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H NH2
R
CO2H
R = sidechain
!- Amino Acid
20 common amino acids
19 are 1°-amines, 1 (proline) is a 2°-amine
19 amino acids are “chiral”
1 (glycine) is achiral (R=H)
The configuration of the “natural” amino acids is L
1
H
CHO
OH
CH2OH
D-glyceraldehyde
HO
H
H
CHO
H
OH
OH
CH2OH
D-arabinose
HO
CHO
H
CH2OH
H2N
CHO
OH
H
H
CH2OH
L-arabinose
H2N
CO2H
H
R
L-alanine
L-glyceraldehyde
H
HO
HO
CO2H
H
CH3
H2N
H
CO2H
H
OH
CH3
L-theronine
(2S,3R)
H2N
H3C
CO2H
H
H
CH2CH3
L-isoleucine
(2S,3S)
2
1
Dipolar Structure
Zwitterion:
H2N
H3O+
+ R
_
H3N
CO2
H
R
CO2H
H
HO
pKa2
R
+ R
H3N
CO2H
H
pKa1
low pH
_
_
CO2
H2N
H
high pH
Isoelectric point (pI): pH at which the amino acid exists in a neutral,
zwitterionic form (influenced by the nature of the sidechain)
3
Amino acids are classified according to their sidechains
1. Hydrophobic:
+NH3
(S)-(+)-Alanine (Ala, A)
COO–
COO–
COO–
COO–
+NH3
(2S,3S)-(+)-Isoleucine (Ile, I)
+NH3
(S)-(+)-Valine (Val, V)
+NH3
(S)-(–)-Leucine (Leu, L)
COO–
–
N+ COO
H H
(S)-(–)-Proline (Pro, P)
COO–
COO–
S
+NH3
(S)-(–)-Methionine (Met, M)
+NH3
(S)-(–)-Phenylalanine (Phe, F)
+NH3
N
H
(S)-(–)-Tryptophan (Trp, W)
2. Uncharged polar groups
–OOC
COO–
+NH3
+NH3
Glycine (Gly, G)
(S)-(–)-Serine (Ser, S)
+NH3
(2S,3R)-(–)-Threonine (Thr, T)
pKa ~ 13
HS
COO–
+NH3
(R)-(–)-Cysteine (Cys, C)
COO–
OH
COO–
HO
O
(S)-(–)-Tyrosine (Tyr, Y)
pKa ~ 13
+NH3
(S)-(–)-Asparagine (Asn, N)
pKa ~ 10.5
O
COO–
H2N
HO
+NH3
H2N
COO–
+NH3
(S)-(+)-Glutamine (Gln, Q)
pKa ~ 8.3
4
2
3. Acidic
O
-O
COO–
O
COO–
-O
+NH3
+NH3
(S)-(+)-Glutamic Acid (Glu, E)
(S)-(+)-Aspartic Acid (Asp, D)
pKa ~ 4.1
pKa ~ 3.9
4. Basic
+NH3
N
H
COO–
+
N
H
+NH3
(S)-(+)-Lysine (Lys, K)
COO–
+NH3
(S)-(–)-Histidine (His, H)
N+
H
NH2
+NH3
(S)-(+)-Arginine (Arg, R)
pKa ~ 6.0
pKa ~ 10.5
COO–
HN
H
pKa ~ 12.5
5
Amino Acid Synthesis:
New unnatural amino acids with altered properties
new therapeutics (lead compounds)
mechanistic probes
CO2H
HO
HO
NH2
L-DOPA
6
3
Traditional Amino Acid Syntheses:
R-CH2-CO2H
Br2, PBr3
Br
O
R C CO2H
H
N
K
NaN3
O
NH3
Ph3P
N3
R CH CO2H
KOH, H2O
H2N
-orH2, Pd/C
O
R C CO2H
H
O
N
R C CO2H
H
7
Strecker Synthesis
C
NC
CN
O
R
H
R
OH
H
cyanohydrin
NH4Cl
O
R
C
H
CN
NH2
KCN
R
C
NC
R
H
NH2
C
HO2C
H3O
H
R
NH2
C
H
Amidomalonate Synthesis
AcHN
CO2Et
C
H
EtO
CO2Et
AcHN
Na
RCH2
RCH2X
CO2Et
C
CO2Et
H3O
- CO2
HO2C
RCH2
NH2
C
H
Reductive Amination
NH4Cl
O
R
C
CO2H
H2, Pd/C
NH2
R
C
CO2H
HO2C
R
NH2
C
H
8
4
Azlactone Synthesis
O
O
O
Ac2O
(excess)
O
EtO
O
HN
NH3
N
O
Na
O
azlactone
O
O
RCH2X
O
N
RCH2
N
RCH2
OH
O
EtO
N
Na
O
H
H3O
O
H
NHAc
C
RCH2
CO2H
NH2
C
CO2H
O
O
-H2O
R
R
RCHO
O
N
O
N
azlactone
H3O
R
CO2H
H
1) H2, Pd/C
NHAc
RCH2
2) H3O
NH2
C
CO2H
9
These are racemic syntheses!!
