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BiBBi:* 3 S 2 S : f - H (O-S-ESri gaswb^jfc ...... fflssa i 3 Total Synthesis of Nephritogenic Glycopeptide, Nephritogenoside • • • Sift ZB 22 rnf-r—tffc-tSx^rf-Hwti^Rjefc-ovvc • • • a e®. »*ss 2 e S3S: a / F/\ • • ■ *«&** 3 5 i 7r-f yy^invx^nmm ■ ■ ■ #JH IE, ffl# m 4 2 5 y SE^9 2= A ...... 5*FSS 5 1 Chemistry of Lantibiotics ...... $ 5 9 S4S : ^7f-F«flS£jj?ifr fab»*»yr • ■ •eUSX, TBSM 8 5 ...... mwmw. 9 0 S5S : < 7" f-F <948 *51$ ##K*5i$&$f FE f <9 @m ...... ±er* 9 7 meg : nmm t mm%M ...... ±® $$ 109 A low-cost,high-efficiency solar cell based on dye-sensitized colloidal Ti02 films ■ ■ • B.0'Regan & M.Gratzel 12 0 Development of Specific Immunoadsorbent Containing Immobilized Synthetic Peptide of Acetylcholine Receptor for Treatment of Myasthenia Gravis • ■ ■ i 2 3 jiimeil 12 7 I? m : s 1. (ac&ic Tb^TrS ao "37 0, 7 ; y#&Lb#W4-(synthon)6 LT, me7% &, wb#j (Dfct ixm L%, #A^7; y ^^tr<77 r #^ac, m ©fffli§7bN b ©## t) °J£b o £££!£!+ L LT^ < Ci(t #&©A#c## 2) 7©^# twmgT&ao #C#38%%#^^^mLT, Zb) 7&fcZ *)&j£L&&RJimti -1 - #L'/:6C6T^6o t Lrfijffl ltv< ±X' 4. / 4) LT lr^y*r-t LT^fiJffl^-e^>B)o m&3#m r/<7f , p i - 3, 5) ^##$46 LTOfUffl D) <-??- Kte£&3£.iz&HktB>W*4T £> B> C - 2 - C0>I *2 ’ EE tl: ** Quantitative Analysis and Prediction of Hydrophobicity of Oligopeptides Miki Akamatsu* and Toshio Fujita* The log P value used for the hydrophobicity parameter, where P is the partition coefficient in a 1-octanol/pH 7.0 buffer system , of a number of di—pentapeptides was analyzed with physicochemical parameters for the side chain substituent of component amino acids. The log P value was shown to be governed not only by the “intrinsic” hydrophobicity but also by the steric effect of side chain substituents as well as intramolecular-type solvation and “polar proximity ” effects for polar side chains. The yff-turn conformational parameters of compo nent amino acids devised from the Chou-Fasman propensity index were nicely applied for the analysis of log P value of tetra- and pentapeptides. Using the results of the correlation analyses for logP of oligopeptides, we proposed a new effective hydrophobicity scale. na, for unionizable amino acid side chains. This scale and the hydrophobicity of oligopeptides would be useful not only for the process of peptide synthesis but also for the quantitative analysis of the structure-activity relationship of bioactive peptides. Key words : Oligopeptides ; Hydrophobicity : Partition coefficient; Peptide synthesis ; Drug design. # s ti-c v &4-5,0 1. -M-th ROD K3,£ V z-tzzt nTfg & 6 If, WrLi'ES, ammaUBWKa tU> X n i: £ O X £ tzo iis 20-30 6 ^ 6 v yf- L, 6 Ztlt2 -cw&o S .1 * (t606 c t icfca-e * Faculty of Agriculture. Kyoto University (Kitashira- kawa Oiwake-cho, Sakyo-ku, Kyoto 606, Japan) Hansch &«'. — 3 — ( 41 ) y v y^yy 837 Free-Wilson j£9 >£(Z £ tlfijffl 7r=log px—log P„ (1) s/vcs/jo ^yy m^<7>E5yyft£-timz, z > utz&&, mmitb L^;i%n^ L/zB# • #60 2(7)Z9 — 4 — 838 (1991) ( 42 ) 9X, < %z>n) 0 muzr i (jU) 4. v-, h V'Cff E*^?)#%U: 1 l:(i(3T#L< . CtUi, i-r, $JS!±USilES£&A:&V'7 S y &, fiD*> i£ 5 ymmmemfo e?(r,. i=n, '£lZM&m*fttz%^&&7 5 y#, gpt, Try, Trp, m, C)1±, +-n+-'tu7)BEtc^tt^T 5 y MSI — 5 — ( 43 ) * v rfXY^- T# 839 Table 2 Regression coefficient of indicator variable terms. Regression Corrected2 nb Amino Acid Side Chain Coeffcient Coefficient Ser -CH,OH 1.67 0.8 —1.1 2 Thr -CH(CH,)OH 1.67 0.8 —1.1 2 Met -ch2ch2sch3 0.64 0.64 3 Asn -ch2conh2 1.75c 0.9 —1.2C 3 Gin -ch2ch2conh2 1.16c 0.3—0.6C 4 Trp "*XO 0.35 0.35 4 H Tyr —CH2-^ ^-OH 0.17 0.17 6 aThe value is “corrected” by subtracting the intramolecular bridgingsolva- tion factor. bThe number of bonds separating the polar hetero atom in the polar group from thearcarbon of the peptides. c The value for N-acetyl-peptide-amides. YW.b I I I ,.* OR JiliOJ:-) tz, v-, h V^Yft KO logP $r^$ili- Hj:—on* —oN Hj:—ch,—oXh Hj:—ch,—c< flJEo # Z.btfX%tz 0 L 9=° ROH Q=o-~*-o( RQH c=o --h o;'_ L — L R —L ^L-r h-7-, /<>?/<7^ KOlogP li, v-, h I I I Yf- V'o V-, h V^YY Kti£2o£rr a >£ Fig. 1 Intramolecular bridging-solvation of the hydroxyl group of Ser residue and the carboxyl group on the backbone. R is either 1-Oct or H. Th7^Yf- KW.±(Z&&t. m2 - 6 - 840 $49 ##9-5 (1991) ( 44 ) &2izfc&Ltz & o 7>z(j& 6)"'. log P=0.94 2^-0.58 Ipep + 0.55 E'C(R„) + 0.30[EE^c(Rm) + Esc(Rc)] + 0.52 Iturn + 0.76 log fl + z + 0.14 IY +0.38Iw+0.66 I„ + 1.58 (Is + It) -0.81 Ip(N)—0.35IP( # pep)—3.87 n= 124 s=0.21 r=0.97 ( 6 ) Itum iitf b*I $£££bf- h 7-, ^y yKt:*t LX 1 t Ip(N), fp(#pep)Ufflffl N Pro z (7Pro 6# Fig. 2 /9-Turn structure (Type I). Reproduced from Dickerson, R.E., Takano, T., $ztkx&b0 zti-b - 7 - ( 45 ) * V 841 [0] x 10’3 [0] x 10 Ile-Ala-Ala-Ile 240 nm 240 nm. wavelength wavelength Fig. 3 CD Spectra of Ile-Pro-Gly-Ile and Ile-Ala-Ala-Ile. (------) in TFE, (...... ) in aqueous 0.1 M sodium phosphate/phosphoric acid buffer (pH 7) Jta = [' K(D#^-e t u-c, N-T-t-f-Jl"*’?’?- K7 5 K<7) log P 240 nm wavelength fifeo-C, t F l/j ^ < y msi (^;6){i# - 8 - 842 ^49 ##9 -# (1991) ( 46 ) Table 3 Hydrophobicity scales of amino acid side chains. Amino HI(K.D.)d HP(W.)e HS(C.)f /T (F.P.)* A^°(N.T.)h f(R.y Acid Nb MCC Gly 0 0 0 0 0 0 0 0 Ala 0.19 0.24 2.2 -0.45 0.02 0.31 0.5 0.53 Val 0.49 0.82 4.6 -0.40 0.18 1.22 1.5 1.46 Leu 0.92 1.28 4.2 -0.11 0.10 1.70 1.8 1.99 lie 0.72 1.17 4.9 -0.24 0.22 1.80 - 1.99 Phe 1.35 1.57 3.2 -3.15 0.14 1.79 2.5 2.24 Tyr 0.78 1.01 -0.9 -8.50 -0.38 0.96 2.3 1.70 Trp 1.72 1.93 —0.5 -8.27 -0.12 2.25 3.4 2.31 Met 0.67 0.93 2.3 -3.87 0.05 1.23 1.3 1.08 Ser -0.08 0.04 -0.4 -7.45 -0.21 -0.04 -0.3 -0.56 Thr 0.07 0.25 -0.3 -7.27 -0.19 0.26 0.4 -0.26 Gin -0.51 -0.30 —3.1 -11.77 -0.71 -0.22 - -1.09 Asn -0.51 -0.26 -3.1 -12.07 -0.48 -0.60 - -1.05 Pro j j -1.2 - -0.30 0.72 — 1.01 * From ref. 14, calculated from eq. 6. bFor //-terminal residues. c For central and C-terminal residues. d Hydropathy index of Kyte-Doolittle (31). Reference was shfted to Gly. e Hydration potential of Wolfenden et al. (32). f Logarithm of hydrophobic scale of Chothia (33). 8 7r value of Fauchere-Pliska (34) from log P values of //-acetyl amino acin amides. h Relative free energy of transfer from either ethanol or dioxane to water (kcal/mole) of Nozaki-Tanford (35). 1 f value of Rekker (36). j?ra(location, number of residues) of proline ;,7a(N, 2) : 0.35, ,Ta(MC, 2) : 1.16, ;ra(N, 3) : 0.00,— a(MC, 3) : 0.81, z-„ (N, 4) : -0.34,/Ta(MC, 4) : 0.46. -1 0 1 2-1 1 2 -1 0 1 2 lta(MQ Jia(MC) *a(MC) ° 1 Jta(MC) 2 _1 0 '..(MC,2 •' ° 1 Xa(MQ 2 Fig. 5 Relationships between ^0(MC) and other hydrophobicity parameters. - 9 - ( 47 ) y v =f/ (D*^SW6Si$&^omT66^* ;uy -^iz Zfrgj-X-fZ < T&ffl $ fix^h 8) 8, o gfc< 3' < Nozaki-Tanford O^bS:351, ©Rekker log(BR) = a^-+/ocr + 5‘S+const. ( g ) > 13 fzytl^Ofiy, ttZJCat ^^15 tc^i"0 a, />, ^ (i'E-tL^tLO^mo —£ LXfc>±'b X 7 tz, na 7 ZzZX na *-9 mmmtlzmi-ZmTlZXvXMff-fZ Free-Wilson & emv'-c. yo&m, * Nisato btfL-y IES&JeWf LtzWK Zas lavsky TV'SOT*, Kyte-Doolittle Oil i> t TkStS / > Leu 7. K7 7 7'fif'f>^ffl Orn ' D-Phe $o^ib^m%o#m±oaf -10- 844 #*&&&%# 49 ## 9 (1991) (48) j3zcm*# >J, 1986, tm E] 6 C0«fc ^ tl D-Phe-Pro (Offifr P.129 id mm s, 9, 571(1984) 12) M. Akamatsu, Y. Yoshida, H. Nakamura, M. ZthZ &?)%##%(: t v^tL-Cv^ Asao, H. Iwamura, T. Fujita, Quant. Struct. -Act. 6^0 #4r $i Orn £ Lys (C, D-Phe £ $ t £ d-T Relat,. 8, 195 (1989) S logP -11- ( 49 ) *') 845 37) D„ Nisato. J. Wagnon . G. Callet, D. Mettefeu, Biochem. Pharm., 31. 3757 (1982) J.-L. Assens, C. Plouzane. B. Tonnerre. V. 39) M. Asao, H. Iwamura, M. Akamatsu, T. Fujita,/. PliSka. J.-L. Fauchere. J. Med. Chem., 30. 2287 Med. Chem.. 30. 1873 (1987) (1987) 40) M. Aoyagi , S. Lee, N. Izumiya, J. Mol . Graphics, 38) B. Yu. Zaslavsky . N.M. Mestechkina, L.M. 5, 35 (1987) Miheeva. S.V. Rogozhin. G. Ya. Bakalkin. G.G. 41) n-lU 1991 Rjazhsky . E.V. Chetverina. A.A. Asmuko. J.D. Bespalova. N.V. Korobov , O.N. Chichenkov, 1991 , p.198 -12- es: Ky?KCDSfiE&ffi =B*E6|! '<■//- K^Effc^/tr 67/(3 K©5H6 • 7=;i 50 BS r%, 9 $i$ «£ <15 £ 6 (3 & ^ r e /c0 $6(3, e* li&lNfii, io K |iSOH6®^@^Tl%S T^5o //////////////////////////////////////////////////////////////////////////////////////////////////////////////////////////////////////////////. (i D#>(3 90 %t3??/: 6 -< 7> KJk^oE% oES #/£t‘t>''m&&n~%imzhx£tza ttz, '&fa Saburo Aimoto, (f565 3 - 2 ) [Institute for Protein Research, Osaka Uni versity, Yamadaoka, Suita, Osaka 565, Japan] Chemical Synthesis of Protein [k«r word i [SaKOXk^aE] (i^^/SS] h’/f h'^] —13 — 215 jzi6o-<7* 9-74jl/s;W h 4=- y ^7 /> tr* - /L- (Fmoc) fb 7 5 y E ; y EoS'n^EOf5&{bE i. k, ga%(D^-j52 ^%8s?o& t^hplc -e ko^E^iz, M^$K-CA'P)OE&^ 61j>-t*^7> K7):mg^%70^A^2^LL^'#^EL'C <, Boc I'^i'Z 9^#-^, ^v„ -^, ^/set^ee-c, vtDi^mfi'boWL yt^g-CB^tJ^^Z < ^0 ffi-tz cbiitTb^-C®^ 0 ta LC#7K7 y {b*%o Z 5 6 L ft v T&a, Boc-NHCHR „ CO-OCHs-P AM-gj H I CF3COO- • +NH,CHR „CO-OCH2-PAM-£Jia ©❖fa l nh 2chr „co -och 2-pam -®e ©7 2 /##;&& 1 Boc-NHCHRn^CO-NHCHR^O-OCHr-PAM-^E ®, ©, ®<7)< 9SL 1 Boc-NHCHRtCO-NHCHR^O-NHCHRaCO...... NHCHRn_1CO-NHCHR„CO-OCH2-PAM-^|§ ©SfS^ib(7)Sz^ttiL i ©gfi^tl^SiiS i • ©«£i I NH:CHR1CO-NHCHR2CO-NHCHR3CO-NHCHR4CO--NHCHR„-2CO-NHCHR„_1CO-NHCHR„COOH 0 1. Boc 7 5 ;®$rfflV'5^y7- F —14 — 216 Vol. 37 No. 3 (1992) Ltz&^x, t-f B-Ci^TLfz fr^5o ^C^Rs^yf- Ktl'A42%^Z < ^SL-CV 7 t&mtm < y/^p-7^7 7^-,IfgoHP- ^-O-a-E HIV-l 7 PT7-4:^ > CE LC, -f yh 73c# ^ p -? h ^7 7 ^ — SrEtiEL'C, t 76sgE-c^7'f- xig*r£j£r TBoW^MU -f £i bfi'V&b'h' ktzoizo *R-, -f^-p^y8 (IL-S, 72 o-a-E^N Nutt x&o, #E%-7:t# T5 5)o #(^6 5” o [#%ej] HIV-l 7^7^'yi/P77-i: (HI Kent l?it, $%(=. V-1 7" p T 7 - -E) O^E 67 95 {&0 7 7 7 4 ygl%t-a-7 ; / ES(Aba) ^#SE*eoS6So-a-E^^V'-t io «1. [Aba 67,95] -HIV-1 yof7-f(0M }) 1 15 4%^ Pro-CIn-Ile-Thr-Leu-Trp-GIn-Arg-Pro-Leu-Val-Thr-Ile-Arg-lle- mm K irE&PE (^mol) i±7S&a) ##ZS 30 B5& Gly-Gly-GIn-Leu-Lys-Glu-AIa-Leu-Leu-Asp-Thr-Gly-Ala-Asp-Asp- i phe-mm 300 - = 100% - 45 3 y-mm 211 - 70% - Thr-Val-Leu-Glu-Glu-Met-Asn-Leu-Pro-Gly-Lys-Trp-lys-Pro-Lys- 4 154 51% = 100% 60 5 Sephadex G-50 0.14 Met-1le-GIy-Gly-lle-GIy-GIy-Phe-11e-Lys-VaI -Arg-GIn-Tyr-Asp- 6 #% HPLC 15 0.29 5% 10% 67 75 6 fSiSSE 1.00 Cln-Ile-Pro-Val-Glu-lle-Cys-Gly-His-Lys-Ala-lle-Gly-Thr-Val- 7 -f 5b) 1.00<> 1.8% 3.5% vi/y7^- 90 Lcu-Val-Gly-Pro-Thr-Pro-Val-Asn-IIc-Ile-Gly-Arg-Asn-Lcu-Lcu- a ) = : Abz-Thr-Ile-Nle-Phe(NO);-Gln- 95 99 Arg • 7 1 K (Abz ; 2-7 5 / ^7-/ Thr-GIn-IIc-Gly-Cys-Thr-Leu-Asn-Phe < yu) i®9iilX : pH 6. 5, 0.1 m MES, 1 m NaCi0 El 2. t h&f3R\£1M /V7-1 (HIV-1) 077/-?7* b) ^ 50 m go 7®7o-f7—tV)7 ; / c ) f — 7^-—/<-% : ^ 5/#o -15- 5 6 m o Ik i= -S' 217 (Ac-Ser-Leu-Asn-Pne-^T [CH(OH)-CH2N]-Pro-Ile- emam (hbS) o^ei9) Val-OMe, Val-Ser-Gln-Asn-Leu-?F[CH(OH)-CH2]- ES6iZ, y y K-^ y y v h olWE-^jlltR Val-Ile-Val) > O MW1’1* e^7 ; / So##d:##cfT%x.a;% ^ ^ L, Nagai K© £ 9 - y-Sr&L*:- = v BPTI &%mz < 6 C 6 ^Lfc^, Lrf‘LE HIV-1 y 7 y 7 -4)L mboyjmxa, * ^^^'y^moMn^mk^ntz 5 c kti'X^tzfi'^iztzg), WHO * y/ugg^o 72}-MBL*C < tlMb'b ofc0 < ^7 7 A ^ 7T b y? 7 / c:Z, 2ooM#7):&-,yCa S l or=9E^;z, WH*/^ -l COEPg-CiELvSB K4=y / V h^r© ^z-5^-e£-fi-SfcSLf-io, ^ p-r h ^ —e&fc ^ctcfcDi-6. Hiv-i y^t7- em%-^ bfrtfi-Ut'ftbtzti'^iz c £-c£>Oo #2 oft5 -tfOZ 5 l-2S^1rSEi‘»S6Sf-ib'V'-Cit, 2##: J@*fi, 7^^7^ygfy^^ ; y^ES&r$'< 4>o ^o^TS-Cc e>S-x§B^mM75^6E-CS)5 = -t y > y h t?it, ^^c^^TK&oiEv^ES ^ i o ; 66ES© HIV-1 y,~T7--i£(D'&J&t $SBkik 2. g|9at^L/;^7'f * a.y*4 1 y /v-So^^6^66^ > i' $ Ac^i'"C, iE^ 6K —16 — 218 a k mm m ^ Vol. 37 No. 3 (1992) 4 -methy I benzhyd ry I am i ne &3i (NH2-&jH) Sit, 0.1 m NaOH tKS^E'CSE^ 1 Boc-Gly-SCHaCHsCOOH + DCC coSKag&fht, - /io dnamemam chbS) d < z‘Noc-Leu-Ser-Lys(Boc)-Lys(Boc)-Asp-Ala-Thr-Lys(Boc)-Ala-Val-Asp-Ala-Val- Phe-Asp-Ser-Ile-Thr-Glu-Ala-Leu-Arg-Lys(Boc)-Gly-SCH 2CH2CONH2 HBs 36, Z v -> XT): 7 A .-T-:^ ->^;t z N'oc-Asp-Lys(Boc)-VaI -Gin-Leu-1 le-Gly-Phe-Gly-Asn-Phe-Glu-Val - Arg-Glu- Arg-Ala-Ala-Arg-Lys(Boc)-Gly-SCH 2CH2CONH2 z",Noc-[Lys(Boc)41,59]-HBs(40-60)-SCH 2CH2C0NH2 (3) L, ■h^c-ho^y^- F"-e Z / v F ^ at 6 Arg-Asn-Pro-CIn-Thr-Gly-Glu-Glu-Met-GIu-1 le-Pro-A la-Ser-Lys(Boc)-Val - Pro F"£P,Z>{$fl:it, ~7 ~7 =7 J W S (: Noc) £7*^ 77/^0^ L^^^EL-C^- Pj-f, 430 AMSEtrSrfflv, Boc 7 £ /BSSrtfH'a DCC+ HPLC cz ommi-SC 6^-c#- HOBt m 5 y’* v + f:3 hplc boc-onsu pi-x^f/Z^ 10-15% Zkfi'-C£tz0 *C Boc S^^Ai“o^lt%^-yi-x.^7/uSit^*C.t) KO^Et OET&vim - -17- W 6 S O it ¥ ^ fig 219 (1) (2) (3) (4) (Boc) 1 (Boc) 4 (Boc) 2 (Boc) 5 Boc H ’ 15] SR iNoc 116 .' '-<39 | SR iNoc 1-tO: < v'„ :^60l SR H |6l- -;90 | OH AgNOj-rHONp AgNOs-rHONp AgNOa-rHONp TFA, HPLC zNocpO/ " ■ 90 1 QH (5) I Boc-ONSu ______I Zn/AcOH „ TFA. '-HPLC zNoc|i6//-, / .T.90 1OH (6) - Boc-ONSu ______Zn/AcOH TFA, HPLC H R ' ' : />»"' ■ • ■ ■ ■ -• 901 OH (7) m 6. HBs (1-90) #-&### 3 it, - hc7 = 7-;^4T- -f Kco-<7 - h P y HBs (1-90) 4 £ Eir L, mm HBs (40-90) SC-it HPLC "Cm# -C #, C £ ,*51 &dc#_kO Boc B8-7 HPLC X^MLiz, #tJ 50% Boc Ltz ?a> —18 — I •4 44 Ik A m -x ik 0 V 6 X X X =\ •4 8 oo it m m V" 55 % •w o V x V o 4; AJ '6 2o oooooooooo oooo •y i & 4 V It oooooooooo 8 ooooo <0 O X X H K G >0 E if 41; Q 4a 5% •*-( o oooooooooo o oooooo n 44 ft 4? V o oooooooooo o It- X M % Ut 4S o ooooooo xk 4? x 4 11 E m m >0 P o oooooooooo oooooooo # K? 3 5 E oooooooooo 8 ooooooo V u •" vO :4 4a A IR >0 „V| o • ©<>♦□■<◄>► oooooo X A) H u V u. J, G k 0 •K1 o oooooooooo o ooooo Y m -R V CO oooooooooo K> IK b ft '-e xk 55 4>< <: y 4 iR -\ -8 oooo H {D u m X oooooooooo § ooo ft ft » J X W W m J w M? >J I y to & 3 X X M XI U X) 41 v ^ p 3 y k' i — & X mtlt® $ Xl, 96 S[§|0 ^7 t F 64, 3074-3077 (1990) g WiXr'n&'C #" 6 & rfiJS e Zl*c l' 2) W u, C.-R., Wade, J. D., Tregear, G. W. : Int. J. Peptide Protein Res., 31, 47-57 (1983) 6. 20 mmo^l LtzM^ 4 7> K 96 1 3) Khomura, M., Nio, N., Ariyoshi, Y. : Agric. aM@g-e#m±ce^-c & %, - -20- gas #m. mm Vo 1. 37 No. 3 (1992) mura, S. : Bull. Chem. Soc. Jpn., 62, 524-531 23) Geysen, H. M., Melon, R. H., Barteling, S. (1989; J.: Proc. Natl. Acad. Sci. USA, 81, 3998- 20) Fodor, S. P., Read, J. L., Pirrung, M. C., 4002 (1984) Stryer, L., Lu, A. T., Solas, D.: Science, 24) Geysen, H. M., Barteling, S. J., Melon, R. 251, 767-773 (1991) H.: Proc. Natl. Acad. Sci. USA, 82, 178- 21) Houghten, R. A.: Proc. Natl. Acad. Sci. USA, 182 (1985) 82, 5131-5135 (1985) 25) Papsidero, L. D., Shue, M., Ruscetti, F. W.: 22) Ruggeri, Z. M., Houghten, R. A., Russell, S. J. Virol., 63, 267-272 (1989) R., Zimmerman, T. S.: Proc. Natl. Acad. Sci. 26) Schnorrenberg, G., Gerhardt, H.: Tetrahed USA, S3, 5708-5712 (1986) ron, 45, 7759-7764 (1989) -21- Tetrahedron Letters. Vol. 33. No. 3. pp. 363-366.1992 00404039/92 $3.00 + .00 Printed in Great Britain Pcrgamon Press pic TOTAL SYNTHESIS OF NEPHRTTOGENIC GLYCOPEPTIDE, NEPHRITOGENOSIDE Tadashi Teshima, Kiichiro Nakajima, Minor! Takahashi, and Tetsuo Shiba* Protein Research Foundation, 4-1-2 Ina, Minoh, Osaka 562, Japan Key Words: nephritogenosidc; glycopeptide; allyloxycarbonyl group; palladium complex; nephritogenic activity Abstract: Nephri togenosidc (1), proposed as a nephritogenic triglycosylhenicosapeptide with asparagine N-a- glycoside linkage, was first synthesized by the coupling of triglycosyldipeptide active ester with the nonadecapeptide (3-21) using allyloxycarbonyl group as the final protecting group. A {5-anomeric glycopeptide was also prepared. Nephri togenosidc was isolated as an active principle of nephritogenicity from the basement membrane of rats by S. Shibata in 198 11). The structure was determined by S. Shibata and his collaborators in 1988 to be a glycopeptide in which the trisaccharide composed of three glucose moieties is linked to the peptide of 21 amino acids via N-glycoside bond on the asparagine residue.2) Although syntheses of the trisaccharide part connected with an amino acid or a short peptide have been reported,3) a total synthesis of whole structure of nephri togenosidc has not yet been accomplished. In order to confirm the proposed structure and to elucidate the biological activity in molecular level, we performed the first total synthesis of nephritogenoside. NHCO I 5 H2NCHCO-Pro-Leu-Phe-Gly- 10 15 20 lle-Ala-Gly-Glu-Asp-Gly-Pro-Thr-Gly-Pro-Ser-Gly-lle-Val-Gly-GIn-OH Structure of Nephritogenoside (1) In our synthetic strategy, allyloxycarbonyl (Aloe) group 4) which is removable by palladium complex under neutral conditions was chosen as the final protecting group of amino and hydroxyl groups, taking account of general instability of glycopeptide under acidic and basic conditions. No existence of basic amino acid residues in the peptide chain makes possible the free peptide to be coupled with glycosyl moiety. The -22- 364 peptide used in this study was synthesized by ABI 430A peptide synthesizer. Boc group was used as protection for a-amino group, and cyclohexyl ester for co-carboxyl group of Asp and Glu. The final deprotec tion and cleavage from the resin were carried out by the treatment with HF - p-cresol (8:2). After purification by HPLC, the peptide obtained was directly used for the coupling with the glycosyl moiety. Heptaacetylisomaltosyl fluoride (2) was prepared from isomaltose by peracetylation followed by treatment with 60% HF in pyridine, and then coupled with 2,3,4-triacetylglucopyranosy 1-a-azide (3)3b) to give trisaccharide azide 4a as shown in Fig. 1. First, a formation of N-glycoside linkage of this trisaccharide with asparagine moiety was attempted. Thus the compound 4a was reduced by catalytic hydrogenation using Pd- C, and then coupled with Aloc-Asp(OH)-(yBu 5) to give an anomeric mixture of ZV-glycosides 5a and 5(3. Under reduction conditions, an anomerization at C-l carbon occurred to give a mixture of diastereomers which were separated and used for the following synthetic steps respectively. Replacement of acetyl group in compound 5a or 5(3 with Aloe group gave 6a or 6(3, which was converted to N-hydroxysuccinimide (OSu) ester 7a or 7(3 by TFA treatment followed by active esterification as shown in Fig. 2. However, the active ester 7a or 7(3 did not react with the eicosapeptide (2-21) at all but cyclized itself to give the succinimide derivative. In order to avoid the succinimide formation of the active ester 7a or 7(3 during the coupling reaction, we next tried a coupling reaction of glycosyldipeptide active ester with free nonadecapeptide (3-21) as shown in Isomaltose (3) OAc ----- O NHCO 2) Aloc-Asp(OH)-OtBu NHCO HOBt,WSCD*HCI Aloc-HNCHCOOfBu Aloc-HNCHCOOtBu (5(3) 68.3% Fig-1 -23- 365 AlocO OAloc AlocO 1)EfeN OAloc MeOH-H,0 1) TFA OAloc 2) Aloc-CI 2) HOSu. AlocO DMAP OAloc NHCO WSCD-HC1 a: 33.9% P: 23.9% OAloc | Aloc-HNCHCOOf Bu NHCO Peptide —O (2-21) t CH2 — No Coupling Product OAloc | NH-Aloc Aloc-HNC HCOOSu OAloc (70,70) Fig. 3. The glycosyl dipeptide can be prepared from glycosylasparagine and the protected proline residue by another coupling method rather than the active ester method. Thus, the compound 5a was treated with TFA to remove z-butyl ester and then coupled with H-Pro-OrBu by l-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride (WSCD-HC1) and 1-hydroxybenzotriazole (HOBt) method. Acetyl group of triglycosyldi- peptide (8a) thus obtained was replaced with Aloe group to give 9a. r-Butyl ester group in 9a was cleaved -O 1) Et3N 1)TFA Me0H-H20 5a,5(3 -NHCO ------» 2) H-Pro-O/Bu 2) Aloc-CI CH2 DMAP HOBt OAc | WSCD-HCI Aloc-HNCHCO-Pro-OfBu a: 42.0% (8a, 8p) P: 40.9% •O 1) TFA -NHCO 2) HOSu, I WSCD-HCI OAloc Aloc-HNCHCO-Pro-OSu a: 97.1% NHCO p: 97.4% (10a,10p) CH, OAloc | (9a, 9p) Aloc-HNCHCO-Pro-OfBu 1) Peptide (3-21) ►- Nephritogenoside and its p-Anomer 2) Pd(PPh3)4i PPha a: 33.7% dimedone" p: 23.5% Fig. 3 -24- 366 with TFA and converted to the active ester (10a). Finally, 10a was reacted with the nonadecapeptide (3-21) to give us a desirable protected nephritogenoside without cyclization at asparagine residue as in compound 7. After deprotection of all Aloe groups by palladium complex, the crude product was purified by HPLC to give free nephritogenoside. 6) Moreover, the P-anomer of nephritogenoside 7) was also synthesized from 5(3 by the same manner as that of nephritogenoside. Biological tests of nephritogenoside and its (3-anomer are now being undertaken. In this study, we established the general strategy for the synthesis of glycopeptide with N-glycosyl bond of asparagine which is linked to a relatively long peptide chain: (i) The glycosyl moiety is first coupled with asparagine residue, (ii) The glycosylasparagine in a protected form is coupled with amino acid or peptide 9 ), (iii) The glycosylpeptide thus obtained is coupled with the longer peptide by the active ester method, (iv) The Aloe group is used as the final protecting group. REFERENCES AND NOTES 1) S. Shibata and K. Miura, J. Biochem., 1987, 89, 1737-1749. 2) a) S. Shibata, T. Takeda, and Y. Natori, J. Biol. Chem., 1988, 263, 12483-12485. b) T. Takeda, M. Sawaki, Y. Ogihara, and S. Shibata, Chem. Pharm. Bull., 1989, 37, 54-56. 3) a) T. Ogawa, S. Nakabayashi, and S. Shibata, Carbohydr. Res., 1980, 86, C7-C10. b) T. Takeda, Y. Sugiura, C. Hamada, R. Fujii, K. Suzuki, Y. Ogihara, and S. Shibata, Chem. Pharm. Bull., 1981, 29, 3196-3201. c) T. Takeda, A. Utsuno, N. Okamoto, Y. Ogihara, and S. Shibata, Carbohydr. Res., 1990, 207, 71-79. d) A. J. Ratcliffe, P. Konradsson, and B. Fraser-Reid, J. Am. Chem. Soc., 1990, 112,5665-5667. 4) a) H. Kunz,' Angew. Chem. Int. Ed. Engl., 1987, 26, 294-308. b) Y. Hayakawa, H. Kato, M. Uchiyama, H. Kajino, and R. Noyori, J. Org. Chem., 1986, 51, 2400-2402. 5) Aloc-Asp(OH)-OfBu was prepared from H-Asp(OBzl)-OH through three steps. [1) Aloc-Cl, NaHC03, 2) (CH3)2C=CH2, H2S04, 3) NaOH, total yield: 75.4%] 6) Amino acid analysis 83 ), Asp(2): 2.00, Thr(l): 0.92, Ser(l): 0.91, Glu(2): 1.96, Pro(3): 2.90, Gly(6): 5.91, Ala(l): 0.96, Val(l): 0.58, Ile(2): 1.56, Leu(l): 1.00, Phe(l): 0.98, NH3(2): 2.35; PD-MS8b ), M+H : 2471.3, M+Na : 2493.7; 1H-NMR&:), 0.78-0.95 (24H), 1.05-1.23 (3H), 1.19 (3H, d, 6.3 Hz), 1.37 (3H, d, 7.3 Hz), 1.35-1.60 (5H), 1.70-2.48 (24H), 2.75-4.55 (59H), 4.92 (1H, d, 3.7 Hz), 5.60 (1H, d, 5.4 Hz), 7.20-7.39 (5H); HPLC8d ): 15.7 min. 7) Amino acid analysis 83 ), Asp(2): 2.00, Thr(l): 0.95, Ser(l): 0.97, Glu(2): 2.04, Pro(3): 3.03, Gly(6): 5.87, Ala(l): 1.04, Val(l): 0.57, Ile(2): 1.50, Leu(l): 1.01, Phe(l): 1.00, NH3(2): 2.67; PD-MS8b ), M+H : 2471.6, M+Na : 2493.9; lH-NMR8c ), 0.78-0.95 (24H), 1.05-1.20 (3H), 1.19 (3H, d, 6.4 Hz), 1.33 (3H, d, 7.1 Hz), 1.35-1.60 (5H), 1.65-2.50 (24H), 2.75-4.55 (59H), 4.89 (1H, d, 3.9 Hz), 4.98 (1H, d, 9.0 Hz), 7.20-7.39 (5H); HPLC8d ): 15.7 min. 8) a) Hydrolysis conditions: 6M HC1, 110°C, 22 h. Under this condition, only about 60 % of Ile-Val in the peptide sequence was hydrolyzed. Theoretical values of amino acid ratios were shown in parentheses, b) Plasma desorption mass spectrometry. The calculated molecular weight, M+H: 2470.5, M+Na: 2492.5. c) 5(ppm)(270 MHz ^H-NMR in D20). The peak of HDO was used as the reference (8=4.70 ppm), d) Retention time of HPLC (Cosmosil 5C%g, 10 x 250 mm, CH3CN-H2G containing 0.1% TFA, gradient 10 - 60 % CH3CN (25 min), flow rate: 3 ml/min, detection: 210 nm). 