 129.0, 128.9, 128.8 (d, JCP = 3.1 Hz), 128.7, 128.4, 79.9, 66.5, 66.1 (d, JCP = 23.0
Hz), 53.0, 30.4, 28.4, 27.5; 31P{1H} NMR (162 MHz, Chloroform-d) δ -16.5; HRMS (ESI) m/z calcd for
C36H39NO6P [M+H]+ 612.2510, found 612.2488.
(S)-4-((tert-butoxycarbonyl0amino)-5-((2-(diphenylphosphaneyl)benzyl)oxy)-5-oxopentanoic acid (Boc- L
Glu-C6H4PPh2) (4e). Boc-Glu-OH (0.300 g, 1.20 mmol), PPh2C6H4CH2OH (0.35 g, 1.20 mmol), DCC (0.25 g,
1.20 mmol), DMAP (0.015 g, 0.120 mmol) were used following the above general procedure; Purification of this
crude product by flash column chromatography (silica gel, 0–60% ethyl acetate/hexanes), Rf = 0.1 (silica gel,
1
EtOAc/Hexanes, 1:1); Colorless oil, Yield 68% (0.43 g); H NMR (400 MHz, Chloroform-d) δ 7.39 – 7.35 (m,
1H), 7.29 – 7.23 (m, 7H), 7.15 – 7.10 (m, 5H), 6.87 (ddd, J = 7.7, 4.4, 1.4 Hz, 1H), 5.29 (s, 2H), 5.01 (d, J = 8.2
Hz, 1H), 4.14 – 4.04 (m, 1H), 1.96 – 1.84 (m, 3H), 1.72 – 1.65 (m, 1H), 1.36 (s, 9H); 13C{1H} NMR (101 MHz,
Chloroform-d) δ 175.7, 172.7, 155.9, 140.2 (d, JCP = 25.0 Hz), 137.0 (d, JCP = 16.1 Hz), 136.7 (d, JCP = 9.3 Hz),
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134.3, 134.2, 134.0, 132.3 (d, JCP = 10.2 Hz), 130.0, 129.3, 129.1, 129.0, 128.9, 128.8, 128.7, 80.5, 65.7 (d, JCP
=
1
2
3
4
22.3 Hz), 53.0, 29.9, 28.5, 27.3; 31P{1H} NMR (162 MHz, CDCl3) δ -16.17. HRMS (ESI) m/z calcd for
C29H33NO6P [M+H] + 522.2040, found 522.2025.
5
6
7
8
2-(Diphenylphosphanyl)benzyl ((benzyloxy)carbonyl)-L-isoleucyl-L-alaninate (Cbz-L-Ile-L-Ala-C6H4PPh2)
(4f). Cbz-L-Ile-L-Ala-OH (0.120 g, 0.357 mmol), PPh2C6H4CH2OH (0.110 g, 0.357 mmol), DCC (0.073 g, 0.357
mmol), DMAP (0.040 g, 0.357 mmol) were used following the above general procedure; Purification of this
crude product by flash column chromatography (silica gel, 0–30% ethyl acetate/hexanes), Rf = 0.22 (silica gel,
EtOAc/Hexanes, 7:3); Colorless oil, Yield 70% (0.150 g); 1H NMR (400 MHz, Chloroform-d) δ 7.61 – 7.33 (m,
3H), 7.32 – 7.22 (m, 12H), 7.20 – 7.19 (m, 1H), 7.17 – 7.14 (m, 2H), 6.90 – 6.85 (m, 1H), 5.99 (d, J = 7.6 Hz,
1H), 5.41 – 5.28 (m, 2H), 5.23 (d, J = 11.2 Hz, 1H), 5.03 (s, 2H), 4.23 (t, J = 7.2 Hz, 1H), 3.89 (t, J = 7.6 Hz,
1H), 1.77 – 1.70 (m, 1H), 1.40 – 1.33 (m, 2H), 1.07 (d, J = 7.2 Hz, 3H), 0.85 – 0.78 (m, 6H); 13C{1H} NMR (101
MHz, Chloroform-d) δ 171.0, 169.4, 155.2, 138.4 (d, JCP = 25.1 Hz), 135.9, 135.3, 135.2, 133.1 (d, JCP = 5.7 Hz),
132.8, 132.6, 128.5, 128.2, 128.0, 127.9, 127.8, 127.7, 127.6 (d, JCP = 3.2 Hz), 127.5, 127.2, 127.1, 127.0, 66.0,
65.0 (d, JCP = 23.7 Hz), 58.5, 47.0, 36.7, 23.7, 17.1, 14.4, 10.4; 31P{1H} NMR (162 MHz, Chloroform-d) δ -16.5;
HRMS (ESI) m/z calcd for C36H40N2O5P [M+H] + 611.2669, found 611.2660.
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1-(2-(Diphenylphosphanyl)benzyl) 5-methyl ((benzyloxy)carbonyl)-L-valyl-L-glutamate (Cbz-L-Val-L-
Glu(OMe)-C6H4PPh2) (4g). Cbz-L-Val-L-Glu(OMe)-OH (0.210 g, 0.53 mmol), PPh2C6H4CH2OH (0.160 g, 0.53
mmol), DCC (0.110 g, 0.53 mmol), DMAP (0.070 g, 0.53 mmol) were used following the above general
procedure; Rf = 0.12 (silica gel, EtOAc/Hexanes, 7:3); Colorless oil, Yield 70% (0.250 g). This reactant was used
1
as crude for further ligation reaction; Crude H NMR (400 MHz, Chloroform-d) δ 7.38 – 7.33 (m, 1H), 7.30 –
7.20 (m, 12H), 7.19 – 7.08 (m, 5H), 6.92 – 6.85 (m, 1H), 6.67 – 6.63 (m, 1H), 5.46 – 5.23 (m, 3H), 5.09 (s, 2H),
4.28 – 4.14 (m, 1H), 3.95 – 3.83 (m, 1H), 3.52 (s, 3H), 2.16 – 2.03 (m, 3H), 1.66 – 1.57 (m, 2H), 0.87 – 0.80 (m,
3H), 0.79 (d, J = 7.6 Hz, 3H). 13C{1H} NMR (101 MHz, Chloroform-d) δ 173.5, 171.5, 171.5, 156.5, δ 139.4 (d,
JCP = 25.0 Hz), 136.4, 135.3, 134.2, 134.0 (d, JCP = 2.0 Hz), 133.8 (d, JCP = 4.4 Hz), 129.4, 129.0, 128.9, 128.8,
128.8, 128.7, 128.7, 128.7, 128.5 (d, JCP = 4.5 Hz), 128.3 (d, JCP = 1.8 Hz), 128.2 (d, JCP = 3.4 Hz), 67.5, 67.2 (d,
JCP = 9.5 Hz), 60.4, 52.0, 34.1, 30.1, 27.1, 25.1, 19.4, 19.2; 31P{1H} NMR (162 MHz, Chloroform-d) δ -17.2.
HRMS (ESI) m/z calcd for C38H42N2O7P [M+H] + 669.2724, found 669.6716.