Resolution: separation of enantiomers
H
RCH2
NH2
C
CO2H
H
N
N
H
(-)-sparteine
(chiral base)
H
RCH2
H
NH2
C
H
H
CO2
+
N
H
H2N
N
N
RCH2
N
H
C
CO2
H
H
Diasteromeric salts
(separate)
H3O
H
RCH2
NH3
C
CO2
H3O
H3N
RCH2
H
C
CO2
10
5
Asymmetric Synthesis of Amino Acids
H
R
H H
H
S
H
H
NHAc
CO2H
pro-chiral
2
HO2C
CO2H
R
NHAc
1
NHAc
H 3 R
R 3 H
pro S-face
H H
H
R
R
2
CO2H
1
AcHN
pro R-face
CO2H
NHAc
H
H
H2, Pd/C - heterogeneous catalysis
H2, (Ph3P)3RhCl (Wilkinson’s catalyst)- homogeneous catalysis
CO2H
Ph
HN
P
OMe
MeO
*P
PPh2
PPh2
P
Rh (I) L*, H2
HN
(95% ee)
O
S
CO2H
Ph
Ph
Ph
O
Rh
P
S
*
CO2Me
CO2H
NHAc
O
BINAP
HO
O
DIPAMP
NH2
OH
L-DOPA
11
O
Br
Br2, PBr3
RCH
R-CH2-CO2H
Br
R C CO2H
H
Br
Br
H
R
H
S
H
Br Br
H
Br
CO2H
O
R
Br
Br Br
H
R
R
H
R
Br
C
R
CO2H
NaN3
SN2
N3 H
R
C
S
CO2H
CO2H
Br
H
H
H2N
R
H
C
S
CO2H
12
6
Chiral Auxiliaries
O
HN
H2N
O
OH
Ph
Ph
O
HN
H2N
O
OH
Ph
Ph
H
O
O
N
R
S
H
Br
H
H
Br
CO2R*
O
R
O
H N
O
H
X
O
X
Br
N
R
R
CO2R*
Br
H
H
O
13
Li
O
R
R
Cl +
N
N
O
R
LDA
O
N
R
N
N3-
R
N
O
1) LiOH
2) H2, Pd/C
O
N3
Br
N
O
SO2N3
Ph
O
O
LDA, THF
OH
D- amino acids
O
O
O
R
NH2
Ph
Ph
~ 95 : 5
R
O
O
O
O
O
Ph
Ph
Ph
NBS
O
O
O
O
O
R
N
O
N3
Ph
14
7
Peptides
- H2O
+
H2N
H2N
CO2H
CO2H
H
N
H2N
O
N-terminus
Val
Ala
CO2H
C-terminus
Ala - Val
(A - V)
- H2O
H
N
H2N
N-terminus
By convention, peptide sequences are written left
to right from the N-terminus to the C-terminus
CO2H
O
C-terminus
Val - Ala
(V - A)
O
H
N
_
R2
N
H
R1
H
N
O
H
N
O
R1
+
N
H
R2
C=N double bond character
due to this resonance structure
H
N
O
restricts rotations
resistant to hydrolysis
amide bond
15
Peptide Coupling: need for protecting groups
Pn
N
H
OH
peptide
coupling
+
OPc
H2N
O
O
- H2O
N
H
H
N
H2N
O
H
N
OPc
O
Ala - Val
(A - V)
Val
Ala
selectively
remove Pn
Pn
O
peptide
coupling
(-H2O)
OPc
O
Ala - Val
(A - V)
Pn
Ph
H
N
Ph
Pn
N
H
O
N
H
H
N
O
OPc
O
OH
O
Phe (F)
Phe - Ala - Val
(F - A - V)
Repeat
peptide synthesis
16
8
C-protecting groups (Lloyd-Williams et al., p. 11)
O
R
O
O
Ph
R
benzyl (bn)
Ph
O
O
Ph
R
removed with mild acid
O
t-butyl
benzhydryl
O
R
O
insoluble solid support
(resin)
linker
N-protecting groups (Lloyd-Williams et al., p. 10)
O
O
O
Ph
NH
O
O
NH
O
R
R
NH
R
O
O
tert-butylcarbamoyl
(t-BOC)
O
benzyloxycarbamoyl
(cBz)
fluorenylmethylcarbamoyl
(FMOC)
O
O
O
O
O C O C O
Ph
O
O
Cl
removed with
mild acid or by
hydrogenolysis
removed with
mild acid
Cl
removed with mild base
(piperidine)
17
Solid-Phase Peptide Synthesis (SPPS)
(Lloyd-Williams et al., Chapter 2, pp. 19-92)
• peptides up to ~ 100 amino acids can be synthesized
in a laboratory
• laboratory synthesis is from the C-terminus to the N-terminus
• nature synthesizes peptides from N to C.
