Peptido~ The Road To Becoming A Peptide Master ~4th Edition

塩
基
脱
保
護
流
極
意

Katayama lab,
Department of Applied Chemistry,
Faculty of Engineering,
Kyushu University, Japan
Released in December 2015

Table of Contents
1. Fmoc/tBu Solid Phase Synthesis

3

2. Checking Circumstances

4

5. Analysis of Peptide Extension
5-1. Quantiﬁcation of introduction ratioof amino acid

2-1. General reagents

5-2. Kaiser test

2-2. Instruments

5-3. Acetylation

3. Preparation For Reagents
And Instruments

59

6. Peptide Cleavage from Resin& Deprotection

10

6-1. Cleavage and deprotection
by strong acid

3-1. Selection of resin
3-2. Selection of column

6-2. Cleavage of protected peptide
by diluted acid3-3. Swelling of resin
3-4. Fmoc amino acid7. Special Protected Amino Acid

3-5. Preparation of coupling reagents

7-1. Fmoc-Lys(ivDde)-OH

3-6. Preparation of Kaiser test reagents

7-2. Fmoc-Cys(Acm)-OH

4. Peptide Extension

12

7-3. Fmoc-Cys(tButhio)-OH

77-4. Fmoc-Cys(Mmt)-OH

4-1. Introduction to Fmoc protected resin
4-2. Introduction to hydroxy resin
based on DCC/DMAP method

8. Column

14

4-3. Introduction to active trityl resin

9. References & Acknowledgements

179-1. References

4-4. Peptide extension

9-2. Authors and editors

4-5. Ending of peptide extension

Basic action in Peptido

Addition of solvent

Mixing-2-

Removal of solvent

Fmoc/tBu Solid Phase Synthesis
1-1. Reaction① Fmoc deprotection by secondary amine (piperidine)② Extension reaction of peptide sequence by coupling reagent
Scheme. Fmoc deprotection ① & extension reaction ②

Scheme. Deprotection & cleavage from resin by strong acid（TFA）※ Fmoc is not deprotected by TFA.

1-2. Molecular design
The functional group in C terminal（Y）depends on the type of resin. After cleavage
from resin by TFA, Y becomes -NH2 and -OH in the case of carboxamide and peptide
acid, respectively.
On the other hand, the functional group in N terminal（X）becomes amine group（H-）after Fmoc deprotection. Therefore, it is possible to acetylate（X = CH3CO-）or modify carboxyl group（X = HOOC-(CH2)2-CO-）to N terminal.
Fig．Peptide structure

N terminal

Sequence of extension reaction（R1,R2,…,Rn）

C terminal

1-3. Solid phase synthesis
Unlike liquid phase synthesis, we can synthesize a large amount of peptide with easy
reaction steps and high reaction eﬃciency in solid phase synthesis. We can also get
the peptide with high purity if we optimize the reaction steps（handling, coupling
reagents, etc）.
In addition, we can synthesize not only linear peptide, but also circular peptide andpeptide which side chain is modiﬁed. As for selective deprotection of amino acid (AA)side chain, please refer to chapter 7 and solid phase synthesis handbook1.

-3-

Checking Circumstances2-1. General reagents

Table. Reagents used in general synthesis

R eag en t n am ed ich lo ro m eth an e
N,N -d im eth ylfo rm am id e
N -m eth ylp yrro lid o n em eth an o lp ip erid in e

A bb
D CM
DMF
NMP
M eO HPPD

A p p licatio n
S w ellin g resin
R eactio n so lven t
R eactio n so lven t, resin sto rag e so lven t
S h rin kin g resin
Fm o c d ep ro tectio n2 -(1 H -B en zo triazo le-1 -yl)1 ,1 ,3 ,3 -tetram eth ylu ro n iu m
h exaflu o ro p h o sp h ate

H B TUC o u p lin g ag en t（co n d en sin g ag en t）

1 -H yd ro xy-1 H -b en zo triazo le,m o n o h yd rate

H O BtC o u p lin g ag en t（activatin g ag en t）

CO M UC o u p lin g ag en t（co n d en sin g ag en t）

d iiso p ro p yleth ylam in e
p h en o l
n in h yd rin
eth an o l0 .0 0 1 3 % K C N /p yrid in e
acetic an h yd rid e

D IE AO rg an ic b ase
K aiser test
K aiser test
K aiser test
K aiser test（d o m estic ch em ical）
A cetylatio n o f u n reacted p o in ts

triflu o ro acetic acidT FA

triiso p ro p ylsilan e
eth an ed ith io ld ieth yl eth er

T IS
ED T
E t2 O(1 -C yan o -2 -eth o xy-2 o xo eth ylid en am in o o xy)d im eth ylam in o -m o rp h o lin o -carb en iu m
h exaflu o ro p h o sp h ate

E tO HA c2 O

D ep ro tectio n o f p ro tectin g g ro u p ,C leavag e fro m resin
S caven g er fo r d ep ro tectio n o f p ro tectin g g ro u p
S caven g er fo r d ep ro tectio n o f p ro tectin g g ro u p
E th er p recip itatio n

Table. Reagents used in special cases

R eag en t n am e
A bb
d icyclo h exylcarb o d iim id e
D CC
N,N -d im eth yl-4 -am in o p yrid in e D M A P
b en zo ic an h yd rid ep yrid in eh yd razin e m o n o h yd rate
2 -m ercap to eth an o lio d in e
I2