2-(Diphenylphosphaneyl)benzyl ((benzyloxy)carbonyl)-L-isoleucylglycylglycinate (Cbz-L-Ile-Gly-Gly-
C6H4PPh2) (4h). Cbz-L-Ile-Gly-Gly-OH (0.250 g, 0.66 mmol), PPh2C6H4CH2OH (0.200 g, 0.66 mmol), DCC
(0.140 g, 0.66 mmol), DMAP (0.080 g, 0.66 mmol) were used following the above general procedure;
Purification of this crude product by flash column chromatography (silica gel, 0–60% ethyl acetate/hexanes), Rf =
0.32 (silica gel, EtOAc/Hexanes, 1:1); Colorless oil, Yield 70% (0.300 g); 1H NMR (400 MHz, Chloroform-d) δ
7.66 – 7.62 (m, 1H), 7.56 – 7.48 (m, 2H), 7.45 – 7.38 (m, 2H), 7.36 – 7.31 (m, 1H), 7.26 – 7.23 (m, 11H), 7.16 –
7.10 (m, 4H), 6.87 – 6.83 (m, 1H), 6.00 (d, J = 8.4 Hz, 1H), 5.30 (s, 2H), 5.01 – 4.91 (m, 2H), 3.93 – 3.80 (m,
2H), 3.47 (d, J = 5.6 Hz, 2H), 1.80 – 1.76 (m, 1H), 1.50 – 1.34 (m, 2H), 0.85 (d, J = 6.8 Hz, 3H), 0.79 (t, J = 7.2
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Hz, 3H); C{1H} NMR (101 MHz, Chloroform-d) δ 172.4, 169.8, 169.4, 156.9, 139.5 (d, JCP = 25.2 Hz), 137.0
(d, JCP = 16.4 Hz), 136.5 (d, JCP = 9.0 Hz), 136.3, 132.5 (d, JCP = 2.8 Hz), 134.3, 134.1, 133.9, 133.8, 133.0,
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132.5, 132.3, 132.2, 129.3, 129.1, 129.0, 128.8, 128.8, 128.7, 128.7, 128.7, 128.5, 128.4, 128.2, 128.2, 67.4, 66.3
(d, JCP = 22.3 Hz), 60.2, 43.1, 41.4, 37.3, 25.0, 15.7, 11.5. HRMS (ESI) m/z calcd for C37H41N3O6P [M+H]+
656.2789, found 656.2780.
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2-(Diphenylphosphaneyl)benzyl (tert-butoxycarbonyl)glycylglycinate (Boc-Gly-Gly-C6H4PPh2) (4i). Boc-
Gly-Gly-OH (0.084 g, 0.165 mmol), PPh2C6H4CH2OH (0.050 g, 0.165 mmol), DCC (0.04 g, 0.17 mmol), DMAP
(0.020 g, 0.165 mmol) were used following the above general procedure; Purification of this crude product by
flash column chromatography (silica gel, 0–40% ethyl acetate/hexanes), Rf = 0.1 (silica gel, EtOAc/Hexanes,
1:1); Colorless oil, Yield 71% (0.060 g); 1H NMR (400 MHz, Chloroform-d) δ 7.41 – 7.34 (m, 1H), 7.33 – 7.19
(m, 7H), 7.15 – 7.12 (m, 5H), 6.88 (dd, J = 7.7, 4.3 Hz, 1H), 6.36 (t, J = 5.5 Hz, 1H), 5.35 (d, J = 1.1 Hz, 2H),
5.20 (t, J = 5.8 Hz, 1H), 3.71 (d, J = 5.9 Hz, 2H), 3.48 (d, J = 5.4 Hz, 2H), 1.37 (s, 9H); 13C{1H} NMR (101
MHz, Chloroform-d) δ 169.8, 169.4, 156.2, 139.5 (d, JCP = 25.1 Hz), 137.1 (d, JCP = 16.4 Hz), 136.4 (d, JCP = 9.4
Hz), 134.3, 134.1, 133.9, 130.1 (d, JCP = 5.3 Hz), 129.3, 129.1, 129.1, 128.8, 128.7, 80.5, 66.4 (d, JCP = 21.6 Hz).,
60.6, 40.9, 28.5; 31P{1H} NMR (162 MHz, CDCl3) δ -16.18. HRMS (ESI) m/z calcd for C28H32N2O5P [M+H]+
507.2043, found 507.2019.
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2-((2-((2-(diphenylphosphaneyl)benzyl)oxy)2-oxoethyl)amino)-2-oxoethan-1-aminium chloride (HCl.NH2-
Gly-Gly-C6H4PPh2) (4i′). Compound 4i was stirred in HCl/dioxane for 1 h at room temperature. After
completion of the reaction as monitored by TLC, the solvent was removed under reduced pressure and the crude
product was washed with diethyl ether to afford crude 4i′, which was used as such for next step without any
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purification. H NMR (400 MHz, DMSO-d6) δ 8.76 – 8.73 (m, 1H), 8.26 – 7.99 (m, 2H), 7.52 – 7.35 (m, 6H),
7.19 – 7.15 (m, 3H), 6.87 (dd, J = 7.6, 4.4 Hz, 1H), 5.29 (s, 2H), 3.64 – 3.51 (m, 4H). 31P{1H} NMR (162 MHz,
DMSO-d6) δ -16.61.
General procedure for the synthesis of α-azido amides/dipeptidyl esters (5a, 5c-5i). To a solution of α-azido
acid18 (1.0 equiv), DCC (1.0 equiv) in dry DCM/DMF was added. After stirring for 10 minutes, p-toluidine (1.0
equiv) or α-amino ester (1.0 equiv) and NEt3 (1.0 equiv) were added and the stirring continued at room
temperature for 16 h. After 16 h, the reaction mixture was filtered through celite and the dried reaction mass was
subjected to flash column chromatography using ethyl acetate/hexanes gradients to afford pure α-azido amides
(5a, 5c-g)/ α-azido dipeptidyl esters (5h-i).
(S)-2-Azido-3-phenyl-N-(p-tolyl)propanamide (N3-L-Phe-p-tol) (5a). Purification of product by flash column
chromatography (silica gel, 0–10% ethyl acetate/hexanes), Rf = 0.3 (silica gel, EtOAc/Hexanes, 1:9),White solid,
mp: 90-91 ºC; 1H NMR (400 MHz, Chloroform-d) δ 7.92 (brs, 1H), 7.27 – 7.15 (m, 7H), 7.01 (d, J = 8.0 Hz, 2H),
4.20 – 4.16 (m, 1H), 3.30 (dd, J = 14.1, 4.3 Hz, 1H), 3.00 (dd, J = 14.1, 8.1 Hz, 1H), 2.21 (s, 3H); 13C{1H} NMR
(101 MHz, CDCl3) δ 166.9, 136.1, 136.1, 134.7, 134.6, 134.3, 129.8, 129.5, 129.5, 128.7, 127.3, 120.6, 120.5,
115.3, 65.6, 38.7, 20.9. HRMS (ESI) m/z calcd for C16H17N4O [M+H] + 281.1397, found 281.1405.
2-Azido-N-(p-tolyl)acetamide (N3-Gly-p-tol) (5b) This compound was synthesized by literature reported
method.13 Purification of product by flash column chromatography (silica gel, 0–10% ethyl acetate/hexanes), Rf =
0.5 (silica gel, EtOAc/Hexanes, 2:8), Light yellow solid, mp: 103-104 ºC (Lit.13 mp: 106-107 ºC); 1H NMR (400
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MHz, Chloroform-d) δ 8.08 (s, 1H), 7.42 (d, J = 8.4 Hz, 2H), 7.15 (d, J = 8.2 Hz, 2H), 4.11 (s, 2H), 2.34 (s, 3H);
13C{1H} NMR (101 MHz, Chloroform-d) δ 164.7, 134.9, 134.4, 129.7, 120.4, 53.1, 21.1.