O
linker
O
NH2 +
O
H
N
HO
R1
O
coupling
reagent
linker
FMOC
O
R1
R2
R2
H
N
O
O
purify:
wash & filter
remove
FMOC
O
R1
N
H
O
linker
R2
H
N
O
R1
deprotect
sidechains
H
N
O
linker
O
cleave from
solid support
O
N
H
H
N
FMOC
R2
NH2
purify:
wash & filter
O
purify:
wash & filter
N
H
H
N
HO
FMOC
FMOC
R3
coupling reagent
remove
FMOC
Repeat Cycle
R3
final
purification
N
H
18
9
Mechanism of Peptide Coupling (Lloyd-Williams et al., p. 48)
19
Mechanism of stereochemical scrambling
Additives can suppress the scrambling
(Lloyd-Williams et al., pp. 120-121)
Peptide coupling reagent (one-pot):
N-protected carboxylic acid, C-protected amine
DCC, HOBT
20
10
Newer coupling reagents:
(Lloyd-Williams et al., p. 53-55)
uronium salts:
(salts of urea, not uranium)
phosphonium salts
PF6
N
PF6
N
N
N
O
N(CH3)2
P
N(CH3)2
(CH3)2N
(H3C)2N
N
N
P
N
O
N
N
N
N(CH3)2
N
N(CH3)2
BOP
N(CH3)2
N
N
O
PF6
N
N
PyBOP
O
HATU
actual structure
21
Why not N to C peptide synthesis?
(Lloyd-Williams et al. pp. 116-119)
R1
linker
N
H
O
H
N
O
R3
N
H
R2
linker
N
H
linker
O
R1
O
R2
N
H
O
H
N
O
R2
R3
N
H
X
O
O
O
H
N
R1
activation
OH
+
N
H
R3
H
VERY acidic
easily racemized (scrambled)
22
11
Importance of maintaining stereochemical integrity during
the coupling step:
O
O
O
NH2 +
O
H
N
HO
R1
coupling
reagent
FMOC
H
N
O
R2
R1
FMOC
O
R2
R1
O
H
N
HO
FMOC
H
N
O
R3
coupling reagent
R2
H
N
O
R1
O
N
H
O
H
N
O
N
H
O
R1
R3
FMOC
R2
R2
H
N
O
FMOC
N
H
O
H
N
O
O
R2
H
N
O
FMOC
O
N
H
O
H
N
FMOC
R3
FMOC
O
R3
R2
H
N
O
R1
O
N
H
O
H
N
O
R1
R3
R2
H
N
O
FMOC
O
O
N
H
H
N
FMOC
R3
Number of possible stereoisomers = 2n where n= # of chiral centers
A peptide w/ 10 AA residues has 210 possible stereoisomers
23
Standard α-amino protecting group is FMOC
removed (deprotected) with base (piperidine)
O
H
N
H
piperidine
+
O
O
_
NH
+
R
O
NH
R
O
O
NH2
+
+
N
H2
R
O
an unusually acidic
carbon acid
+
OH
N
Orthogonal Protection Strategy: if the α-amino group has a baselabile protecting group, then the C-terminus and the side
chains require base-stable protecting groups
24
12
Sidechain Protecting Groups: (Lloyd-Williams et al., pp. 23-39)
for nitrogen sidechain functional groups
• NH2 of lysine (Lloyd-Williams et al., pp. 24-26)
2+Cu
+
NH3 +
_
NH3
O2C
4
Cu2+
_
O
4
+
NH3
O2C
O2C
H
N
4
OtBu
O
BOC group removed with acid
4
+
NH3
H
N
4
_
2
NHFMOC
NHBOC
HO2C
+
NH3
1) (tBuOCO)2O
2) H2S
+
NH3
O
FMOC-Cl
_
NH
O
_
Ph
O2C
H
N
4
O
O
O
Alloc: selectively removed w/ Pd(0)
cBz: removed w/ H+