2-2. Instruments

A p p licatio n
In tro d u ctio n o f am in o acid to h yd ro xy resin
O rg an ic b ase
B en zo ylatio n o f u n reacted p o in ts
C leavag e fro m S ieb er, 2 -C h lo ro trityl resin
D ep ro tectio n o f ivD d e
D ep ro tectio n o f tB u th io
D ep ro tectio n o f A cm & Trt、-S S - fo rm atio n

Table. Instruments and consumable items

In stru m en t n am e

K o m ag o m e p ip ette/p ip ette

vo rtex m ixer

d iap h rag m p u m p /su ctio n b o ttle

A p p licatio n
A d d itio n o f so lven t(2 m L-K o m ag o m e is u sefu l.)
M ixin g o f co lu m n(S h o u ld h ave resistan ce fo r o rg an ic so lven t)
R em o val o f so lven t(S u ctio n b o ttle: m ad e b y g allo n b o ttle & ru b b er sto p p er)

g lass tu b e/h o t p late

K aizer test IW A K I 9 8 3 0 -1 0 0 7

d esiccato r/vacu u m p u m p

D ryin g sam p le

U V ab so rp tio m eter

Fm o c q u an tificatio n

cen trifu g e

C o llectio n o f d ep ro tected p ep tid eevap o rato r

D istillin g aw ay so lven t-4-

Selection & Preparation of Resin
3-1. Selection of resin
Generally speaking, it is better to use「Rink Amide AM resin HL」thanks to good costeﬀectiveness and high yield. In the case of long and bulky peptide or α-helix peptide
synthesis, it may be diﬃcult to extend peptide. In that case, you should use the resin
which has low reaction point density and PEG chain.
There are some types of resin which we donʼt need 4-1 <Fmoc deprotection> step.
Before using, please check resin structure.
R esin n am e
P ep tid e acidFm o c-A A -W an g resin
N o vaS yn T G A resin

Table. Type of resin and method for utilization1
R eactio n p o in t d en sity*

B ase m aterial P rice /g

1 st lo ad in g C leavag e

1 .0 m m o l/g
0 .2 m m o l/g

PS
P E G -P S

¥ 9 ,5 0 0

p re-lo ad ed
4 -2 .

6 -1 .
6 -1 .

P ep tid e carb o xam id e
R in k am id e P E G A resin
N o vaS yn T G R resin
R in k A m id e A M resin LL
R in k A m id e A M resin H L
R in k A m id e N o vaG el

0 .2 m m o l/g
0 .3 m m o l/g
0 .4 m m o l/g
0 .6 m m o l/g
0 .7 m m o l/g

P E G -A A m
P E G -P SPSPS
P E G -P S

¥ 2 1 ,0 0 0¥ 1 5 ,0 0 0¥ 8 ,0 0 0¥ 8 ,0 0 0¥ 1 3 ,6 0 0

4 -1 .
4 -1 .
4 -1 .
4 -1 .
4 -1 .

6 -1 .
6 -1 .
6 -1 .
6 -1 .
6 -1 .

P ro tected p ep tid e acid
2 -C h lo ro trityl ch lo rid e resin
N o vaS yn T G T alco h o l resin

1 .3 m m o l/g
0 .2 m m o l/g

PS
P E G -P S

¥ 6 ,4 0 0¥ 1 4 ,8 0 0

4 -3 .
4 -2 .

6 -2 .
6 -2 .

P ro tected p ep tid e carb o xam id e
S ieb er A m id e resin

0 .6 m m o l/g

P E G -P S

¥ 1 8 ,1 0 0

4 -1 .

6 -2 .

* It depends on Lot. Check the value on the bottle.

3-2. Selection of column

Table. Synthesis column

C o lu m n n am e
P D -1 0 E m p ty C o lu m n s
E co n o -P ac C h ro m ato g rap h y C o lu m n s

O p tim u m scale~ 0 .1 m m o l~ 0 .3 m m o l

P rice\ 1 9 ,8 0 0 /5 0
\ 2 7 ,0 0 0 /5 0

Select column according to reaction scale (the amount ofsolvent). We should choose the optimal column which isenough for resin to soak in reaction solvent. Although we
can reuse the column, should be careful about
degradation of ﬁlters and caps. Especially, the bottom
cap tends to crack due to repeated vortex mixing. Before
addition of strong acid for deprotection or cleavage, you
should check carefully (You can also use the cap for
PetiSyzer (p.16)). If the ﬁlter ﬂoats, reset it.

O th er in fo .
(A ) G E , 1 7 -0 4 3 5 -0 1
(B ) B IO R A D , 7 3 2 -1 0 1 0

(A)

(B)

3-3. Swelling of resin

Selected resin is weighed, transfer into the column, andsoaked in DMF overnight. You can also use DCM which
has higher swelling coeﬃcient (2-3 hours). Please notethat DCM is easy to evaporate and the resin which has
low reaction point density tends to ﬂoat on solution level(Here, swelling time is longer than usual. It is acceptable
to soak resin in DCM for 30 min).