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(S)-2-Azido-N-(p-tolyl)propanamide (N3-L-Ala-p-tol) (5c). Purification of product by flash column
chromatography (silica gel, 0–10% ethyl acetate/hexanes), Rf = 0.2 (silica gel, EtOAc/Hexanes, 1:9), Colorless
semi-solid; 1H NMR (400 MHz, Chloroform-d) δ 8.01 (s, 1H), 7.34 (d, J = 8.4 Hz, 2H), 7.05 (d, J = 8.4 Hz, 2H),
4.11 (q, J = 7.2 Hz, 1H), 2.23 (s, 3H), 1.54 (d, J = 6.8 Hz, 3H); 13C{1H} NMR (101 MHz, Chloroform-d) δ 168.0,
143.9, 134.5, 129.5, 120.3, 115.3, 59.3, 20.9, 17.1. HRMS (ESI) m/z calcd for C10H13N4O [M+H] + 205.1084,
found 205.1072.
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(S)-2-Azido-4-methyl-N-(p-tolyl)pentanamide (N3-L-Leu-p-tol) (5d). Purification of product by flash column
chromatography (silica gel, 0–10% ethyl acetate/hexanes), Rf = 0.2 (silica gel, EtOAc/Hexanes, 1:9), Colorless
semi-solid; 1H NMR (400 MHz, Chloroform-d) δ 8.11 (s, 1H), 7.43 (d, J = 8.4 Hz, 2H), 7.15 (d, J = 8.2 Hz, 2H),
4.09 (dd, J = 9.0, 4.2 Hz, 1H), 2.33 (s, 3H), 1.95 – 1.65 (m, 3H), 1.02 (d, J = 4.5 Hz, 3H), 1.01 (d, J = 4.6 Hz,
3H); 13C{1H} NMR (101 MHz, CDCl3) δ 168.1, 134.7, 129.7, 120.3, 63.5, 41.6, 25.2, 24.8, 23.2. HRMS (ESI)
m/z calcd for C13H19N4O [M+H]+ 247.1553, found 247.1548.
(S)-2-Azido-3-methyl-N-(p-tolyl)butanamide (N3-L-Val-p-tol) (5e). Purification of product by flash column
chromatography (silica gel, 0–10% ethyl acetate/hexanes), Rf = 0.3 (silica gel, EtOAc/Hexanes, 1:9), White
microcrystals, mp: 80-82 ºC; 1H NMR (400 MHz, Chloroform-d) δ 8.14 – 7.99 (m, 1H), 7.45 (d, J = 8.5 Hz, 2H),
7.20 – 7.12 (m, 2H), 4.01 (d, J = 3.8 Hz, 1H), 2.50 (dd, J = 6.9, 3.9 Hz, 1H), 2.35 (s, 3H), 1.17 (d, J = 6.9 Hz,
3H), 0.99 (d, J = 6.8 Hz, 3H); 13C{1H} NMR (101 MHz, Chloroform-d) δ 167.3, 134.7, 134.5, 129.6, 120.4, 71.0,
32.4, 21.0, 19.8, 17.0, 16.9. HRMS (ESI) m/z calcd for C12H17N4O [M+H]+ 233.1397, found 233.1395.
(S)-2-Azido-3-(4-hydroxyphenyl)-N-(p-tolyl)propanamide (N3-L-Tyr-p-tol) (5f). Purification of product by
flash column chromatography (silica gel, 0–20% ethyl acetate/hexanes), Rf = 0.2 (silica gel, EtOAc/Hexanes,
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2:8), Hygroscopic solid; H NMR (400 MHz, Chloroform-d) δ 7.86 (brs, 1H), 7.25 (d, J = 8.4 Hz, 2H), 7.10 –
7.00 (m, 4H), 6.71 (d, J = 7.9 Hz, 2H) 4.22-4.18 (m, 1H), 3.24 (dd, J = 14.2, 4.3 Hz, 1H), 2.97 (dd, J = 14.2, 7.8
Hz, 1H), 2.23 (s, 3H); 13C{1H} NMR (101 MHz, Chloroform-d) δ 167.3, 155.5, 135.1, 134.1, 130.9, 129.8, 127.7,
120.7, 115.9, 66.2, 38.2, 21.1. HRMS (ESI) m/z calcd for C16H17N4O2 [M+H]+ 297.1346, found 297.1337.
Benzyl (S)-(5-azido-6-oxo-6-(p-tolylamino)hexyl)carbamate (N3-L-Lys(Cbz)-p-tol) (5g). Purification of
product by flash column chromatography (silica gel, 0–10% ethyl acetate/hexanes), Rf = 0.2 (silica gel,
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EtOAc/Hexanes, 2:8), Colorless gelly solid; H NMR (400 MHz, Chloroform-d) δ 8.10 (s, 1H), 7.39 (d, J = 8.5
Hz, 2H), 7.35 – 7.25 (m, 5H), 7.10 (d, J = 8.2 Hz, 2H), 5.04 (s, 2H), 4.88 – 4.85 (m, 1H), 3.16 (q, J = 6.3 Hz,
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2H), 2.28 (s, 3H), 1.96 – 1.84 (m, 2H), 1.55 – 1.39 (m, 4H); C{1H} NMR (101 MHz, CDCl3) δ 167.4, 156.7,
136.8, 134.77, 134.6, 129.7, 128.7, 128.3, 128.2, 120.3, 66.8, 64.5, 40.7, 31.9, 29.7, 22.6, 21.1, 14.4. HRMS
(ESI) m/z calcd for C21H26N5O3 [M+H]+ 396.2030, found 396.2025.
Ethyl ((S)-2-azido-3-(1H-indol-3-yl)propanoyl)-L-phenylalaninate (N3-L-Trp-L-Phe-OEt) (5h). This product
was used without further purification. Yellow oil; 1H NMR (400 MHz, Chloroform-d) δ 8.24 (s, 1H), 7.59 (d, J =
7.8 Hz, 1H), 7.26 (d, J = 6.8 Hz, 1H), 7.14 – 7.02 (m, 5H), 6.98 (d, J = 2.4 Hz, 1H), 6.72 (d, J = 7.9 Hz, 2H), 6.59
(d, J = 8.2 Hz, 1H), 4.73 (dt, J = 8.2, 5.9 Hz, 1H), 4.18 (dd, J = 7.3, 4.3 Hz, 1H), 4.04 (qd, J = 7.2, 1.2 Hz, 2H),
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3.34 (dd, J = 14.9, 4.3 Hz, 1H), 3.13 (dd, J = 14.9, 7.3 Hz, 1H), 2.87 (dd, J = 13.8, 5.8 Hz, 1H), 2.75 (dd, J =
13.8, 5.9 Hz, 1H), 1.12 (t, J = 7.1 Hz, 3H); 13C{1H} NMR (101 MHz, Chloroform-d) δ 171.1, 168.8, 136.3, 135.6,
129.4, 128.6, 127.5, 127.2, 123.8, 122.4, 119.9, 118.9, 111.5, 110.2, 64.8, 61.8, 53.1, 37.9, 28.5, 14.2. HRMS
(ESI) m/z calcd for C22H24N5O3 [M+H]+ 406.1874, found 406.1883.