9) The C-terminal amino acid of the glycosylpeptide is recommended to be either Gly or Pro to avoid the racemization in further fragment condensation. (Received in Japan 17 October 1991) 430 63(10), 430—437 (1989) yof K^H^Etbtcocx^ ^ m*, s f u/cws&iCck a^yf %u6o comet, y v’z&zttbK, m^a#m©##^m *irra, %#%©y- mte v'/f A/-L-y □ -yyu-y y -y^r- v Ke) As&r&^tta c 1 & cable .k¥u y-^-H y/j-:*E^;/j^yy koj&em.; 7"DTT--t/|t, 7 V'<9¥tJP'* **-f-J- V ICftH/U ‘efeaci^.iiEb/co f ©m, yy x h v y-y y/f y UT, / /So cnt-eic, • ya 7-r —\£&■&&&sv^rnm^,© 5 Hi l- HJ y h 7 7 yi©3c^ 10), yy^T-f yEj£ %%m btz? y/^7 6a©m#©*s-6^#%fb"'"\ JIJ4:^J/l]Uct®T'*«o izt'UIL, i*i'rt, :S/lb©?iM- jo J:0*. 7 y yy r y y ylM^Xf KAb t hZ5f y y lc jd id- £ WJ^M y o f 7 - -tf ©*W >2), f - X © M ^ u y^^©^m@#^yy k©^e 13-15) tc^icb, (c#jo#5 y y- y $) A^Mi/y-y 41, tf—yutfijc ^©mAEj^^WrA6C6D, 7°drT —M jd(i5 7 V'*9 527E$iWjlh (DtzbbCD chillproofing en k a $na zyme t y©m 5>, ESnnn t Condensation Reaction of Dipeptides by Proteases * Tetsuo Mu RO *|SmiCXaSBf%iJf £&fl3*aiPF2fcfl SfMi (536 1-6-50) ** Yoshio Tominaga r.i] .h $8fi Sh9 Voi.63 — 26 — 431 E 2 RSiC^jf 6 V * * » * 7U A 7 i K ©M161 [R,-COOH][R2-NH2j [R,-CO-NH-R2] ■yDf^-t;i/A7': K ii jistf 4 ishli'A (O.OSM Z-Afk) 2.1 mm T t >6% 7b (0.5M Leu-Nil2) a) [E>0.1mM, b V x-vu-< >KS®K (pH6.5). Mbil b v y\cX6 N-^vi/ju 37"C, 20D.fBJ£l£: t 5- istl Jl'tf — Jl' -L-7 -L-a 4 *> J^T i KO b) [E>10/*M. (pH5.5), 0.05 M KCN. ImM EDTA. 37eC. 208?I?UStC Og%n.!f, b V 7°y y^-y--^ v y yicJ; 5N-^ y ;l/t'-yH.-7 i - ^7 7 - (85 #) 0 i,4-y^ yy^--^©^ iJUXttkidT'ZjmtteQ, * L/c (m Do k*7n L/Co t/c, b V yyy&avxi/< y\C£& N-^yv J;u^- + y/7 7i/.+r^;L/-L-7^4:r 1 M&mtL, m# k L. 50# 7 / -7- VU.I-. A, a 7 ■ K /; ;u-V- y T ; y&%# (#) (DfttEX, L~C 4 — 5 W. I: ft blc^J (M) (M) iT5c6erneu-cua ce 2)« l^u Z-Arg Leu-NH2 b v yy y=) vn’/n0^ yb) ^/ -^, mmfe& Vol.63 — 27 — 432 CtlZ> 7k#t ; / (E #, K0Wj9kcm%/j:%)m:©# 3)0 CCDZ7IC, ^6%K^IC^#6#^CD@#S;(&IC6 $ ft, mtcffl 7°n TT--tf Z-Phe Gly-NH 2 <10 Z-Phe Ala-NH; 12 Z-Phe Pro-NHz 0 Z-Phe Val-NH2 70 Z-Phe Leu-NHz 71 e 20 - Z-Phe Ile-NH2 75 CE]=0.2mM, H V H (pH7.0), 37* c. 20B$|3]££ C (H,N) (COOH) Sz s, S', Sz El 2 y y &&±xm Vol.63 28 433 4 2.5^ y^yyKi^t^yofT-t' 4 SCC (D scc (Dtm >?^7*y K W' /DfT-^' SCC KA6%(C^U-6K^#m Leu-Gly 2 - te^^b^(cy^,/cyj(c, 36^6 L-r^«©^:->'^ Leu - Ala 1 - yy r&muTRj%fr(,\ o-T h Leu-Ser 2 - y7 7^-!Cj;D^#U/:o -€-©##, E 4 iC^ktJzo Leu-Val 1 - w$, &5uam^©^## (r Leu-Met 1 + ; / #) RfWc 1 ~2 W-y b K U xtf- y h Gly-Leu 2 - Gly-Ile 1 - 5fi/c29)0 £/c, %*#©T % / f&fr z>uz> •>'^ Gly-Phe 2 - yy KT&a Phe-Val, Val-Phe. Leu-Met U Ala-Leu 1 - \&, Mmvmi\cx-o xmma Phe-Val 1 + su E 5 ##&#©7* o y y - 4f (C j; 5 Plgi t Plg 2 ©fTR% 7 yvN°7fi PLG1 + PLG2 PLGl+pLG2b) 7° □ t t - y (PA) (U/mZ) (AiM/min) PA 7° o y T —lz SCC (5. cellulosae) 25 250 10 a-7y h V 7°v'y 1,000 10 1.0X10"2 /■fv'ezf y 1,000 4.2 4.2X10"3 y —tu y y 1,000 13 1.3X10-2 7°nyM A (Streptomyces No.1033) 1,000 49 4.9X10" 2 h' H" 7° 7 —tf (B. subtilis var. Biotecus) 5,000 0 0 x yy v y y bpn* 10,000 0 0 T A/fj *J 7”o y T—If (S. griseus) 10,000 0 0 7*o 7T—If A (A. oryzae ) 10,000 0 0 cptl-y o X 7—tf (S. griseus) 10,000 0 0 b) 0.2M Leu-Gly fcSHlCffit-'/:: i *© PlG i t Plg 2 t ©£fi£9)a@©:to£^re Vol.63 — 29 — 434 1M b b3 /csb, %IC, CO vc £ Leu-Gly Vc = P[A]2/{( K, + [A])( Ku+ [A])} (4) ccx, vmilS&fcOhk-XMfZ'Cb V), k [EL I cm "i-#-5o ([E]. El 3 CD Vc \c\l\?Z>UVA' [A] / Vc = ( /KaCD6&, . '- < < + II 0.2 0.4 0.6 a (6) [Leu-Gly] (M) 6, ^ /7x i; x —y yf- ySic/j:^, [A]/vc El 3 (Leu-Gly),, (Leu-Gly)s, Leu «LCX Gly CD £CA]£©115]lcm%BWd##-C^5o LJTO Leu-Gly ©©£©&$ moT, Ei5&ic^zoA]>o.i3 55t$i±. /fa($f: Leu—Gly Leu—Gly [A]V vc = (Ka/l/)-( Ab + [A]) (7) £/cte, [A] [A]3/ Vc = (Kb/K).(K, + [A]) (8) Protease SCC £KK), o-fftbl A]2/vc ^fill Vol.63 — 30 — 5.2 Leu-GlyLeu-NH 2 tit stem 0.13M Leu-Gly b 0.2M Leu-NH2 imim'iT©^^ Leu-Gly-Leu-NHa (Plgln ) 'SrX: ElT 5ifrSiriXlE £, Leu-Gly £#fi¥LT Leu 6 Gly 6JjU/jx^WJxlc.cKVm\C Mu-Tvr, cilZWU L'C. 5.1 TftGnA:. O U) Leu-Gly W^aitvUlc'4 (Ka L Ki.) © mm h ICA^WX" -6 vO^-S" [ A ] (M) (DWx&% t ca £ UT##L/:o t&BEZ (6.25mM) © 7,^m^m^(C6^6[A]/rc-[A]yo 7 Ha)6 m^rmmnmcis^6 [A]Vvc~[A]/n7 Kb) Leu-Gly -fj'-i'i; [v(Ci>'V’>T, Plgln ©fTDlcW CE]=1.4X10-*M (?c) Leu-NHz © @0; ©# o ei 6 tcTjvr^tc.so mM m 7°n .y h6tbftTF.£n, ##© 3cA ck 0 Toissit, uc t Leu-NHz (Dfimb^mmi, ; Ku (£/c{2Ka)= 0.016M ii x IJ 7. — 7 y t y ©lYdto llh $X Ltzfr, 100 mM El 3 (Cj-J^T, 'JC^'c'/]'-; L/c uc © Ji - YU, 5t 4 (C ft. h©,:‘:^,H!):mM^(i;luni'!L',lv©^^^^^GnA:.. - /Ca(t/:!i/Ch) = 0.65M, Kb(£/c(3Ka) = 0.016 -^M© Leu-NH2 m\:. K(C^J©r, VC {C.tkiz M, y=2.4xi0-' M s-' ^^^©{^©Li'Cft f Leu-Gly on/cfn'i^juu^ 50mM Leu-NI L iV-iL K’C(L 25 mMU K©^m:ulitii"C iiii^/jcjr/ivtcj;<-g(LT^oc b iCffimt £ *> ©X& £ bfr\ctz>tt (IT # 7a i/^o -ec-c, c comm & m t>frKtz>iz#> KBromfc 50 100 150 £fro /;0 50 100 150 [Leu-Gly] ( mM ) El 7 Leu-Gly-Leu-NH 2, Leu &XV Gly ©/|J# WM& (vc b VH) Leu-Gly © 100 200 300 [I.eu-NH2] : 25mM (O). 50mM (#). 100mM (A). 200mM (□), 300m M (▲) [E]=2.1X10-5M 100 200 300 irCT, vc b Leu-NHz $>&!<'& Leu-Gly ©Sffi [Leu-Mllg] ( mM ) 6omm^mn-tz>\ttitzv, $z, El 6 Leu-Gly-Leu-NH 2, Leu £><££* Gly ©£E "C tb 7' lb b SaiSilC Leu-Gly^, tb 7'lb -< b SV (C wmic&juzir Leu-NHz 0^©^# Leu-NH2iL Leu-Gly #: Leu-Gly-Leu-NH 2 (D!llJ$.t)iWSi. (vc). -Leu-NHz U# O : Leu 3f:l± Gly (DHHS.WWS. (vh ). [E]=2.1X10-5M, [Leu-Gly]=6.25mM ^RZCD^gm^b LT%t 9^#GfL6o ?4'T-i-X^ Vol.63 — 31 — 436 j^i/Mii!rcd3oo coMJRZt), 0 8 J:-) ic, ## i'c = V [A] [B]/((/f, + [ A ]) ( A\ + [B])} (9) h S2i S,tc Leu-Gly CCV, Kxt Kzii, Ztlfftl Leu-Gly £ Leu-NH2 y<5%^CD#m^% A';,(=A',) (i 0.65M, -*;--/ +M h Utz. S.’cl S..’(C Leu-Gly /;WiA^oCDWit'-ii$^i,(d. [A]i[B](i, Leu-Gly £ Lcu-NHZ 0.01GM V A o C £ yjiljlJ G ©' 6 o /: o v\t, m&Kfco&x&mzniro (D-f 7'->)• 4 Y Si’ £ S2\ (t, V" -f h Sz6 S, (Cljy< &tMak(DZL'D © [B]^-%in?, l^maka:, ££&tzo bn/:teiiJ: <9, ^ 1W\ it 9 (2, vc = ( V [B] / [ K, ( K2 + [B])}) • C A 3 (11) o„ mm ^ ^ k#*), vc 6[A]6G>R9A# -oyo-rT —t: -nh2#."£tt i/vc^i/ib]t°d .y hktzv, m £ it £K) Kz=0.008M m?f h ntZo £ fz, %%#CD $ 1) K.Hayashi, M.Terada, K.Mogi, Agric. Biol. A Z*] it 12 t)mz>txiZo Chem., 34, 627 (1970) 2) H.Sekine.. //;/J., 40, 703 (1976) (A, + [A]) / 1/ [A] = 6.70 x 106M‘' - min (12) 3 ) B.Foltmann, “Methods in Enzymologv, ” Vol. 19, CCT'[A]=6.25X10-3M-C£>5o —1j, ©CD#A, vc p. 421 (1970, Academic Press, New York) tiAitvrfi\citmig^Bsb t>ntz mi)0 ttz, m 4 ) K.Arima, S.Iwasaki, G.Tamura, Agric. Biol. 'EBWv^ESb ^ftteKB]- 50mM (Dk£, m#CDf§& Chem., 31, 540 (1967) £*9 it 13 tm^titzo 5) L.Wallerstein, U.S. Patents 995, 824 (1911) 6) &raBE, vofi-r . —p.205 y[B]/(A,(A 2 + [B])} = 2.08x 10"5 min'1 (13) (1983, y ?~) Lfctfo-C, it 12ctit 13J; >0 Leu-Gly CD#%^^[ 7 ) W.W. Sawyalow, Pflujers Arch., 85, 171 (1901) UT 0.6M (bV'9###&W=o&CD CD#(i, 8 ) M.Bergmann, H.Fraenkel-Conrat, J. Biol. Chem., 5. 1 7f>£^/ciig&6U£ £V)%btliz 2ocomSE JQ 119, 707 (1937) CD#(0.65M6 0.016M) IWilt Vol.63 -32- 437 (1979) 23) K.Morihara, ibid., 41, 175 (1974) 15) K.Morihara, T.Oka, H.Tsuzuki, Y.Tochino, T. 24) K.Morihara, Seikagaku, 46, 949 (1974) Kanaya, Biochem. Biophys. Res. Commun., 92, 25) K.Morihara, T.Oka, J. Biochem., 89, 385 (1981) 396 (1980) 26) K. Morihara, T.Oka, FEBS Lett., 33, 54 (1973) 16) H.Tsuzuki, T.Oka, K.Morihara, J. Biochem., 88, 27) K.Morihara, T.Oka, Arch. Biochem. Biophys., 178, 669 (1980) 188 (1977) 17) T.Oka, K.Morihara, ibid., 84, 1277(1978) 28) T.Muro. Y.Tominaga, S.Okada, Agric. Biol. 18) T.Oka, K.Morihara, ibid., 88, 807(1980) Chem., 48, 1223 (1984) 19) G.A. Homandberg, J.A. Mattis, M.Laskowski Jr., 29) T.Muro, Y.Tominaga, S.Okada, ibid., 48, 1231 Biochemistry, 17, 5220 (1978) (1984) 20) I. Schechter, A.Berger, Biochem. Biophys. Res. 30) T.Muro, Y.Tominaga, S.Okada, J. Biochejn., 99, Commun., 27, 157 (1967) 1625 (1986) 21) I. Schechter, A. Berger, ibid., 32, 898 (1968) 31) T.Muro, Y.Tominaga, S.Okada, AgrzY. Biol. 22) J.S. Fruton, Adv. EnzymoL, 53, 239 (1982) Chem., 51, 2657 (1987) -33 * -u r53 13 q Wf CO E 360 Reprinted from Accounts of Chemical Research, 1992, 25. Copyright © 1992 by the American Chemical Society and reprinted by permission of the copyright owner. Stereoselective Routes toward the Synthesis of Unusual Amino Acids Y asufumi Ohfune Suntory Institute for Bioorganic Research, Shimamoto-cho, Osaka 618, Japan Received January 22, 1992 More than 700 amino acids that are the so-called Scheme I unusual, unnatural, or nonproteinous amino acids have Epoxidation of 2-Amino-3-butenol Derivatives been found in nature in the free zwitterionic form or NHR WCPBA/CHyClj NHR as constituents of peptides. These amino acids have OH attracted much attention from scientists due to their 6*R-Boc 12» (syn) 12b (anti) important biological activities as antibiotics, metal 6b 2R epimw R - Boc: syn/anti - «yi H - Ts: syn/anli -1.6/1 NHSoc chelators, neurotoxins, enzyme inhibitors, etc.1 Because 6d R-COCF, R - COCF3: syn/anti - 2/1 in many cases only minute quantities have been isolated NHBoc and because their structures are unique, they are in H-O teresting synthetic targets. 2-4 (2S,3S)-13 A feature common to many of these unusual amino acids is the 1,2- or 1,3-amino hydroxyl system. In NHB oc particular, such amino acids are often found as con TBSO' stituents of peptides. Our research group has been (2S.3S)-13 involved in the development of new methods for the TBSO' elaboration of such 1,2- and 1,3-amino hydroxyl sys tems. Our chief focus has been on the total synthesis (TBSO^^JjCufCNJU NHBoc of the peptide antibiotics echinocandins (l)5,6 and ga- lantin I (2: the structure has been revised to 5 as a result of our synthesis), 7-11 which have a variety of new amino acids. Echinocandins, isolated from Aspergillus useful synthetic building blocks because of the presence ruglosus or nidulans, exhibit potent antifungal and of chirality and appropriate functionalities accessible anti-yeast activities.5 The structures of la—c are com to a variety of chemical transformations. Using the posed of a highly hydrophilic cyclic peptide and a hy above synthons, their diastereoselective conversions to drophobic linoleyl moiety. Galantin I is a metabolite syn and anti 1,2- and 1,3-amino hydroxyl systems of Bacillus puluifaciens with antibacterial activity. 7 (methods a-f) were examined as summarized in Figure Galantin I has two new amino acids, named galanti- 2. The total synthesis of lc and the right-half equiv- namic acid (Glm, 3a assigned as its primary structure) and galantinic acid (Gla, 4b: isolated as an anhydro form 4a) (Figure l).11 (1) (a) Amino acids. Peptides and Proteins; The Chemical Society: Cambridge, 1968-1991; Vols. 1-22. (b) Hunt, S. Chemistry and Bio In recent years, great progress in the asymmetric chemistry of the Amino Acids; Barrett, G. C., Ed.; Chapman and Halt synthesis of both the 1,2- and 1,3-amino hydroxyl sys London, 1985; p 55. (c) Wagner, I.; Musso, H. Angew. Chem., Int. Ed. tems has been reported. The available methods for the Engl. 1983, 22, 816. (2) Coppola, G. M.; Shuster, H. F. Asymmetric Synthesis, Construc synthesis of the 1,2-amino hydroxyl system are the tion of Chiral Molecules Using Amino Acids; John Wiley & Sons: New following: (i) nucleophilic opening of an epoxide with York, 1987; pp 127, 204, and 267. amines, isonitriles, and azides; (ii) reduction of amino (3) Williams, R M. Synthesis of Optically Active a-Amino Acids; Pergamon Press: London, 1989; pp 1, 167,186, 208, and 304. ketones or hydroxyl imines; (iii) nucleophilic addition (4) a-Amino Acid Synthesis; Tetrahedron Symposium-in-Print; O’ of organometallic reagents to amino ketones; and (iv) Donnell, M. J., Ed. Tetrahedron 1988, 44, 5253. coupling of an achiral or a chiral glycine equivalent with (5) (a) Benz, F.; Knusel, F.; Nuesch, J.; Treichler, H.; Voser, W.; Ny- feler, R; Keller-Schierlein, W. Helv. Chim. Acta 1974, 57, 2459. (b) aldehydes. 3,12 However, several basic problems re Keller-Juslen, C.; Kuhn, M.; Loosli, H.-R; Petcher, T. J.; Weber, H. P.; garding stereocontrol, racemization, protecting groups, von Wartburg, A. Tetrahedron Lett. 1976, 4147. etc. still remain. (6) (a) Kurokawa, N.; Ohfune, Y. J. Am. Chem. Soc. 1986, 108, 6041. (b) Kurokawa, N.; Ohfune, Y. J. Am. Chem. Soc. 1986, 108, 6043. (b) Our approach to the 1,2- and 1,3-amino hydroxyl Evans, D. A.; Weber, A. E. J. Am. Chem. Soc. 1987, 109, 7151. systems is the diastereoselective introduction of a hy (7) Shoji, J.; Sakazaki, R; Wakisaka, Y.; Koizumi, K.; Mayama, M.; droxyl group into the allyl or homoallyl amines 6-11, Matsuura, S. J. Antibiot. 1975, 28, 122. (8) (a) Ando, T.; Terashima, S,.; Kawata, M.; Teshima, T.; Wakamiya, which are readily available from commercial a-amino T.; Shiba, T. Peptide Chemistry 1980; Okawa, K., Ed.; Protein Research acids. These unsaturated amines can be viewed as Foundation: Osaka, Japan, 1981; p 113. (b) Wakamiya, T.; Ando, T.; Teshima, T.; Shiba, T. Bull. Chem. Soc. Jpn. 1984,57, 142. (c) Waka Yasufumi Ohfune was bom in Asahkawa. Japan, in 1948. He received miya, T.; Terashima, S.; Kawata, M.; Teshima, Tj Shiba, T. Bull. Chem. his B.Sc. Degree from Hokkaido University in 1971 and his Dr.Sc. degree Soc. Jpn. 1988, 61, 1422. from the same university in 1976, where he worked with Professor Takeshi (9) Ohfune, Y.; Kurokawa, N. Tetrahedron Lett. 1984, 25, 1587. Matsumoto. After a postdoctoral position with Professor Paul A. Grieco at (10) Hori, K.; Ohfune, Y. J. Org. Chem. 1988, 53, 3886. the University of Pittsburgh, he was appointed to the position of Researcher (11) (a) Sakai, N.; Ohfune, Y. Tetrahedron Lett. 1990, 31, 3183. (b) at the Suntory Institute for Bioorganic Research in 1979 and is currently Sakai, N.; Ohfune, Y. Tetrahedron Lett. 1991, 32, 4151. (c) Sakai, N.; Chief Researcher. His research interests include the development of new Ohfune, Y. J. Am. Chem. Soc. 1992, 114, 998. synthetic methods for bioactive amino adds and peptides, biomime tic reac (12) Zurczak, J.; Golebiowski, A. Chem. Rev. 1989, 89, 149. Other tions. and studies on excitatory amino add receptors in mammalian CNS. references are cited therein. 0001-4842/92/0125-0360S03.00/0 © 1992 American Chemical Society -35- Stereoselective Synthesis of Unusual Amino Acids Acc. Chem. Res., Vol. 25, No. 8, 1992 361 Scheme II Ecftnocandins (1) Epoxidation of Hydroxymethyl Allylamines (R..R..OO h -^ ' {B, - OH B, . H) VJ Vo 1C 0 (R, • Rj • H) CH, PhCH?OCH2/\v_rZ^c 1 8 (syn/anti « 1/2S) O^nal struct^* o* gaartn I: 2j (n - 3). 2> (a . 4) Oh OH O CM, ' n T~ m V ’■QXQju^u. 7b - H. R; - TBS 19 Rn - H. R2 - TBS (syn/anti • 40/1) 20 R, • Rj - acetonide 21 R, - Rj - acetonide galantinamc aod Gim (3a) gaianwK aod: GU (4a) "'j— NBoc Revised srucaxe of gaiaron I. 5a (n • 3). Sb (n . 4) o« C« O CM, M 0 C=M* M o ^'Yr’S'Y 23 (syn/anti - 1/1) absolute tirudur* Of G>m (3b) revised ssicaxe of Ga (46) SoefO): revsed sequence Figure 1. Syntr»*l» e/ l^-amioe hydroxyi tymtmm. (syn) 6^ (ml.) OH (c)T which are constituents of 1 and 2, respectively (Scheme HHCO jTBS I). R.OSOCH^riR’-CIefOCCiPh. Epoxidation of Hydroxymethyl (Z)-Allylamines 6oc - CO-#6*. TBS . 5/n(M**/s o/ I.X^mtno hydrojyl eytlwn. Epoxidation of the allyl alcohol 17, which has a methyl group at C4, has been shown to give anti-ep NHCOjTBS oxide 18 with high stereoselectivity (syn/anti = 1/25).17 The mechanism involves less hindered side attack (re face on C3) of an internal chelate complex of MCPBA on the C-C double bond (Scheme II, B). Contrary to this, epoxidation of the hydroxymethyl (Z)-allylamine (a). (t>) «oo««s*on w«fl MCPBA: (c| SU tree cycle eaoamaa fcmaw (CL (•) Se, lye# eye be careen*# kymeon; (l) rubucenezxen. 7b yielded syn-epoxide 19a (syn/anti = 40/1), stereo- Figure 2. selectively. 10,18 This example indicated that MCPBA attacked from the more hindered si face on C3. The alent of la,b and 5 has been accomplished on the basis epoxidation of 20, in spite of the lack of an amide hy of these methods. drogen, was also syn selective to afford syn-epoxide 21 exclusively. The protection of the hydroxyl group of Epoxidation of 2-Amino-3-butenol Derivatives 20 with the TBS group resulted in a decrease in both Both enantiomers of N-(tert-butoxycarbonyl)-2- yield (>20%, 3 days) and product ratio (— 3/!)- amino-3-butenol (6), a masked form of chiral serine, Therefore, the high syn selectivity in the epoxidation have been synthesized from L- or D-methionine. 13 14 15 of hydroxymethyl (Z)-allylamine was attributed to the Compound 6a, upon treatment with 3-chloroperoxy- fact that the epoxidation proceeded through a chelation benzoic acid (MCPBA), underwent stereoselective ep complex C. The (E)-allyl alcohol 22 provided a 1/1 oxidation to give syn-epoxide 12a (syn/anti = 40/1). mixture of syn- and anti-epoxides 23.llc Thus, ep The reaction likely proceeds through an internal che oxidation of hydroxymethyl (Z)-allylamine proved to lation of MCPBA with the primary hydroxyl group, be a potential method for the preparation of syn-1,2- since epoxidation after protection of the hydroxyl group amino alcohols. of 6a with the tert-butyldimethylsilyl (TBS) group re quired a prolonged reaction time (~1 week) and re Sn 2 Type Cyclic Carbamate Formation from sulted in reduced syn selectivity (syn/anti = 3/1).14,15 tert-Butyldimethylsilyl Carbamate The bulky tert-butoxycarbonyl (Boc) group may hinder iV-tert-Butoxycarbonyl (Boc) and iV-benzyloxy- the undesired chelation with the amino group (A). carbonyl (Z) groups are the most common amino pro The syn-epoxide 12a proved to be useful as a pre tecting groups used for the synthesis of amino acids, cursor for the synthesis of various /3-hydroxy a-amino amino sugars, and peptides. 19 These groups can be acids. For example, the reaction of the acetate 13 with transformed into the Ar-(tert-butyldimethylsilyl)oxy- the appropriate cuprate resulted in regioselective nu cleophilic opening of the epoxide to give syn-1,2-amino (16) (a) Golebiowski, A.; Kozak, J.; Juiczak, J. J. Org. Chem. 1991, 56, alcohols 14 and 16, respectively, which were converted 7344. (b) Ikota. N. Heterocycles 1991, 32, 521. (c) Takahata, H.; Banba, to /S-hydroxyhomotyrosine IS6® and anhydro Gla 4a,9,16 Y.; Tajima, M.; Momose, T. J. Org. Chem. 1991, 56, 240. (d) Kano, S.; Yokomatsu, T.; Shibuya, S. Heterocycles 1990, 31, 13. (e) Golebiowski, A.; Kozak, J.; Jurczak, J. Tetrahedron Lett. 1989, 30, 7103. (13) Ohfune, Y.; Kurokawa, N. Tetrahedron Lett. 1984, 25, 1071. (17) Nagaoka, N.; Kishi, Y. Tetrahedron 1981, 37, 3873. (14) (a) Rao, A. S.; Paknikar, S. K.; Kirtane, J. G. Tetrahedron 1983, (IS) Kogen, H.; Nishi, T. J. Chem. Soc., Chem. Commun. 1987, 311. 39, 2333. (b) Narnia, A. S. Tetrahedron Lett. 1981, 22, 2017. (19) (a) Bodanszky, M.; Bodanszky, A. The Practice of Peptide Syn (15) (a) Umbreit, M. A.; Sharpless, K. B. J. Am. Chem. Soc. 1977, 99, thesis; Springer-Verlag: Berlin, 1984; pp 7 and 151. (b) Greene, Y. XV. 5526. (b) Sharpless, K. B.; Verhoeven, T. R. Aldrichimica Acta 1979, 12, Protective Group in Organic Synthesis; Wiley: New York, 1982; pp 232 63. and 239. -36 362 Acc. Chem. Res., Vol. 25, No. 8, 1992 Ohfune Scheme III Scheme IV SeN- Cyclic Carbamate Formation TBDMSOTV2.6-luiidin« RNHCOj-t-Su (Boc) RNHCOpSid-BulMe, O RNHCOzChy ’h (2) NHCOjTBS TBSH/Pd(OAc), (equivalent to RNHCOj ) PhCHjO Pt>CHjO H/D.or M.OH RNH* * X - Cl: AgF (sytvwti . 3n. 76%)* RNHCOjSid-BuJMej AgF/Pd(ll) (Kjrn/ami -8 /1. 81%)* 27b (anti) RNHCOjR- R7C (FT - ikyl. benzyl: X - Cl. Br) 33 X - OBz: nBu.NF/PdIO) (lyivar* - VI. 78%)' (1) TBSOT!/2.6-lu1.dine /V-Boc-L-VaHOMe (2*) ------W-Z-L-Val-OM* (2S. 85%) (2) n-Bu.NF/Pt>CHjB» 30 31 (sy#vanti«1S:1) statin# (32) *2 of A5F/CH3CN *2 #qu*v Agf. 0.1 #qtw ot atytpaladium(U) Chbcda dim##, 0.3 equiv Pl^PvCH^CN 27b (anti) '' O C1.2 #qum o< ft-Bu,NF. 0.1 #qutv ot (PtVjP),Pd(0yTHF synthesis of the 1,2- and 1,3-amino hydroxyl systems 26b (ami) OMj 27a (syn) ^ O as a cyclic carbamate. AgF was chosen in order to ac tivate both the silyloxycarbonyl and the allyl chloride groups. Compound 9 with AgF underwent cyclic car bamate formation in an S^ manner to give a mixture of syn- 27a and anti-27b (AgF, syn/anti = 3/1). The 28c 3fl apimw cl 28a use of AgF in the presence of a Pd(II) catalyst (0.1 equiv carbonyl group 20 by treatment of the iV-Boc derivative of allylpalladium(II) chloride dimer and 0.3 equiv of with tert-butyldimethylsilyl trifluoromethanesulfonate triphenylphosphine) was superior to AgF in view of its (TBSOTf) in the presence of 2,6-lutidine and the N-Z syn selectivity (syn/anti = 8/1). As an additional ex compound with tert-butyldimethylsilane (TBSH) in the ample, treatment of the allyl chloride 30 with AgF or presence of a catalytic amount of Pd(OAc)2, respec AgF/Pd(H) gave cyclic carbamate 31 (AgF, syn/anti = tively. 21 The silyl carbamate is stable under ambient 5/1; AgF/Pd(II), syn/anti = 15/1). The carbamate 31 conditions and can be activated by fluoride ion to un was readily converted into statine (32), a constituent dergo various electrophilic substitution reactions. In of pepstatine, which is a well-known potent renin in hibitor. 23,25 the presence of an alkyl halide, the silyl carbamate yielded the corresponding alkyl carbamate. For exam Allyl esters are more attractive than allylic clorides ple, the iV-Boc amino ester 24 was converted, efficiently, as internal electrophiles in view of their ease of prepa to the corresponding N-Z amino ester 25 via the silyl ration and stability. 26 Also, the use of stoichiometric carbamate. Thus, the silyl carbamate prepared from amounts of AgF can be avoided. Thus, treatment of an N-Boc or N-Z compound is a useful intermediate the benzoate (Bz) 33 with a catalytic amount of tetra- which can be converted to a variety of urethane-type kis (triphenylphosphine) palladimn(O) and 1 equiv of n-Bu4NF gave syn- 27a as the major product (syn/anti compounds. 