or w/ hydrogenolysis
25
• Imidazole of Histidine (Lloyd-Williams et al., pp. 28-31)
R
N
N
N
H2O
N+
X
FMOCHN
R
N
R
N
O
FMOCHN
O
CPh3
N
FMOCHN
N
HO2C
O
BOC
N
FMOCHN
N
HO2C
O
FMOCHN
HO2C
Boc
trityl (Tr)
OH
FMOCHN
S
O
N
N
tosyl (Ts)
stable to mild base, removed with acid
• Indole nitrogen of tryptophan (often not protected)
(Lloyd-Williams et al., p. 31)
CO2H
N
BOC
CO2H
NHFMOC
N
O
H
NHFMOC
stable to mild base
removed with acid
26
13
• Amides of asparagine and glutamine (Lloyd-Williams et al., pp. 32-33)
usually not protected- could dehydrate to -C≡N
NH2
NH
O
X
n
FMOCHN
n
O
OH
O
FMOCHN
Ph
O
n N
H
HO2C
Benzyl (Bn)
N
FMOCHN
O
n N
H
HO2C
O
FMOCHN
O
FMOCHN
C
n
CPh3
stable to mild base,
removed with acid
Trityl (Tr)
• Guanidine group of arginine- often not protected
+
NH2
H2N
(Lloyd-Williams et al., pp. 26-28)
NH
N
FMOCHN
O
X
FMOCHN
NH2
NH2
+
O
FMOCHN
HO2C
H
N
3
HONO2,
H2SO4
NH2
FMOCHN
HO2C
FMOCHN
H
N
3
NH2
N
NH2
HO2C
H
N
3
NH2
N
NO2
removed with hydrogenolysis
or with Zn(0) in acetic acid
Ts
27
removed with HF
for oxygen sidechain functional groups
• carboxylate groups of aspartate and glutamate
(Lloyd-Williams et al. pp. 33-35)
FMOCHN
O
FMOCHN
n O
HO2C
Ph
tBu
n O
HO2C
Benzyl (Bn)
FMOCHN
O
HO2C
n O
allyl
t-butyl
removed with acid or
hydrogenolysis
O
removed with acid
removed with Pd(0)
• alcohols of serine, threonine and tyrosine (Lloyd-Williams et al. pp. 35-35)
FMOCHN
HO2C
FMOCHN
O
Ph
Benzyl (Bn)
removed with acid or
hydrogenolysis
HO2C
FMOCHN
FMOCHN
O
tBu
HO2C
O
CPh3
t-butyl (tBu)
trityl (Tr)
removed with acid
removed with acid
HO2C
O
O
acetate (Ac)
removed with acid
28
14
Sulfur of Cysteine (Lloyd-Williams et al. pp. 39-40)
FMOCHN
FMOCHN
S
HO2C
Ph
FMOCHN
S
HO2C
S
HO2C
CPh3
t-butyl (tBu)
trityl (Tr)
removed with acid
FMOCHN
FMOCHN
FMOCHN
S
HO2C
removed with acid
Benzyl (Bn)
removed with acid
tBu
S
HO2C
HO2C
S
S
O
NO2
NO2
o-nitrobenzyl
removed photochemically
H
N
stable to acid
removed with HO- or Hg(II)
removed with Ph3P
or with HOCH2CH2SH
Disulfides of cysteine (cystine) redox active amino acid side chain
(Lloyd-Williams et al., Chapter 5, pp. 209-236)
HO2C
NH2
H2O
1/2 O2
NH2
2
S
HO2C
SH
S
CO2H
NH2
29
Solid-Phase Peptide Synthesis: The solid support (resin, bead, etc.)
(Lloyd-Williams et al., pp. 19-21, 41-46)
Merrifield Resin: R. Bruce Merrifield, Rockefeller University, 1984 Nobel Prize in Chemistry:
for his development of methodology for chemical synthesis on a solid matrix.