-5-

Fig. synthesis column

Preparation of Amino Acid, Coupling Reagents, Kaiser Test Reagents
3-4. Fmoc amino acid

3 eq. mol (relative to reaction point on the resin) of a AA is taken in a tube.
The separated AA is recommended to keep in dry and without light.
Table. Main Fmoc AA we use (AAs shown in red are kept at -20℃.)
AA
A bb
MW
AA
A bb
MW
AA
A bb
A la
A
3 1 1 .3
LeuL
3 5 3 .4
Lys(b io tin )Kb
A rg (P b f)R
6 4 8 .8
Lys(B o c)
K
4 6 8 .6
Lys(ivD d e)Ki
A sn (T rt)N
5 9 6 .7M et
M
3 7 1 .5
Lys(M tt)
Km
A sp (O tB u ) D
4 1 1 .5
PheF
3 8 7 .4
A sp (O d m ab )Do
C ys(T rt)
C
5 8 5 .7
P roP
3 3 7 .4
S er(P O (O b zl)O H ) p S
G ln (T rt)
Q
6 1 0 .7
S er(tB u )S
3 8 3 .5
T h r(P O (O b zl)O H ) p T
G lu (O tB u )E
4 2 5 .5
T h r(tB u )T
3 9 7 .5
T yr(P O (O b zl)O H ) p Y
G lyG
2 9 7 .3
T rp (B o c) W
5 2 6 .6
C ys(A cm )Ca
H is(T rt)
H
6 1 9 .7
T yr(tB u )Y
4 5 9 .6
C ys(tB u th io )Ct
IleI
3 5 3 .4V alV
3 3 9 .4
C ys(M m t)
Cm

MW
5 9 4 .7
5 7 4 .7
6 2 4 .8
6 6 6 .8
4 9 7 .4
5 1 1 .5
5 7 3 .5
4 1 4 .5
4 3 1 .6
6 1 5 .7

3-5. Preparation of coupling reagents
Coupling reagents are prepared at the time of use according to reaction scale ofsynthesis. When you use sonication to dissolve these reagents in solvents, prevent
water being mixed into prepared solutions. If you want to store them temporarily, keepthem in refrigerator away from the light, after treating them with nitrogen gas. You can
use them for about 1 month. However, you should use up them in one synthesis
because these reaction activities are gradually decreasing. In order to avoid decrease ofreagents activities due to repeated opening and closing of a cap, you should separate
these reagents into several bottles at ﬁrst.
Table. Recipe for coupling reagents
0 .4 5 M co u p lin g co cktail
H B TU
6 .1 g3 .0 5 g
H O B t･H 2 O
2 .5 g
1 .2 5 g
DMF
32 m l16 m lA vailab le n u m b er o f reactio n tim es w ith 0 .1 m m o l scale~ 4 5 tim es ~ 2 0 tim es
0 .9 M D IE A
D IE ANMP
A vailab le n u m b er o f reactio n tim es w ith 0 .1 m m o l scale

5 .5 m l
2 9 .5 m l~ 4 5 tim es

2 .7 5 m l
1 4 .2 5 m l~ 2 0 tim es(Additive amount of each solution for one reaction in 0.1 mmol scale：700 µL)

3-6. Preparation of Kaiser test reagents
We can purchase reagents 1~3 from domestic chemical company (product number:
2590077, Reagents for Kaiser Test, \9,000/set). We can also prepare reagents 1 and 2
by ourselves because these are relatively safe. If the color of reagent 1 turns to dark,
prepare it again. Since it takes time to prepare for reagent 2, you should check
whether it is enough or not before your synthesis. After preparation of them, conﬁrm
whether these reagents make resin which has amine groups blue.
Table. Recipe for Kaiser test reagents
K aiser test reag en t
S o lu te
S o lven t
1 . n in h yd rin /E tO H
0 .5 g n in h yd rin
1 0 m l E tO H
2 . p h en o l/E tO H
8 g p h en o l
2 m l E tO H
3 . K C N /p yrid in e
0 .1 3 m g K C N
1 0 m l p yrid in e

-6-

First Amino Acid Loading
4-1. Introduction to Fmoc protected resin

We can introduce AA to the resin according to the same protocol as
4-4. Peptide extension.

4-2. Introduction to hydroxy resin based on DCC/DMAP method (0.1 mmol scale)〈Coupling〉

Synthesis column

25 ml Eggplant ﬂask

Swell overnight

Stir for 30 min at 0℃※ If DCC is not dissolved in DMF,
you can use DIC instead of DCC.
※ If the amount of solvent is too small to stir,
add DMF more as you think proper.

0.05 mmol DMAP
Stir for 1 h at RT〈Removal of impurities &Masking of unreacted points〉

0.5 mmol Fmoc-AA-OH
0.5 mmol DCC
in DMF (amount of dissolution,less than 5 ml)

0.1 mmol Hydroxy resin
in DCM or DMF

2 ml DMF wash ×3
2 ml DCM/EtOH (50/50) wash ×3
2 ml DCM wash ×3
2 ml DMF wash ×3
0.5 mmol benzoic anhydride in 2mL 20% pyridine/DMF
Stir for 1 h at RT
DMF wash ×3
DCM wash ×3※ Calculate introduction eﬃciency
according to 5-1. Fmoc quantiﬁcation