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Ethyl (2-azidoacetyl)-L-phenylalaninate (N3-Gly-L-Phe-OEt) (5i). This product was used without further
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purification. Light yellow solid, mp 50-52 C (Lit.19 mp: 57 ºC); 1H NMR (400 MHz, Chloroform-d) δ 7.24 –
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7.12 (m, 3H), 7.06 – 17.02 (m, 2H), 6.85 – 16.77 (m, 1H), 4.77 (dt, J = 8.2, 6.1 Hz, 1H), 4.14 – 4.04 (m, 2H),
3.80 (s, 1H), 3.09 – 12.98 (m, 2H), 1.17 – 11.13 (m, 3H); 13C{1H} NMR (101 MHz, Chloroform-d) δ 171.1,
166.6, 135.7, 129.3, 128.7, 127.3, 61.7, 53.2, 52.4, 37.9, 14.1.
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General Procedure for the synthesis of peptides (6) via SPL
Phosphinyl benzyl ester (4a-h, 4i′) (1.0 equiv) was dissolved in DMF (5 mL) and α-azido amide (5a, 5c-g)/ α-
azido dipeptidyl ester (5h-i) (1.1 equiv) and NEt3 (1.0 equiv) (Note: 2.0 equiv of NEt3 in case of 4i′) were added
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at room temperature. The reaction mixture was stirred for 12 h at 40 C, after which water (0.5 mL) was added
and the mixture was further stirred for 30 minutes. The reaction was then quenched with crushed-ice and
extracted with ethyl acetate (3 x 10 mL). The organic layer was washed with brine (3 x 10 mL), dried over
anhydrous sodium sulfate, filtered and dried under reduced pressure to afford crude product, which were further
purified by column chromatography.
Benzyl (S)-(2-oxo-2((1-oxo-3-phenyl-1-(p-tolylamino)propan-2-yl)amino)ethyl)carbamate (Cbz-Gly-L-Phe-
p-tol) (6aa). Cbz-Gly-OC6H4PPh2 (0.100 g, 0.207 mmol), N3-L-Phe-p-tol (0.060 g, 0.23 mmol), Et3N (0.05 mL,
0.33 mmol) were used following above general ligation procedure; Purification of this crude product by flash
column chromatography (silica gel, 30–60% ethyl acetate/hexanes), Rf = 0.52 (silica gel, EtOAc/Hexanes, 1:1);
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White solid, Yield 90% (0.080 g), mp 164-165 C; H NMR (400 MHz, DMSO-d6) δ 9.93 (s, 1H), 8.20 (d, J =
8.0 Hz, 1H), 7.59 – 7.55 (m, 1H), 7.45 (d, J = 8.4 Hz, 2H), 7.43 – 7.39 (m, 1H), 7.36 – 7.32 (m, 4H), 7.28 – 7.22
(m, 4H), 7.21 – 7.16 (m, 1H), 7.10 (d, J = 8.4 Hz, 2H), 5.03 (s, 2H), 4.67 (q, J = 5.2 Hz, 1H), 3.67 (dd, J = 16.8,
6.1 Hz, 1H), 3.57 (dd, J = 16.8, 6.1 Hz, 1H), 3.04 (dd, J = 13.6, 5.4 Hz, 1H), 2.94 – 2.83 (m, 1H), 2.25 (s, 3H);
13C{1H} NMR (101 MHz, DMSO-d6) δ 169.9, 169.3, 156.9, 137.8, 137.4, 136.5, 132.8, 131.8, 129.5, 129.3,
129.2, 128.7, 128.5, 128.1, 128.0, 126.7, 119.9, 65.9, 55.0, 43.8, 38.3, 20.8; HRMS (ESI) m/z calcd for
C26H28N3O4 [M+H]+ 446.2074, found 446.2060.
Benzyl (2-oxo-2-((2-oxo-2-(p-tolylamino)ethyl)amino)ethyl)carbamate (Cbz-Gly-Gly-p-tol) (6ab). Cbz-Gly-
OC6H4PPh2 (0.160 g, 0.33 mmol), N3-Gly-p-tol (0.070 g, 0.36 mmol), Et3N (0.05 mL, 0.33 mmol) were used
following above general ligation procedure; Purification of this crude product by flash column chromatography
(silica gel, 30–60% ethyl acetate/hexanes), Rf = 0.43 (silica gel, EtOAc/Hexanes, 1:1); White solid, Yield 95%
(0.110 g), mp 205-206 oC; 1H NMR (400 MHz, DMSO-d6) δ 9.77 (s, 1H), 8.24 (t, J = 6.0 Hz, 1H), 7.54 (t, J = 6.0
Hz, 1H), 7.48 (d, J = 8.0 Hz, 2H), 7.39 – 7.28 (m, 5H), 7.10 (d, J = 8.0 Hz, 2H), 5.05 (s, 2H), 3.89 (d, J = 5.6 Hz,
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2H), 3.69 (d, J = 6.0 Hz, 2H), 2.24 (s, 3H); C{1H} NMR (101 MHz, DMSO-d6) δ 169.6, 167.3, 156.6, 137.0,
136.2, 132.2, 129.1, 128.3, 127.8, 127.7, 119.2, 65.6, 43.6, 42.6, 20.4; HRMS (ESI) m/z calcd for C19H22N3O4
[M+H]+ 356.1605, found 356.1598.
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Benzyl (S)-(2-oxo-2-((1-oxo-1-(p-tolylamino)propan-2-yl)amino)ethyl)carbamate (Cbz-Gly-L-Ala-ptol)
(6ac). Cbz-Gly-OC6H4PPh2 (0.100 g, 0.207 mmol), N3-L-Ala-p-tol (0.050 g, 0.23 mmol), Et3N (0.05 mL, 0.33
mmol) were used following above general ligation procedure; Purification of this crude product by flash column
chromatography (silica gel, 30–60% ethyl acetate/hexanes), Rf = 0.36 (silica gel, EtOAc/Hexanes, 1:1); White
solid, Yield 95% (0.070 g), mp 165-166 oC; 1H NMR (400 MHz, DMSO-d6) δ 9.84 (s, 1H), 8.17 (d, J = 7.6 Hz,
1H), 7.98 – 7.56 (m, 1H), 7.51 – 7.47 (m, 2H), 7.36 – 7.27 (m, 5H), 7.10 (d, J = 8.0 Hz, 2H), 5.04 (s, 2H), 4.44
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DMSO-d6) δ 171.3, 169.3, 157.0, 137.5, 136.8, 132.7, 131.9, 129.5, 128.8, 128.2, 128.1, 119.7, 65.9, 49.3, 43.9,
20.9, 18.8; HRMS (ESI) m/z calcd for C20H24N3O4 [M+H]+ 370.1761, found 370.1763.