Ph
H3COCH2Cl
ZnCl2
styrene
initiator
Ph
Ph
Ph
Ph
Ph
Ph
Ph
CH2Cl
+
polymerization
Ph
Ph
Ph
Ph
divinylbenzene
(crosslinker, ~1 %)
Ph
Ph
in the range of 10% of
the available phenyl
groups are functionalized
30
15
Amide-linked resins
O
N - K+
1)
CH2Cl
O
CH2NH2
2) H2NNH2
O
HN
NHCBz
Commercially
available
R1
Amide Linked
Ester-linked Resins
K+
O
_
NHCBz
O
CH2Cl
O
R1
O
NHCBz
R1
CF3CO2H
O
Commercially
available
O
NH2
31
R1
Other Resins:
Merrifield
resin
O
R1
X
linker
NH2
Kaiser (oxime) resin
(Lloyd-Williams et al., pp. 144-145)
O
Cl
O
O2N
N OH
H2NOH•HCl
- H2O
AlCl3
NO2
NO2
O
R1
N O
NH2
NO2
Wang Resin
(Lloyd-Williams et al., pp. 143-144)
O
OH
CH2Cl
O
O
O
NH2
R1
32
16
Rink (amide) resin
(Lloyd-Williams et al., pp. 45-46)
H2N
H2N
R1
R1
HN
HN
O
O
H3CO
CH3
OCH3
Tanta gel
+
styrene
divinylbenzene
(crosslinker, ~1 %)
+
HO
O
initiator
O
O
O
OH
n
polymerization
O
OH
n
polyethylene glycol (PEG)
O
O
O
O
n
O
R1
NH2
Solublizes the synthetic peptide
Particularly good for the synthesis of long peptides
33
Deprotection of the peptide
sidechain protecting groups
cleavage from the solid support
(Lloyd-Williams et al., pp. 71-75)
Acid hydrolysis: CF3CO2H, HF
anisole or p-cresol is added as an alkylation scavenger
Purification
High performance liquid chromatography (HPLC)
electrophoresis
Analysis
mass spectrometry
34
17
Ribonuclease A- 124 amino acids
catalyzes the hydrolysis of RNA
Solid-phase synthesis of RNase A:
B. Gutte & R. B. Merrifield, J. Am. Chem. Soc. 1969, 91, 501-2.
Synthetic RNase A: 78 % activity
0.4 mg was synthesized
2.9 % overall yield
average yield ~ 97% per coupling step
LYS
GLN
SER
LYS
LYS
LEU
LYS
ASN
ILE
LYS
GLN
GLU
ASP
GLU
HIS
SER
SER
PRO
ALA
ASN
CYS
THR
TYR
ALA
GLY
ALA
THR
MET
SER
ARG
VAL
ASP
VAL
TYR
ASP
PRO
ASN
ASN
SER
ALA
ASP
ASN
ASN
ASN
VAL
ALA
GLN
CYS
ASN
LYS
PRO
VAL
ALA
SER
TYR
LEU
THR
GLN
CYS
SER
ARG
CYS
HIS
TYR
ALA
SER
CYS
THR
PHE
ALA
LYS
TYR
GLU
ALA
ILE
VAL
LYS
THR
ASN
LYS
VAL
VAL
ASN
SER
THR
TYR
ILE
PRO
PHE
SER
GLN
ASP
HIS
CYS
GLY
THR
GLY
LYS
VAL
VAL
GLU
ALA
MET
ARG
GLU
SER
GLN
MET
SER
THR
ALA
HIS
ARG
ALA
MET
CYS
SER
GLN
THR
SER
SER
THR
CYS
PHE
His-119 A
His-119 B
His-12 A
His-12 B
pdb code: 1AFL
35
Linear vs. Convergent Synthesis
SPPS- linear synthesis of peptides, many steps, low overall yield,
inefficient for long peptides and proteins
Convergent Synthesis (segmental coupling strategy)- make short
peptides by SPPS then couple the short peptides, in solution,
to give longer ones. Less linear steps and higher overall yield
if the segmental coupling is efficient.