21 = 9/1) (Scheme IV). Furthermore, the silyl carbamate can be trapped by an internal electrophile with complete inversion of The syn selectivity using AgF can be understood from configuration. Treatment of the syn-mesylate 26a with the examination of the hypothetical cyclic intermediates n-Bu4NF gave the cyclic carbamate 27b having an D-l and D-2. Due to the presence of severe allylic anti-amino hydroxyl system. The anti-mesylate 26b strain27 in the transition-state conformer D-2, the re gave the syn-carbamte 27a. This method proves to be action proceeds via the thermodynamically more fa useful when the Mitsunobu reaction is not effective.22 23 vored transition-state D-l to give syn-27a. Using For example, under Mitsunobu conditions the conver AgF/Pd(II) or n-Bu4NF/Pd(0), the major isomer 27a sion of the syn-amino alcohol 28a to the corresponding could be derived via (Tr-allyl)palladium complex F-l anti isomer 28c was not effective due to the bulky na from the thermodynamically more favored conformer ture of the neighboring iV-Boc group. However, the silyl E-l, which has less allylic strain than E-2 (rate-deter carbamate method was successfully applied to this mining step) (Scheme V).28 It is noted that the proline conversion to give the desired anti cyclic carbamate 29 derivative 34 gives a mixture of cyclic carbamates 35a,b [4,5-trans (syn)/4,5-cis (anti) = 3/2] in good yield. The (Scheme III).10,23 decreased syn selectivity can be attributed to the re Scn ' Cyclic Carbamate Formation via Silyl duced steric bulkiness of the conformationally con Carbamate strained CH2 group in a five-membered ring (G-l and Because of its high reactivity, a silyl carbamate can G-2) when compared to the freely rotating CH2R of 33 be viewed as an N-carboxylate ion equivalent. Thus, (25) (a) Umezawa, H.; Aoyagi, T.; Morishima, H.; Matsuzaki, M.; intramolecular trapping of this reactive species in an Hamada, H.; Takeuchi, T. J. Antibiot. 1970, 23, 2569. (b) Rich, D. H. Proteinase Inhibitors; Barrett, A. J.; Salvensen, G., Eds.; Elsevier New ScN- manner24 provides a stereoselective method for the York, 1986; p 179. (c) Omura, S.; Inamura, N.; Kawakita, K.; Mori, Y.; Yamazaki, Y.; Masuma, R.; Takahashi, Y.; Tanaka, H.; Huang, L.-Y.; (20) Breederveld, H. Reel. Trav. Chim. Pays-Bas 1962, 81, 276. Woodruff, H. B. J. Antibiot. 1986, 39, 1079. (21) (a) Sakaitani, M.; Ohfune, Y. Tetrahedron Lett. 1985, 26, 5543. (26) (a) Trost, B. M. Angew. Chem., Int. Ed. Engl. 1989, 28, 1173. (b) (b) Sakaitani, M.; Hori, K.; Ohfune, Y. Tetrahedron Lett. 1988, 29, 2983. Trost, B. M.; Verhoeven, T. R. Comprehensive Organometallic Chem (c) Sakaitani, M.; Ohfune, Y. J. Org. Chem. 1990, 55, 870. istry; Wilkinson, G., Sir, Stone, F. G. A_, Abel, E. W., Eds.; Pergamon (22) Mitsunobu, 0. Synthesis 1981, 1. Press: Oxford, 1982; p 799. (23) (a) Sakaitani, M.; Ohfune, Y. J. Am. Chem. Soc. 1990, 112,1150. (27) (a) Johnson, F. Chem. Rev. 1968, 68, 375. (b) Hoffmann, R. W. (b) Sakaitani, M.; Ohfune, Y. Tetrahedron Lett. 1987, 28, 3987. Chem. Rev. 1989, 89, 1841. (24) Stork, G.; Schoofs, A. R. J. Am. Chem. Soc. 1979, 101, 5081. (28) Anh, N. T.; Eisenstein, 0. Nouv. J. Chim. 1977, 1, 61. -37- Stereoselective Synthesis of Unusual Amino Acids Acc. Chem. Res., Vol. 25, No. 8, 1992 363 Scheme V Scheme VI Proposed Mechanism of ScN- Cyclic Carbamate Formation Cyclic Carbamate Formation from Secondary Allyl Esters H 1, (0-1) (0-2) R - CHjOCHjPh I minof I ralb of syn/anli AgF: 27a/27b . 3/1 27a (syn) AgF/Pd(ll). X . Q: 27a/27b - 8/1 27b (anti) nBu.NF/Pd(0). X - OBz: 27a/27b - 9/1 37a/37b » 20/1 37«: 4.5-trans (syn)-E 37b: 4.5-cis (anti>Z AgF/Pd(ll) or nBu.NF/Pd(0) 39a: 4.5-trans (syn)-Z 39b: 4.5-cis (anti)-E 35a 4.5-trans (syn) 35b 4.5-cis (anti) Scheme VII Six-Membered-Ring Cyclic Carbamate Formation G-1 G-2 reduced alfylic strain compared to E-1 R - CO,Me (syrVanti - 3/2. 63%) AgF/Pd(») (syrVanti ■ 1/1.72%) (the structure is shown in Scheme IV).29 The use of secondary allylic esters is of interest since Syntheses of Glm (3b) and Gla (4b) there are four possible products considering both the Before this work, only the primary structure of Glm ring stereochemistry and double-bond geometry. From (3a) was reported. 8 The structure determination of 3a the (S,S) isomer 36, the 4,5-trans (E) product (syn) 37a required the synthesis of eight diastereomers from L- was produced with high stereoselectivty (37a/37b = or D-lysine. The methods elaborated in our laboratory 20/1), while (Srfi)-38 gave the 4,5-cis (E) adduct (anti) (Figure 2a-d) were used for the synthesis of the key 39b as the major product (39b/39b = 7/1). In each intermediates 43 and 44. The hydroxymethyl (Z)-al- case, the other stereoisomers were not detected. For lylamine 41 on epoxidation (method b, Figure 2) gave mation of the syn adduct 37a from the (S,S) isomer 36 the syn-epoxide 42, which upon subsequent reduction and primarily the anti adduct 39b from the (SJi) isomer with LLAIH4 afforded, regioselectively, the desired syn 38 suggests that these transformations proceed via amino diol 43. Upon Sn 2 type cyclic carbamate for double inversion (net retention): (1) the leaving group mation using method c (Figure 2), the mesylate 28b and silyloxycarbonyl group are placed on the same face provided the anti cyclic carbamate 44, exclusively. in the ground-state conformers (36a and 36b, 38a and Each isomer was then converted to the corresponding 38b) (the major or exclusive isomer is produced from Z and E unsaturated esters 45a-d using a Wittig or the conformers, 36a and 38b, having the least A1-3 Horner-Emmons type olefination. 30 Upon osmium strain); (2) palladium(O) attacks from the back side of tetraoxide oxidation of 45a-d, each gave a 1/1 mixture the leaving group; and (3) attack of the N-carboxylate of diols 46a-h, a total of eight diastereomers. Thus, ion species on the (Tr-allyl)palladium complex gives the eight diastereomers with unambiguous stereochemistry cyclic carbamates 37a and 39b, respectively. These were prepared. Spectroscopic comparisons of the tri transformations provide stereoselective access to the acetates 47a-h prepared from 46 with the natural syn- or anti-1,2-amino hydroxyl systems (Scheme VI).29 product 3b indicated that (2S,3E,5E,6S)-47 derived The present method was also employed for the syn from 45d was identical with the natural triacetate in all thesis of a 1,3-amino hydroxyl system (method e, Figure respects except the sign of its optical rotation. Thus, 2). The chloromethyl homoallylamine 10 produced a the absolute structure of Glm was confirmed to be mixture of six-membered cyclic carbamates 40a,b. The (2E,3S,5S,6E)-3b. Finally, the synthesis of the natural syn/anti ratio was ~ 1/1. However, greater syn selec form 3b was accomplished in a straightforward manner tivity was observed when the reaction was applied to starting from D-lysine (43 — 44 —- 45d —3b) (Scheme the peptide system (vide infra) (Scheme VII).23 VIII).10 (29) Spears, G. W.; N'akanishi, K.; Ohfune, Y. Tetrahedron Lett. 1990, 31. 5339. (30) Maryanoff, B. E.; Reitz, A. B. Chem. Rev. 19S9, S9. 863. -38- 364 Acc. Chem. Res., Vol. 25, No. 8, 1992 Ohfune Scheme VIII Scheme X Synthetic Structure Determination of Glm (3b) Halolactonization of 2-Substituted 4-Pentenoic Acids L-Lysine 51a R - MewPn 52 R - We or PH. X - I. csttans - 3.2 51b R - OH orOCHjPn 53 R - OH orOCHjPn. X - Br. ravans - 2n 11a R - NHBoc 54R. NHBoc. X. Br. canrans - an (82%) OH OH OM* OIBi 11bR -NHZ 55R-NHZ.X-Br.bveans.an (80%) lie R - NHTs 56 R - NHTs. X - Br. ovtrans - 8 an (100%) ZHN(CHj) _ ZHN(CH,L^A^J __ _ 2HN(CH,)4 11d R - NPht 57 R - NPhl X - Br. bVSare - 6n (81%) <3 (syn) MHBoc 28b NHBoc t’) TBSCH *, (lnti) Scheme XI R -O (H-1) 60 R - CHjOH (1) TFA (21 Ba(OH)j Scheme IX "NHBoc Synthesis of Revised Structure of Gla (4b) (-)-Bulgecinine (61) (1) 1-BuOOH/Trton B HOjC 59a (78%. cis/eans - 6371) ■ NBoc ^ V NHBOC 59b (88%. bveans -17/1) 62a R . CHjOH O-o' 62b R - CHjOTBS AgOTf/THF/ Cl -Ag" 1 2.6-lubdine (3) TBSQ 63a R - CHjCl 63b R - CHjCl (Z isomer) Ah (92% from 63a) „ N (73% from 635) revised struaue ol Gla (4b) 64 cis/trans - Synthesis of the reported structure of galantin I (2) et al. reported that 2-amino derivative lib gives cis-y- and comparison with the natural product showed that butyrolactone 55 as the major isomer (cis/trans = the two components were not identical. The structure 4.6/1).37 Since this method appeared to be potentially of galantin I was then revised to 5 with the change of useful for the synthesis of the anti-1,3-amino hydroxyl the structure of Gla to 4b from 4a.ll8,c systems (method f, Figure 2), we reexamined the bro- The revised structure of Gla (4b) and its C3 epimer molactonization of 2-amino-4-pentenoic acids with was synthesized, stereoselectively, starting from epoxide jV-bromosuccinimide (NBS) in order to optimize the 21. The epoxide was converted to the unsaturated 8- reaction conditions with respect to improving both the lactone 48, which upon epoxidation followed by re yield and stereoselectivity. The use of anhydrous tet- ductive cleavage of the resulting epoxide 31 regio- and rahydrofuran (THF) as the solvent afforded satisfactory stereoselectively gave the alcohol 49. The desired 3S results (cis/trans = >8/1, >80%). The product ratio isomer 50 was obtained from 49 by an oxidation/re was independent of the nature of the N protecting duction sequence. 32-33 Removal of the protecting groups group (Scheme X).68,38,39 with trifluoroacetic acid (TFA) afforded Gla (4b) It has been proposed that the reaction proceeds through an initial bromination of the amino group and (Scheme IX). a subsequent internal bromonium ion transfer to the Electrophilic Lactonization of C-C double bond (Scheme XI, H-1 via path A) to give 2-Amino-4-pentenoic Acid Derivatives cis-7-butyrolactone as the major product. 37 However, Halolactonization of 3-substituted 4-pentenoic acid despite the fact that the N-phthaloyl group of lid derivatives has been shown to be an efficient entry for cannot be brominated, the cis selectivity remains un the stereoselective construction of either 3,4-syn or -anti changed (cis/trans = 6/1). This fact led to an alter stereochemistry. 34 35However, 36 2-substituted 4-pentenoic native mechanism (path B) in which the C2 amino acid derivative 51a produced a mixture of 2,4-disub- group would stabilize, stereoelectronically, the putative stituted 7-butyrolactones 52 with poor stereoselectivity halonium species of the cis transition state (H-2). (cis/trans = ~3/2).35,36 On the other hand, Witkop In order to determine the favored pathway, the hal olactonization of 5-substituted (Z)-allylglycine 58 was (31) Miyashita, M.; Suzuki, T.; Yoshikoshi, A Tetrahedron Lett. 1987, examined: the reaction of 58 would produce the 7-bu- 28, 4293. tyrolactone (5f?)-59a via H-1 (path A) or (5S)-60 via H-2 (32) (a) Mancuso, A. J.; Swem, D. Synthesis 1981, 165. (b) Smith, A. B., Ill; Levenberg, P. A. Synthesis 1981, 567. (path B). Determination of the stereochemistry of the (33) Hausler, J. Liebigs. Ann. Chem. 1983, 982. resulting lactone should give an answer. (34) (a) Dowle, M. D.; Davis, D. I. Chem. Soc. Rev. 1979, 171. (b) Bartlett, P. A.; Richardson, D. R.; Myerson, J. Tetrahedron 1984, 40, (37) Izumiya, N.; Witkop, B. J. Am. Chem. Soc. 1963, 85, 1835. 2317. (c) Chamberlin, A. R.; Dezube, M.; Dussault, P.; McMills, M. C. (38) Ohfune, Y.; Kurokawa, N. J. Synth. Org. Chem., Jpn. 1986, 44, J. Am. Chem. Soc. 1983, 105, 5819. 647. (35) Bartlett, P. A.; Myerson, J. J. Am. Chem. Soc. 1978, 100, 3950. (39) Ohfune, Y.; Hori, K.; Sakaitani, M. Tetrahedron Lett. 1986, 27, (36) Ohfune, Y.; Kurokawa, N. Unpublished results. 6079. -39- Stereoselective Synthesis of Unusual Amino Acids Acc. Chem. Res., Vol. 25, No. 8, 1992 365 Treatment of the (Z)-aHyl alcohol 58 with NBS in Scheme XII THF gave a mixture of cis 2,4-disubstituted 7-butyro- cydic carbamate lactone (either 59a or 60) and its trans isomer (cis/trans = 8.8/1, 95%). The structure of the major isomer was determined by its conversion to the amino acid bulge- cinine (61), a constituent amino acid of the glycopeptide bulgecine. 39 "41 Thus, the structure of the lactone ob tained from 58 was shown to be 60. It was concluded that the bromolactonization of 2-amino-4-pentenoic acid derivatives proceeded via H-2 (path B). This mecha 66* R, - H. R, - TBS nism was further supported by the halolactonization of 66b R, - Na. R, - TBS 62a having a 5(E)-substituent. This yielded cis-7- 66c R, -Na. R,- H lactone 59a as the major product (cis/trans = 6.3/1), 54 4- H*Uveony>-aftyi£tycine{Boc)-OH which indicated that the reaction proceeded through intermediate I. Protected alcohol 62b showed greater 66b,c . - HN cis selectivity to afford 59b (cis/trans = 17/1).39 In addition to a bromonium cation, an allyl cation 67a R - TBS could also be stabilized by the C2 amino group. (osirans . S9/1 ) Treatment of either the (E)- or (Z)-allyl chlorides 63a,b with silver trifluoromethanesulfonate (AgOTf) gave the TBSO, same cls-4-vinyl 7-butyrolactone 64 as the major product (cis/trans = 6/1). The mechanism of this transformation appears to involve intermediate J.39 Thus, halolactonization of 2-amino-4-pentenoic acid (l)TBSOTI " 65 (syrvanii .12/1) derivatives proved to be useful for the synthesis of the 'OTBS (2) AgF/CH,CN chiral lactones which are equivalent to the anti-1,3- amino hydroxyl systems. "OTBS Stereoselective Conversion of a Simple ined halolactonization of the structurally rigid analogue Tripeptide to the Echinocandin Right Half 68 in which the jV,0-acetonide constrained the structure Equivalent to be U-shaped (7-turn conformation). 43 The reaction Diastereoselective synthesis of the constituent amino provided czs-lactone 69 as the major product (cis/trans. acids of echinocandins (1) followed by the coupling of = 30/1). These results suggested that the bulky silyloxy these constituents has led to the successful total syn group of 66b constrained the conformation to be U- thesis of echinocandin D (lc).6 In conjunction with this shaped (Scheme XII, K), where the bromonium cation study, we also examined the synthesis of the right half was stabilized by a cooperative stereoelectronic effect equivalent (tripeptide 65) from a simplified tripeptide of the neighboring amide and carbonyl groups. Un 66. Our idea was to examine the applicability of protected 66c might have a linear conformation which methods e and f (Figure 2) in the peptide system. results in a decrease of cis selectivity. The desired 67a Moreover, it was expected that this novel strategy might was then converted to the protected tripeptide 70. provide information concerning chemo- and stereose Although the silyl carbamate method e (Figure 2) was lectivity induced by the peptide functionality and/or shown to give poor 1,3-syn selectivity (10, syn/anti = peptide conformation. 42 The key transformation was —1/1, Scheme VII), this method (e) was applied to a stereoselective introduction of the requisite 7-hy introduce a hydroxyl group into the allyl chloride 71 droxyl group into both the N- and the C-termini of prepared from 70. Surprisingly, successive treatment allylglycyl moieties. of 71 with TBSOTf and AgF gave the desired syn cyclic Halolactonization of 66 was highly dependent on the carbamate 65 as the major product (cis/trans = 12/1). reactivity of the COOH vs CONH group. Using the free The AgF/Pd(II) system was not effective in this case. carboxyl compound 66a, peptide bond cleavage occur Both participation from the proximal functional groups red at the N-terminal of threonine to give cis-7- and steric effects derived from the peptide conforma butyrolactone 54 and H-threonyl-iV-Boc-allylglycine- tion at the reaction site may have contributed to the OH. On the other hand, the sodium salt 66b, which is high stereoselectivity observed. Thus, the conversion more nucleophilic than the free carboxyl, provided the of a simple tripeptide 66 into the right half equivalent desired czs-7-butyrolactone 67a, exclusively.- The cis of echinocandins 65 was accomplished. 44 selectivity (>99/1) was much greater than that of N- Boc-allylglycine 11 (cis/trans = 8/1). On the other Conclusions hand, unprotected 66c showed a decrease in cis selec The basic stereochemical consequences of hydroxy- tivity (cis/trans = 4/1). In order to examine the ste lation of allyl- or homoallylamines which lead to the syn reochemical outcome of this transformation, we exam and/or anti 1,2- and 1,3-amino hydroxyl systems have been described. The synthetic methods developed in (40) Imada. A.; Kintaka, K.; Nakao, M.; Shinagawa, S. J. Antibiot. 1982, 35, 1400. our group have also been used for the structure deter (41) (a) Wakamiya, T.; Yamanoi, K.; Nishikawa, M.; Shiba, T. Tet mination and synthesis of several unusual amino acids. rahedron Lett. 1985, 26, 4759. (b) Bashyal, B. P.; Cho, H.-F.; Fleet, G. In spite of these advances, more efficient methods are W. J. Tetrahedron Lett. 1986, 27, 3205. (42) (a) Izumiya, N.; Francis, J. E.; Robertson, A. V.; Witkop, B. J. Am. Chem. Soc. 1962, 84, 1702. (b) Wilchek, M.; Patchornik, A. J. Am. (43) Chon, P. Y.; Fasman, G. D. J. Mol. Biol. 1977, 115, 135. Chem. Soc. 1962, 84, 6079. (44) Sakaitani, M.; Ohfune, Y. Tetrahedron Lett. 1989, 30, 2251. -40- 366 still required for the synthesis of such amino acids due unusual amino acids is focused not only on chemistry to their important biological activities and medicinal but also on the impacts on interdisciplinary scientific interests.45 Furthermore, it is suggested that a hydroxyl fields. These amino acids are expected to function as group placed on the backbone of an amino acid is an useful probes to investigate molecular mechanisms of active site when bound to a receptor protein or a a variety of biological functions. 1,46 biomembrane. When such amino acids are incorporated It is a pleasure to acknowledge the contribution of my col into peptides, the hydroxyl group plays an essential role leagues: their names are recorded in the references. I am grateful in constraining the peptide structure into a specific to Professor Koji Nakanishi for his continuous encouragement. conformation through intramolecular or external hy The financial support of a grant-in-aid from the Ministry of drogen bonding. la-b’25 Recent interest in the family of Education, Sciences, and Culture, Japan, is appreciated. (45) (a) Shimamoto, K.; Ohfune, Y. Tetrahedron Lett. 1988, 29, 5177. (46) (a) Axelrod, J. Science 1971, 173, 598. (b) Williams, D. H. Acc. (b) Sakai tani, M.; Hori, K.; Ohfune, Y. Tetrahedron Lett. 1988, 29, 2983. Chem. Res. 1984, 17, 364. (c) Shimamoto, K.; Ishida, M.; Shinoz&ki, H.; (c) Spears, G. W.; Nakanishi, K.; Ohfune, Y. Synlett 1991, 91. Ohfune, Y. J. Org. Chem. 1991, 56, 4167. 41 182 W-MMESUBEtc «fc t 77 4 ift ;i/X-\(7)flJffl * ;n z", m# w" Af 5 c 6-e, 1. It U V) (C TK^bSS, @5#-NaBr &iW:%^g##©##T6S. ##$E#W&gRNi (TA-NaBr-MRNi-U) &'& btl m, maf ^ + 7 6. 3 * Tadashi Kikukawa (^)±— K9f%@rTJ$gf%S- #e#± y:WK^i%^^y7K(ioomi)T2[g%y#-#-a,. ia±©E MIS^> 1976^fi*^jf±gRS4>ii. -2 DSt-C — 42 — Vol. 34 No. 3 1992 TO— 2.2.3 RNi-U Table 1 Comparison of fR, .R)-TA-NaBr-MRNi and (R, R)-TA-NaBr-MRNi-U in the enantio- differentiating hydrogenation of 1,3-diketones. OO Hj OH O h2 OH OH OH OH step-1 R^^R step-2 R ^R R-^^R (/?*. 5*) (R*, R*) I E n ( meso) V Reaction Product E.e of Isolated yield conditions 1^ ratio^ yc) of optical! Substrate pure y d) R Catalyst 7/G t/h I e m N 96 96 Configuration Me TA- NaBr-MRNi 100 24 0:20:10:70 90 21 (2R, 4R) TA-NaBr-MRNi-U 100 4 0: 7: 7:86 91 60 (2R, 4R) (Me)jCH TA-NaBr- MRNi 100 192 1:17:16:66 85 32 (3S. 5S) 110 48 c) TA-NaBr-MRNi-U 100 60 0: 6:22:72 90 59 (3S.5^) O OO CD 110 12 O 89 62 (35,55) ») Reaction conditions: substrate (100 g); tetrahydrofuran THF (100 cm3); catalyst (15.9 g); initial H2 pressure 100 kPa. b) Determined by means of GC. c) Determined by polarimetry, [a] d 20—54.7‘(C 10, EtOH) for (2R, 4RPpentanediol and [a] D20—64.5*(C1.0, MeOH) for (35, 55)-2,6-dimethyIpentanc-3,5-diol after separation of each component by MPLC. d) Two succesive recrystallizations of the reaction products from diisopropyl ether gave optically pure IV. e) Dehydration of II took place under these conditions. -43- 184 mu ie, bb# m pd c±£&\c, &&&&&&-&%& SltSST& ZC %:7Ft. CtlbOfrfcttm Lf:7%9% 0 base HkRi jl + Cl) Ri R2 OH OH YXr‘ Nu Rg N u *R2 1) PCC 2) Base 3) Ac20 H2/Lindlar 2) TiCI4 HVXH H'^H OAc OAc 1) HCH0/BF3-Et20 2) H2/Pt02 C2)'" 3) aq KOH/MeOH I nhoffen - Lyt hgoediol (1) Me Li OH C3)") Ph Ph (I) - Phosphine latic O C4)") OH 0 (5) 16) — 44 — Vol. 34 No. 3 1992 X 7 r -* >7 i X^x^cofm 185 Br2AlH ■v Q Cl jH 23 LiNEt2 o o O 0 (7)'" o OH (8) m R=Me(PD) or i-Pr(DMHD) (9) ’°> H V" 1) Et2Zn/CH2i2 ?>|0H- 1) Na/MeOH HgOAc 2) CHCIs/NaOH yZ p 2) Hg (OAc)2 "‘r-ci Cl . 1) L1AIH4 ^ 1) Na/MeOH 2) BuONO/H+ >( ] 2) ijy^nnr (+) -T7 V A/-yu (2) WltTF*. DMHD *-J -J\S3.-T)U(D is? a/p 100#®d.e-Z'Mrr-fZ (3; 5. TA-NaBr-MRNi-Ulc^S 3-zj-drV^ (8)). JuX-r ;u® *S^€>^155: V ;>T;i/3- ,u, h g^® A 6% 7"D .y 7 — 45 — 186 mu ie, h# m m m Table 2 Comparison of (R, R)-TA-NaBr-MRNi and (R, R)-TA-NaBr-MRNi-U in the enantio-differentiating hydrogenation of methyl 3-oxoalkanoates. 0 O OH 0 R/U E. e. of S / Ca) Reaction6^ product^ Substrate Catalyst (g) (g) time/ h (*) Configuration Me TA-NaBr-MRNi 100/16 5 80 (R) TA-NaBr-MRNi-U 100/16 2.5 86 (R) 15000/160 8 81 (K) «-c7hi6 TA-NaBr-MRNi 10/1.6 144 83 d) (R) TA-NaBr-MRNi-U 10/1.6 36 89 (R) n-CeHie TA-NaBr-MRNi 10/1.6 192 86 d) (R) TA-NaBr-MRNi-U 10/1.6 48 91 (R) n-CuHgj TA-NaBr-MRNi 10/1.6 192 86 d) (R) TA-NaBr-MRNi-U 10/1.6 48 94 (R) a) S: substrate, C: catalyst. b) Reaction conditions: THF as solvent, initial pressure of H2 100 kPa: temperature 100‘C. c) Determined by polarimetry [cc] D20—22.4 (neat) for (R)-Methyl 3-hydroxy- butanoate. d) Determined by NMR spectroscopy with a chiral shift reagent. ®50 / *- h 7 R)-TA-NaBr-MRNi-U tin ft m 3-1 kd+-«o7 $ ymtm&X'tto (160g), THF(10 /), T-fe ^A/(15kg)£E 6. yrfy^r^il'X^MiUO 100 kg/cm 2, lOOCTTkSfb^fTO. ^ 8 3- b K □ 5 6 (R)-3- b Ko4-> 7'7 3-1Fo+^ ^w(81~-83% e. e) #(s-#) (D&^mx'n.wi*PT£fot LT#&u, cn 5.2 3-b TA-NaB r- MR Ni- U led: >5 TkSfbTf# £ ft 5 3- 7'd y 7 & b K n + 81 — 95 # x y * ESiJlxJL'CD^F^Z®t LT7 7 d y — 46 — Vol. 34 No. 3 1992 7 T d 's*T 187 OAcO © © OHO 00 och 2ch ,ncch ,)8.ci 83) Myrmicacin (3) Pahutoxin (4) 341 (CH2)6CH3 Serrataniolide (6)36) Lipid A from bacterial cell wall (5) 86 ) O CHj 0 II I /\^\>»-OC-C=CH-CH2OC CHj CH, O iH, O HOHjC - Empossfilin (7) Biopolymer (8) 88 ) Fig. 2 3-Hydroxyalkanoic acid moiety in natural products. Ph-C-N Vs, J (S) OH 0 H T ------IT A ___ CSO CO.CH, -^^OH - W 'a / or R Fig. 3 Various optically active natural products derived from 3-hydroxybutanoic acid. — 47 — 188 mi ie, a# m OH O CH3(CH2)n/Jx'/tixOH Fig. 4 Natural products derived from 3-hydroxyalkanoic acids. I47xO^y0773^l'7'f KJ (12), xyi/* h ■7 7*7 7®&i5#;c7'y/\/ OH OH /yCOzBu 22a) OH CHSI CH 3 CH s A>C02Bu ------(ID") Base OH OH CO2BU 22b) CHs CH3 RNu OH O o^ H RV"H TT-oh — AAo - ^CO*H (12)") OH OH x/x. Br ZnCH2CO?Et A CH2OH + n-C7HliCHO /k/COzEt (13)") OH TiCI4 G7H1; 1 COzEt M-CyHi; — 48 — Vol. 34 No-3 1992 JS&}—7 r A 's>T $ 189 ;i/ii^.