Move on to 4-4. Peptide extension

4-3. Introduction to active trityl resin (0.1 mmol scale)〈Coupling〉

25 ml Eggplant ﬂask (or synthesis column)
0.1 mmol 2-Chlorotrityl chloride resin
in DCM (amount of soaking)Stir for 10 min at RT
0.15 mmol Fmoc-AA-OH※ If AA is not dissolved in DCM,
0.5 mmol DIEA (80 μl)
you can add the small amount of DMF
in DCM (amount of dissolution)
as you think proper.
Stir for 2 hs at RT〈Inactivationof
unreacted
points〉

Synthesis column
2 ml DCM/MeOH/DIEA=17/2/1 wash ×3
2 ml DCM wash ×3
2 ml DMF wash ×3
2 ml DCM wash ×3※ Calculate introduction eﬃciency
according to 5-1. Fmoc quantiﬁcation

Move on to 4-4. Peptide extension※ In 4-2・4-3, you should select the kind of solvent which can make AA conc. from 0.3 to
0.5M.

-7-

Peptide Extension
4-4. Peptide extension (0.1 mmol scale)〈Fmoc
deprotection〉

Fmoc-AA0-･･･-resin
2 ml 20% PPD/DMF
2 ml 20% PPD/DMF

Mix with vortex mixer for 1 min※ You should wash the solvent andresin on the upper and the bottom
caps carefully, otherwise Fmoc may
be deprotected during storing.

Stir for 10 min at RT
2 ml DMF wash ×5〈 Coupling 〉

H-AA0-･･･-resin
0.3 mmol Fmoc-AA1-OH※ After mixing and dissolving all of
0.3 mmol Coupling cocktail 700μl
t h e m c o m p l e t e l y, a d d t h e
0.6 mmol DIEA
mixture to resin.
700μl

2 ml DMF wash ×5

Kaiser test
positive※〈Swelling resin〉

positive

Stir for 15 min at RT

negative

Acetylation

2 ml DCM wash ×3
2 ml DMF wash ×3※ You can change the reaction
time, additive amount ofreagents and the number ofreaction times. Optimize thembased on analysis.
If Kaiser test shows positivea f t e r 2nd – 3rd r e a c t i o n ,acetylate unreacted points.
※ When you keep your peptide in
the middle of your synthesis
overnight, you should stop yoursynthesis in this step, soak theresin in DCM or NMP, and keep
it in refrigerator.

Fmoc-AA1-･･･-resin
Repeat※ When you change your reaction scale, the mol ratio in 〈Coupling〉step should be FmocAA-OH : Coupling cocktail : DIEA : H2N-(Resin) = 3: 3 : 6 : 1.
And also, the amount of solvent in〈Fmoc deprotection〉or each washing step should be
about 1.5 eq. (relative to the resin volume) .
※ You need to remember that coupling reagents such as HBTU or COMU, are for activating
the carboxyl groups.
In the common peptide extension, AA after being activated is added to the free aminegroups on the resin. In an opposite case, in which an amine compound is added tocarboxyl groups on the resin, coupling reagents should be directly added to the resin.
Then in the second step amine compound will be added to the activated resin.

4-5. Ending of peptide extension〈Fmoc
deprotection〉

Fmoc-AAn-･･･-resin
2 ml 20% PPD/DMF
2 ml 20% PPD/DMF

Mix with vortex mixer for 1 min

Stir for 10 min at RT
DMF wash ×5

H-AAn-･･･-resin

Acetylation

2 ml DCM wash ×5
2 ml MeOH wash ×5

Remove the cap and dry in a desiccator over 2 hrs-8-

When you synthesize thepeptide whose N terminal isa c e t y l a t e d , yo u s h o u l dacetylate it in this step.

Analysis of Peptide Extension
5-1. Quantiﬁcation of introduction ratio of amino acidIf we assume that 100 % Fmoc group is deprotected, the introduction ratio of the AA
can be estimated by the amount of Fmoc group which is eluted in〈Fmocdeprotection〉step. You should quantify the amount of Fmoc group at least for 1st,
middle and ﬁnal AAs during the waiting time in＜Coupling＞step.
Protocol・Collect all ﬁltrate in 4-4.〈Fmoc deprotection〉step and measure that volume withmeasuring cylinder (The volume will be about 14 mL in 0.1 mmol scale, so you can
use a 15 ml-centrifuge tube for measuring the volume).
・Dilute collected ﬁltrate 10~100 times with DMF.
・Measure absorbance of the prepared sample at 301 nm.
Blank absorbance should be obtained with DMF.
・Calculate the amount of Fmoc group by using ε301nm = 7,800 M-1cm-1.

260

280
300
Wavelength (nm)320

Three Abs peaks can beobserved from 260~320 nm.

5-2. Kaiser test
It can be checked whether there are unreacted amine groups or not based onninhydrine reaction.
Protocol・Mix each of 20 μl Kaiser test reagents in a glass tube
・Add very tiny amount of resin to the glass tube
・Heat the glass tube with boiling water for 1 min
・Check the resin or solution color
dark red~blue：positive / yellow：negative
Fig. Kaiser test result
Left：positive / Right：negative

5-3. Acetylation
Acetylation enables us to mask unreacted amine groups or amine groups in N
terminal. If Ac2O is old, its pH may be lower than usual due to hydrolysis. Thus, be
careful if you use the resin which cleaves peptides with dilute acid.