(Lloyd-Williams et al., Chapter 3, pp. 95-137, Chapter 4, pp.139-207)
36
18
Must activate the C-terminus of a peptide segment
recall there are problems with the N to C peptide synthesis
(Lloyd-Williams et al., pp. 116-120)
O
H
N
R1
N
H
R2
O
X= OH
R2
N
H
X
O
N
H
N
O
couple at glycine,
R1=H, no stereochemistry
couple at proline- unstable
azlactone, little scrambling
of stereochemistry
O
O
R2
R2
Scrambling of stereochemistry
R1
H
X= activated acid
N
H
N
O
O
H
N
X
37
Couple at cysteine (Kent, Tam)
Native peptide ligation
O
O
PhCH2S
H2NNH2
_
O
SCH2Ph
H2N
SCH2Ph
+
HS
NH2
N
H
N3
H2N
Thioester: a less reactive
activated acid
O
O
H3N
O
HONO
NHNH2
H2N
O
H2N
(Lloyd-Williams et al., pp. 190-195)
O
CO2
H3N
N
H
SH
H
N
CO2
O
38
19
More general peptide ligation strategy
O
O
HS
O
HN
+
SCH2Ph
H3N
O
R
H3N
CO2
N
H
R
N
O
H
N
CO2
O
HS
Zn(0), AcOH
O
H3N
R
N
H
H
N
CO2
O
O
O
O
N
H
NH-FMOC
Br
HO
O
O
R
O
NH2
O
O
Br
N
H
DCC, HOBT
R
(sidechains protected)
HO2C
O2N
S
S
O
O
NH2
O
O
SN2
N
H
H
N
O
S
S
Ar
1) deprotect peptide
sidechains
O
2) HOCH2CH2SH
R
O
O
N
H
H
N
SH
O
R
39
Staudinger reaction
R N PPh3
+
R N N N
-N2
PPh3
H2O
R NH2
+ O PPh3
R N PPh3
Staudinger Ligation:
O
H3N
O
O
+
S
PPh2
R
N3
N
H
CO2
H3N
R
N
H
H
N
CO2
+
HS
O
O
PPh2
40
20
Cyclic Peptides
HO
H3C
CH3 O
N
N
O
CH3
O
H H
N
N
CH3 O
CH3
N
O
R2
H2N
O
R1
O
H
N
NH
HN
OH
R1
O
H3C
O
O
R2
H3C
N
O
Diketopiperizine
N
H
O
H
N
O
N
CH3
O
N
H
N
O
Cyclosporin A
41
Cyclic Peptide Synthesis
Problems with the solution-phase cyclization reaction
• stereochemical scrambling
- use acyl azide, acyl thioester, HATU or PyBop in the
coupling reaction
• dimerization
- high dilution conditions favors cyclization
SPPS
O
HO
O
R1
O
R1
Rn
N
H
X
R1
R1
NH2
O
NH2
Rn
O
NH
Rn
O
Rn
O
O
undesired
desired
42
21
Intramolecular Native Peptide Ligation Strategy
O
SCH2Ph
R1
R1
O
NH2
NH
SH
SH
O
O
O
R1
SCH2Ph
R1
HN
O
O
SH
N
Rn
O
R1
Zn(0), AcOH
SH
O
NH
Rn
O
Rn
O
O
Solves the stereochemical scrambling problem but not the dimer
formation issue
43
Cyclization on the solid support will solve the dimerization problem
Attached the first amino acid through the side chain
applicable for Asp, Glu and Lys
Requires a carboxylate protecting group that is removed under
conditions other than acid or base → Allyl
Ph
C-Cl
Ph
CH2Cl
O
CH2O
NH-cBz
n
O
n = 1, 2
O
Ph
C NH
Ph
NH-cBz
O
O
44
22
Attached the first amino acid to the solid support via
the α-amino group
NH2
OCH3
CH2O
CH2Cl
O
R1
CHO
O
NaB(CN)H3
OCH3
FMOCHN
OCH3
OH
R2
O
CH2O
O
N
H
OCH3
OCH3
O
O
O
CH2O
N
PyBOP
OCH3
R1
O
R1
NHFMOC
R2
45
On-suppport cyclization
OCH3
1) Pd(0), Et3SiH
2) piperidine
O
O
CH2O
N
OCH3
O
NHFMOC
R1
Rn
O
NH
R2
OCH3
HO
PyBOP
O
CH2O
N
OCH3
O
NH2
R1
Rn
O
NH
R2
OCH3
H
N
R1
CH2O
N
O
Rn
O
H
N
R1
CH3CO3H, HF
anisole
HN
O
Rn
O
OCH3
O
O
NH
NH
R2
R2
46
23