©x>!r-;u7 7 l\ 4#(i 3- t K Lett., 1003 (1990) u^i/Wd&v&tfeMt LT&Kffi&t&fflfe&St&t Lte 19) T. Sugimura, M. Yoshikawa, T. Futagawa, A. I'MRNi (C j:6*#. Chem. Commun. (in press) 21) T. Sugimura, T. Futagawa, A. Tai, Chem. 7. is 7b V Id Lett., 2291, 2295 (1990) TA-NaBr-MRNi-U©^^T:C, 1. 3- 'Jt- 22) a) T. Kikukawa, Y. Iizuka, T. Sugimura, T. Harada, A. Tai, Chem. Lett., 1267 (1987); 3- b KD.*>@EE©£/&^‘SBlC/ao/c. cn b) N. Nakahata, M. Imaida, H. Ozaki, T. •Mh£$I©!IEliSi£D , Harada, A. Tai, Bull. Chem. Soc. Jpn., 55, ^ li C ©Ste - - Xic^x. , Jc 0 2186 (1982) 23) H. Schildknecht, K. Koob, Angew. Chem. £4ffi (cca^ibA#! ^a#$4"C lU^D-t^©^ Int. Ed., 10, 124 (1971) f}£il26T^6. 24) M. Yoshikawa, T. Sugimura, A. Tai, Agric. mmic^A < sw ' Biol. Chem., 53, 37 (1989) L t-f. 25> s ea, 46, .501 (1988).. 26) D. G. Bishop, et al., J. Lipid. Res., 4, 81 ■£ ffi. (1963) 1) B8# Bft, SB, 29, 963 (1991) 27) W. Herz, R. P. Sharma, J. Org. Chem., 41, 2) A. Tai, T. Harada, “Tailored Metalcatalysts ”, .1015 (1976) ed. Y.. Iwasawa, Reidel Publishing Company 28) mil IE, H# m, 44, 1740 (1986), p. 265 (1991) 3) A. Tai, T. Kikukawa, T. Sugimura, Y. Inoue, 38, hi (1990) T. Osawa, S. Fujii, Chem. Commun., 795 29) A. I. Meyers, R. A. Amos, J. Am. Chem. (1991) ' . Soc., 102, 870 (1980) 4) K. Ito, H. Harada, A. Tai, Bull. Chem. Soc. 30) H. Tsutsui, O. Mitsunobu, Tetrahedron Lett., Jpn., 53, 3367 (1980) 25,2159(1984) 5) A. Alexakis, P. Mangeney, . Tetrahedron: 31) W. Seidel, D. Seebach, Tetrahedron Lett., 23, Asymmetry, 1,477 (1990) 159 (1982) 6) P. A. Bartlett, W. S. Johnson, J. D. Elliott, 32) T. Kitahara, K. Koseki, K. Mori, Agric. Boil. J. Am. Chem. Soc., 105, 2088 (1983) Chem., 47, 389 (1983) 7) W. S. Johnson, R. Elliott, J. D. Elliott, ibid., 33) K. Mori, Tetrahedron, 37, 1341 (1981) 105, 2904 (1983) 34) K. Mori, K. Tanida, Tetrahedron, 37, 3221 8) J. D. Elliott, V. M. F. choi, W. S. Johnson, J. (1981) Org. Chem., 48, 2295 (1983) 35) P. J. Maurer, M. J. Miller, J. Am. Chem. Soc., 9) A. Mori, K. Ishihara, H. Yamamoto, Tetra 105, 240 (1983) hedron Lett., 27, 987 (1986) 36) B#gP861-18791 10) A. Mori, J. Fujihara, K. Maruoka, H. Yama 37) A. Griesbeck, D. Seebach, Helv. Chim. Acta, moto, ibid., 24, 3367 (1983) 70, 1320 (1987) 11) S. D. Lindell, J. D. Elliott, W. S. Johnson, 38) G. Frater, Helv. Chim. Acta, 62, 2829 (1979) ibid., 25, 3947 (1984) 39) T. Kikukawa, A. Tai, Chem. Lett., 1935 12) A. Ghriki, A. Alexakis, J. F. Normart, ibid., (1984) 25, 3038 (1984) 40) M. Kiso, Y. Ogawa, S. Tanaka, H. Ishida, A. 13) W. S. Johnson, J. D. Elliott, G. Hanson, /. Hasegawa, J. Carbohydr. Chem., 5, 621 Xm. C/zem. Soc., 106, 1138 (1984) (1986) 14) T. Hosokawa, T. Yagi, Y. Ataka, S. Mura- 41) B. Lammek, W. Neugebaner, D. Perkowska, hashi, Bull. Chem. Soc. Jpn., 61, 3380 (1988) G. Kupryszewski, Poly. J. Chem., 52, 756 15) K. Funakoshi, N. Togo, K. Sakai, Tetra (1978) hedron Lett., 30, 117 (1989) 42) A. Tai, N. Morimoto, M. Yoshikawa, T. Sugi 16) M. Kaino, K. Ishihara, H. Yamamoto, Bull. mura, T. Kikukawa, Agric. Biol. Chem., 54, Chem. Soc. Jpn., 62, 3736 (1989) 1753 (1990) 17) A. Mori, H. Yamamoto, J. Org. Chem., 50, 43) E37J B#r, Nippon Nogeikagakukaishi, 64, 5444 (1985) 1741 (1990) 18) M. Yoshikawa, T. Sugimura, A. Tai, Chem. 44) D. Seebach, S. Roggo, J. Zimmermann, — 49 — 190 m\ ie. a# m Mi m “Stereochemistry of Organic Transforma Helv. Chim. Acta, 70, 448 (1987) tions ”, eds. W. Bartmann, B. Sharpless, VCH, 46) T. Basile, E. Tagliovini, C. Trowbini, A Weinheim (1987), p. 55 Umani-Ronchi, J. Chem. Soc., Chem. Com- 45) D. Seebach, R. Imwinkelried, G. Stucky, mun., 596 (1989) Asymmetrically Modified Heterogeneous Catalyst; Its Application for the Synthesis of Optically Active Fine Chemicals Tadashi KIKUKAWA* and Akira TAI** *NARD Institute, LTD, 2-6-1, Nishinagasucho, Amagasaki, Hyogo 660 Japan **Faculty of Science, Himeji Institute of Technology, 1479-1, Kanaji, Kamigori, Hyogo 678-12, Japan Tartaric acid-NaBr modified Raney nickel catalyst(MNi) is an unique heterogeneous catalyst for the enantio-differentiating hydrogenation of prochiral ketones. This catalyst system has recently been significantly improved by the use of ultrasound irradiated Raney nickel catalyst. In the first part of this report, preparation procedures of the new MNi and methods to obtains optically pure 1,3-diols of C^ symmetry and 3- hydroxyalkanoic acids by the use of MNi were mentioned and in the last part, utilizations of optically active 1,3-diols and 3- hydroxyalanoic acids for the synthesis of fine chemicals were reviewed. (© 1992 Catalysis Society of Japan) -50 5S/=S 1 . (i C*l= K#^b¥£4 ca-c(±- (l) tTSKSx&'t5 /seStoy&yy-s-jiK.T v - -1- -51- (2) (g?2/''?S®^6SJ) . o jry o) #»ts xs^x x d- 2. -S-J$tf V-<7'f K(ii*:<7) J: 7 ii-^TnJ. (1 ) JE@to(c UftSOT S X®h>- -yyxSrS-o^ U-^7> K^-g-Sr-S 5 C j:. (2) $$*■£ v> (2 ffl#:*T-a --^>) v ? x £ if <7>$|il $££$:>) 7 4C i. (3) &-o%¥r£t£ft#*x-*>z> ;t. (4) 3 1XXTV t. C « 4; vyi&T-tHst L-r«^x>j5-fe*£ijm§tt.*M#-e^rasaxij ?-e4;iS8*ltlt. 01 cSta^«Mttii«SH:4 7#X!ST5 fiKU iX8S©7 5 X m t Z&bvx* VimsH-fi z tlz X t) . 3fc Nonnatural Amino Acids Carrying Aromatic Groups : HjNCHCOOH 2-1. - -®ex*/v^---esff - - f7f)K b'l^xyi,, * 5 mi? V f- >; 7 vx* '; KtJ'^figsn*. ch.i3li-ik7n6tiicSfl'JSB?iJL/i%SE%-fe$*-S-ff i: IT. « ^3fcx*A'*'-0 *££!*# i £ ££fr$&S;S}-^ btli. Wxlf. 7 > 1- V ^8$-to -oy —7yxtf >/ K (I) (2, j§S4>T-0 2coJ:7C7yh >; ;ir$z)'dt v -<7‘f- K±flH dO^V -y 7 Xlc?6-3rSim(cE5i| LTV'^Sz)'. R^M (CD) x-^X I- Vr^ti'bBJi b *>tc $ -2- — 52 — -es- -c - (II) o Z‘l'0«“ 0^-0£=u CH3X /3eH N eH3 0 A 03 eh 3 2H3 Z(EH3) EH30-r(03H3HN)—03H3HN-0(03H3HN)-03H3HN—u(03H3HN)H. ^ ^y a o (ii) x A ^y ^ A ^ - ^^ (d '#^-/i.q) (a '#/A-^^^/^) -----¥ %e .%Ig^J. ^ Y- a 4 y.^< -4.^^ A ^ '3-3 9 'fH -4/.^ A ^(^3 El^4= ^$gC;JL]Tl#c.qa^ -4.3:^ A .4^^^A^^^^I^2@9:^T(^#'i/A4/ m = 2 @3. K P %X5.5 A#fL7 (/'&#-& (m = 0, 2) . 1 0 , 9.4A(7)%^ (m=l) = 2(7)^v-^y-f < . ^f8&i@L/z&#T&&C -3^. 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'<'/ f- K (4 4 ■ Mttco&ffltfx-Z ZZ. t&m Zix-ti (@8) . fitJSfitffSfSfi- U'icD%.&f5 ^ (OSI&jrS-oTvScO-C. •?" c £ T\ tx^S yKffi4i'«4(^Rj5S cfc -7- — 57 — [}#%o 4 . tffl# icftu*. . ?>;’g-X\$>Z (09) . 3 #» < #4-t-§ z,mkx-z>itzio. m? '/; n t i "Thb14«Sv^w-p^5 -8- — 58 — Chemistry of Lantibiotics Tetsuo Shiba Peptide Institute, Protein Research Foundation, 4-1-2 Ina, Minoh, Osaka 562, Japan SUMMARY: Lantibiotics are biologically active lanthionine peptides isolated as products of microorganisms. The first lantibiotic, nisin, was found in 1928 and is now in practical use as a food preservative. Another type of lantibiotic was recognized in ancovenin, which is an angiotensin I converting enzyme inhibitor. We elucidated the chemical structures of nisin and ancovenin by chemical analyses. The structure of lanthiopeptin with an antiviral lantibiotic was also determined in our laboratory. It was confirmed that lanthiopeptin is the same compound as cinnamycin and Ro 09-0198 whose structures had not been established at that time. A classification of all lantibiotics in view of those structures is presented. We achieved the first total chemical synthesis of nisin of 34 amino acids involving five lanthionine or methyllanthionine loops and three dehydroamino acids. In this study, new methodologies for the preparation of lanthionine peptide and dehydroamino acid peptide are presented. Related to the biosynthesis of lantibiotics, a novel preparative method of lanthionine peptide from synthetic precursor by enzyme for coupling of thiol group with double bond is introduced. 1. INTRODUCTION Nature sometimes shows rather peculiar behavior in peptide chemistry. A disulfide linkage in cystine residue is an obligatory clamp to fix a peptide chain to the definite conformation of proteins in plants and animals. In some microorganisms, a monosulfide linkage is utilized plausibly for the same purpose. A thioether amino acid corresponding to cystine is called lanthionine, which was originally found in wool. 1) On the other hand -59- 430 T. Shiba lanthionine peptides have been found as microbial products possessing various biological activities. Recently, it has been proposed that a group of biologically active lanthionine peptides should be called lantibiotics. An antibiotic-like activity of nisin was first reported in 1928 slightly earlier than the discovery of penicillin,2) but it was not isolated until 1947. 3) Since then, more than ten different kinds of lantibiotics have been found, all from microorganisms. Because of the multiple biological activity and unique structure of lantibiotics, much attention has been paid from many points of view, particularly concerning biosynthesis, chemical structure, chemical synthesis, active mode, fermentative production and even practical application. During the past ten years, we performed structural studies on nisin, ancovenin and lanthiopeptin as well as the synthetic study of nisin at Osaka University and the Protein Research Foundation. The results are summarized and presented herein. 2. STRUCTURE OF NISIN, ANCOVENIN, AND LANTHIOPEPTIN The establishment of the molecular structure of the lantibiotic is necessary for the further development of biology and chemistry of this unique bioactive peptide. It must be based on a precise analysis of the components including stereochemistry and an exact determination of the amino acid sequence involving the lanthionine ring moiety. The structural study of nisin was initiated by Gross and Morell in 1971. 4) They proposed the whole structure of nisin (Fig. 1) which is composed of 34 amino acids including five thioether linkages of meso- lanthionine and r/zreo-p-methyllanthionine as well as two residues of dehydroalanine and one residue of dehydrobutyrine. Similar structural features were soon recognized in another lantibiotic, subtilin (Fig. 2), although this has 32 composite amino acids.5) A B C D E lteTHisTVaJYDhan.ys)oH ProjfGly [AsnlMet Fig. 1 Structure of nisin. — 60 — Chemistry of Lantibiotics 431 A B C D E Fig. 2 Structure of subtilin. Before starting our synthetic study of nisin, we were forced to reinvestigate the structure of this lantibiotic, because details of its structure determination had never been published. We purified commercially available nisin6) by preparative HPLC method (Nucleosil 7Cig, 35 % acetonitrile-0. 3 % HCOONH4 (pH 4.5)) to obtain the completely pure sample. The amino acid analysis confirmed the proposed amino acid composition of nisin. FAB-MS gave a molecular ion peak of 3352.7 (M+H+) corresponding to the proposed structure. 1) OH 2) H+ + Ph-NH-C = NH Sx zCH-Rm 11 O CH3 Rn NH3 + 0=C-C0-NH-CH-C0-- Fig. 3 Edman degradation of dehydroalanine peptide. — 61 — 432 T. Shiba For sequential analysis of nisin including dehydro amino acid, it must be kept in mind that, as shown in Fig. 3, the Edman degradation does not go over the dehydro amino acid residue by loosing an amino terminal. Furthermore, when phenyl isothiocyanate reacts with the counterpart of lanthionine residue, the thiazolinone ring after cleavage of the peptide bond, links to another part of the lanthionine moiety by thioether linkage, and will be liberated after Edman degradation proceeds to this residue, as shown in Fig. 4. R, Ph-N=C=S + H2N-CH-CO|-NH-CH-CO...... ■’NH-CH-COI-NH-CH-CO...... CH-R CH-R' V______J (R, R'=H or CH3) 1) OH 2) H+ R. R, Ph-NH-C = NH H2N-CH-CO...... NH-CH-CO-NH-CH-CO...... I I S CH-CH-R CH-R’ 9 k____ ------/ O S=C—NH HN — C=S I I I I R, Ph-N CH-CH-R R’-CH-CH N-Ph + H2N-CH-CO— ^ / V______/ \ / 9 9 O o U (not detected) Next ^Cyclej Fig. 4 Edman degradation of lanthionine peptide. In fact, we first reduced the intact molecule in the presence of palladium black under 8-9 atm of hydrogen at 55 °C to obtain the reduced nisin in which the double bonds in Dha (dehydroalanine) and Dhb (dehydrobutyrine) were saturated, and meso-Lan (meso-lanthionine) was converted to two Ala (alanine) residues by desulfurization, and MeLan (p- methyllanthionine) to Ala and Abu (a-aminobutyric acid) residues. The -62- Chemistry of Lantibiotics 433 reduced nisin was subjected to Edman degradation, which proceeded as far as 15 steps, as shown in Fig. 5. I le-Abu-Ala- lie-Ala-Leu-Ala-Abu-Pro-Gly-Ala-Lys -Abu-Gly-Ala- - - Fig. 5 Edman degradation of reduced nisin. D E ABC 25 min Cosmosil 5C18 (4 x 125 mm) Fig. 6 Elution pattern of BrCN degradation products of nisin on HPLC. We then tried a fragmentation of the original molecule of nisin by means of cyanogen bromide degradation to obtain mainly two fragments cleaved at methionine residues which were separated as shown in Fig. 6. -63 434 1/2S 1/2S 1/2S 1/2S 1/2S 1/2g Entry I I I 5 10 I I 15 I Peptide Modification H-Ala-Val-GIn-Ala-Ala-Dha-Phe-Gly-Pro-Leu-Abu-Trp-Ser-Ala-Asp-Gly-Asn-Abu-Lys-OH Ancovenin Intact 1 1 Reduction of Dha 5 Ala Z Mem \ , Addition of HSCH2COOCH3 5 X 10 \X=Cys J 2 to Dha o Proline Specific Endopeptidase 10 d Digestion 4 Reduction and Desulfurization 5 Ala Cha 10 15 Fig. 7 Sequence analysis of intact and modified ancovenin. Chemistry of Lantibiotics 435 One corresponds to the A-B-C ring part where ring C is opened at the methionine position, and the other to the D-E cumulative ring part. These two fragments were analyzed sequentially after reduction as mentioned before. The results of the sequence analysis were quite satisfactory being coincident to the proposed sequence. An assignment of thioether linkages at lanthionine and methyllanthionine was carried out according to the same principle mentioned later in the structure determination of ancovenin. Consequently, a correctness of the structure of nisin proposed by E. Gross was assured by our cautious reinvestigation of the structural analysis.7 ) Parallel to the structural study of nisin, we attempted a determination of the structure of a novel lantibiotic, ancovenin. Ancovenin was isolated from a culture broth of Streptomyces sp No. A647P-2 as an inhibitor of angiotensin I converting enzyme at Fujirebio Inc., Japan in 1983.8) IC50 value of the inhibition activity for a rat lung angiotensin I converting enzyme is 8.7 x 10"7 M, which is between those of the natural potentiator C and the synthetic antihypertensive agent, captopril, in strength. Although this lantibiotic exhibits quite different biological activity from that of nisin, ancovenin also contains Lan, Melan and Dha in the molecule, which is composed of 19 amino acid residues. The sequential analysis of ancovenin was carried out according to the same principle as that for nisin.9 ) The results are summarized in Fig. 7. The Edman degradation to an intact molecule of ancovenin gave only phenylthiohydantoin derivatives of the 2nd and 3rd amino acids and stopped there (Fig. 7). Therefore we reduced ancovenin under 1 kg/cm 2 hydrogen atmosphere in the presence of Pd at room temperature, then repeated the Edman degradation (Entry 1). In this experiment, Ala appeared at the 6th position indicating Dha in the original form, but the reaction stopped at the 7th residue. Next we treated ancovenin with methyl thioacetate which was added to the double bond of Dha. The Edman reaction of this product went as far as the 10th position (Entry 2) showing a Pro residue at the 9th residue. Taking advantage of this information, we digested the original molecule with proline-specific endopeptidase to secure a new amino terminus for further sequencing (Entry 3). The Edman degradation now gave us whole amino acid residues as far as the C -terminus. In order to make sure of the sequential assignment, we finally attempted a drastic reduction under 10 kg/cm 2 hydrogen atmosphere in the presence of Pd at 50 °C to saturate all double bonds and remove sulfur atoms from thioether linkages. The result is shown in Entry 4. — 65 — 436 T. Shiba 1/2S 1/2g 1/2g 1/2g 1/2g 1/2g I I I 5 10 I I 15 I H-Ala-Val-GIn-Ala-Ala-Dha-Phe-Gly-Pro-Leu-Abu-Trp-Ser-Ala-Asp-Gly-Asn-Abu-Lys-OH Entry 1 | Dns derivative B : Edman degradation Entry 2 —- JDn^dedvativ^Ajj t....( | : Dansylation Entry 3 —- ■- Dns derivative B Fig. 8 Determination of position of meso-lanthionine. The determination of the positions of thioether linkages for Lan and MeLan moieties was carried out as follows (Fig. 8 ). First, the molecule of ancovenin was dansyl(Dns)ated followed by acid hydrolysis to give Dns- threo-P-MeLan (Dns derivative B in Fig. 8 ) indicating that the counterpart of 1st Ala residue should be Abu (Entry 1). Second, after three steps of Edman degradation, the same procedures as above were applied to give Dns-mejo-lanthionine (Dns derivative A) showing that the counterpart of the 4th Ala residue should be Ala (Entry 2). Finally, after four steps of Edman degradation followed by dansylation and then hydrolysis gave Dns- threo-P-MeLan again (Entry 3). It was thus concluded that the 14th Ala residue is clearly linked to the 4th Ala residue. Ancovenin Deamination and cleavage of prolyl peptide bond 1) NaN02 2) proline specific endopeptidase digestion Edman degradation ( ——) 1/2g 1/2g ^ 1*S H-Hpr-Val-GIn-Ala-Ala-Dha-Phe-Gly-Pro- Leu-Abu-Trp-Ser-Aia-Asp-Gly-Aan-Abu-Lya-OH X 4 ...... S--...... 14 ...... s...... ^ *Hpr: 2-hydroxypropionic acid tbreo-p-methyilanthionine disappeared H-A|a-Val-Gln-Ala-Aja-Dha-Phe-Gly-Pro-Leu-Ayu-Trp-Ser-Ala-Asp-Gly-Asn-A^u-Lys-OH 14 Fig. 9 Assignment of sulfide bridge belonging to r/ireo-p-methyllanthionine. — 66 — Chemistry of Lantibiotics 437 Assignment of the remaining two thioether linkages of MeLan was rather tricky. First the original molecule was treated with NaNC>2 to change the amino terminus to hydroxyl group. We then applied proline endo- peptidase digestion followed by Edman degradation (Fig. 9). Acid hydrolysis of the product after the second step of the Edman degradation gave no more p -MeLan. This means that Ala5 and Abu18 are not linked to each other, otherwise both residues would give intact p- MeLan after these treatments. The conclusion is that Ala1 must be linked to Abu18 and Ala5 to Abu11, as shown in Fig. 9.9) 1 HOTLys Fig. 10 Structure of ancovenin. Fig. 11 Conformation of ancovenin. 438 T. Shiba Steric configurations of all component amino acids were determined gas-chromatographically 10) on chiral capillary column 11) for TFA-amino acid isopropyl esters obtained from acid hydrolyzate of either ancovenin or desulfurized ancovenin. 9 ) The results showed that all amino acids except Lan and MeLan are of L-forms, one Ala residue of Lan and two Abu residues are of D-forms. Therefore, we deduced that ancovenin has one meso-Lan and two MeLan of L-Ala D-Abu form. The whole structure of ancovenin was thus determined as shown in Fig. 10.9 ) This structure of ancovenin is quite unique and peculiar having triply cumulative sulfide rings. Conformation analysis of this lantibiotic was performed by NMR measurements including COSY and NOESY in H%0 and D 2O. For an elucidation of the tertiary structure, distance geometry calculations were used. One of the conformations of the lowest value of target function is shown in Fig. 11,12) The structure-activity relationship of this particular compound is worthy of attention. We attempted specific cleavages of rings B and C by 2-(2-nitrophenylsulfenyl)-3-methyl-3-bromoindole and proline-specific endopeptidase respectively (Fig. 12). Surprisingly both products lacking either ring B or C maintained significant inhibitory activity comparable to that of the original ancovenin. Based to this result, only ring A seems to be responsible for the exhibition of enzyme inhibition. 1 .AlalAla Proline specific Endopeptidase SerlTrp IC<0 = 8.5 x 10 M Ancovenin IC<„= 1.4 x 10"6M BNPS-Skatole : \ AlalAla 2- (2-nitrophenylsulfenyl)- 3- methyl-3-bromoindole SerVH *Oia : oxyindolylalanine Fig. 12 Ring opening of ancovenin. — 68 — Chemistry of Lantibiotics 439 OH H-Ala-Arg-GIn-Ala-Ala-Ala-Phe-Gly-Pro-Phe-Abu-Phe-Val-Ala-Aslj-Gly-Asn-Abu-Lys-OHA Fig. 13 Structure of lanthiopeptin. The third lantibiotic we studied was an antiviral compound, lanthiopeptin, found at Bristol-Myers Research Institute, Japan.13) Lanthiopeptin was isolated from Streptoverticillium No. L337-2 and shown to be effective against Herpes simplex virus I and some other bacteria. An amino acid analysis showed again the presence of meso-Lan and threo-$- MeLan. Besides these, eryf/zro -(3-hydroxyaspartic acid (Asp(OH)) and lysinoalanine were found as unique amino acid components. FAB-MS and Edman degradation were carried out in a manner similar to that for nisin or ancovenin. The structure of lanthiopeptin deduced from the structural analysis was shown to be very similar to that of ancovenin (Fig. 13). The only significant differences are in a replacement of Asp with Asp(OH) and the existence of a lysinoalanine residue which corresponds to the positions of Dha6 and Lys 19 in ancovenin.I 4) OH r °\ H-Ala-Arg-GIn-Ala-Ala-Phe-Phe-Gly-Pro-Ala-Abu-Phe-Val-Ala-Asp-Gly-Asn-Abu-Lys-OH Fig. 14 Structure originally proposed for Ro 09-0198. -69- 440 T. Shiba Meanwhile, the structure of an immunopotentiating lantibiotic termed Ro 09-0198 was proposed by Kessler et a/.,15) as shown in Fig. 14. As seen in this structure, this lantibiotic is quite similar to that of lanthiopeptin except for the position of a counterpart of Ala residue of lysinoalanine. We compared samples of lanthiopeptin and Ro 09-0198 directly by NMR and HPLC and found that both are exactly the same compound.I 4) At the same time Kessler et al. revised the structure of Ro 09-0198 to the correct one, making it identical to that of lanthiopeptin. 