Protocol (0.1 mmol scale)
2 ml DCM wash ×3
2 ml 25% Ac2O/DCM

Mix with vortex mixer for 30 sec(Stir for 5 min at RT) ← In the case of acetylation for N terminal
2 ml DCM wash ×3
2 ml DMF wash ×5-9-

Peptide Cleavage from Resin & Deprotection
6-1. Cleavage and deprotection by strong acid
Protection groups on the side chains of Fmoc AAs can be deprotected by TFA withscavenger to avoid side reactions. Since deprotection is exothermic
reaction,
deprotection cocktail and TFA should be cooled in advance.
Table. Selection of deprotection cocktail
D ep ro tectio n co cktail
V o lu m e ratio
V o lu m e in 0 .1 m m o l scale (μl)
If p ep tid e h as C ys (T rt) o r M et
T FA /w ater/T IS /E D T
9 4 /2 .5 /1 .0 /2 .5
2 3 5 0 /6 2 .5 /2 5 /6 2 .5

If p ep tid e d o es n o t h ave C ys (T rt) o r M et
T FA /w ater/T IS
9 5 /2 .5 /2 .5

Protocol 2〈Cleavage・Deprotection〉

Resin in synthesis column
Deprotection cocktail (Cooled in advance)
Keep for 90 min at RT (Stir every 30 min)Collect ﬁltrate into a 50 ml centrifuging tube
1.5 ml cooled TFA ×3→ Collect each ﬁltrate into a 50 ml centrifuging tube
Wash cooled Et2O as you think proper

Crude peptide solution in a 50 ml centrifuging tube
〈Ether precipitation〉

Protocol 1

2 3 7 5 /6 2 .5 /6 2 .5

40 ml cooled Et2O
Stir wellCentrifuge (4℃, 3,500×g, 5 min)Discard supernatant

Repeat 3 times

Keep it in hood for about 10 min to evaporate Et2O
Dry in desiccator over 2 hrs

Crude peptide〈Reference 1〉Peptide puriﬁcation
Check crude yield by weighing crude peptide. If crude yield is too low,
there are possibilities such as･ Introduction eﬃciency of each AA is low.
･ There are still protected peptides on the resin due to the lack of the deprotection.
We can solve these problems by Fmoc quantiﬁcation and redoing deprotection, respectively.
Prepare crude peptide solution with about 10 mg/ml with 0.1% TFA solution.
If there is precipitate, try the following methods to dissolve it.
･ Add more TFA till its conc. becomes 1% in the case of basic peptide･ Dissolve into mixture of acetonitrile/water
After ﬁltration, check whether target peptide has been synthesized by MALDI-TOF-MS.
Sort target peptide by using RP-HPLC of water/acetonitrile. It can be puriﬁed by Sephadex
G-10~25 in advance. Then, you should care about changing of solubility with pH.
Lyophilize and obtain target peptide powder after removal of acetonitrile by an evaporator(Obtained peptide is TFA salt. If it contains acetonitrile, it may be diﬃcult to lyophilize).
According to peptide sequence, there is optimal peptide storage solution. You should
check it out in advance.

- 10 -

Cleavage of Protected Peptide
6-2. Cleavage of protected peptide by dilute acid (0.1 mmol scale)2Protocol 1

Protected peptide on resin in synthesis column〈Cleavage〉〈Swell with DCM〉
Repeat 10 times.
After that, transfer all ﬁltrate
Collect ﬁltrate into test tube (5 ml) from 10 test tubes to one 200
ml eggplant ﬂask.
with 2 ml 10% pyridine/MeOH

2 ml DCM ×3
2 ml MeOH ×3
2 ml DCM ×3
2 ml MeOH×3

Collect each ﬁltrate into eggplant ﬂask
※ You can check the end of the elution by TLC.

Protected peptide solution〈Reprecipitation〉

Protocol 1

2 ml 1% TFA/DCM
Mix with vortex mixer for 2 min

Condense by an evaporator
40 ml cooled H2O
Filter precipitate and wash it with cooled H2O
Dry in a desiccator over 2 hrs

Protected peptide
Basically, protected peptides cannot be puriﬁed by HPLC. Thus, peptide extension
reaction should be done eﬃciently. For example,
・ To make molar ratio and reaction time in〈Coupling〉stepØ Fmoc-AA-OH : Coupling cocktail: DIEA : H2N-(Resin) = 4: 4 : 8 : 1
Ø 10 minin order to avoid side reactions.
・ Whichever Kaiser test result shows, acetylate N terminal.
The protocol for deprotection of side chain is the same as 6-1.

〈Reference 2〉Head-to-tail cyclic peptide synthesis

According to 6-2, you can obtain protected peptide acid whose N terminal is notprotected. When low-concentrated this peptide is exposed to amide coupling reaction,
you can get intramolecularly-cyclized peptide.