Benzyl (S)-(2-((4-methyl-1-oxo-1-(p-tolylamino)pentan-2-yl)amino)-2-oxoethyl)carbamate (Cbz-Gly-L-Leu-
p-tol) (6ad). Cbz-Gly-OC6H4PPh2 (0.100 g, 0.207 mmol), N3-L-Leu-p-tol (0.060 g, 0.23 mmol), Et3N (0.05 mL,
0.33 mmol) were used following above general ligation procedure; Purification of this crude product by flash
column chromatography (silica gel, 30–60% ethyl acetate/hexanes), Rf = 0.35 (silica gel, EtOAc/Hexanes, 1:1);
White solid, Yield 80% (0.070 g), mp 149-150 oC; 1H NMR (400 MHz, Chloroform-d) δ 8.66 (s, 1H), 7.31 (d, J =
8.0 Hz, 2H), 7.26 – 7.20 (m, 5H), 7.04 (d, J = 8.0 Hz, 1H), 6.96 (d, J = 8.1 Hz, 2H), 5.64 (t, J = 5.8 Hz, 1H), 5.01
(s, 2H), 4.56 (td, J = 8.4, 5.7 Hz, 1H), 3.86 – 3.77 (m, 2H), 2.19 (s, 3H), 1.74 – 1.64 (m, 1H), 1.63 – 1.45 (m, 2H),
0.85 (d, J = 6.3 Hz, 3H), 0.82 (d, J = 6.0 Hz, 3H); 13C{1H} NMR (101 MHz, Chloroform-d6) δ 170.4, 170.0,
157.0, 136.3, 135.4, 134.2, 129.6, 128.7, 128.5, 128.3, 120.3, 67.5, 53.0, 42.5, 41.0, 25.1, 23.7, 23.1, 21.0; HRMS
(ESI) m/z calcd for C23H30N3O4 [M+H]+ 412.2231, found 412.2229.
Benzyl (S)-(2-((3-methyl-1-oxo-1-(p-tolylamino)butan-2-yl)amino)-2-oxoethyl)carbamate (Cbz-Gly-L-Val-
p-tol) (6ae). Cbz-Gly-OC6H4PPh2 (0.100 g, 0.207 mmol), N3-L-Val-p-tol (0.050 g, 0.23 mmol), Et3N (0.05 mL,
0.33 mmol) were used following above general ligation procedure; Purification of this crude product by flash
column chromatography (silica gel, 30–60% ethyl acetate/hexanes), Rf = 0.52 (silica gel, EtOAc/Hexanes, 1:1);
White solid, Yield 80% (0.066 g), mp 180-182 oC; 1H NMR (400 MHz, DMSO-d6) δ 9.96 (s, 1H), 7.93 (d, J = 8.8
Hz, 1H), 7.50 – 7.42 (m, 3H), 7.39 – 7.25 (m, 5H), 7.10 (d, J = 8.0 Hz, 2H), 5.03 (s, 2H), 4.31 (t, J = 8.0 Hz, 1H),
3.70 (d, J = 6.4 Hz, 2H), 2.25 (s, 3H), 2.08 – 1.95 (m, 1H), 0.89 – 0.87 (m, 6H); 13C{1H} NMR (101 MHz,
DMSO-d6) δ 170.1, 169.5, 156.9, 137.5, 136.6, 132.7, 129.5, 128.7, 128.1, 128.0, 119.8, 65.8, 58.8, 43.8, 31.3,
20.8, 19.5, 18.6; HRMS (ESI) m/z calcd for C22H28N3O4 [M+H]+ 398.2074, found 398.2061.
Benzyl (S)-(2-((3-(4-hydroxyphenyl)-1-oxo-1-(p-tolylamino)propan-2-yl)amino)-2-oxoethyl) carbamate
(Cbz-Gly-L-Tyr-ptol) (6af). Cbz-Gly-OC6H4PPh2 (0.100 g, 0.207 mmol), N3-L-Tyr-p-tol (0.070 g, 0.23 mmol),
Et3N (0.05 mL, 0.33 mmol) were used following above general ligation procedure; Purification of this crude
product by flash column chromatography (silica gel, 30–60% ethyl acetate/hexanes), Rf = 0.30 (silica gel,
EtOAc/Hexanes, 1:1); Pale yellow solid, Yield 92% (0.090 g), mp 122-124 oC; 1H NMR (400 MHz, DMSO-d6) δ
9.91 (s, 1H), 9.18 (s, 1H), 8.13 (d, J = 8.0 Hz, 1H), 7.47 – 7.39 (m, 3H), 7.38 – 7.25 (m, 5H), 7.09 (d, J = 8.0 Hz,
2H), 7.03 (d, J = 7.6 Hz, 2H), 6.63 (d, J = 8.4 Hz, 2H), 5.02 (s, 2H), 4.60 – 4.55 (m, 1H), 3.67 (dd, J = 16.8, 6.1
Hz, 1H), 3.57 (dd, J = 16.8, 6.1 Hz, 1H), 2.89 (dd, J = 12.4, 3.6 Hz, 1H), 2.79 – 2.72 (m, 1H), 2.24 (s, 3H);
13C{1H} NMR (101 MHz, DMSO-d6) δ 170.2, 169.3, 156.9, 156.3, 137.5, 136.6, 132.8, 131.9, 131.8, 130.5,
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129.5, 129.4, 128.8, 128.2, 128.1, 127.9, 119.9, 115.4, 65.9, 55.4, 43.8, 37.6, 20.9; HRMS (ESI) m/z calcd for
1
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C26H28N3O5 [M+H]+ 462.2023, found 462.2003.
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4
Benzyl
(S)-(2-((6-(((benzyloxy)carbonyl)amino)-1-oxo-1-(phenylamino)hexan-2-yl)amino)-2-oxo
5
6
7
8
ethyl)carbamate (Cbz-Gly-L-Lys(Cbz)-p-tol) (6ag). Cbz-Gly-OC6H4PPh2 (0.100 g, 0.207 mmol), N3-L-
Lys(Cbz)-p-tol (0.090 g, 0.23 mmol), Et3N (0.05 mL, 0.33 mmol) were used following above general ligation
procedure; Purification of this crude product by flash column chromatography (silica gel, 30–60% ethyl
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o
acetate/hexanes), Rf = 0.26 (silica gel, EtOAc/Hexanes, 1:1); White solid, Yield 88% (0.100 g), mp 187-188 C;
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1H NMR (400 MHz, DMSO-d6) δ 9.90 (s, 1H), 8.10 (d, J = 8.0 Hz, 1H), 7.50 – 7.45 (m, 3H), 7.38 – 7.26 (m,
10H), 7.26 – 7.21 (m, 1H), 7.10 (d, J = 8.0 Hz, 2H), 5.03 (s, 2H), 4.99 (s, 2H), 4.42 – 4.35 (m, 1H), 3.68 (d, J =
5.6 Hz, 2H), 2.99 – 2.92 (m, 2H), 2.24 (s, 3H), 1.76 – 1.66 (m, 1H), 1.64 – 1.53 (m, 1H), 1.48 – 1.36 (m, 2H),
1.36 – 1.17 (m, 2H); 13C{1H} NMR (101 MHz, DMSO-d6) δ 170.3, 169.0, 156.5, 156.1, 137.2, 137.0, 136.3,
132.3, 129.0, 128.3, 127.8, 127.7, 127.6, 119.3, 65.4, 65.1, 53.2, 43.4, 31.9, 29.1, 22.6, 20.4; HRMS (ESI) m/z
calcd for C31H37N4O6 [M+H]+ 561.2708, found 561.2697.