16) Furthermore, cinnamycin, another antibiotic isolated by Dworidi et al. in 1954 but of unknown structure,17) was found to be the same compound as lanthiopeptin and Ro 09-0198. In addition it was soon realized that duramycinlB) has a very similar amino acid composition to lanthiopeptin. The only difference was found at position 2. In duramycin, Arg 2 of lanthiopeptin is replaced by Lys 2. Therefore, a similar amino acid sequence was again suggested. 14) Leucopeptin was also found to be identical to duramycin. I9 ) In summary, the structures of the lantibiotics so far found may be classified into four groups with respect to structural features,20) as shown in Table 1. Table 1 Classification of lantibiotics. Number of Amino Acids Number of Thioether Rings Nisin Tvpe Nisin 34 5 Subtilin 32 5 Ancovenin Tvpe Ancovenin 19 3 Cinnamycin 19 3 /LanthiopeptinX yRo 09-0198 j Duramycin 19 3 (Leucopeptin) Epidermin Tvpe20) Epidermin 22 3 Gallidermin 22 3 Others20^ Pep 5 34 3 Mersacidine 20 4 Actagardine 19 4 -70- Chemistry of Lantibiotics 441 3. SYNTHESIS OF NISIN For the synthetic study of lantibiotics, synthetic methods for the preparation of lanthionine loop and introduction of dehydroamino acid in the peptide chain must be exploited and established. As for the synthetic route of lanthionine peptide, there are three possible ways, as shown in Fig. 15: (a) ■NHCHCO...... OH H...... NHCHCO- I I CHo------S------CHR -NHCHCO...... NHCHCO- I I CHo------S------CHR (b) -NHCHCO...... -NHCCO- I II CH2—SH CHR -S (c) •NHCHCO...... NHCHCO Oxidation -NHCHCO...... NHCHCO- I I I I CH2—SH HS—CHR ch2—s- •S------CHR Fig. 15 Synthetic routes for lanthionine peptide. Route (a) seems to be the most accessible way to build the lanthionine peptide, and this route was actually employed by Photaki et all 1) However, this route may encounter severe difficulty in the selection and control of protection of four functional groups in lanthionine residue. Route (b) is interesting from the viewpoint of biosynthesis of lantibiotics. 22) A crucial point in this route is the inevitable disadvantage that the addition of a thiol group to dehydroamino acid may not give a desirable enantiomer as a single product but a mixture of diastereomers. In contrast, route (c) seems to be very promising, since the cyclic disulfide peptide is readily available according to the usual peptide synthesis using the protected cysteine residues. For desulfurization from cystine peptide by a particular reagent, P(NEt2)3 may be employed. 23-24) -71 442 T. Shiba Md/KHCO, NH2 (_NMe3 \ CH, Z CH2 CH2 — NH-C^-CO- II -NHCHCO—- NMe2 —NHCCO- i * A2pr V HCHO CH, Mel(leq) / H Dha NaBHiCN ..... NHCHCO — KHCO3 Fig. 16 Synthesis of dehydroalanine peptide through Hofmann degradation. : B C f 0 \ ,------S------\ H-lle-Dhb-o-Ala-lle-Dha-Leu-AlaiD-Abu-Pro-Gly-Ala-Lys o-Abu-Gly-Ala-Leu-Met-Gly-Ala-Asn-Met (1 -7) (8 - 12) (13 - 21) Segment i Segment II Segment III D Lys-D-Abu-Ala-o-Abu-Ala-His-Ala Ser-lle-His-Val-Dha-Lys-OH (22 - £8)” (29 - 34) Segment IV Segment V Fig. 17 Five segments for the total synthesis of nisin. Much caution is required for introducing a dehydroamino acid whose double bond may cause degradation or polymerization of the peptide. We exploited a new synthetic method for dehydrtialanine peptide from a,p- diaminopropionic acid (A]pr) peptide by application of Hofmann degradation as shown in Fig. 16.25* 2^) Dehydrobutyrine (Dhb) residue was successfully prepared by dehydration of threonine residue in peptide with carbodiimide-CuCl. 27) -72- Chemistry of Lantibiotics 443 D-Cys lie A2pr Leu Cys Acm Boc- •ONSu H OMe Acm Boc- ^OMe c HCI HClyTHF Acm Boc ONSu H- k-OMe Acm Boc- ^-OMe HCI HC1/THF Acm Boc ■ONSu H- ^-OMe Acm Boc- ^OMe Trt HCI HC1/THF Acm Boc- L ONSu H- OMe Trt Acm 6 Ij/MeOH Boc- L ^-OMe r A Boc-A a K -A 3'OMo P(Et2N)3 Boc \L OMe Fig. 18 Synthesis of the lanthionine intermediate of ring A in segment I. For the total synthesis of nisin, we adopted a synthetic strategy in which five segments containing A, B, C, D-E rings and C-terminal chain as shown in Fig 17 are successively coupled. According to this strategy, we can check the identity of synthetic coupled segments I-II and IV-V with authentic samples of nisin (1-12) and nisin (22-34), respectively, which are obtained as degradative products from the whole nisin molecule either by enzymatic or BrCN degradation. -73- 444 T. Shiba A -S- Boc-D-Afa-lle-A2pr(Z)-Leu-Ala-OMe 1) Pd/HCQ2NH4 -Ala-( Boc-D-Ala-lle-A2pr(Me2)-Leu-Ala-OMe 2) HCHO/NaBH3CN 90% (2 steps) Mel/KHCC^ ^ r "S------\ 1)TFA /------S------\ Boc-o-Ala-lle-Dha-Leu-Aia-OMe------HClH-o-Ala-l le-Dha-Leu-Ala-OMe DMF (+MeOH) 2)HCl/MeOH 91% Z-lle-Thr-0 H /------S------\ EDCHCl/CuCl Z-lle-Thr-D-Ala-lle-Dha-Leu-Ala-OMe EDC/HOBt DMF-CH2CI2 (1:1) DMF-DMSO (2:1) 74% 83% (3 steps) ------< z' S \ 1m NaOH/Dioxane 1 t------S------V Z-lle-Dhb-D-Aia-lle-Dha-Leu-Ala-OMe * Z-lle-Dhb-o-Ala-lle-Dha-Leu-Ala-OH ...... 50% Fig. 19 Synthesis of segment I containing ring A. Synthesis of the intermediate to segment I was carried out as shown in Fig. 18 25) by elongation of the peptide chain followed by desulfurization of cystine derivative to lanthionine derivative. For removal of the NP-Z group of A2pr residue, catalytic transfer hydrogenation was applied to give a free (3-amino group, which was then permethylated using DMF as solvent in the final step of the methylation with CH3I (Fig. 19). p-Elimination of the quaternary ammonium intermediate in Hofmann degradation was accelerated by the addition of methanol to the reaction mixture to afford Dha derivative. After removal of the Boc group, the ring A part was coupled with Z-Ile-Thr-OH. Dehydration at Thr residue was carried out by 1 -ethyl-3 -(3-dimethylaminopropyl)carbodiimide (EDC)*HCl/CuCl and final ly the segment I of A-terminal part was obtained after saponification. — 74 — Chemistry of Lantibiotics 445 Synthesis of segment II containing ring B was performed as shown in Fig. 20.28) For this synthesis, f/zreo-3-methylcysteine is required as the starting amino acid. This thioamino acid was prepared from D-Thr through aziridine ring intermediate stereospecifically, as shown in Fig. 21.29) The original stereoconfigurations in D-Thr were maintained in the final product of 3-methylcysteine since double S%2 reactions occurred both at ring closure and opening steps. threo 3-Me-o-Cys Pro Gly Cys Trt Acm Boc- Z-OSu H- ■OH Boc- ■OSu HCIH- OBzl Trt Acm 87% 82% Z—OBzl Boc- ■OH Boc- /Tn DCC/HOSu Acm HCl/AcOH Boc- ■OSu* HCIH- k-OBzl Trt Acm 27% z ^-OBzl Boc- -s------s- 90% Boc -d-Abu a-OBzl P(NEt2)3 ------s------41% Boc- ■OBzl 1) HF-Anisole (9:1) 74% / £*) k l WVVvJU B T roc-o-Abu-Pro-Gly-Ala-OH + HCIH-Lys(Z)-OBu r EDC/HOBt in DMF 90% Troc-D-Abu-Pro-Gly-Ala-Lys(Z)-OBu f l)Zn/AcOH 86 % 2)HC1/THF ------S------\ 12 HCIH-D-Abu-Pro-Gly-Ala-Lys(Z)-OBu f Fig. 20 Synthesis of segment II containing ring B. -75- 446 T. Shiba Desulfurization reaction proceeded satisfactorily to give methyl- lanthionine derivative (Fig. 20). In this desulfurization, two plausible routes as shown in Fig. 22 are considered. However, actually, the reaction went through via route (a) to lead to the desired threo form, perhaps due to an electron donating effect of the methyl group; otherwise an inversion may occur via route (b) to give the wrong configuration. Saponification of the methyl ester at C-terminus resulted in an undesired decomposition. Therefore, we chose benzyl ester for C-terminal blocking in this case. After the simultaneous removal of Boc and Bzl groups, the amino terminus was reprotected with Troc group. Finally, the ring B part with Lys moiety gave us the segment II derivative, as shown in Fig. 20. H / 1) CH3NH2/CH3OH 2) I2 Trt = (C6H5)3C , Ms = CH3SO2, Z = C6H5CH2OCO Fig. 21 Synthesis of f/zreo-3-methylcysteine from D-threonine. — 76 — 447 P(NEy 3 s“ \0 /X h3c-c-h ch2 I H3C—C-H ch2 -—NHCHCO — --NHCHCO- (a (25,35) (A) —NHCHCO — -NHCHCO — S s Z I *1 fftreo-Methyilanthionine H3C—C—H CH2 NHCHCO •§—NHCHCO— (25,35) (*) threo -3-Methyl-D-cystelne P(NEt2)3 (b) H3C-C-H —NHCHCO...... NHCHCO —- (25,37?) (R) -—NHCHCO e/ylh/u-Methyilanthionine Fig. 22 Plausible desulfurization pathways in ring B formation. C Boc-o-Abu-Gly-Ala-Leu-Met-Gly-^la-OBzlr -s- Nps-Asn(Mbh)-Met-OBur 1) HF-Anisol (9:1) 80% 2) TrocOSu HC1 in THF 96% e------°------\ T roc-D-Abu-Gly-Ala-Leu-Met-Gly-Ala-OH + HCIH-Asn(Mbh)-Met-OBuf EDC/HOBt in DMF 90% Troc-D-Abu-Gly-Ala-Leu-Met-Gly-Ala-Asn(Mbh)-Met-OBu f 1) Zn/AcOH; 2) HC1 58% isr------s------\ 21 HCIH-D-Abu-Gly-Ala-Leu-Met-Gly-Ala-Asn(Mbh)-Met-OBu t Fig. 23 Synthesis of segment III containing ring C. -77- 448 threo threo 3-Me-o-Cys Ala 3-Me-o-Cys Cys His Cys 78% quant 91 % quant 70% 78% 1) TFA; 2) HCl/MeOH r Boc-Lys(Z)-OH + HCIH-D-Abu-Ala-D-Abu-Ala-His(Tos)-A!a-OMe V J EDC/HOBt in DMF 69% (3 steps) /------S------\ Boc-Lys(Z)-p-A ^u-Ala-D-Abu-Ala-His-Ala-OMe NH2NH2H20 89% Boc-Lys(Z)-D-Abu-Ala-D-Abu-Ala-His-A)a-NHNHj 22 V------S------J 28 Fig. 24 Synthesis of segment IV containing ring D-E. -78- Chemistry of Lantibiotics 449 In a similar way, segment III involving ring C was synthesized as shown in Fig. 23.3°) An acid amide group of Asn residue was protected by the 4,4'-dimethoxybenzhydryl (Mbh) group. Removal of the Troc group gave the desired segment III. A cumulative D-E ring moiety was prepared by a simultaneous one- step desulfurization reaction from the corresponding biscystine peptide as shown in Fig. 24.3U After coupling with Lys moiety, the segment IV derivative was obtained, which was converted to a hydrazide for coupling with segment V by azide method. 29 34 Ser lie His Val Dha Lys Troc josOH- - Boc-Aapr-OH H—^-OBzl Troc Z 82% Boc- Z ^OBzl Jroc Boc- -OH H- z ^OBzl DCC/HOBt Troc Z 75% Boc- ^OBz i) Zn/AcOH ii) Mel Z 73% Boc- Dha OBzl Jos Boc- ^-OH H ^-OBzl Jos i) DCC/HOBt ii) TosCl Z 72% Boc- Z ^OBzl Jos Boc- •OH H- z ^-OBzl Jos i) DCC/HOBt ii) TosCl Z 80% Boc z OBzl lXBZl Jos Boc- k—OH H Z ^-OBzl Bzl Jos i) DCC/HOBt ii) TosCl Z 62% Boc- Z. Z ^-OBzl HOBt (6eq) Bzl Z 81% Boc 1Z. ^-OBzl Fig. 25 Synthesis of segment V of C-terminus. -79- 450 T. Shiba Segment V, corresponding to the C-terminal linear hexapeptide was synthesized as shown in Fig. 25.32) Dha residue in this part was also prepared from A2pr residue by Hofmann degradation. Couplings of the five segments I to V prepared above were carried out as shown in Fig. 26.32) The synthetic intermediate obtained by the coupling of segment I and II was confirmed by comparison with benzyloxycarbonyl derivative of nisin (1-12) which was derived from natural nisin by trypsin digestion. The coupling product of segment IV and V was also identified with the authentic sample of nisin (22-34), the BrCN degradation product from the original nisin, after deprotection. The final coupling of the synthons I-II-III and IV-V followed by deprotection with HF in the presence of anisole afforded the crude product which was purified by HPLC. The synthetic pure product thus obtained was completely identical to natural nisin in all respects including 1H-NMR, FAB- MS, HPLC, and antibacterial activity. 1 r------°------\7 Z-lle-Dhb-D-Ala-lle-Dha-Leu-Ala-OH l)EDC/HOBt Segment I inDMF 2)TFA 8f ----- »------\ 12 , 55% l)EDC/HOBt HCIH-D-Abu-Pro-Gly-Ala-Lys(Z)-OBu r in DMF Segment II 2)TFA 40% 13r------o ------\ 21 , HCIH-D-Abu-Gly-Ala-Leu-Met-Gly-Ala-Asn(Mbh)-Met-OBu f l)EDC/HOBt in DMF Segment III 2)HF-Anisole Nisin (9:1) 28 Isoamyl nitrite 10% Boc-Lys(Z)-D-Abu-Ala-D-Abu-Ala-His-Ala-NHNH 2' 1) TEA v-----g. HC1/THF Segment IV in DMF 2) Boc20 3) TFA 29 34 TFA 4) HC1 Boc-Ser(Bzl)-lle-His-Val-Dha-Lys(Z)-OBzl Segment V 25% (4 steps) Fig. 26 Couplings of segments I to V for the total synthesis of nisin. -80- Chemistry of Lantibiotics 451 Thus we achieved the first total synthesis of one lantibiotic, nisin, sixty years after its discovery. This synthetic work also provides a novel synthetic method for the preparation of dehydroalanine peptide as well as lanthionine peptide. In a recent study, we investigated the structure-activity relationship of nisin employing NMR conformational analysis. 33) Besides several synthetic segments for total synthesis of nisin, we prepared more than ten kinds of fragments or analogs for this purpose. Biological tests for those compounds suggested that the minimum structure required for the exhibition of antibacterial activity is the ^-terminal part (1-19). Furthermore, Dhb2 and Dha9 cannot be replaced with the corresponding saturated amino acids without loss of the activity. Although we succeeded in the total synthesis of the typical lantibiotic nisin, tremendous efforts were needed particularly for the preparation of the sterically pure methyl cysteine, desulfurization step, and introduction of the double bond in amino acid residue. Such disadvantages strongly inhibit the further development of the chemistry and biology of lantibiotics by limiting the supply of necessary samples. N N N Fig. 27 Biosynthesis of lantibioitics. 452 T. Shiba On the other hand, a biosynthesis of the lantibiotics has been studied in relation to that of other peptide antibiotics. However, it was earlier suggested that the 1 antibiotic is not formed by a multiple enzyme system like usual peptide antibiotics, but synthesized on a ribosome from a precursor peptide as in protein biosynthesis. 34,35) Lanthionine or methyl- lanthionine part is formed between cysteine and serine or threonine residues on propeptide, which is connected to a leader peptide at the N- terminal side. After a thioether ring formation in the propeptide between cysteine and serine or threonine, and also dehydration at serine and threonine residues, a process to remove the leader peptide will occur to complete the biosynthesis of the lantibiotics. 20) We are now attempting to utilize this concept of biosynthetic process as a tool for the preparation of the desired peptide. The idea is to achieve an enzymatic synthesis of lanthionine peptides from artificial precursor peptides possessing cysteine, serine and threonine at appropriate positions but without the leader peptide part. Such precursor peptides can be easily prepared using techniques for conventional peptide synthesis. In fact, we investigated an enzymatic formation of lanthionine or methyllanthionine from synthetic peptide substrates involving cysteine and serine or threonine using cell-free extracts of 35 different kinds of microorganisms. As a result, some species of Streptomyces and Str ept ov e rticillium certainly produced 3-methyllanthionine in the hydrolyzate of a product from the substrate corresponding to the nisin precursor. We are now developing this strategy to establish a novel and general preparative method for the lanthionine peptide in collaboration with Prof. H. Yamada, Faculty of Agriculture, Kyoto University. Acknowledgments The author expresses sincere gratitude for the invaluable contributions of excellent collaborators to this study. They are Dr. T. Wakamiya, Dr. K. Fukase, Dr. T. Teshima, Dr. K. Inami, and many students at Osaka University. References 1. N. J. Horn, D. B. Jones, and S. J. Ringel, J. Biol. Chem., 138, 141 (1941). 2. L. A. Rogers and E. O. Whittier, J. Bad ., 16, 211, 321 (1928). 3. A. T. R. Mattik, and A. Hirsh, Lancet , ii, 5 (1947). 4. E. Gross, and J. L. Morell, J. Am. Chem. Soc., 93, 4634 (1971). -82- Chemistry of Landbiotics 453 5. E. Gross, H. H. Kiltz, and E. Nebelin, Hoppe-Seyler's Z. Physiol. Chem., 354, 810 (1973). 6. The sample of natural nisin was produced in Aplin & Barrett Ltd., Beaminster, Dorset, U. K. and supplied by the laboratory of the late Dr. Gross. 7. K. Fukase, Doctor’s Thesis, Faculty of Science, Osaka University , 1987. 8 . Y. Kido, T. Hamakado, T. Yoshida, M. Anno, Y. Motoki, T. Wakamiya, and T. Shiba, J. Antibiot., 36, 1295 (1983). 9. T. Wakamiya, Y. Ueki, T. Shiba, Y. Kido, and Y. Motoki, Bull. Chem. Soc. Jpn ., 63, 1032 (1990). 10. E. Bayer, E. Gil-Av, W. A’ Konig, S. Nakaparksoin, J. Oro, and W. Parr, J. Am. Chem. Soc., 92, 1738 (1970). 11. T. Saeed, P. Sandra, and M. Verzele, J. Chromatogr., 186, 611 (1979). 12. M. Nishikawa, T. Teshima, T. Wakamiya, T. Shiba, Y. Kobayashi, T. Okubo, Y. Kyogoku, and Y. Kido, Peptide Chemistry 1987, pp 71-74. 13. N. Naruse, O. Tenmyo, K. Tomita, M. Konishi, T. Miyake, and H. Kawaguchi, J. Antibiot., 42, 837 (1989). 14. T. Wakamiya, K. Fukase, N. Naruse, M. Konishi, and T. Shiba, Tetrahedron Lett.,29, 4771 (1988). 15. H. Kessler, S. Steuernagel, D. Gillessen, and T. Kamiyama, Helv. Chim. Acta, 70, 726 (1987). 16. H. Kessler, S. Steuernagel, M. Will, G. Jung, R. Kellner, D. Gillessen, and T. Kamiyama, Helv. Chim. Acta, 11, 1924 (1988). 17. W. Dworidi, O. L. Shotwell, R. G. Benedict, T. G. Pridham, and L.A. Lindenfelser, Antibiot. Chemother., 4, 1135 (1954). 18. O. L. Shotwell, F. H. Stodola, W. R. Michael, L. A. Lindenfelser, R. G. Dworschak, and T. G. Pridham, J. Am. Chem. Soc., 80, 3912 (1958). 19. S. Kondo, M. Sezaki, M. Shimura, K. Sato, and T. Kara, J. Antibiot., 17, 262, (1964). 20. G. Jung, Angew. Chem., 103, 1067 (1991); Angew. Chem. Int. Ed. Engl., 30, 1051 (1991). 21. I. Photaki, S. Caranikas, I. Samonilidis, and L. Zervas, J. Chem. Soc. Perkin 1, 1980, 1965 . 22. L. Ingram, Biochem. Biophys. Acta, 224, 263 (1970). 23. D. N. Harpp and J. G. Gleason, J. Org. Chem., 35, 3259 (1970). 24. D. N. Harpp and J. G. Gleason, J. Am. Chem. Soc., 93, 2437 (1971). 25. T. Wakamiya, K. Shimbo, A. Sano, K. Fukase, and T. Shiba, Bull. Chem. Soc. 56, 2044 (1983). 26. S. Nomoto, A. Sano, and T. Shiba, Tetrahedron Lett., 1979, 521. 27. M. J. Miller, /. Org. Chem., 45, 3131 (1980). — 83 — 454 T. Shiba 28. K. Fukase, T. Wakamiya, and T. Shiba, Bull. Chem. Soc. Jpn., 59, 2505 (1986). 29. T. Wakamiya, K. Shimbo, T. Shiba, and K. Nakajima, Bull. Chem. Soc. Jp%.,55, 3878 (1982). 30. K. Fukase, M. Kitazawa, T. Wakamiya, and T. Shiba, Bull. Chem. Soc. ypn., 63, 1838 (1990). 31. K. Fukase, Y. Oda, A. Kubo, T. Wakamiya, and T. Shiba, Bull. Chem. Soc. 63, 1758 (1990). 32. K. Fukase, M. Kitazawa, A. Sano, K. Shimbo, H. Fujita, S. Horimoto, T. Wakamiya, and T. Shiba, Tetrahedron Lett..,29, 795 (1988). 33. D. E. Palmer, D. F. Mierke, C. Pattaroni, M. Goodman, T. Wakamiya, K. Fukase, M. Kitazawa, H. Fujita, and T. Shiba, Biopolymers, 28, 397 (1989). 34. A. Hurst, Adv. Appl. Microbiol., 27, 85 (1981). 35. C. Nishio, S. Komura, and K. Kurahashi, Biochem. Biophys. Res. Commun ., 16, 751 (1983). — 84 — : K© FAB-f wmmM 0^ H ______ fab • mam Am By E* •S69f-$6SWm^ „ __ 1 . (1UAC 01 fab i£¥, g5^&0%mcl± B LV't i), 6azo, #LW t y v -t v >f Application of fast atom bombardment mass spec *?ii ;u^'+'>^y^^-df (CPase) CZ trometry to analysis of protein primary structures. tfo/_- <>L. Toshifumi TAKAO, Yasutsugu SHIMONISHl Institute for Protein Research. Osaka University (40kDa-) mag^^rnfm^mg^-cii, — 85 — (42) Vol.32 No. 1 (1992) fZl:Z 07x//>Mt> ^ii'i^EiiPJI" Z^t'CT (ESI Ml##) ZxKvy** ..\ iui* ii _ ifz. -i!i | m 2 k • m&ftv-wmmvf. DNA^0@^iJ^m^6C ^l:ZcT^(D7 5 7@g@d ifz, Vi>'S5tiX\''Z>^ti>£>2>(7) ^iJcoSEiilf^tiTv^T)', $6H<0S^iifltcijV' -e, nv h/77 j -^;i:z o TliSS££lt/.:M?n LlZUZM-f*- £ tfil K£i£S£ LZjEtcMfeZIfoWJ: v\ 7<7!Km^. FAB Effi:5HrrtcZ SS6E5?- &6Wi, DNA j^g£8^"ij ?)> 6 $£ £ ft /j 7 5 ;mz o y \d j. — 7 — (c Z fii>$, [u]/ii L, 7 % 1> 20- T4 JE wiwWhjIwiW 300 1 000 3000 3 ;k:l$S"Cji4i£ftA: bhdy^-XxD >-oA <7) b 'J 77 ym«<7) FAB HSX^7 b ;u5). — 86 — FAB K • m&fl (43) #j^fT^^%:16C tT7 ; y B#LT4u&f&7 7?V > MSreM£tifz7?X* yb'tt'siiB?1 $fta aCt^T^a. |5]C#^-C%%fe^:tf^7 77yyF^l>(7)^#^KimclfactCZ»), t 7 7 7y>Mf>ezomm:WffaB t(7)/<77 K<7)7 5 ymznZikxiX'^Z. 77^ %-c, 2 &(7)rmmmtz? >h^77li, -tec, MSfta&ECJ: r)g]4 C #7):UfdfLldffflvubtLTv^a. C#btU>r- mf Z i, C^^$tiTv^75'10), EtII $ fta 7 y 7 ?im#c*m-c#adf, y > h ^ 7 7(7)a^, MT 7 y-fili^T'T- K<7)t£H -M&ftmtr-j: t ES-cin^-e £ a t $xm C<£ o *Ct)'£ 0 OWi'hh. а. HI 5 C^i& 7 □ 7' V 7 L BO Achromobacter 7*D t б. ma 7 - -t? M ik# Tiwb^-SI LZ:/<7’7 KO CAD/linked - scan 7^? p ;l/£^; l/j. ±C, y. 6 V V-X7>7 7 y^7h7^7^E$ij$K -E-fi^(7)Ram(7 iZffimtb C t t)?-C £, ^ ttf)^77 K07 5 ##&<7Xk#^%m cti act t)'-c # a (7)Tfm#s!i(7)#7cdu 0 f?n ctri ai 6j C, a, />2 C; dj W Cj a. mi tv*GTP&^zgaH (h7>xfA- El 4 CAD/linked-scan &U&tt * ^77 F0 7 7 i/>) r^fzL-y h cti(tasss^<7)^rrH) c oV'Tili^a. Thr-Ser-Thr-Ser-Pro-Xle-Val-Lys (204 — 211) MH + 832.4 , , Thr [ t Ser i [ Pro , Xle Val 1X1. H--§?-r - - -H- - -Tt>-r— h— - 5-er.. .^.. Thr d Thr . Ser V4 X) e b8 9 9D c 200 400 600 800 m/2 El 5 y D 7 V > L Ifl^) Achromobacter 7d x 7 —tf mik#T)'6j(l%LX:^77 F (204-211) (7)CAD/ linked-scan X-^<^ h 7Un). -87 (44) £ ## # Vol.32 No. 1 (1992) >, &(/, S£%Li)'b, t>i£—C ^?S^7'f-b'7 x V7 > N (Leu- $r u-c Lys-Gly-Gly-Cys) Ufl^&vVi £ ft, yt^- v^ma^T, e(39k), /?(35k), y(8k) (030(7) h(DC^%7 ; Cys-7(W%/u t7i-7 of7i-7 Mi, it e^VTV'6t<7)^#x^ft^. -E-CC. /? yh^&TTGTP^^mc^ ^#ia^%6 7)4:V6 gmro, 2(7)C^%^yf- K t&zixfzmz, hm <7)FABme^m^^tfc/:. (a6)mm^fi.^% # (m/z: 681.438) (i^7'ftKe#^:f6 5^3g(0 /?, y4f-fzL- y h tCOV'Tti:, ZtlhfD&MftM, 7 681.438 681.438 476.242 205.196 Leu-Lys-Gly-Gly-Cys Sesquiterpene (Farnesyl) 1 10 20 Pro-Val-Ile-Asn-Ile-Glu-Asp-Leu-Thr-Glu-Lys-Asp-Lys-Leu-Lys-Met-Glu-Val-Asp-Gln- 30 I------1 40 Leu-Lys-Lys-Glu-Val-Thr-Leu-Glu-Arg-Mec-Leu-Val-Ser-Lys-Cys-Cys-Glu-Glu-Phe-Arg- 50 60 Asp-Tyr-Val-Glu-Glu-Arg-Ser-Gly-Glu-Asp-Pro-Leu-Val-Lys-Gly-Ile-Pro-Glu-Asp-Lys- Asn-Pro-Phe-Lys-Glu-Leu-Lys-Gly-Gly-Cys 06 06 77m^GTP#^9ra% ( b7>Xf ju-7>) y ^yjL- .y h60C*^^7-/- K (66-70) CO FAB h ;uM). Rl>\ ytra-7 1 co -88- fab t y’-f- K - (45) 6109. 3 ) Gibson, B. W„ Biemann, K. (1984) Proc. Nall. Acad. 62 t I:Z 0 2(DUmffi&CDtkTzZfto/j. Sci. U. S. A. 81, 1956-1960. 7. tibU (C 4 ) Fukuhara, K., Tsuji, T., Toi, K„ Takao. T.. Shimonishi. FAB^it^CZa^y^F, $6^(7)-^:#^ Y. (1985) J. Biol. Chem. 260. 10487-10494. 5 ) Takao, T„ Kobayashi. M„ Nishimura, 0.. Shimonishi. Y. (1987) J. Biol. Chem. 262, 3541-3547. 6 ) Carr, S. A.. Biemann, K. (1984) “Methods in Enzymolo- K, gy ”, ed. by Wold, F., Moldave, K.. Academic Press. 4i&, London 106, 29-58. 7 ) Takao, T.. Yoshino, K„ Suzuki, N.. Shimonishi, Y. (1990) C, (fmol) oma^crr Biomed. Environ. Mass Speclrom. 