Cyclization of RGD peptide (RGDfX)
H-Arg(Pbf)-Gly-Asp(OtBu)-Dphe-Xxx-OH〈Crudepuriﬁcation〉〈Coupling〉

ProtocolDMF (peptide ﬁnal conc. ≦ 5mM)
5 eq. NaHCO3
3 eq. DPPA
Stir for 24 hrs
Filter solid NaHCO3
Remove DMF by oil pump
Precipitate with cold water
Suction ﬁltrate & wash precipitate with water and ether
Dry in a desiccator over 2 hrs-Arg(Pbf)-Gly-Asp(OtBu)-Dphe-Xxx-- 11 -

Special Protected Amino Acid (0.1 mmol scale)
7-1. Fmoc-Lys(ivDde)-OH3

ivDde group is stable against 20% PPD/DMF and TFA, and deprotected by hydrazine
selectively. Therefore, you can synthesize branched peptides by using ε amine group of
Lys as bifurcation. Please note that hydrazine deprotects not only ivDde but also Fmoc.
IvDde deprotection step can be monitored by measurement of Abs at 290 nm as well as
Fmoc quantiﬁcation.
Ac-AA-･･･-Lys(ivDde)-･･･-resin in synthesis column〈ivDdedeprotection〉

Protocol2 ml 2% hydrazine monohydrate/DMF
Mix with vortex mixer for 1 min
2 ml 2% hydrazine monohydrate/DMF
Stir for 30 min at RT
2 ml DMF wash ×5

Ac-AA-･･･-Lys-･･･-resin in synthesis column
Extension reaction
from ε amine group※ Check that Kaiser testshows positive.
The color should be weakblue.

7-2. Fmoc-Cys(Acm)-OH
Acm group is stable against 20% PPD/DMF and TFA, and deprotected by I2 selectively. At
that time, it makes disulﬁde bond. Moreover, in the case of Fmoc-Cys(Trt)-OH which isused in general Fmoc solid phase synthesis, Trt is also deprotected by I2 and Cys makes
disulﬁde bond. One of the main diﬀerences between these AAs is stability for acid.
ProtocolIodine oxidation on resin4〈Fmoc deprotection
Removal of〈Iodineoxidation〉
remained HI〉

Fmoc-AA-･･･-Cys(Acm)-･･･-Cys(Trt)-･･･-resin in synthesis column
1 mmol I2 (250 mg) in 2 ml DMF
Keep for 4 hrs at RT (Mix every 1hr)
2 ml DMF wash ×10
2 ml DCM wash ×3
2 ml DMF wash ×3
2 ml 20% PPD/DMF
2 ml 20% PPD/DMF

Mix with vortex mixer for 1 min

Stir for 15 min at RT
2 ml DMF wash ×5

H-AA-･･･-Cys-･･･-Cys-･･･-resin in synthesis column

SSDonʼt use EDT, which cleave disulﬁde bond, as deprotection scavenger.
If reaction point density is high, there may be disulﬁde bond between molecules.
You should adjust introduction eﬃciency of 1st AA or choose an optimal resin.

〈Reference 3〉Iodine oxidation in liquid phase5
Cys(Acm) containing crude peptide is dissolved in water/acetonitrile =1/5, and delivered
I2 solution by drops with stirring till solution shows color. After keeping for 30 min,ascorbic acid sodium salt is added to quench I2. After removal of solvent, it can be
puriﬁed by RP-HPLC.
The selectivity of disulﬁde bond formation within or between molecules should becontrolled by peptide concentration.

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Special Protected Amino Acid (0.1 mmol scale)
7-3. Fmoc-Cys(tButhio)-OH6

tButhio group is deprotected selectively by reduction with thiol group.
･･･-Cys(tButhio)-･･･-resin in synthesis column〈tButhio
deprotection〉

Protocol2 ml 20% 2-mercaptoethanol/DMF
Stir for 4 hrs at RT
2 ml DMF wash ×5-Cys-･･･-resin in synthesis column

7-4. Fmoc-Cys(Mmt)-OH6

It can also be used for Fmoc-Lys(Mtt)-OH.

Mmt group is deprotected by dilute acid reversibly. It can be deprotected without side
reactions by using TIS as scavenger. Mmt deprotection step can be monitored bymeasurement of Abs at 460 nm as well as Fmoc quantiﬁcation. If it is diﬃcult todeprotect Mmt、Mtt, repeat deprotection step.
･･･-Cys(Mmt)-･･･-resin in synthesis column〈Mmt
deprotection〉

Protocol2 ml TFA/TIS/DCM=1/5/94
Stir for 5 min at RT

Repeat 3 times

2 ml DCM wash ×3･･･-Cys-･･･-resin in synthesis column〈Reference 4〉Disulﬁde bond formation on resin by in situ activation with Npys6
Disulﬁde bond formation on resin can be monitored by using activated thiol formation
by 2,2ʻ-dithiobis(5-nitropyridine)（DTNP）(Npys group) and 7-3,4.
･･･-Cys(Mmt)-･･･-Cys(tButhio)-･･･-resin in synthesis column
Protocol 7-3.
〈Npys group〉･･･-Cys(Mmt)-･･･-Cys-･･･-resin in synthesis column
2 ml DCM wash ×3
1.0 mmol DTNP (310 mg) in 2ml DCM
Stir for 1 hr at RT
2 ml DCM wash ×5〈-SS- formation〉･･･-Cys(Mmt)-･･･-Cys(Npys)-･･･-resin in synthesis column
2 ml TFA/TIS/DCM=1/5/94
Stir for 5 min at RT
2 ml DCM wash ×3

Measure Abs of ﬁltrate.
Repeat till Abs of DTNP at
386 nm disappears.