Benzyl ((S)-1-oxo-1-(((S)-1-oxo-1(p-tolylamino)propan-2-yl)amino)propan-2-yl)carbamate (Cbz-L-Ala-L-
Ala-p-tol) (6bc). Cbz-L-Ala-OC6H4PPh2 (0.100 g, 0.20 mmol), N3-L-Ala-p-tol (0.050 g, 0.22 mmol), Et3N (0.03
mL, 0.22 mmol) were used following above general ligation procedure; Purification of this crude product by flash
column chromatography (silica gel, 30–60% ethyl acetate/hexanes), Rf = 0.22 (silica gel, EtOAc/Hexanes, 1:1);
White solid, Yield 87% (0.070 g), mp 225-226 oC; 1H NMR (400 MHz, DMSO-d6) δ 9.82 (s, 1H), 8.08 (d, J = 7.3
Hz, 1H), 7.48 (d, J = 8.6 Hz, 3H), 7.39 – 7.27 (m, 6H), 7.10 (d, J = 8.1 Hz, 2H), 5.02 (d, J = 2.8 Hz, 2H), 4.39 (p,
J = 7.0 Hz, 1H), 4.08 (p, J = 7.2 Hz, 1H), 2.24 (s, 3H), 1.29 (d, J = 7.0 Hz, 3H), 1.21 (d, J = 7.1 Hz, 3H);13C{1H}
NMR (101 MHz, DMSO-d6) δ 172.9, 171.3, 156.3, 137.4, 137.4, 136.8, 132.7, 131.9, 129.6, 128.8, 128.1,
119.7, 65.9, 50.5, 49.4, 20.9, 18.7, 18.5; HRMS (ESI) m/z calcd for C21H26N3O4 [M+H]+ 384.1918, found
384.1912.
Benzyl ((S)-1-oxo-1-(((S)-1-oxo-1(p-tolylamino)propan-2-yl)amino)propan-2-yl)carbamate (Boc-L-Trp-Gly-
p-tol) (6cb). Boc-L-Trp-OC6H4PPh2 (0.100 g, 0.17 mmol), N3-Gly-p-tol (0.040 g, 0.19 mmol), Et3N (0.03 mL,
0.22 mmol) were used following above general ligation procedure; Purification of this crude product by flash
column chromatography (silica gel, 30–60% ethyl acetate/hexanes), Rf = 0.32 (silica gel, EtOAc/Hexanes, 1:1);
White solid, Yield 80% (0.070 g), mp 165-166 oC; 1H NMR (400 MHz, DMSO-d6) δ 10.82 (s, 1H), 9.67 (s, 1H),
8.37 (t, J = 5.8 Hz, 1H), 7.59 (dd, J = 11.7, 6.2 Hz, 1H), 7.50 (d, J = 8.2 Hz, 2H), 7.33 (d, J = 8.0 Hz, 1H), 7.18
(d, J = 2.3 Hz, 1H), 7.11 (d, J = 8.1 Hz, 2H), 7.06 (t, J = 7.5 Hz, 1H), 6.98 (t, J = 7.4 Hz, 1H), 6.93 (d, J = 7.6 Hz,
1H), 4.25 – 4.18 (m, 1H), 3.91 (dd, J = 16.3, 5.8 Hz, 1H), 3.84 (dd, J = 16.6, 5.6 Hz, 1H), 3.14 (dd, J = 14.6, 4.6
Hz, 1H), 2.95 (dd, J = 14.6, 9.4 Hz, 1H), 2.25 (s, 3H), 1.31 (s, 9H); 13C{1H} NMR (101 MHz, DMSO-d6) δ 173.0,
167.9, 156.0, 136.6, 136.6, 132.8, 129.6, 127.8, 124.2, 121.3, 119.7, 118.9, 118.7, 111.8, 110.6, 78.8, 55.8, 43.3,
28.6, 28.0, 20.9; HRMS (ESI) m/z calcd for C25H31N4O4 [M+H]+ 451.2340, found 451.2328.
Benzyl (S)-4-((tert-butoxycarbonyl)amino)-5-oxo-5-((2-oxo-2-(p-tolylamino)ethyl)amino) pentanoate (Boc-
L-Glu(OBn)-Gly-p-tol) (6db). Boc-L-Glu(OBn)-OC6H4PPh2 (0.100 g, 0.16 mmol), N3-Gly-p-tol (0.035 g, 0.18
mmol), Et3N (0.03 mL, 0.22 mmol) were used following above general ligation procedure; Purification of this
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crude product by flash column chromatography (silica gel, 30–60% ethyl acetate/hexanes), Rf = 0.30 (silica gel,
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2
1
EtOAc/Hexanes, 1:1); White solid, Yield 90% (0.070 g), mp 100-101 oC; H NMR (400 MHz, DMSO-d6) δ 9.69
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4
5
6
7
8
9
(s, 1H), 8.24 (t, J = 5.6 Hz, 1H), 7.48 (d, J = 8.4 Hz, 2H), 7.39 – 7.29 (m, 5H), 7.13 – 7.07 (m, 3H), 5.09 (s, 2H),
4.04 – 4.34 (m, 1H), 3.92 – 3.80 (m, 2H), 2.45 (t, J = 8.0 Hz, 2H), 2.24 (s, 3H), 2.09 – 1.91 (m, 1H), 1.87 – 1.77
(m, 1H), 1.38 (s, 9H); 13C{1H} NMR (101 MHz, DMSO-d6) δ 172.7, 172.4, 167.7, 156.0, 136.6, 132.7, 129.5,
128.8, 128.4, 128.3, 119.5, 78.8, 65.9, 54.1, 43.1, 30.5, 28.6, 27.4, 20.9; HRMS (ESI) m/z calcd for C26H34N3O6
[M+H]+ 484.2442, found 484.2429.
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60
Benzyl
(S)-4-((tert-butoxycarbonyl)amino)-5-oxo-5-(((S)-1-oxo-1-(p-tolylamino)propan-2-
yl)amino)pentanoate (Boc-L-Glu(OBn)-L-Ala-p-tol) (6dc). Boc-L-Glu(OBn)-OC6H4PPh2 (0.100 g, 0.16
mmol), N3-L-Ala-p-tol (0.040 g, 0.18 mmol), Et3N (0.03 mL, 0.22 mmol) were used following above general
ligation procedure; Purification of this crude product by flash column chromatography (silica gel, 30–60% ethyl
o
acetate/hexanes), Rf = 0.36 (silica gel, EtOAc/Hexanes, 3:2); White solid, Yield 88% (0.072 g), mp 108-110 C;
1H NMR (400 MHz, Chloroform-d) δ 8.78 – 8.75 (m, 1H), 7.41 (d, J = 6.0 Hz, 2H), 7.36 – 7.21 (m, 6H), 7.05 (d,
J = 8.0 Hz, 2H), 5.66 – 5.62 (m, 1H), 5.09 (s, 2H), 4.68 – 4.62 (m, 1H), 4.25 – 4.18 (m, 1H), 2.52 – 2.42 (m, 2H),
2.27 (s, 3H), 2.18 – 2.11 (m, 1H), 2.00 – 1.95 (m, 1H), 1.46 – 1.38 (m, 12H); 13C{1H} NMR (101 MHz,
Chloroform-d) δ 173.1, 172.3, 170.3, 156.1, 135.8, 135.4, 134.0, 129.5, 129.5, 128.7, 128.5, 128.4, 126.3, 120.4,
120.3, 80.6, 66.8, 54.4, 49.8, 30.6, 28.4, 27.6, 21.0, 18.0; HRMS (ESI) m/z calcd for C27H36N3O6 [M+H]+
498.2599 , found 498.2586.