19, 705-712. < smsm&fcznm^tzfz&izu, 8 ) Biemann. K.. Martin, S. A. (1987) Mass Spedrom. Rev. Sib^FTk-e^*), yittii^fa titi&M%f -89- -mi & & ft A molten globule state has been assumed to be a major intermediate of protein folding. The conformational stability of the acidic molten globule state of several proteins and the corres ponding state of a synthetic model polypeptide is determined by the balance of the electrostatic repulsive forces, which favor the extended unfolded state, and the opposing forces mainly hyd rophobic interactions, which favor the compact molten globule state. molten globule state/protein folding/acid denaturation/cytochrome c/amphiphilic polypeptide bfih. ^S«bl±R-fe14 (CD) x h -y y h 7 D - Hmm't'Xa 7^ h7;uy$ > #b X: )\/t's • tf JL — ;u (molten globule) vm t & if til>~4> s 6 ftvmxt&i&vftm zmzmt S&S£Et£l± V Vf-A &(±C#f <%#?)(,'< od' n*.btix^z>. <0S6ST*<)EiIiJ$tL, 3 2 ^ d^ $tLfz. 1. ;ux> • -;mra &&^E;u-r > • ynif *-;u -90- > * yn (123) t#it<>$7fcS£&h H-NMR ^(Eo-CPI^fbnZc. f- h ? 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Z. -95- H5S: 'C75LK©£SS14 (m 3B^f KJA^ESEii) ##a^g{b## ±56* 1. ktbtotc ^-J=f- K A^t"£ 2. «fc <5m6^,T(''d«k ■) tc, #* V Km^fSttESE©®!#©—o i:4ofc4\ -e©ESE®@T-©'^? K©iFSKE©iH#ttft^©E5EtcAS4'BS%4- xfc. #*3 3©E%4 6x (i@#) -etui ©£3?£tttfpiMCtfcl*S*i,TV'5. -®totcti:jE®K# - #E# • IS# • m#« • * s#43* tcA®stir^4\ 3j%6©m#c*f 4yf^ym 4i:©-@B©#E#A%&MM%©A©9-y'v h h UTtti Stlfc. (t*i:'ts 3tT,6©E5E$raCT*l3 • Jf 7 3ittit5#XM7S t' 3. m%kmA#©mt^ #@4#A&6 a ©ffiEfEffl&ffi®Ttotfc'*4 4-BT-Hfb#&#^K47^0-^C4 V 3^6©%#(cM-#-f 6±gaK#%4u < ■oA'Sl'i jiT 3tl?)ffl4'4(ctt^A^ K#{bo'%ti#& ^l-T £> U x MAKFerrichrome-^ A f A#©4 3 4siderophorettflfi#)#: ^©^S-f A>©Eatcl$4$S]%iSDTl'-5„ 1 — 97 — —3^ 6phytotoxinKA#©##^#Mm©#^@©*%Bf 1: L T###%#©chemical determinanti: UTlSc#< ©WSEMdASi-5„ -E"tv-> fflWStc ^ k mrnrnAtm* 6dt6 , KS-e-©Stt^#®#1±kifeai,Sll©M«65g-$-ti>i)xT-S$41lb^'ti>':S6t" SfflT% if phytotoxinfflffi&tco t'TM^-5 t i: =fa KR i-dt6©3%f%&%&t©lco l'T®t5. &A Fig.l f 6fb; 3 — 1. ##P#jK#©7#3l##ft (Host-specificity of plant pathogenic fungi)3’ m) 5 ^ O-Ol/X : j!y\3 V'=b*>S? : -Y 4- : g £tfS 2. k < C###%-pl4#M#(c*LT|m|—(SSttsusceptible, S) SttkffiSttt (resistant, R) 6#A'4)5 . #j) :%+me (s> , mtas (r> V : -i > K • -r’J (S) , H$ (R) x>JR-7 Victoria blight# : 'd-f’/MJf (Pc2, S). mm (P c 3 8, R) 3 • BattSf* : =»xu i>5-=>:7T-f hTV*i>>©4-6-S; (RSffiSiStS) 4. mm&&m -. =>H S T, -9-y v -y =>E*SfitS{E©Flk =>@3EOTtcS^t$W»!$-5©r-tt4'(■'*'? 2 -98- 3 — 2 . #### (Phytotoxin) $\) ivA lv U >, t?7'/>E Marasmin, Tentoxin, Marformin, Cyl 2 (H s T) : Host-specific toxin(Host-selective toxin) 2. 3. 4. ^tcnsT^. 5 . H S T ^ k (#) . #J) AM-toxin 1, 2) : U > ternaria mali ) AK-toxin 1, 2) : ± i>#%#(Aiternaria kikuchiana) HMT-toxinn : h n u f> ilTifc l&^&tlffiiHelminthosporium maydis race T) HC-toxin 1) : h 0 HrO u i> ^^{’t^tl^iHelminthosporium carbonum) Victorin(HV-toxin) 5, 6>6, 7>7, 8-8,9, 9 ’ 10, 11. 12) : x y )\>y Victoria blight^ (Helminthosporium victoriae) W.fy)(D]mMifot LT^, K • 7^^^7X7 • &BS (A^f'J7) ^phytotoxinT^^^- KM 4 — 1 . Coronatine : Pseudomonas syringae pv. coronafaciens 4a##: vibotoxine ^coronafacic acid, coronamic acid/z Id T‘Id:##ti:& 0 coronatine 3 -99- 4—2. Rhizobitoxine : Rhizobium japonicum {X 4 X(DW&M) : irreversible inhibitor of ethylene biosynthesis j£jg : H NH H NH rhizobitoxine 4 — 3. Tabtoxin &MM : Pseudomonas syringae pv. tabaci : jS — LactamSiS HN- *OH CH- R I CH. CHOH h2n-ch-co-nh-ch-co2h R=CH^ tabtoxin R=H (2-serine)tabtoxin 4 — 4. Phaseolotoxin : Pseudomonas syringe pv. phaseolicola : irreversible inhibitor of carbamoyl transferase : phosphosulfamyl tripeptide ? nh-p(oh)-o-so,nh2 t^N-CH-CO-ala -h. arg Phaseolotoxin 4 -100- 5 . 6 phytotoxin A)###### 5 — 1. Fusaric acidhPicolic acid %£.MM : Fusarium oxysporum f. sp. lycopersici Pyricularia oryzae R = (ch 2)3 ch 3 R = (ch2)2 ch= ch2 R = (CH2)2 CH(OH)CH3 R = H 5 — 2. Marasmin OeMM * Fusarium oxysporum f. sp. lycopersici R, Rz NH-----CH2- - CH ---- R co 2h ch 2conh 2 co 2h ch 2ch (nh 2)co 2h co 2h ch 2co 2h ch2—-CH NH co 2h H H CH(C02H)CH2C02K co2h co2h r2 C02H CH(C02H)CH2C02H 5 — 3. Tentoxin &MW : Alternaria tenuis : chloroplast#%f H Ph 5 -101- 5 — 4. Malformin : Aspergillus niger : % • ##(malformation) I 0 0 L D L I NH — CH — CO — NH — CH — CO — NH — CH — CO — NH — CH — CO — NH — CH — CO CH, CH, CH CH- / \ CH, CH, ch3 c2h5 CH / X CH, CH, 5-4. Cyl-2 AAc-MM : Cylindrocladium scoparium yy * — i> 3 > & tent ox in (egg UTIf'So HC-toxinCQJjf £:,iL CH] /CH3 CH 0 H ,X,= /=x / v4!--" \ 2—/>—(CH2)n""C—H d) y (3) H—C—-R ^ \ y' \ / -c. y (2) x n - 0^ X ■ X Y z n R Toxicity(ug/mi ) 1 AM-toxin I 0 =ch2 0CH3 3 ch3 0.002-0.0002 2 AM-toxin II 0 =ch2 h 3 ch3- 0.02 3 AM-toxin III 0 =ch2 OH 3 ch3 . 0.02-0.002 4 (L-Ala2)AM-toxin I 0 -CHj , ••••H och3 3 ch3 >100 5 (D-Ala2)AM-toxin I 0 -H ,■••• CH^ 0CH3 3 ch3 5-10 6 (L-Tyr-MeV)AM-toxin I 0 =ch2 .0CH3 1 ch3 10-20 7 (L-Tyr-Me]l-Ala 2)AM-toxin I 0 -CH ,— H • 0CH3 1 ch3 >100 8 (L-Tyr-Me]o-Ala 2)AM-toxin I 0 -H,—CH. 0CH3 1 ch3 >100 9 (L-Amb^)AM-toxin I 0 =CH2 0CH3 2 CH3 0.01 10 (L-Arah^)AM-toxin I 0 =ch2 0CH3 4 ,1.0 , 4 CH3 11 (L-VaV)AM-toxin I NH »CH2 0CH3 3 ch3 0.1 12 (L-Tyr-Me]l-Val 4)AM-toxin I NH =ch2 0CH3 1 0.1 , *5 CH3 13 (L-Abu )AM-toxin I 0 0CH3 3 CH3CH2 0.1-0.01 / 3 =CH2 14 (l-Leu. )AM-toxin I 0 =ch2 0CH3 3 ch2ch2ch(ch3)2 0.01 , n 15 (L-Phe )AM-toxin I 0 : =ch2 och3 3 CH2Ph 0.001 6 -102- : U y'zM&M-MMiAlternaria mali) %# 5 — 6 . AK-toxin 1, 2) : i~ i/HkffiM(Alternaria kikuchiana ) m#m (K+(D#^) AK-toxin [2,Z,2] ^Z@^^ftTn^^,AF- toxinTkmW#m##&a [2,2,Z] , [2,Z,2] , [2,2,2] #3######m#;E2ft Tf)U, -eft^(Dnfftt^^|^^(D^V^@^%&^7(7)r, Zft6m^fFlK#^^m ^#&& u (D^fbk 6 ft, z ZCDcko^mAf)^, foftfoft^AK-toxinll (lb) ^^U±^T,^fb#^7^a 1) t. Kndp b V ^yijJi^yMcoyji^r^- Jiffiftitty j^y • yx> • h’J^y^T'ti Z-tjffi&rfm&iDtl, -£ (Dfe&te z (DMffTigtni- £ (DT% T)\s>T — M^brt'5, 2) ^%(D^;bzK^x;b^^%.%7MbUTt%%c^^{b^^v\. zcdz t\$Wfyifc\H 3) ##%#%%=#, k:Ka4rt/3:^jrX^;i/^>m(D8,m(D%^@dg (%S) 7 -103- <6> 5 „ 4) 75 JWffiftC O R'o COOH COOH (la) AK-toxin I : R = CH3 (2a) AF-toxinl :R = COCH(OH)C(CH3)zOH (lb) AK-toxin 11 : R = H (2b) AF-toxin II : R = H (2c) AF-toxin III : R = COCH(OH)CH(CH3)2 R OH 0 COOH (3a) ACT-toxin lb : R = OH (3b) ACT-toxin lib : R = H (S3, t: F h3rtS^a^]JU;Tx>^;£'at;'S-^<:yd-n — <7 (D mi#,#) (^^) 8 —104 — 1. R 2 R i Configuration Toxicity of C i and C 2 to Plant Ac-L-Phe -CH=CHCOOMe= R, 5 + Ac-D-Phe -CH=CHC00Mez R, S + Ac-L-Phe -CH=CHC00Mez R, R — Ac-D-Phe -CH=CHC00Me= R, R — Ac-L-Phe -(CH=CH)2C00He‘ R, S + Ac-D-Phe -(CH=CH)2C00Me‘ R, S + Ac-L-Phe -(CH=CH)2C00Me * R, R — Ac-D-Phe -(CH=CH)2C00Me‘ R, R — Ac-L-Phe -(CH=CH)3C00Me‘ R, S + + Ac-D-Phe -(CH=CH)3C00Me’ R, S + + Ac-L-Phe -(CH=CH)3C00Me‘ R, R — ' Ac-D-Phe -(CH=CH)3C00Me‘ R, R — Ac-L-Phe -(CH=CH)3C00«e' ' R, S + + Ac-D-Phe -(CH=CH)3C00Me* 1 R, S + + Ac-L-Phe -(CH=CH)3C00Me' * R, R — Ac-D-Phe -(CH=CH)3C00Me'x R, R — ( + ) «i 0 - = mo 1, (+ + ) a 1 0 _4mo 1 necros i 0 ( 1 ) tttrans.trans-transJb'ctt/trans-trans-transi^o ( 1 z) ti: trans-ci s~trans££0 9 5 — 7. HC-toxin §£jOM : h id^tlM(Helminthosporium carbonum ) SiE : j: 3 (aK CH, H I \ . u A °vN' l 'V CHj-C ■ H H C— (CHjjs— C — CH — CHj /°\ \ / \ H-C —(CH,),-C —CH —CH, "'%-4-»'s’ — C-H H-C — \ / C^ HC-toxtn H-C V revlsad by // "~~C" 0 1 H HC - toxin Walton «/ of. (l982) CH 3 proposed by Llesch Qro• a «f of.98 2) .1 Of. (1982) Pop* «f of.^1983) Kawil ./ of.(l983) Rich .f of. (1983) \ C- < H N" V N" / \ A Q-CH,—C H H C (cH , ] j— C — CH — CH h\ CH. /-A Cyl-2 Chlamydocln CH, Hlrota »f of. (1973) Closse «f of. (l9 74 j OCH, H \H 0 z° CH, o o \ Z X II / \ II / \ H — C —(CH, J, - C — CH — CH, CH CH, — C — H H — C — (, CH, j s- C — CH — CH / CH, u/< H C Cyl-1 o I H WF 3161 CH, Tekayam a tr of. ^19 8 4 ) Umehahara «f of. (1983) OCH, 1 0 -106 5 — 8. Victor in (HV-toxin) : x yA ^Victoria blight^S (Helminthosporium victoriae ) : glyoxylic acidu^^^U =>[EI4#M Victor in C-receptor 6Q#]2jg13) 125I-Victorin Cobinding protein(lOOKDa) & R, S # u t 2 - 2 & # ^ c ^3 n % ( ?) HO-C-OH COOH NH Me' CHCI 1 Vlctorin C ECjq = 37 nM 6 . -ic CD# 6 — 1 . bennzoxazinone#^$: #3 phytoalexin avenalunim I dianthalexin (benzoxazinone) (benzoxazinone) — ^ — ^ 3 >(-TT >-3#) 1 1 -107- 6-2. HERBICIDES containinmg C-P bond O HO-P-CH2-NH-CH2-COOH OH glyphosate synthesis inhibitor of 5-enolpyruvylsikimate(EPSP) synthase 0 ho -p-ch 2-ch 2-ch -cor 1 z z I OH NH2 bialaphos analogs natural products inhibitor of glutamine synthase(GS) ch 3 ch 3 R = NH-CH-CO-NH-CH-COOH bialaphos Streptomyces hygroscopics R = OH L-isomer : phosphinothricin (active phytometabolite) DL-form : glufosinate (active phytometabolite) H3C, .CH3 CH ch 3 ch 2 R = NH-CH-CO-NH-CH-COOH phosalacine Kitasatosporia phosalacinea O O OH HoC-P-CH-COOH synthesis CoH50-P-0+NH4' synthesis J i CONH2 CH3 fosamine inhibitor of keto-acid reductoisomerase 7. t>"?Zf (phytotoxin) -5 #### 1) {b#, 39, 161-167(1984) 2) ±^m^, ISSG^g^#-, 72-83^-iv. 3) (fb#'l#fJl 1 4 ) 13-22(1988). 4) R, P, Scheffer and R. S. 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Hlyasaka6101 % Chi a ©S#EBt, LB K&jSOS n-Sn02 *$k;8«U, 12* TJfcSSIrtHSJLfe. 3.2 5nn7-(M K-f 5<-?-a!)fc«?lS] Tang k Albrecht"’lt^*a«±IC«*a-eSiaSnfc (Chi a - 2HaO)n ®-ktC(±SB8jK©/J'S% A1 6KSltlCtraT«JSbfc'tl->K'f Ag/ Chl/Al SftiMKUfc. Dodele16'91 It Ag/Chl/Al -fe;i/©7 n n 7-f VI/7 * - A ©S v&ge< t/?-© Chi a t•)t>-7jcfaS-6-fl: (cht a• 2ii20)n ©*6^i6v$ #s$6*&#^4r kl:#WLfc. totifctv^’itt*^'; k'-;u7;vn-;i/7 7;i/At h>SS»'6*-vxh UTt#fc Chi a Sfflk®StlS-9->H'l' y?"lzVl' Au/PVA/Chl a'Au $c^#L, S at^fflkyefi«*&«aiLfc. chi a/pvA#mi:/\f ( @7) 3.3 *#f***-##6# ®fi*SK«j8a(bS^TW6K#astb4rKU tfn-;iz, rKU-N-^vi/fcrn-vi/, rKV (N-k'-;i/A;Vr 650 mV, 190 nA (}I6H81 t=0. 17 mW cm"2) , 7-f 71/77 77- 0.25, *#%##* 0.018 X &£*.£. -#, ^fcMt'fcS^l*B8@IIS«i8«E 560 mV, 56 nA Oti568tr=0. 17 mW cm2) , 7 7 71/ 7 7 7 7 - 0. 23, %*#ft #* 0.001 x r*U, )t****EkUTtt l©*fi:)tx-c#&A% k U-CttffiATillvfcgMt&jjt't. 011 (a,b) ®77’>a >7.^7 t'71/©Jfc«A'e>, l©SEtt PMeT/ lC (57/A1 #SJfcl*Mk bT« < fc©^#-Ac#«£Jt!t4^.fck %X>=h& (012) . c©ckl±*ai&#©afG8&#$#l:&^-Ct'&c:k# 4. fcfcVlc )t ^ sa» S: ^ r 71/k-r S « « 8 ft 3d ? © ff 38 tt * A--t © »i: 71 > fc « A> U r * ■£ AS tl3kkS < %«A'S3^Stv5flf38^5T-6 5. 6tv6tv(t±#Af © @ #B&#mLT%m»7k-#ft%ft%?AEi//<7i/?©K9;#m&#ftL, ttv&7 z/t7S;fc'J:tJV$fclt£&liic@5gl,, S6tc-ttvS:1ISKJ;r,r*ffiSSWfc li«H$«^E»SllcE0 • &kt'3#&ft-cm:&m&j:3k UTt'5. L BK&l:A't&©@f}f #ftk%#*%/\©E0 • B&8 ©WmA'g$ tv*. *fcmS£(&lc,*oTtS6ivSMM*ife£E©iB#E#ftkAE3dE • /W ^3d?/\©fGmtft#A'tT&. * n*> tv it m k - t” s ti ic w# 63d&*^##?ftfti/t#ftr.ftf!imA;#%A:*m8ft©#T&6c:k&aaL fcAS Z©3d?&%Al&&©j:7& 100 X lc)Sv'i6S)te«$fti8i*, SSA©t>©lc -fSfcfttcli, ft#©#0&#% LTD: A'»Wtv«% (,»!'. ^tv6®E3EI±M 89tc*U>*raft*©MIE&'kt)i5;dt)©-e64. -112- mx® 1) C. S. French et al., Plant Physiol. 49, 421 (1972). 2) K. Uehara et al., Chem. Lett. 1981 . 1643; Photochem.Photobiol. 48* 725 (1988). 3) K. Uehara et al. , Chem. Lett. 1991 . 909. 4) J. Deisenhofer et al. , Nature 318, 618 (1985). 5) Y. Yasuda et al., Thin Solid Films 210/211, 733 (1992). 6) L. L. Shipman et al. , Proc. Natl. Acad. Sci. U. S. A. 73, 1791 (1976). 7) K. Uehara et al. , Chem. Lett. 1982 . 1445; Pho tochem. Pho tob iol. 53, 371 (1991). 8) K. Uehara et al., Chem. Lett. 1985 . 897. 9) H. Toribuchl and M. Calvin, Photochem. Photobiol. M, 95 (1971). 10) T. Miyasaka et al. , J. Am. Chem. Soc. 100, 6657 (1978). 11) C. W. Tang and A. C. Albrecht, J.Chem. Phys. 62, 2139 (1975). 12) J. P. Dodelet et al. , Photochem. Photobiol. 29, 1135 (1979). 13) K. Uehara et al., Chem. Lett. 1984 . 1499. 14) K. Uehara et al., Chem. Express 2, 129 (1987); 1987, 2236; J.Polym.Sci. Part C Poylm.Lett. 26, 95 (1988). 15) K. Uehara et al., Denki Kagaku 57, 1121 (1989); 47, 909 (1990). 16) K. Uehara et al., Proceeding of 2nd Internal Inal Simposlum on Chemistry of Functional Dyes (1992.8. Kobe) in press, 17) K. Uehara et al., Thin Solid Film 215, 123 (1992). -113- cooch3 COOR COOR (Phytyl) 0 1 7 a a y j A: Chl a (r3 = ch3) ; Chi b (r3 = cho ) B : >'C^rU*^nn7^;i/b (BC) (BP) t* Mg Wavelength,nm 677.2 8.6 669.4 9.8 661.5 It.l 683.6 9.9 649.2 11.7 693.3 13.0 r 702.5 Error X8.I9 —.—i------j—|—1------'—f—1------'------r —i— 160 155 150 145 140 Wavenumber, cm"1 XIO"3 @2 Scenedesmus ® 0 O D 7 V X b fr 6 <1 6 tl tz 7 V T T 0 a a V -f JUS 6 fc $ ffl iSiRX'-t? Hi/" —114 — @3 m 4 9 51 600 a Absor bonce . n HU (b) BSA v 4 (A) ...... 650 Wavelength ;u Wavenumber Error a * /^Mr)\yzf Bel R. ( 700 x V 10 (kK) ir He2 (nm) id ) 750 is Ee3 i MU He (B) 4 (a) (6 v/v BC Wavelength Wavenumber Error x 700 BP 7-b ( hvtf) P« x Ob (nm) 750 8 # (kK) ) ro«iRx (b) 0—CM (a) (b) 0 5 n D 7^1/O^fil/ (a) P700 Katz661 ©t r;i/ (R’=H) (b) P680 A-683 BfW (e = Hg) A E SEMICONDUCTOR ELECTROLYTE 06 @2 9 0 o 7-f j:&**B#©jRW Chi: VB: CB: E*W, Ep: Red: i#A#j©#? (/"VP, Ox: -116- Transparent Electrode Chi a pva -h 2o Chi a Glass Plate @7 Chi a/PVA / A (&$) r ♦ xe xEt^N* undoping ------► <------xEt4NCl04 doping 08 PMeT (K-f) ®JRje**-A PMeT 3 : R-CN 4 : R-CH Glass Plate @9 yKKD7itJ/>J!»# (1-5.) ait&m&t PHeTZ (1-5) -fe/U©*l! -117- @10 3» : » % Au/PMeT/(l)/Al o C \ as T3 Arb. unit 3C CD T3 . T JB W 3C M CD ■£ —i X *£ > < (D 0*' 3 H X ) (A rv 3> c t -r ^ = T # m % V m" Au/PMeT/(5)/At 3> C ten 00 9- Arb. Unit \T' cv I n> P / t 3 o N o Current / |iA V) » < ) (B (D Kr *-» m 3 i rr ©S3E-SE49tt tfl 009 V ft i ° j>» a 3* o ten X 700 3* o | >. X »- 3 T 800 p i o O Vi !° X (a) A1 \ (b) \ PMeT A1 @12 Au/PMeT/ (1)/A1 (a) t Au/PMeT/(5)/Al (b) -119- LETTERS TO NATURE 10-7 S m2 J~' for H2P and ZnP respectively at room temperature. maximum jump times of 0.6 and 2 ps for solid ZnP at room The presence of the central zinc atom therefore seems to increase temperature and the mesophase at 100 °C, respectively. the conductive properties of the solid slightly. Our results confirm that the transport of charge can occur In Fig. 2, (Acr/D)0 is plotted as a function of temperature. very rapidly within an organized self-assembly of porphyrin For H2P, a single, sharp decrease occurs at the transition from moieties even in a relatively flexible, liquid crystalline phase. the solid to the isotropic liquid. No conductivity could be Such peripherally hydrocarbon substituted compounds may detected in the isotropic phase. For ZnP, the conductivity therefore be considered to provide model systems which decreases abruptly, but does not disappear completely, at the crudely mimic the channelling of energy and charge found in solid to liquid-crystal transition temperature. The conductivity the highly varied antenna chlorophyll arrays of photosynlhclic in the mesophase is a factor of —3 less than in the solid with membranes. ■ ' ■ . ' ■ • □ (Atr/£>)„"= 0.5 x 10-7 S m2J~'. This increases slightly with fur ther increase in temperature in the mesophase and then suddenly Received 9 July; accepted 13 September 1991. drops to zero at the clearing point of ZnP. 1. Gregg. 0. A_ Fox. M. A. 4 Bard. A. J. J An chem. See. Ill, 3024-3029 (1989). We conclude that the long-range columnar order of porphyrin 2. Weber. P. Guidon. D. 4 Skoulios. A. Ua CrysL 9. 369-382 (1991L ' '' moieties in the solid and liquid-crystalline phases is essential 3. War man. J. M. 4 dc Haas. M. P. In Pulse Radiolysis. Qu. 6. ted. Tabala. YJ 101-133 (CRC, Boca Raton. 1991). for the conductive properties observed. In the isotropic liquid 4. Warmnn. J. M. 4 do Haas. M. P. RatSaL Phys Chen 34, 581-586 (1989)." phase, any molecular organization that might occur must be of 5. Elcy. O. D. Atofcc. CrysL in CrysL 171.1-21 (1989). f . ' 6. Simon. J. 4 Andrt. J-J. Molecular ScrmayxAxlors (Springer. Berlin. 1985). such a limited temporal or geometrical extent that it cannot 7. Pietro. W. J. Marks. T. 14 Rainer. M. A. 2 Am. chcm Soc 107. 5387-5391 (1985). support charge conduction. The results provide definitive 8. War man. 1M- de Haas. M. P. van dor Pol. 1F.4 Orcnlh. W. Chen i Phys Lett. 164,581-586 (19891. 9. War man. AM. In The Study of Fast Processes and Transient Species by Electron Pulse Radbtysls evidence, therefore, for the general supposition that electrons (eds Baxcndale. 1 H 4 Bust FJ 433-533 (Rddd. Dordrecht. 1982). and/or holes can move rapidly along the axis of column-stacked 10. Schmidt W. F. 4 Allen. A. 0.1 Phys Own 72. 3730-3736 (1968). " 7r-systems 5,6. The decrease in conductivity on going from the 11. Shinsaka. K. 4 Freeman. C R. Can 2 chem 52. 3495-3506 (19741 • • 12. Cox. C. A. 4 Knight P. C. 1 Phyi C 7. 146-156 (1974). crystalline solid to the mesophase is attributed to a lower charge 13. Molt N. F. Conduction in Non&ystaHine Materials. Ch. 6 (Clarendon. Oxford. 1987). mobility. This is probably due to an increase in positional ACKNOWLEDGEMENTS. We thank M. Northotl and S. 1 Pkhen (AKZO Research Laboratories. Arnhem) disorder of the porphyrin moieties7 on melting of the hydrocar for X-ray diffraction analysis of the ZnP compound and for helpful dscusskms. The synthetic work bon mantle. was sporfsored by the Texas Advanced Technology Program. ' We have found a similar decrease in the radiation-induced conductivity at the solid to mesophase transition of octa-alkoxy- "substituted phthalocyanines. An isotropic liquid phase of these compounds is not, however, attainable within the temperature A low-cost, high-efficiency solar range of our equipment. They could not therefore be used to cell based on dye-sensitized carry out the conclusive experiment of completely melting the material to sec if long-range order was essential for conduction. colloidal TiOz films The experiments on octa-n-alkoxy substituted phthalocyanines have, however, shown that the lifetime of the conductivity Brian O’Regan* & Michael Gratzelf transient increases exponentially with the length of the alkyl tails*. This is taken to be strong evidence that the mobile charge Institute of Physical Chemistry, Swiss Federal Institute of Technology, carriers responsible are restricted to rapid diffusional motion CH-1015 Lausanne, Switzerland along the phthalocyanine axis of a stack, with eventual charge recombination requiring stack-to-stack electron tunnelling The large-scale use of photovoltaic devices for electricity gener . through the hydrocarbon mantle. ation is prohibitively expensive* at present: generation from existing The absolute value of (Acr/D)0 is related to the mobilities, commercial devices costs about ten times more than conventional /x, of the charge carrcrs, Y. V- — (m(~) + m(+)). and the average methods'. Here we describe a photovoltaic cell, created from low- energy required to produce one charge-carrier pair, £p (in cV), to medium-purity materials through low-cost processes, which by 3-4 exhibits a commercially realistic energy-conversion efficiency. The device is based on a 10-p.m-thick, optically transparent film of (Acr/D)0-2>/£p (1) titanium dioxide particles a few nanometres in size, coated with For organic compounds, the total initial yield of electron-hole a monolayer of a charge-transfer dye to sensitize the film for light pairs corresponds to .a value of £p of —25 eV for high-energy harvesting. Because of the high surface area of the semiconductor radiation9 "11. Only a fraction of the initial electrons and holes film and the ideal spectral characteristics of the dye, the device will escape rapid (subnanosecond) geminate recombination* 9 harvests a high proportion of the incident solar energy flux (46%) and diffuse to separate columns, thus being observed in the and shows exceptionally high efficiencies for the conversion of present experiments. Using a value of £p = 25:cV together with incident photons to electrical current (more than 80%). The overall the experimentally determined value of (Acr/D)0 will therefore light-to-clcctric energy conversion yield is 7.1-7.9% in simulated yield a lower limit to the charge-carrier'mobilities. For the solid solar light and 12% in diffuse daylight. The large current densities phase of ZnP at room temperature, assuming electrons and holes (greater than 12 mA cm"2) and exceptional stability (sustaining to have almost equal mobilities'2, this gives jjl> at least five million turnovers without decomposition), as well as 2.6 x 10-A m2 V-1 s~' and for the mesophase, it gives p.> the low cost, make practical applications feasible. 0.6 x 10-6 m2 V-1 s_l. Solar energy conversion by photoelectrochcmical cells has This order of magnitude of the mobility indicates small been intensively investigated 2"". Dye-sensitized cells differ from polaron motion 13 corresponding to the hopping of a more or the conventional semiconductor devices in that they separate less localized charge between neighbouring sites with an average the function of light absorption from charge carrier transport. jump time between sites of Tj. The mobility measured in the Ini the case of n-type materials, such as TiOz, current is generated present, randomly orientated columnar materials is related to when a photon absorbed by a dye molecule gives rise to electron Ti by injection into the conduction band of the semiconductor, Figi 1. To complete the circuit, the dye must be regenerated by Tj = edf/6k0Tfj. ' " (2) where is the distance moved along the columnar axis per jump (4.9 for the present systems). The minimum values of • Present address: Department of Chemistry. University of Washington. Seattle. Washington 98195, A USA. the carrier mobilities given above correspond therefore to t To whom correspondence shtxid be addressed. NATURE • VOL 353 - 24 OCTOBER 1991 737 120- LETTERS TO NATURE electron transfer from a redox species in solution which is then easily. Films of 10 pun thickness consisting of particles with an reduced at the counter electrode. The monochromatic current average size of 15 nm gave linear photocurrent response up to yield full sunlight and were used throughout. A cubic close packing 7)i(A) = LHE(A)x (/)inJx rjc (1) of 15-nm-sizcd spheres to a 10-p.m-lhick layer is expected to produce a 2.000-fold increase in surface area. where LHE (light harvesting efficiency) is the fraction of the Absorption spectra obtained for such nanostructurcd Ti02 incident photons that arc absorbed by the dye, d>inj is the quan films arc shown in Fig. T J) % m films n re-bran spa remand colour tum yield for charge injection and rjc is the efficiency of collecting less displaying the fundamental absorption edye of anatase the injected charge at the back contact, expresses the ratio of (band-gap 3.2 eV) in the ultraviolet region. Deposition of a measured electric current to the incident photon flux at a given monolayer of the trimcric ruthenium complex' 5-1'’, RuL2(p.