･･･-Cys-･･･-Cys-･･･-resin in synthesis column
SSDonʼt use EDT, which cleave disulﬁde bond, as deprotection scavenger.
If reaction point density is high, there may be disulﬁde bond between molecules.
You should adjust introduction eﬃciency of 1st AA or choose an optimal resin.

- 13 -Column
Is that reagent fresh?
Almost all reagents used in peptide synthesis are prone to be deteriorated by moisture or O2.
Reagents should be purchased with consideration for frequency of synthesis and also separated
into some bottles at ﬁrst as necessary.
It depends on the kinds of reagents or frequency of opening and closing of their cap, but
reagents should be used within 1 year after purchasing.
Donʼt buy too much amount of reagent with a large bottle even if it is cheaper!
Method of storage and using is important. For example, reagents which is kept in refrigerator or
freezer such as protected AAs, should be kept at RT about 30 min before using in order to avoid
moisture. DIEA which tends to be deteriorated by oxidation or CO2 should be stored under N2
gas.
Method of reagent storage is directly related with peptide yield or purity! Manage reagentsproperly!

When should I prepare for amino acids?
If necessary AAs are weighed the day before using, you can use well the waiting time forreaction on the day for synthesis. Needless to say, the bottles of AAs should be put at RT in
preparation for recoupling.
On the other hand, in the case of peptide which has only 1~3 AAs in the sequence, it is not
diﬃcult to weigh AAs and dissolve them during the waiting time for〈Fmoc deprotection〉step.
It depends on your experimental style to use the 10 minute waiting time eﬃciently.
Synthesis by hand vs PetiSyzer
It is much easier to synthesize by PetiSyzer than by hand. One of the merits of PetiSyzer is ability
to control temperature. However, a usable amount of resin in PetiSyzer is lower than that insynthesis by hand because of thin synthesis column for PetiSyzer.
By hand：~ 0.3 mmol PetiSyzer：~100 mg
Except for quantity synthesis, it is recommended to use PetiSyzer.
Trick in the synthesis by PetiSyzer
If there are a lot of synthesis columns (over 4?), it is faster to add washing solvents by washingbottles. Even if molecular sieve is in the bottle, it is not hamper unlike Komagome pipette whichis inserted into a solvent bottle.
When Kaiser test & Fmoc quantiﬁcation are not enough for you to have conﬁdence …
You can cleave small amount of intermediate peptide from resin and analyze it.
The same amount of resin for Kaiser test is taken from synthesis column to an Eppendorf tube.
Then, small amount of cleavage cocktail is added to the tube. After about 30 min, ether
precipitation is repeated a few times by using a desk centrifuge. After drying and dissolving inwater, it can be analyzed by MALDI-TOF-MS or RP-HPLC.
This step is not troublesome. Therefore, it may relieve you when your resin has color due tointroduction of dye or peptide chain is long.
Is it really easy to synthesize by an automatic peptide synthesizer?
It is really easy to synthesize peptides by a peptide synthesizer. If you want to synthesize a lotof kinds of peptides with small scale, peptide synthesizer is useful. However, peptide purity withpeptide synthesizer tends to be low because the synthesizer doesnʼt do Kaiser test andrecoupling. Especially, if you use a peptide synthesizer whose operation rate is low with little
maintenance, it may be diﬃcult to purify the target compound by RP-HPLC, which results in ahuge amount of time in the puriﬁcation step.
Even though it is a little troublesome to synthesize peptides by hand, it is better to do so withgood purity, which results in saving time in puriﬁcation step and acetonitrile.

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Troubleshooting
When the yield of your target compound is low …
There may be problems in the steps of Fmoc deprotection, peptide extension reaction or
cleavage.
Predictable reasons could be ① mixing steps are not enough, ② activity of coupling reagentsdecreases, ③ it is diﬃcult for an AA to react due to its steric hindrance. Troubleshooting methods
may lead to ① change the size of reactors, the amount of the solvent, mixing time or strength,② remake coupling reagents, ③ change the kind of the coupling reagent ( such as HATU or
COMU which has higher reaction eﬃciency than HBTU7). Whichever step you have a trouble in,you can analyze and improve it based on Fmoc quantiﬁcation. In terms of troubleshooting in thecleavage step, please refer to 「Cleavage」as below.
When you use COMU as a coupling reagent, COMU 3.1 g/DMF 18 ml (0.4M)とDIEA 2.1 ml /NMP 18 ml (0.6M)
Kaiser test
If the washing is not enough, the color of the solvent may turn to blue, red or purple. However, it
should be negative if the color of the resin doesnʼt change. In addition, it often tends to be
negative when the color of the resin is red (especially in the case of using active trityl resin).
Storage of the resin
The resin should be kept in ① the solvent or ② the dry state. In the case of ①, it is better to use
DCM or NMP. Please note that DCM is easy to evaporate. DMF is not recommended, because DMF
is degraded during long storage and it produces amine groups, which may result in undesired
Fmoc deprotection. ② is suitable for long storage of the resin. Please note that you need to swell
the dried resin with DCM for about 30 min before you restart your experiment.
CleavageThis step is one of the reasons of decrease in yield. Because the cleavage reaction is exothermic
reaction, it is better to cool cleavage cocktail and TFA in advance. Depending on your peptide
sequence, you need to select the optimal kind of the deprotection cocktail or adjust the reaction
time. Since some peptide sequences show diﬃculty for deprotection of protection groups andneed some scavengers in peptide cleavage step to avoid side reactions. Moreover, if the amountof TFA or cooled Et2O is too low or too high in the reprecipitation step, the yield may be low.
Thus you should check the yield every time. Rough standard is TFA/Et2O=1/20 v/v.
Fluorophore labeling
Generally speaking, ﬂuorophores are more expensive than Fmoc AAs. Therefore you should careabout your reaction scale.
If it is a reaction with 1.2 eq. mol (relative to reaction point) of a ﬂuorophore, it is possible that
almost 100% reaction is succeeded by increase of the ﬂuorophore conc. and reaction time andtemperature. However, if the ﬂuorophore conc. is too high, sometimes it is diﬃcult to dissolve it
or mix it.
Know-how of synthesis① Optimize reaction conditions! (conc., pH & the kind of catalysts, the amount of solvents,
mixing time, reaction temperature)② Think about the purpose of each step! (reaction, washing, cleavage etc…each handling has
each optimal way.)③ Donʼt neglect to check the progress of each reaction! (Fmoc quantiﬁcation, MS analysis, kaizer
test, weighing the yield etc)
For example, you can check the progress of each reaction and activities of your reagents by
Fmoc quantiﬁcation!
HPLC
Peptide samples are dissolved in the A/B mixture. The ratio of solvent B should be lower than theinitial value of the solvent gradient. Dusts or impurities should be removed by ﬁltration or
centrifuge. If the sample cannot be dissolved in the A/B mixture, the other solvents can be usedsuch as DMSO. If you use the other solvents, please check that it is not insolubilized in thecolumn.
MS
Peptide solution and matrix solution are mixed with the ratio of 1:1, 1:10, 1:100. When it iscrystalized after drying, it is easy to be ionized. If it cannot be dried, you should change the kindof the matrix or solvent (CHCA, DHBA, HPA etc).