(S)-4-((tert-Butoxycarbonyl)amino)-5-oxo-5-((2-oxo-2-(p-tolylamino)ethyl)amino)pentanoic acid (Boc-L-
Glu-Gly-p-tol) (6eb). Boc-L-Glu-OC6H4PPh2 (0.100 g, 0.19 mmol), N3-Gly-ptol (0.04 g, 0.21 mmol), Et3N (0.03
mL, 0.2 mmol) were used following above general ligation procedure; Purification of this crude product by flash
column chromatography (silica gel, 100% EtOAc), Rf = 0.2 (silica gel, 100% EtOAc); White solid, Yield 73%
o
1
(0.055 g), mp 128-130 C; H NMR (400 MHz, DMSO-d6) δ 9.99 (s, 1H), 8.20 (t, J = 6.0 Hz, 1H), 7.48 (d, J =
8.4 Hz, 2H), 7.07 (d, J = 8.0 Hz, 2H), 6.36 (d, J = 7.2 Hz, 1H), 3.80 (d, J = 5.6 Hz, 2H), 3.73 (q, J = 6.4 Hz, 1H),
2.23 (s, 3H), 2.20 – 2.14 (m, 2H), 1.92 – 1.85 (m, 1H), 1.82 – 1.75 (m, 1H), 1.36 (s, 9H); 13C{1H} NMR (101
MHz, DMSO-d6) δ 174.6, 172.8, 167.9, 155.2, 136.5, 132.2, 129.1, 119.4, 77.8, 54.3, 42.8, 31.9, 28.3, 20.5;
HRMS (ESI) m/z calcd for C19H26N3O5 [M+H]+[-H2O] 376.1872, found 376.1886.
Ethyl ((benzyloxy)carbonyl)-L-isoleucyl-L-alanyl-L-tryptophyl-L-phenylalaninate (Cbz-L-Ile-L-Ala-L-Trp-
L-Phe-OEt) (6fh). Cbz-L-Ile-L-Ala-OC6H4PPh2 (0.050 g, 0.08 mmol), N3-L-Trp-L-Phe-OEt (0.040 g, 0.09
mmol), Et3N (0.03 mL, 0.22 mmol) were used following above general ligation procedure; Purification of this
crude product by flash column chromatography (silica gel, 1-5% DCM/MeOH), Rf = 0.1 (silica gel, DCM:
MeOH, 9:1); White solid, Yield 79% (0.045 g), mp 120-121 oC; 1H NMR (400 MHz, DMSO-d6) δ 11.00 (s, 1H),
8.68 (brs, 1H), 8.34 (d, J = 6.6 Hz, 1H), 7.86 – 7.80 (m, 1H), 7.78 – 7.70 (m, 1H), 7.64 – 7.58 (m, 1H), 7.51 –
7.36 (m, 5H), 7.35 – 7.25 (m, 3H), 7.23 – 7.13 (m, 3H), 7.11 – 7.08 (m, 2H), 6.89 (t, J = 7.4 Hz, 1H), 5.02 (d, J =
12.7 Hz, 1H), 4.98 (d, J = 12.7 Hz, 1H), 4.63 – 4.50 (m, 1H), 4.46 – 4.31 (m, 2H), 4.06 – 3.92 (m, 2H), 3.87 (t, J
= 8.5 Hz, 1H), 3.12 – 2.96 (m, 2H), 2.95 – 2.85 (m, 1H), 2.77 (dd, J = 13.8, 8.2 Hz, 1H), 1.66 – 1.60 (m, 1H),
1.39 – 1.33 (m, 1H), 1.14 – 1.07 (m, 4H), 1.04 (t, J = 6.9 Hz, 3H), 0.79 – 0.63 (m, 6H); 13C{1H} NMR (101 MHz,
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DMSO-d6) δ 171.9, 171.6, 171.5, 156.4, 140.2, 137.5, 136.7, 135.4, 133.5, 130.2, 129.3, 128.8, 128.7, 128.2,
128.0, 127.1, 125.5, 121.6, 119.1, 118.9, 111.9, 65.8, 65.3, 63.8, 61.0, 59.1, 47.7, 37.0, 36.9, 24.7, 17.1, 15.5,
14.3, 11.2; HRMS (ESI) m/z calcd for C39H48N5O7 [M+H]+ 698.3548, found 698.3543.
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3
4
5
6
7
8
Ethyl
(5S,8S,14S)-14-benzyl-5-isopropyl-8-(3-methoxy-3-oxopropyl)-3,6,9,12-tetraoxo-1-phenyl-2-oxa-
4,7,10,13-tetraazapentadecan-15-oate (Cbz-L-Val-L-Glu(OMe)-Gly-L-Phe-OEt) (6gi). Cbz-L-Val-L-
Glu(OMe)-OC6H4PPh2 (0.100 g, 0.15 mmol), N3-Gly-L-Phe-OEt (0.050 g, 0.16 mmol), Et3N (0.03 mL, 0.22
mmol) were used following above general ligation procedure; Purification of this crude product by flash column
chromatography (silica gel, 100% ethyl acetate), Rf = 0.32 (silica gel, 100% EtOAc); White solid, Yield 80%
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10
11
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47
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49
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60
o
1
(0.075 g), mp 154-155 C; H NMR (400 MHz, DMSO-d6) δ 8.29 (t, J = 7.1 Hz, 1H), 8.22 (d, J = 7.8 Hz, 1H),
8.15 – 8.08 (m, 1H), 8.00 (d, J = 7.5 Hz, 1H), 7.40 – 7.34 (m, 3H), 7.33 – 7.28 (m, 2H), 7.26 (d, J = 7.2 Hz, 2H),
7.24 – 7.16 (m, 3H), 5.03 – 4.98 (m, 2H), 4.44 (q, J = 7.6 Hz, 1H), 4.34 – 4.21 (m, 1H), 4.01 (qd, J = 7.0, 1.9 Hz,
2H), 3.88 (t, J = 7.8 Hz, 1H), 3.76 – 3.66 (m, 2H), 3.57 (s, 3H), 2.99 (dd, J = 13.7, 6.3 Hz, 1H), 2.91 (dd, J =
13.7, 8.4 Hz, 1H), 2.33 (t, J = 6.8 Hz, 2H), 2.02 – 1.87 (m, 2H), 1.84 – 1.66 (m, 1H), 1.07 (t, J = 7.1 Hz, 3H),
0.84 (d, J = 6.7 Hz, 3H), 0.82 – 0.78 (m, 3H); 13C{1H} NMR (101 MHz, DMSO-d6) δ 173.2, 172.0, 171.8, 171.6,
169.1, 156.8, 137.4, 129.5, 128.8, 128.7, 128.3, 128.2, 127.0, 66.0, 65.9, 61.0, 54.2, 52.3, 51.8, 42.0, 37.4, 30.6,
30.2, 30.1, 27.5, 19.6, 18.7, 14.3; HRMS (ESI) m/z calcd for C32H43N4O9 [M+H]+ 627.3025, found 627.3018.