- wavelength. The photovoltage AV in Fig. 1, generated by the (CN)Ru(CN)L2)2, 1, where Lis 2,2' bipyridine-4,4'-dicarboxylic cell, corresponds to the dillcrcncc between the Fermi level in acid and L' is 2,2'-bipyridine, results in deep brownish-red the semiconductor under illumination and the Nernst potential coloration ofjhc film. The absorption onsetTs shTTtecTTtr?5Q ttttt, of the redox couple in the electrolyte. -• tRe light harvesting efficiency reading almost 100% in the whole Although attempts to use dye-sensitized pholoelectrochcmical visible region below 550 nm.TrUegration oi the spectral overlap cells in energy conversion have been made before, the efficiency ^ctwirn-a snlar_gmission of AMI.5 and this absorption band of such devices has been extremely low and practical applica shows that 46% of the incident solar energy flux is harvested tions have seemed remote. One problem is that of poor light by the dye coated film (AM = 1/sin cr where o is the angle of harvesting. On a smooth surface, a monomolecular layer of incidence of the solar rays at the Earth’s surface). sensitizer absorbs less than 1 % of incident monochromatic light. The optical density of the film at 478 nm corrected for the Attempts to harvest more light by using multilayers of dye have absorption by the conducting glass support was 2.45. Dividing in general been unsuccessful. The remaining option is to increase by the extinction coefficient1'' of 1 (e47,= 1.88 x 107 cm2 mol-1) the roughness of the semiconductor surface so that a larger yields the dye surface concentration, f = 1.3 x 10~7 mol cm-2. number of dye molecules can be adsorbed directly to the surface As each dye molecule occupies an area16 of 1 nm2, the inner and can simultaneously be in direct contact with the redox •surface of the film is 780 cm2 for each 1 cm2 of geometric surface. electrolyte. Matsumura el aO2 and Alonso et al.li have used Thus, the roughness factor is 780, which is smaller than the sintered ZnO electrodes to increase the efficiency of sensitization predicted value of 2,QQ0. The dillcrcncc is attributed to necking by rose bcngal and related dyes. Willig, Parkinson and col between Ti02 particles. In addition, the large size of 1 prevents leagues 14 have reported high quantum yields for the dye sensitiz its access to very small pores, reducing the apparent surface area. ation of SnS2. But the conversion yields from solar light to The photocurrent action spectrum obtained with the dye- electricity remained well below 1% forthesesystems. In addition, coated Ti02 film is also shown in Fig. 3. It closely matches the the instability of the dyes employed presented a severe practical absorption spectrum, indicating that the current is due to elec drawback. By using semiconductor films consisting of tron injection from 1 into the conduction band of Ti02. The nanometer-sized Ti02 particles, together with newly developed photocurrent yield measured at 520 nm was found to depend charge-transfer dyes, we have improved the efficiency and stabil ity of the solar cell. High-surfacc-area Ti02 films were deposited on a conducting glass sheet from colloidal solutions. A transmission electron micrograph of the colloid is shown in Fig. 2. Electronic contact between the particles was produced by brief sintering at 450 °C. The size of the particles and pores making up the film is control led by the size of the particles in the colloidal solution. The internal surface area of the film is determined by the size of the particles and the thickness of the film. These parameters were optimized to obtain efficient light harvesting while maintaining a pore size large enough to allow the redox electrolyte to diffuse semiconductor dye electrolyte conducting glass counterelectrode FIG. 2 Transmission electron micrograph of Ti02 particles used in thin film production. The scale bar represents 10 nm. Particles were prepared by hydrolysis of titanium tetraisopropoxide 18-13 followed by autoclaving for 12 h at 200 °C. To form films the sol was concentrated by evaporation of water in vacuum at 25 *C until a viscous liquid was obtained. Carbowax M-20.000 (40% by weight of Ti02) was added and the viscous dispersion (Ti02 content 20% by weight) was spread on the conducting glass support (Asohi glass, fluorine-doped Sn02 overlayer, transmission 85% in the visible, sheet resist ance 80/square) to give a membrane of 10 p.m thickness. This was heated FIG. 1 Schematic representation ot the principle ot the dye-sensitized under air for 30 min at 450 °C. High-resolution scanning electron microscopy photovoltaic cell to indicate the electron energy level in the different phases. revealed TiOz films to be composed of a three-dimensional network of The cell voltage observed under illumination corresponds to the difference, interconnected nanoscale particles19 . Transmission electron micrographs A % between the ouasi-Fermi level of TiO, under illumination and the of TiO, particles were taken before heat treatment; the annealing of 450 “C electrochemical potential of the electrolyte. The latter is equal to the Nernst did not Induce significant changes in particle size. Before dye coating, a few potential of the redox couple (R/R-) used to mediate charge transfer between monolayers of Ti02 were electrodeposited onto the colloidal Ti02 film from the electrodes. S. sensitizer; S*. electronically excited sensilizer;S*. oxidized a Ti(lll) solution. Detailed description of this procedure will be published sensitizer. elsewhere (L. Kaven. B.O'R., A. Kay and M.G.. manuscript in preparation). 738 NATURE • VOL 353 • 24 OCTOBER 1991 -121 LETTERS TO NATURE on the counter ion of the iodide/triiodide redox electrolyte , increasing from 68% for.tctrapropylammonium to 84% for Li* . After correction for the light absorption by the conducting glass. the yields are 80% and 97%. respectively. This shows that the nanostructured TiO-, films used in conjunction with suitable charge Transfer dyes can achieve quantitative conversion of visible light photons into electric current! Figure 4 shows the current-voltage characteristics obtained • with the thin layer cell under illumination by simulated AM 1.5 solar light. The conversion efficiencies for ond tenth and full sunlight are 7.9% and 7.12%, and the fill factors (maximum output power of cell +■ [short circuit current x open circuit vol tage]) are 0.76 and 0.685, respectively. Similar yields were obtained under direct sunlight (measurements performed in June early in the afternoon on the roof of the institute). Under diffuse daylight the efficiency increased to 12%, indicating that under such conditions the cell performance is better than that of conventional silicon devices. This is because the spectral distribution of diffuse daylight overlaps more favourably with the absorption spectrum of the dye-coated Ti02 film than direct sunlight. The.fill factor of the cell remains above 0.7 even at very low light intensity (<5 W m-2). Conventional photovoltaic cells have a much smaller fill factor (<0.5) under these condi tions. This indicates that loss mechanisms such as recombina tion, normally encountered in semiconductor photoconversion, have been minimized. This result might appear surprising in view of the disordered structure of our film giving rise to defects. But the role of the semiconductor in a dye-sensitized device is merely to conduct the injected majority charge carriers. There are no minority carriers involved in the photoconversion process. • Cell potential (V) Therefore, surface and bulk recombination losses due to lattice FIG. 4 Photocurrent-voltage characteristics of a cell, based on a colloidal defects, encountered in conventional photovoltaic cells, are not Ti02 film sensitized by 1: the film, supported on a conducting glass sheet, observed in such a device. was used in a sandwich-type configuration. The size of the dye coated TiO, The long-term stability of cell performance was tested by photoanode was 0.5 cm2. The counterelectrode, consisting of conducting illuminating the thin TiO, film loaded with 1 with visible (A > glass coaled with a few monolayers of platinum, was placed directly on top 400 nm) light Co l 2 monihUThe change in the photocurrcnt was of the working electrode. A thin layer of redox electrolyte is attracted into less than 10% over this period, during which a charge of the intra-electrode space through capillary forces. The cell was exposed to 62,000 C cm-2 was passed through the device, corresponding to simulated sunlight with AM1.5 spectral distribution, a, light intensity a turnover number of 5 x 106 for the sensitizer. This implies that 83 W m-2, electrolyte: 0.5 M tetrapropylommonium iodide +0.04 M Iodine if any dye degradation had occurred its quantum yield (<£«,.) is in a mixture of ethylene carbonate (80% by volume) with acetonitrile. Fill factor was 0.76: surface area 0.5 cm2 (before multiplication by roughness less than 2 x 10“\ As d\,ec = A'dee/Xk, the rate constant, fcdccs-1, factor). Conversion efficiency was 7.9%. b. light intensity 750 W m“2. elec for excited-state decomposition (due to processes such as ligand trolyte: 0.5 M tetrapropylammonlum Iodide. 0.02 MKI x 0.04 M l2 In the same loss) must be at least 10~* times smaller than XA, the sum of solvent. Fill factor was 0.684: conversion was 7.12%. rate constants for all channels of dye deactivation. Because charge injection is the predominant channel, this sum is practi- cally equal to the rate constant for charge injection, which exceeds 1012 s~' in the case of 1. Therefore, the upper limit for Adec is 2 x 104 s-1, which agrees with the known photophysics of this class ol transition metal complexes' 7. The very fast electron injection observed with dyes such as 1, combined with high chemical stability, renders these compounds very attractive for practical development. □ * 1 11 u3 o.4o Received 19 My: accepted 20 August 1991. 1. Bucher. K. 4 FrkJte. 1 Phys. Zeit 21. 237-244 (19801. 2. Honda. K. 4 Fujishlma. A. Nattre 238. 37-39 (19721.. 3. Tulls. 0. J. el at. Nature 326. 681-683 (1987). 4. Gerischcr. K Bcctrochim Ada 35. 1677 (1990). 5. Licht. S- Modes. G„ Tenne. R. 4 Manassen. J. Nature 326. 863-804 (19117). 6. Heller. A. Acc cheat, ties. 14. 154-162 (1981). 7. Nozk. A. J. Phil. Trans. R Soc Load. A295. 453-470 (1980). 8. Tributsch. H. 4 OenncL J. C. I etectroanaL Chem 81. 97 (1977). Wavelength (nm) 9. Wrighlon. M. S. Acc cheat. Res. 12, 303-310 (19791. 10. Bard. A. J. Science 207. 139 (1980). FIG. 3 Absorption and photocurrent action spectra of Ti02 films supported 11. Memmlng. R. Phil. Tech. ftpv. 38. 160 (1979). on conducting glass. A. absorption efficiency of the bare Ti02 film corrected 12. Matsumura. M.. Nomura. Y. 4 Tsubomura. K Bud cheat. Soc Japan 50. 2533 (1977). 13. Alonso. N_ Beley. V. M, Char tier. P. 4 Ern. V. Rev. Phys. AppL 16. 5 (1981). for conducting glass background: B. absorption efficiency of the same film. 14. Willig. F. Ekhbergcr. R_ Sundaresan. N. S. 4 Parkinson. 0. A. 1 Ant. ptcni Soc. 112. 2702-2707 coaled with a monolayer of 1: full circles, monochromatic current yield at (1990). short circuit as a function of excitation wavelength. Yield is corrected for 15. Amodclll. R_ Argazzl. R_ Olgnozzl C. A. 4 Scandola. F. 1 Am. chem. Soc 112. 7099-7103 (1990). 16. Nazceruddin. M. K_ Llska. P. Moser. J. Vlachopoulos. N. 4 GrStzcl. M. Helv. chint. Acta 73. 15% loss of incident photons through light absorption and scattering by 1788-1803 (1990). the conducting glass support. 17. Arris. A. Oafrani, V. Oariglclti. F. Campagna, S- Oclzcr. 0. Coord Client. Rev. 84. 85 (1908). NATURE • VOL 353 • 24 OCTOBER 1991 739 -122- Proceeding of 4th International Congress of the World Apheresis Association, 1992. 6. 3-5, Sapporo, in press Nakaji S, et al. DEVELOPMENT OF SPECIFIC IMMUNOADSORBENT CONTAINING IMMOBILIZED SYNTHETIC PEPTIDE OF ACETYLCHOLINE RECEPTOR FOR TREATMENT OF MYASTHENIA GRAVIS Shuhei Nakaji/ Kiichiro Oka, Masao Tanihara, Koichi.Takakura, Masaharu Takamori* Kuraray Co., Ltd., Kurashiki, Japan Kanazawa University School of Medicine, Kanazawa, Japan KEY WORDS: Myasthenia gravis, Immunoadsorption , Acetylcholine receptor, Synthetic peptide, Blocking antibody, Introduction Myasthenia gravis (MG) is an autoimmune disorder in which neuromuscular transmission is impaired by antibodies against the nicotinic acetylcholine receptor (AChR) in skeletal muscle. In order to remove anti-AChR antibodies, plasma exchange and double filtration plasmapheresis have currently been used. However, these methods have ' the disadvantages that loss of useful plasma components is inevitable, and supply of the replacement fluid such as fresh frozen plasma or albumin preparations is required. Therefore, recently the clinical significance of the immunoadsorption has increasingly been recognized. Anti-AChR antibodies are classified into two subclasses; binding antibody and blocking antibody. These antibodies cause the acceleration of AChR degradation, complement- mediated lysis of post-synaptic membrane and blockade of ACh-binding with AChR. The blocking antibody is known to prevent the ACh-binding with AChR, thereby inducing MG. Takamori et al.1),2> reported that the #183-200 segment of the Torpedo californica AChR is the ACh-binding site recognized by a blocking antibody. Based on their study, we have developed the new immunoadsorbent column for MG treatment (Medisorba MG) by using the synthetic peptide (Torpedo #183-200) as an affinity ligand to remove specifically the blocking antibodies. Materials and Methods Adsorbent : The synthetic peptide Torpedo californica # 183-200 (Fig. 1) synthesized by a solid-phase procedure was used as a ligand. The peptide was covalently immobilized on porous cellulose beads (dia. 250pm). The 'amount of the immobilized peptide was 35mg/50ml beads. 123- Nakaji S, et al. In vitro study : (Blocking antibody) The patient plasma 200pl was treated with 50mg of peptide-bound adsorbent for 3hr at 37 * C. A glycine-bound adsorbent was used as a control. The blocking antibody titer was measured by a- bungarotoxin binding inhibition assay. (Plasma protein) 5ml of healthy human plasma was treated with 250mg of the pep tide-bound adsorbent for 3hr at 37 * C, and then the plasma protein levels were measured. Immunoadsorbent device : Immunoadsorbent Medisorba MG consists of a small column, packed with 50ml of peptide-bound adsorbent and sterilized by autoclaving. The safety of the device has been confirmed by various toxicity tests including acute and subacute toxicity, cytotoxicity, mutagenicity and immunogenicity tests. Clinical evaluation : 77 treatments of plasma immunoadsorption were performed for 19 patients with MG. The immunoadsorpt ion treatment was carried out 3 times/week.3’ 183 H-Lys-Lys-Gly-Trp-Lys-His-Trp-Val-Tyr-Tyr-Thr-Cys-Cys 200 -Pro-Asp-Thr-Pro-Tyr-Leu-Asp-Lys-Lys-Gly-OH Figure 1 Amino acid sequence of the synthetic peptide corresponding to Torpedo a183-200 Results and Discussion In Fig. 2 and Table 1 are shown the results of in vitro study on adsorption of blocking antibody and on adsorption of plasma proteins, respectively. The clinical results of changes in blocking antibody obtained with the 36 immunoadsorption treatments are represented in Fig . 3. From both in vitro study and clinical evaluation, it was demonstrated that the peptide-bound adsorbent removed specifically blocking antibody without significantly reducing plasma proteins such as albumin and IgG. The removal rates obtained with clinical studies are as follows: blocking antibody 40.2%, binding antibody 12.4%, total proteins 5.7%, albumin 2.4%, IgG 10.2%. Fig. 4 shows the changes in blocking antibody (anti-peptide antibody) and IgG level over the period of 35 days. Blocking antibody titer was reduced remarkably after the treatments contrary to the slight change in IgG level, and remained at low level for the observation period. It is suggested that this immunoadsorpt ion treatment provides promise to the long-term effect without occurrence of rebound . Clinical treatments improved effectively the myasthenic state. The improvement was found in 78% of the cases, and no adverse effects were observed in any case of treatments. —124 — Nakaji S, et al. 75 g Control 3* ^ Adsorbent 50 E .E 25 10 Cut off value 12 3 4 Patient serum Figure 2 Changes in blocking antibody titer by adsorption treatment(in vitro) Adsorbent : synthetic peptide Torpedo a 183-200-bound to porous cellulose beads Control : glycine bound to porous cellulose beads Table 1 Effect of adsorption treatment on plasma protein levels (in vitro) Adsorbent Control Alb (g/dl) 3.8 3.7 IgG (mg/dl) 1060 1120 IgA (mg/dl) 207 196 IgM (mg/dl) 113 105 C3 (mg/dl) 60 60 C4 (mg/dl) 19 22 TP (g/dl) 5.6 5.6 A/G 2.08 2.01 Adsorbent : Synthetic peptide Torpedo a183-200 bound to porous cellulose beads Control : Without adsorbent Conclusions We have developed the new immunoadsorbent containing immobilized synthetic peptide of AChR (Torpedo CX183-200 ) for MG treatment. Medisorba MG showed the specific removal of blocking antibody , one of the antibodies involved in the pathogenesis of MG, without reducing significantly plasma protein levels. The clinical usefulness of the immunoadsorbent has been demonstrated for the MG treatment. References. 1. Takamori M, Okumura S, Nagata M, et al.. Myasthenogenic significance of synthetic a-subunit peptide 183-200 of Torpedo californica and human acetylcholine receptor. J -125- Nakaji S, et al. Neurol Sci 1988; 85: 121-129. 2. Takamori "M, Okumura S, Ide' Y, et al . A synthetic peptide, Torpedo californica (X183-200 of the acetylcholine receptor as a tool for immunosd sorption via plasma perfusion in myasthenia gravis. Artif Organs Today 1991; 1: 53-60. 3. Ide Y, Okumura S, Takamori M, Treatment of myasthenia gravis with a specific immunoadsorbent bound to acetylcholine receptor peptide (X183-200. Therapeutic Plasmapheresis 1991; 9: 147-152. Figure 3 Changes in blocking antibody liter after plasma immunoadsorption on treatments in clinical study (Kanazawa Univ.) Blocking antibody (Anti -peptide antibody) Oftf 5 10 15 20 25 30 35 day Figure 4 Changes in IgG and blocking antibody (anti-peptide antibody) levels Arrows indicate the plasma immunoadsorption treatments -126- V JHMffl S 1. (italic ®8gtt<7 -^- HcoimfctiUTii|g^»‘«9f%<0T7-n-^»>'#t <^n.E> „ ffljeJf. ®Sffl A. #t A&. -eLT. fWShLtnytA-^-yijl^-ya yM'Itl.^ o . XEPSi: LTCji^coSIA^Sk^SAA *lglt LTHacoA^^r A K D ± tf&CUzLt. ^n^cO<7-A HcO^Ak'iiSiatotStgSrWLTti 0 . AKT'A h'aoKif £M <3 *»£Oipi® S: 4 i.& k «t # i <3 it h„ 2. 8l$cO^"E # Lt. 3 0T5y®a±tc^»^i/h-y6libAk LA K##Mt5A L ji<3li m*roartE@k Lt-c. ttc, A^«lc*»L**eS«Brfr^rALt r«tg14^7*f-LW SJEj (:(±##giEn##TA&*A Lfi6%0C0L^. gSl • ^7-AH(:raLTSA cor. AA&. * isst^l<7)2-7<7)^>- 127- 3 . K viE „ 3 -1. 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C J: c T T S /###> 4>fiT W6 3 o r 5 y m&Tcoxm^y 0^ h (7n-ye-fa; seqsons) #::1 LIST OF PEPTIDES WITH LESS THAN 30 AMINO ACIDS. #::2 FORMAT=LIT.NO.:TAXON TAG:PEPTIDE 765024A:Agnatha: fibrinopeptj.de A 1205260A:Agnatha:luliberin 1414306A:Agnatha:somatostatin 14 variant 1406259A:Agnatha:vasotocin 1516329A:Amphibia:alpha melanotropin 710343A:Amphibia:alytesin 0904250A:Amphibia:angiotensin I 0510184A:Amphibia:angiotensin,crinia 1718263A:Amphibia:antimicrobial peptide PGQ 1413199A:Amphibia:atrial natriuretic factor 24 1414243A:Amphibia:atrial natriuretic-like peptide 1509286A:Amphibia:basic tryptophyllin 1608110A:Amphibia:bioact hydrophobic peptide A1 1608110B:Amphibia:bioact hydrophobic peptide B9 710343B:Amphibia:bombesin 720133A:Amphibia:bombesin 730327A:Amphibia:bombesin 1610281A:Amphibia:bombesin related peptide PG-L 1714191A:Amphibia:bombesin-like neuropeptide 700810A:Amphibia:bombinin 1103176A:Amphibia:bradykinin 650766A:Amphibia:bradykinin 690622A:Amphibiarbradykinin 701177A:Amphibia:bradykinin 1103176B:Amphibia:bradykinin 5-9 680356A:Amphibia:bradykinin,1-Val,6-Thr 0510365A:Amphibia:bradykinin,3-Hyp 0504194A:Amphibia:bradykinin,6-Thr 1103176C:Amphibia:bradykinin,des-9-Arg 730327B:Amphibia:bradykininyl-Gly-Lys-Phe-His 1703232A:Amphibia:C type natriuretic peptide 680326A:Amphibia:caerulein 0310191A:Amphibia: caerulein,2-Asn,5-Leu 1508203A:Amphibia:deltorphin 1510394A:Amphibia:deltorphin I 1510394B:Amphibia:deltorphin II 1515244A:Amphibia:dermenkephalin 0704273A:Amphibia:dermorphin 1810298A:Amphibia:dermorphin 1810298B:Amphibia:dermorphin 0704274A:Amphibia:dermorphin,6-Hyp 0311228A:Amphibia:granuliberin R 0812283A:Amphibia:histone H3 1511130A:Amphibia:hydrin 1 1511130B:Amphibia:hydrin 2 1606259A:Amphibia:hydrin 2 0801196A:Amphibia:hylambatin 0712211A:Amphibia:hypothalamic peptide 0308208A:Amphibia:kassinin 0801196B:Amphibia:kassinin,2-Glu 5-Pro 730327C:Amphibia:kinin 0 680668A:Amphibia:kinin peptide III 1104233A:Amphibia:litorin 751393A:Amphibia:litorin 0605192A:Amphibia: litorin,2-Glu(0Et) 0311238A:Amphibia:litorin,2-Glu(0Me) 640051A:Amphibia:mesotocin 1602196A:Amphibia:neuromedin U 1811353A:Amphibia:neurotensin 610522A:Amphibia:oxytocin 610522C:Amphibia:oxytocin 690622B:Amphibia:phyllocaerulein 660391A:Amphibia:phyllokinin 0910155A:Amphibia:phyllolitorin 133- 701175A:Amphibia:phyllomedusin 640455A:Amphibia:physalaemin 0605192B:Amphibia:physalaemin like peptide 1803428A:Amphibiarranakinin 680668B:Amphibia:ranakinin N 0504194B:Amphibia:ranakinin R 1711287A:Amphibia:ranatachykinin A 1711287B:Amphibia:ranatachykinin B 1711287C:Amphibia:ranatachykinin C 1711287D:Amphibia:ranatachykinin D 701177B:Amphibiarranatensin 0504195A:Amphibia:ranatensin R 1112220A:Amphibia:somatostatin 1610281B:Amphibia:tachykinin related peptide PG-KI 1610281C:Amphibia:tachykinin related peptide PG-KII 1610281D:Amphibia:tachykinin related peptide PG-KIII 1610281E:Amphibia:tachykinin related peptide PG-SPI 1610281F:Amphibia:tachykinin related peptide PG-SPII 742295A:Amphibia:thyroliberin 690954A:Amphibia:toxin peptide 4a alpha 690954B:Amphibia:toxin peptide 4a beta 1204212A:Amphibia tryptophyllin 13 1208379L:Amphibia tryptophyllin 13 1208379A:Amphibia tryptophyllin 4 A1 1208379B:Amphibia tryptophyllin 4 A2 1208379D:Amphibia tryptophyllin 4 Ml 1208379C:Amphibia tryptophyllin 4 M3 1208379E:Amphibia tryptophyllin 4 Q 1208379G:Amphibia tryptophyllin 5 1208379H:Amphibia tryptophyllin 5 1208379F:Amphibia tryptophyllin 5 B 1208379J:Amphibia tryptophyllin 5 P 1208379K:Amphibia tryptophyllin 7 1005217A:Amphibia tryptophyllin TPH4 1104246A:Amphibia tryptophyllin TPH4 1104246B:Amphibia tryptophyllin TPH4 1104246C:Amphibia tryptophyllin TPH4 1005217B:Amphibia tryptophyllin TPH5 1104246D:Amphibia tryptophyllin TPH5 1104246E:Amphibia: tryptophyllin TPH7 