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Synthesis by PetiSyzer
Explanation of an equipment
Power button is in the back. You cancontrol 2 independent temperature
between left and right sides by using
the green buttons. Please connect
cords properly!

You should order special columns for HiPep
directly. You can order columns, upper caps andbottom caps separately. Please refer to the catalog
from HiPep when you want to know theseinformation such as price and product number.

Monitor for temperature/Heating unit

Column（5×2）

Cooling water
Removal of solvent
Cooling
Removal of water
solvent
It is connected to suction bottle/diaphragmpump.
When you turn a side ﬁnger grip vertically, you
can suck only your target column.

ShakerDonʼt set this power over 11.
It is dangerous!

ProtocolThe maximum amount of the resin is up to about 150 mg. Ideal amount is 100 mg.
During the coupling reaction, the temperature should be 50℃. From the end of thereaction to the treatment with PPD, the temperature should be decreased to RT withcooling water (If the amount of the solvent is small, it may be meaningless to control
the temperature).
In the cleavage step, column is stirred slowly with upper and bottom caps at 50℃.
When the deprotection cocktail is added to column, it should not be cooled.

Maintenance

It is exposed to organic solvents.
After using, please wipe away the organic solvents and keep clean!
Especially, the shaker and the monitor on heating unit donʼt have resistance fororganic solvents. Therefore please wipe away organic solvents as soon as possible if
these parts are exposed to the solvents.
The plastic part, where column is inserted, for removal of the solvent is removable. If
it gets to be dirty, please wash it in order to avoid choking.
After using, make it sure to turn oﬀ the vacuum pump and water ﬂow.

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References and Acknowledgements
9-1. References
1. B. Dorner et al., novabiochem handbook of solid phase peptide synthesis (MERCK)
2. R. Eritja et al., Tetrahedron 43, 2676 (1987)
3. W. C. Chan et al., Tetrahedron Lett. 39, 1803 (1998)
4. R. Soll et al., J. Peptide Sci. 6, 387 (2000)
5. B. Zeynizadeh et al., J. Chem. Research (S), 564 (2002)
6. A. K. Galande et al., J. Comb. Chem. 7, 174 (2005)
7. A. EI-Faham et al., Chemistry, 15, 9404 (2009)9-2. Authors and editors
Writing and editing：Mr. Yuta Nakamura1 (2nd year of the doctoral program)
Dr. Kazuto Watanabe1 (Alumni)
English translation：Mr. Takanobu Nobori1 (1st year of the doctoral program)
Demonstration：Mr. Takashi Shimomura1 (Alumni)
Technical support：Mr. Akito Hata1 (Alumni)：Dr. Akira Tsuchiya1 (Alumni)
Editorial supervisor：Prof. Takuro Niidome2 (Professor)
Chief advisor：Prof. Yoshiki Katayama1 (Professor)
1: Department of Applied Chemistry, Faculty of Engineering, Kyushu University, Japan
2: Graduate School of Science and Technology, Kumamoto University, Japan

Peptido ~ The Road To Becoming A Peptide Master ~4th edition
Released in December 2015

The purpose of this booklet is to hand down knowledge and technique aboutsolid phase peptide synthesis to the next generation. If you synthesize
peptides according to this book, you can obtain a lot of skills which people on
the Peptido should have. However, still the road to becoming a peptide mastershould be hard and steep. We wish somebody with strong pioneering heart
will ﬁnd new secrets about Peptido and edit a greater book than this one.

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