Benzyl
((2S,3R)-3-methyl-1-oxo-1-((2-oxo-2((2-oxo-2(((S)-1-oxo-3-phenyl-1-(p-tolylamino)propan-2-
yl)amino)ethyl)amino)ethyl)amino)pentan-2-yl)carbamate (Cbz-L-Ile-Gly-Gly-L-Phe-p-tol) (6ha). Cbz-L-
Ile-Gly-Gly-OC6H4PPh2 (0.100 g, 0.15 mmol), N3-L-Phe-p-tol (0.050 g, 0.17 mmol), Et3N (0.03 mL, 0.22 mmol)
were used following above general ligation procedure; Purification of this crude product by flash column
chromatography (silica gel, 100% ethyl acetate), Rf = 0.51 (silica gel, EtOAc); White solid, Yield 85% (0.080 g),
mp 206-208 oC;1H NMR (400 MHz, DMSO-d6) δ 9.89 (s, 1H), 8.30 – 8.19 (m, 2H), 8.08 (t, J = 5.7 Hz, 1H), 7.45
(d, J = 8.4 Hz, 2H), 7.39 – 7.28 (m, 6H), 7.28 – 7.22 (m, 4H), 7.21 – 7.14 (m, 1H), 7.09 (d, J = 8.2 Hz, 2H), 5.03
(d, J = 12.5 Hz, 1H), 4.97 (d, J = 12.6 Hz, 1H), 4.62 (td, J = 8.7, 5.2 Hz, 1H), 3.89 (t, J = 7.9 Hz, 1H), 3.79 – 3.62
(m, 4H), 3.05 (dd, J = 13.7, 5.2 Hz, 1H), 2.88 (dd, J = 13.7, 9.2 Hz, 1H), 2.24 (s, 3H), 1.77 – 1.65 (m, 1H), 1.47 –
1.36 (m, 1H), 1.18 – 1.06 (m, 1H), 0.82 (d, J = 6.7 Hz, 3H), 0.78 (t, J = 7.4 Hz, 3H); 13C{1H} NMR (101 MHz,
DMSO-d6) δ 172.2, 170.0, 169.6, 169.1, 156.7, 138.0, 137.5, 136.6, 132.9, 129.6, 129.5, 128.8, 128.6, 128.3,
128.2, 126.9, 119.9, 66.0, 59.8, 55.3, 42.5, 42.3, 38.2, 36.7, 24.8, 20.9, 15.8, 11.4; HRMS (ESI) m/z calcd for
C34H42N5O6 [M+H]+ 616.3130, found 616.3142.
Ethyl ((benzyloxy)carbonyl)-L-alloisoleucylglycylglycyl-L-tryptophyl-L-phenylalaninate (Cbz-L-Ile-Gly-
Gly-L-Trp-L-Phe-OEt) (6hh). Cbz-L-Ile-Gly-Gly-OC6H4PPh2 (0.100 g, 0.15 mmol), N3-L-Trp-L-Phe-OEt
(0.070 g, 0.17 mmol), Et3N (0.05 mL, 0.33 mmol) were used following above general ligation procedure;
Purification of this crude product by flash column chromatography (silica gel, 1-5% DCM/MeOH), Rf = 0.22
(silica gel, DCM: MeOH, 9:1); White solid, Yield 82% (0.090 g), mp 128-130 oC; 1H NMR (400 MHz, DMSO-
d6) δ 10.79 (s, 1H), 8.46 (d, J = 7.4 Hz, 1H), 8.19 (t, J = 5.7 Hz, 1H), 8.07 – 7.91 (m, 2H), 7.69 – 7.60 (m, 1H),
7.62 – 7.50 (m, 2H), 7.38 – 7.34 (m, 4H), 7.33 – 7.27 (m, 2H), 7.26 (d, J = 6.9 Hz, 1H), 7.23 – 7.16 (m, 3H), 7.11
(d, J = 2.3 Hz, 1H), 7.05 (t, J = 6.9 Hz, 1H), 6.97 (t, J = 7.4 Hz, 1H), 5.04 (d, J = 12.5 Hz, 1H), 4.99 (d, J = 12.6
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Hz, 1H), 4.59 (td, J = 8.6, 4.9 Hz, 1H), 4.44 (q, J = 7.5 Hz, 1H), 4.01 (q, J = 7.1 Hz, 2H), 3.91 (t, J = 7.9 Hz, 1H),
3.75 – 3.68 (m, 2H), 3.71 – 3.56 (m, 2H), 3.10 (dd, J = 14.8, 4.8 Hz, 1H), 3.04 – 2.93 (m, 2H), 2.93 – 2.84 (m,
1H), 1.77 – 1.67 (m, 1H), 1.45 – 1.39 (m, 1H), 1.13 – 1.11 (m, 1H), 1.07 (t, J = 7.1 Hz, 3H), 0.83 (d, J = 6.8 Hz,
3H), 0.79 (t, J = 7.3 Hz, 3H); 13C{1H} NMR (101 MHz, DMSO-d6) δ 172.2, 171.9, 171.7, 169.4, 168.7, 156.7,
137.5, 137.5, 136.5, 132.0, 129.6, 129.4, 128.8, 128.7, 128.2, 128.1, 127.8, 127.0, 124.1, 121.3, 118.8, 118.7,
111.7, 110.3, 66.0, 60.9, 59.8, 54.3, 53.5, 42.5, 37.2, 36.8, 28.2, 24.8, 15.8, 14.4, 11.4; HRMS (ESI) m/z calcd for
C40H49N6O8 [M+H]+ 741.3606, found 741.3614.
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Ethyl glycylglycyl-L-tryptophyl-L-phenylalaninate (NH2-Gly-Gly-L-Trp-L-Phe-OEt) (6i′h). Crude
HCl.NH2-Gly-Gly-OC6H4PPh2 (0.050 g, 0.11 mmol), N3-L-Trp-L-Phe-OEt (0.050 g, 0.12 mmol), Et3N (0.03 mL,
0.22 mmol) were used following the above general ligation procedure; Purification of this crude product by flash
column chromatography (silica gel, 1-10% DCM/MeOH), Rf = 0.1 (silica gel, DCM: MeOH, 9:1); White solid,
Yield 40 % (0.020 g), mp 110-112 oC; 1H NMR (400 MHz, CDCl3) δ 8.43 (s, 1H), 7.62 (s, 1H), 7.53 (d, J = 7.8
Hz, 1H), 7.24 (d, J = 8.0 Hz, 1H), 7.15 – 7.05 (m, 4H), 7.00 (t, J = 7.4 Hz, 1H), 6.91 – 6.78 (m, 4H), 6.66 (s, 1H),
4.66 – 4.61 (m, 2H), 4.00 (q, J = 7.1 Hz, 2H), 3.74 (d, J = 4.3 Hz, 2H), 3.20 – 3.11 (m, 1H), 3.08 – 2.99 (m, 3H),
2.94 – 2.84 (m, 2H), 1.91 (brs, 2H), 1.10 (t, J = 7.1 Hz, 3H); 13C{1H} NMR (101 MHz, CDCl3) δ 171.4, 171.2,
169.2, 136.3, 136.2, 129.5, 128.6, 127.7, 127.1, 124.0, 122.2, 119.8, 118.8, 111.5, 110.1, 61.8, 54.0, 53.7, 44.3,
43.1, 38.0, 27.9, 14.2. HRMS (ESI) m/z calcd for C26H31N5O5Na [M+Na]+ 516.2217, found 516.2202.
Supporting Information
The Supporting Information is available free of charge on the ACS Publications website. Spectral data of
intermediates and products (PDF)
 Copies of 1H, 13C, and 31P NMR spectra and HRMS of 2, 4a-i′, 5a-i, 6aa-6i′h, gif file of A1-7m;
Computational data: coordinates, imaginary frequency and energy data;
Notes
The authors declare no competing financial interest.
ACKNOWLEDGMENT
The authors are grateful for financial support from Department of Science and Technology, India (Grant no.
SB/FT/CS-136/2016). GGP thanks Zastra Innovations for the computing facility and authors thank James Stewart
for the MOPAC 2006 licenses and COSMOlogic GmbH for the TmoleX Client version.
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