751003A:Amphibia:uperolein 1803252A:Amphibia:vasotocin 600015A:Amphibia:vasotocin 610522E:Amphibia:vasotocin 610522F:Amphibia:vasotocin 640487A:Amphibia:vasotocin 671037B:Amphibia:vasotocin 1803252B:Amphibia:vasotocin-related peptide 731315A:Amphibia:xenopsin 1302299A:Animalia0:antho RFamide 1612407A:AnimaliaO:antho RNamide 1805361A:AnimaliaO:antho-RIamide I 1414251A:AnimaliaO:antho-RWamide 0910246A:Animalia0:casomorphin beta 1604340A:AnimaliaO:cionin 1210338A:AnimaliaO:egg assocd peptide A 1210338B:AnimaliaO:egg assocd peptide B 0712245A:AnimaliaO:morphogenetic peptide 1412363A:AnimaliaO:mosact 1404258A:AnimaliaO:neuropeptide 1817216A:AnimaliaO:neuropeptide 1817216B:AnimaliaO:neuropeptide 1717144A:AnimaliaO:neuropeptide Antho-KAamide 1302184A:AnimaliaO:neuropeptide,pennatulid 1001132A:AnimaliaO:peptide Al,sperm activating 1507171A:AnimaliaO:Pol-RFamide -134- 1808186A:Animalia0 Pol-RFamide 1101334A:AnimaliaO resact 1711480A:Animalia0 SALMFamide 1 1711480B:AnimaliaO SALMFamide 2 0804270A:AnimaliaO speract 1708180A:AnimaliaO sperm activating peptide III 1708180B:AnimaliaO sperm activating peptide III 1708180C:AnimaliaO sperm activating peptide III 1708180D:AnimaliaO sperm activating peptide III 1708180E:AnimaliaO sperm activating peptide III 1708180F:AnimaliaO sperm activating peptide III 1708180G:AnimaliaO sperm activating peptide III 1708180H:AnimaliaO sperm activating peptide III 1708180J:AnimaliaO sperm activating peptide III 1708180K:AnimaliaO sperm activating peptide III 1708180L:AnimaliaO sperm activating peptide III 1713215A:AnimaliaO sperm activating peptide TG-1 1713215K:AnimaliaO sperm activating peptide TG-10 1713215B:AnimaliaO sperm activating peptide TG-2 1713215C:AnimaliaO sperm activating peptide TG-3 1713215D:AnimaliaO sperm activating peptide TG-4 1713215E:AnimaliaO sperm activating peptide TG-5 1713215F:AnimaliaO sperm activating peptide TG-6 1713215G:AnimaliaO sperm activating peptide TG-7 1713215H:AnimaliaO sperm activating peptide TG-8 1713215J:AnimaliaO sperm activating peptide TG-9 0705150B:AnimaliaO sperm-activating 9-peptide 0705150A:AnimaliaO sperm-activating peptide 1502213A:AnimaliaO sperm-activating peptide 1502214A:AnimaliaO sperm-activating peptide 1508189A:AnimaliaO sperm-activating peptide b 1508189B:AnimaliaO sperm-activating peptide d 1616199B:AnimaliaO sperm-activating peptide I 1616199T:AnimaliaO sperm-activating peptide I (2,5-Thr,3-Leu,4-Pro,7,10-Ser) 1616199H:AnimaliaO sperm-activating peptide I (2-Phe(Br)) 1616199G:AnimaliaO sperm-activating peptide I (2-Phe(Br),3-Asn) 1616199P:AnimaliaO sperm-activating peptide I (2-Tyr,3,5-Ser,8-Ala,10-Asp) 1616199V:AnimaliaO sperm-activating peptide I (2-Tyr,3-Asn,5-Gly,9-Ile,10-Asp) 1616199R:AnimaliaO sperm-activating peptide I (2-Tyr,3-Asn,7,lO^Asp,8-Arg,9-Ile 1616199F:AnimaliaO sperm-activating peptide I (3,5-Gly) 1616199L:AnimaliaO sperm-activating peptide I (3,5-Ser) 1616199M:AnimaliaO sperm-activating peptide I (3-Ala,5-Gly) 1616199A:AnimaliaO sperm-activating peptide I (3-Asn) 1616199C:AnimaliaO sperm-activating peptide I (3-Asn,4-Ile,5-Gly) 1616199Q:AnimaliaO sperm-activating peptide I (3-Asn,5-Ser) 1616199U:AnimaliaO sperm-activating peptide I (3-Glu,4-Met,5-Gly,7-Thr) 1616199W:AnimaliaO sperm-activating peptide I (3-Gly,5-Ser,9-Ile) 1616199E:AnimaliaO sperm-activating peptide I (3-Ser,5-Gly) 1616199D:AnimaliaO sperm-activating peptide I (5-Gly) 1616199N:AnimaliaO sperm-activating peptide I (5-Thr) 1616199K:AnimaliaO sperm-activating peptide I, Des-6,7-Gly (3-Ala,5-Asp) 1616199J:AnimaliaO sperm-activating peptide I, Des-6,7-Gly (3-Ala,5-Asp,10-Asn) 1802280A:AnimaliaO sperm-activating peptide SV -1 0402232A:AnimaliaO toxin III 1201191A:Arthropoda:adipokinetic hormone 1204166A:Arthropoda:adipokinetic hormone 1515421A:Arthropoda:adipokinetic hormone 1613147A:Arthropoda:adipokinetic hormone 1613152A:Arthropoda:adipokinetic hormone 1708296A:Arthropoda:adipokinetic hormone 1810246A:Arthropoda:adipokinetic hormone 1813270A:Arthropoda:adipokinetic hormone 763125A: Arthropoda-.adipokinetic hormone 1109158A: Arthropoda .-adipokinetic hormone II 1109158B:Arthropoda:adipokinetic hormone II 1203209A:Arthropoda:adipokinetic hormone II -135- 1203209B:Arthropoda:adipokinetic hormone II 1708195A:Arthropoda:adipokinetic hormone III 1402182A:Arthropoda:adipokinetic peptide AKH-G 1413346A:Arthropoda:AKH family neuropeptide 1413258A:Arthropoda:AKH like brain factor BI 1515171A:Arthropoda:allatostatin 1718377A:Arthropoda:allatostatin 1511156A:Arthropoda:allatostatin 1 1511156B:Arthropoda:allatostatin 2 1511156C:Arthropoda:allatostatin 3 1511156D:Arthropoda:allatostatin 4 1707324A:Arthropoda:allatostatin ASB2 1505417A:Arthropoda:allatotropin 670258A:Arthropoda:apamine 1511072A:Arthropoda:apidaecin lb 1010431A:Arthropoda:Balbiani ring 2 gene beta repeat C 1010431E:Arthropoda:Balbiani ring 2 gene beta repeat SR 1103246A:Arthropoda:bombolitin I 1103246B:Arthropoda:bombolitin II 1103246C:Arthropoda:bombolitin III 1103246D:Arthropoda:bombolitin IV 1103246E:Arthropoda:bombolitin V 1412231A:Arthropoda:bombyxin II A 1412231B:Arthropoda:bombyxin II B 1503112A:Arthropoda:bombyxin IV A 1503112B:Arthropoda:bombyxin IV B 1309327A:Arthropoda:bradykinin,6-Thr 754921A:Arthropoda:bradykinin,Ala-Arg 1309327B:Arthropoda:bradykinyl-Lys-Ala,6-Thr 1807377A:Arthropoda:calliFMRFamide 1 1807377K:Arthropoda:calliFMRFamide 10 1807377L:Arthropoda:calliFMRFamide 11 1807377M:Arthropoda:calliFMRFamide 12 1807377N:Arthropoda:calliFMRFamide 13 1807377B:Arthropoda:calliFMRFamide 2 1807377C:Arthropoda:calliFMRFamide 3 1807377D:Arthropoda:calliFMRFamide 4 1807377E:Arthropoda:calliFMRFamide 5 1807377F:Arthropoda:calliFMRFamide 6 1807377G:Arthropoda:calliFMRFamide 7 1807377H:Arthropoda:calliFMRFamide 8 1807377J:Arthropoda:calliFMRFamide 9 1807377P:Arthropoda:calliMIRFamide 1302325A:Arthropoda:cardioactive peptide CCAP 1601326C:Arthropoda:cheraotactic peptide 721419A:Arthropoda:color change hormone 1511082A:Arthropoda:corazonin 1805363A:Arthropoda:corazonin,7-His 1009229A:Arthropoda:crabrolin 1401236C:Arthropoda:Cs gene 1811325A:Arthropoda:diapuase hormone 1803186A:Arthropoda:diuretic peptide Mas-DP2 1508172A: Arthropoda-.FMRFamide like neuropeptide 1413303A:Arthropoda:FMRFamide like peptide 3 1413303B:Arthropoda:FMRFamide like peptide 4 1616410A:Arthropoda:FMRFamide-related peptide 1805364A:Arthropoda:FMRFamide-related peptide 1402211A:Arthropoda:ganglion peptide 1802220A:Arthropoda:gastrin/cholecystokinin-1ike peptide 1709276A:Arthropoda:growth blocking peptide 0712190A:Arthropoda:histamine releasing peptide 0712190B:Arthropoda:histamine releasing peptide 1304184A:Arthropoda:hypertrehalosemic factor II 1211206A:Arthropoda:hypertrehalosemic hormone 1607342A:Arthropoda:hypertrehalosemic neuropeptide 1612381A:Arthropoda:hypertrehalosemic neuropeptide —136 — 1613146A:Arthropoda:hypertrehalosemic neuropeptide 1810301A:Arthropoda:hypertrehalosemic peptide 1810301B:Arthropoda:hypertrehalosemic peptide 1610160C:Arthropoda:hypertrehalosemic peptide Bld-HrTH 1610160D:Arthropoda:hypertrehalosemic peptide Cam-HrTH-II 1610160A:Arthropoda:hypertrehalosemic peptide Pea-CAH-I 1610160B:Arthropoda:hypertrehalosemic peptide Pea-CAH-II 1515421B:Arthropoda:hypotrehalosemic hormone 681037A:Arthropoda:kinin 1301220A:Arthropoda:leucokinin I 1301220B:Arthropoda:leucokinin II 1402209A:Arthropoda:leucokinin V 1402209B:Arthropoda:leucokinin VI 1402210A:Arthropoda:leucokinin VII 1402210B:Arthropoda:leucokinin VIII 1210374A:Arthropoda:leucosulfakinin 1211204A:Arthropoda:leucosulfakinin II 1304197A:Arthropoda:leukopyrokinin 762531A:Arthropoda:light adapting hormone 1510214A:Arthropoda:locusta peptide A 1510214B:Arthropoda:locusta peptide B 1804432A:Arthropoda:locustakinin 1801363A:Arthropoda:locustamyoinhibiting peptide 1612380A:Arthropoda:locustamyotropin 1713291A:Arthropoda:locustamyotropin II 1706255A:Arthropoda:locustapyrokinin 1605188A:Arthropoda:locustatachykinin I 1605188B:Arthropoda:locustatachykinin II 1704380A:Arthropoda:locustatachykinin III 1704380B:Arthropoda:locustatachykinin IV 1709386A:Arthropoda:Lom-AG-myotropin 1208383A:Arthropoda:mast cell degranulating peptide 1312417A:Arthropoda:mast cell degranulating peptide 690923A:Arthropoda:mast cell degranulating peptide 0510099A:Arthropoda:mastoparan 0607189A:Arthropoda:mastoparan 1601326B:Arthropoda:mastoparan 1708155A:Arthropoda:mastoparan 1009229B:Arthropoda:mastoparan C 0710225A:Arthropoda:mastoparan M 0510182A:Arthropoda:mastoparan X 670043A:Arthropoda:melittin 730710A:Arthropoda:melittin 751015A:Arthropoda:melittin 0405213A:Arthropoda:melittin F 720299A:Arthropoda:melittin II 1709388A:Arthropoda:myotropin 1607220A:Arthropoda:neuropeptide Aeal 1607220B:Arthropoda:neuropeptide Aeall 1414190A:Arthropoda:neuropeptide hez-HrTH 1606261A:Arthropoda:neuropeptide led CCI 1606261B:Arthropoda:neuropeptide led CCII 1711289A:Arthropoda:neuropeptide mem-CC 1810245A:Arthropoda:neuropeptide mem-CC 1011173A:Arthropoda:neuropeptide MI 1011173B:Arthropoda:neuropeptide Mil 1301199A:Arthropoda:neuropeptide,hypertrehalosaeraic 1603305C:Arthropoda:omega agatoxin IIA 1107279A:Arthropoda:ORF 1 C term 1103308B:Arthropoda:0RF HB2 750746A:Arthropoda:paragonial peptide PS 1713343A:Arthropoda:paralytic peptide I 1713343B:Arthropoda:paralytic peptide I 1713343C:Arthropoda:paralytic peptide I 1713343D:Arthropoda:paralytic peptide II 1713343F:Arthropoda:paralytic peptide II -137- 1713343G:Arthropoda:paralytic peptide II 1713343E:Arthropoda:paralytic peptide III 0401151A:Arthropoda:peptide Cp,silk fibroin 1501209A:Arthropoda:peptide HR1 1501209B:Arthropoda:peptide HR2 1010330A:Arthropoda:periplanetin CC1 1010330B:Arthropoda:periplanetin CC2 1601433A:Arthropoda:perisulfakinin 1804200A:Arthropoda:pheromonotropic neuropeptide 1304257A:Arthropoda:pigment dispersing factor 1109281A:Arthropoda:pigment dispersing hormone 1211218A:Arthropoda:pigment dispersing hormone 754921B:Arthropoda:pollsteskinin R 1601383A:Arthropoda:polyphemusin I 1601383B:Arthropoda:polyphemusin II 1610280A:Arthropoda:proctolin 754183A:Arthropoda:proctolin 1812292A:Arthropoda:red pigment-concentrating hormone 1413346B:Arthropoda:RPCH family neuropeptide 0405213B:Arthropoda:secapin 1002241A:Arthropoda:secapin 1501299A:Arthropoda:tachyplesin 1601383C:Arthropoda:tachyplesin II 1615222A:Arthropoda:tachyplesin III 0605179A:Arthropoda:tertiapin 0902216A:Arthropoda:toxic 8-peptide 1005287B:Arthropoda:toxin Bot V N term 1601342B:Arthropoda:toxin like peptide II 1104330A:Arthropoda:tropomyosin C term 1314162A:Arthropoda:vasopressin like diuretic hormone 764671A:Arthropoda:vespakinin M 771747A:Arthropoda:vespakinin X 760232A:Arthropoda:vespulakinin 760232B:Arthropoda:vespulakinin 1601326A:Arthropoda:waspkinin 1612286A:Aves:angiotensin I 730699A:Aves:angiotensin I 0410203A:Aves:antigen,major histocompatibility 1413198A:Aves:atrial natriuretic factor 0910250A:Aves:brain peptide 1203416A:Aves:bursin 1704143A:Aves:C type natriuretic peptide 1003829A:Aves:c-myc gene 0504213A:Aves:collagen alphal(I) N term,pro 1007220A:Aves:collagen alphal(II) CBM,pro 0407229A:Aves:fibrinopeptide A 1110284A:Aves:fibrinopeptide A 1110284B:Aves:fibrinopeptide B 1715216A: Aves: gal an in 0701228A:Aves:gastric proventricular peptide 1403328A:Aves:glucagon 1714255A:Aves:glucagon 721287A:Aves:glucagon 721913A:Aves:glucagon 753726A:Aves:glucagon 1604250B:Aves:histone HI 1506448B:Aves:histone H2AF assocd ORF 730530A:Aves:insulin C peptide 0810231A:Aves:luliberin 0906295A:Aves:luliberin 1007246A:Aves:luliberin II 0904299A:Aves:luliberin,8-Gln 701182A:Aves:mesotocin 1403440B:Aves:neurokinin A 1718372A:Aves:neuromedin U 0608263A:Aves:neurotensin -138- 1310315A:Aves:neurotensin 0903220A:Avesrneurotensin related 6-peptide 1314219A:Aves:neurotensin related peptide 1313235A:Aves:ornitho kinin 1313235C:Avesrornitho kininogen 600454A:Aves:oxytocin 640486A:Aves:oxytocin 750800A:Aves:protamine 0311203A:Aves:pyruvate carboxylase biotin peptide 0701221A:Aves:secretin 0507258A:Aves:somatostatin 1403440A:Aves:substance P,3-Arg 753696A:Aves:vasoactive intestinal peptide 600456A:Aves:vasopressin,Arg 600456B:Aves:vasotocin 701182B:Aves rvasotocin 1412334A:Aves:xenopsin related peptide 720476A:Chondrichthyes:aspartocin 720633A:Chondrichthyes raspartocin 741686B:Chondrichthyes:corticotropin like interm lobe peptide 1206345A:Chondrichthyes:endorphin beta 1401182A:Chondrichthyes:gastrin releasing peptide 1112169A:Chondrichthyes:glucagon 1305260A:Chondrichthyes:glucagon 1516142B:Chondrichthyes:glucagon 650080A:Chondrichthyes:glumitocin 650768A:Chondrichthyes:glumitocin 671036A:Chondrichthyes:glumitocin 1301248A:Chondrichthyes:insulin A 1412225A:Chondrichthyes:insulin A 1211262B:Chondrichthyes:insulin C peptide 1709219A:Chondrichthyes:luliberin 1815266A:Chondrichthyes:luliberin 1817182A:Chondrichthyes:luliberin I 700801A:Chondrichthyes:melanotropin alpha 741687A:Chondrichthyes:melanotropin alpha 741687B:Chondrichthyes:melanotropin beta 741687C:Chondrichthyesrmelanotropin beta 690930A:Chondrichthyes:oxytocin 650096A:Chondrichthyes:oxytocin,3-Ser 8-Ile 1301239A:Chondrichthyesrrelaxin A 1301239B:Chondrichthyes:relaxin B 1206256A:Chondrichthyes:scyliorhinin I 1206256B:Chondrichthyes:scyliorhinin II 1412223A: Chondrichthyes-.scyliorhinin II 3-18 1112170A:Chondrichthyes:somatostatin 1703277A:Chondrichthyes:somatostatin 14,5-Ser 1806387A:Chondrichthyesrurotensin II 720476B:Chondrichthyes:valitocin 720633B:Chondrichthyes:valitocin 1311236A:Chondrichthyes:vasoactive intestinal peptide 610242B:Chondrichthyes:vasotocin 764683A:Fungi:alpha factor 1612305A:Fungi:arg-2 assocd ORF 1104318B:Fungi:ATPase 9,F0 1309198B-.Fungi:carbamoylphosphate synthetase ORF 1407245B:Fungi:gene nucl ORF 1 1407245C:Fungi:gene nucl ORF 2 1407245D:Fungi:gene nucl ORF 3 290004A:Fungi:glutathione 721924A:Fungi:inhibitor,galactose oxidase 0401178A:Fungi:mating factor 1302211A:Fungi:mating factor a2 1302211B:Fungi:mating factor a3 1208294A:Fungi:mating factor alpha 762484A:Fungi:mating hormone alphalB -139 1206200A:Fungi:mating pheromone alpha ski 1206200B:Fungi .-mating pheromone alpha sk3 1112155A:Fungi:metallothionein,Cu 1201202A:Fungi:metallothionein,Cu 1408283B:Fungi:ORF 1 1103229A:Fungi:ori region ORF 3 1109166B:Fungi:oxidase I,cytochrome c 1109166C:Fungi:oxidase III,cytochrome c 1003184A:Fungi:pheromone alpha sk2,mating 0812287F:Fungirprotein URFd 1109166D:Fungi:protein varl 0504158A:Fungi:rhodotorucine A 0502206A:Fungi:sillucin 0707174A:Fungi:substance alphaK 771273A:Fungi:substance IA,alpha 762484B:Fungi:substance IB,alpha 0601162A:Fungirtremerogen A10 0705139A:Fungi:tremerogen al3 1005183A:Fungi:tremerogen A9291I 1817362A:ISOTYPE=A:Mammalia:relaxin 1805261A:ISOTYPE=II:Arthropoda:myotropin 1008163A:Mammalia:acrosin light chain 1104244A:Mammalia:adrenal medullary peptide precursor 0911276A:Mammalia:adrenorphin 0410472A:Mammalia:albumin prepiece 1710319A:Mammalia:albuminamide 1608236A:Mammalia:alpha neo-endorphin 732148A:Mammalia:ameletin 1102244A:Mammalia-.amidorphin C term 0804298A:Mammalia:amylase alpha 570121A:Mammalia:angiotensin 1210206A:Mammalia:angiotensin converting enzyme inhibitor 560088A:Mammalia:angiotensin I 560148A:Mammalia:angiotensin I 670366A:Mammalia:angiotensin I 720249A:Mammalia:angiotensin I 560149A:Mammalia:angiotensin II 0411243A:Mammalia:anorexigenic peptide 0803236A:Mammalia:antiarrhythmic peptide 0901253A:Mammalia:antigen,spermatozoal 0604199A:Mammalia:antigonadotropic 3-peptide 1805147A:Mammalia:antioncogene protein pt27 0601225A:Mammalia:ATPase accelerating peptide 1001137A:Mammalia:atria peptide 1004222A:Mammalia:atrial natriuretic factor 1010154C:Mammalia:atrial natriuretic factor III 1010154D:Mammalia:atrial natriuretic factor IV 1002183A:Mammalia:atrial natriuretic peptide alpha 1007202A:Mammalia:atrial natriuretic/vasoactive peptide 1001244A:Mammalia:atriopeptin I 1005179A:Mammalia:atriopeptin I 1005179B:Mammalia:atriopeptin I,des-1,2-Ser 1005179C:Mammalia:atriopeptin I,des-l-Ser 1005179D:Mammalia:atriopeptin I,des-21-Ser 1001244B:Mammalia:atriopeptin II 1005179E:Mammalia:atriopeptin II 1005179F:Mammalia:atriopeptin III 1409308A:Mammalia:bactenecin 1701465A:Mammalia:beta prepro-tachykinin 117-126 0906234A:Mammalia:bombesin like peptide 0906234B:Mammalia:bombesin like peptide 0906234C:Mammalia:bombesin like peptide 1011314A:Mammalia:bombesin like peptide 600196A:Mammalia:bradykinin 620203A:Mammalia:bradykinin 1207241A:Mammalia:bradykinin,3-Ala Lys —140 — 1407164A:Mammalia:bradykinin,3-Hyp 1414313A:Mammalia:bradykinin,3-Hyp 650157A:Mammalia:bradykinin,Met-Lys 1402188A:Mammalia:bradykinin,N-Lys 3-Hyp 1404380A:Mammalia:brain natriuretic peptide 1609081A:Mammalia:C type natriuretic peptide 0311203B:Mammalia:carboxylase biotin peptide,pyruvate 1101255A:Mammalia:casein 6-peptide,immunestimulating 0511270A:Mammalia:casomorphin beta 1815292A:Mammalia:cationic Cys-rich peptide 1815292C:Mammalia:cationic Cys-rich peptide 1406215A:Mammalia:CCK-PZ 8 1012338A:Mammalia:cerebellin 753732A:Mammalia:chemotactic factor 753732B:Mammalia:chemotactic factor 1011260A:Mammalia:cholecystokinin 1103329A:Mammalia:cholecystokinin 22 1108290A:Mammalia:cholecystokinin 22 0604474A:Mammalia:cholecystokinin 4 1304169A:Mammalia:cholecystokinin 5 0409339A:Mammalia:cholecystokinin 8 1108290B:Mammalia:cholecystokinin 8 1408277A:Mammalia:cholecystokinin 8 1812203A:Mammalia:cholinergic neurostimulating peptide 1005178A:Mammalia:chromaffin granule peptide 1005178B:Mammalia:chromaffin granule peptide 1005178C:Mammalia:chromaffin granule peptide precursor 1809219A:Mammalia:chromogranin A-derived peptide WE14 1104274A:Mammalia:collagen alphal III signal peptide 1105272A:Mammalia:collagen II,pro N term 1413205A:Mammalia:complement C3f 754907A:Mammalia:contraceptive peptide 1718167C:Mammalia:corticostatic peptide 3 0611192B:Mammalia:corticotropin like interm lobe peptide 740695A:Mammalia:corticotropin like interm lobe peptide 0707220A:Mammalia:corticotropin releasing 7-peptide 0406187A:Mammalia:CRF active 14-peptide 0310165A:Mammalia:decarboxylase fragment,dopa 1112214A:Mammalia:defensin HNP1 1112214B:Mammalia:defensin HNP2 1112214C:Mammalia:defensin HNP3 1603276B:Mammalia:defensin NP3 0411144A:Mammalia:delicious taste peptide 0412267A:Mammalia:delta sleep inducing peptide 1211230A:Mammalia:DFT stimulating peptide 0712247A:Mammalia:dynorphin 0601259A:Mammalia:dynorphin 13 0811243A:Mammalia:dynorphin 24 0610285A:Mammalia:dynorphin related 8-peptide 750521A:Mammalia:encephalitogenic peptide M 761141A:Mammalia:endorphin alpha 0705147A:Mammalia:endorphin alpha,neo 0705146A:Mammalia:endorphin beta,neo 764744A:Mammalia:endorphin gamma 1412332A:Mammalia:endothelin 0702167A:Mammalia:enkephalin BAM22P 1011313A:Mammalia:enkephalin derived peptide,prepro 1002185A:Mammalia:enkephalin derived peptide,pro 753272A:Mammalia:enkephalin,Leu 761375A:Mammalia:enkephalin,Leu 762848A:Mammalia:enkephalin,Leu 753272B:Mammalia:enkephalin,Met 761375B:Mammalia:enkephalin,Met 762848B:Mammalia:enkephalin,Met 0708293A:Mammalia:enkephalin,N-Ac Leu 0610284A:Mammalia:enkephalinyl-Arg,Leu 141- 0601256A:Mammalia:enkephalinyl-Arg,Met 0601260A:Mammalia:enkephalinyl-Arg-Phe,Met 1109269A:Mammalia:epidermal growth factor receptor 1005225A:Mammalia:ferritin H 0504173A:Mammalia:fibrinogen alpha N term fragment 1006279A:Mammalia:fibrinogen alpha signal 1006279B:Mammalia:fibrinogen beta signal 1006279C:Mammalia:fibrinogen gamma signal 0912289A:Mammalia:fibrinogen N term Bern II 1001180A:Mammalia:fibrinopeptide A 1001180B:Mammalia:fibrinopeptide A 1001180C:Mammalia:fibrinopeptide A 1105261A:Mammalia:fibrinopeptide A 650770A:Mammalia:fibrinopeptide A 650770B:Mammalia:fibrinopeptide A 650770C:Mammalia:fibrinopeptide A 650770D:Mammalia:fibrinopeptide A 650771A:Mammalia:fibrinopeptide A 650771B:Mammalia:fibrinopeptide A 650771C:Mammalia:fibrinopeptide A 650771D:Mammalia:fibrinopeptide A 650771E:Mammalia:fibrinopeptide A 650771F:Mammalia:fibrinopeptide A 650771G:Mammalia:fibrinopeptide A 650771H:Mammalia:fibrinopeptide A 650771J:Mammalia:fibrinopeptide A 650771K:Mammalia:fibrinopeptide A 650771L:Mammalia:fibrinopeptide A 650771M:Mammalia:fibrinopeptide A 650771N:Mammalia:fibrinopeptide A 650771P:Mammalia:fibrinopeptide A 650771Q:Mammalia:fibrinopeptide A 650771R:Mammalia:fibrinopeptide A 650771T:Mammalia:fibrinopeptide A 660906A:Mammalia:fibrinopeptide A 660906B:Mammalia:fibrinopeptide A 660906C:Mammalia:fibrinopeptide A 670960A:Mammalia:fibrinopeptide A 670960B:Mammalia:fibrinopeptide A 670960C:Mammalia:fibrinopeptide A 670960D:Mammalia:fibrinopeptide A 670960E:Mammalia:fibrinopeptide A 671044B:Mammalia:fibrinopeptide A 671044C:Mammalia:fibrinopeptide A 690946A:Mammalia:fibrinopeptide A 701211A:Mammalia:fibrinopeptide A 701218A:Mammalia:fibrinopeptide A 721948A:Mammalia:fibrinopeptide A 721948C:Mammalia:fibrinopeptide A 732189A:Mammalia:fibrinopeptide A 732189B:Mammalia:fibrinopeptide A 754929A:Mammalia:fibrinopeptide A 1004217A:Mammalia:fibrinopeptide A Louisville 1001180D:Mammalia:fibrinopeptide B 1001180E:Mammalia:fibrinopeptide B 1105261B:Mammalia:fibrinopeptide B 1105261C:Mammalia:fibrinopeptide B 630449A:Mammalia:fibrinopeptide B 650770E:Mammalia:fibrinopeptide B 650770F:Mammalia:fibrinopeptide B 650770G:Mammalia:fibrinopeptide B 650771AA:Mammalia:fibrinopeptide B 650771AB:Mammalia:fibrinopeptide B 650771AC:Mammalia:fibrinopeptide B 650771AD:Mammalia:fibrinopeptide B 650771AE:Mammalia:fibrinopeptide B -142- 650771AF:Mammalia:fibrinopeptide B 650771U:Mammalia:fibrinopepti.de B 650771V:Mammalia:fibrinopeptide B 650771W:Mammalia:fibrinopeptide B 650771X:Mammalia:fibrinopeptide B 650771Y:Mammalia:fibrinopeptide B 660906D:Mammalia:fibrinopeptide B 660906E:Mammalia:fibrinopeptide B 670960F:Mammalia:fibrinopeptide B 670960G:Mammalia:fibrinopeptide B 670960H:Mammalia:fibrinopeptide B 670960J:Mammalia:fibrinopeptide B 670960K:Mammalia:fibrinopeptide B 671044E:Mammalia:fibrinopeptide B 671044F: Mammalia'.fibrinopeptide B 690946B:Mammalia:fibrinopeptide B 690946C:Mammalia:fibrinopeptide B 701211B:Mammalia:fibrinopeptide B 701218B:Mammalia:fibrinopeptide B 721948D'.Mammalia:fibrinopeptide B 721948F:Mammalia:fibrinopeptide B 721948G:Mammalia:fibrinopeptide B 721948H:Mammalia:fibrinopeptide B 732189C:Mammalia:fibrinopeptide B 0807231A:Mammalia:fibronectin Cys containing region 1112273A:Mammalia:FMRFamide like 18-peptide 1112273B:Mammalia:FMRFamide like 8-peptide 1001157A:Mammalia:galanin 1712179A:Mammalia:galanin 1718373A:Mammalia:galanin 1802436A:Mammalia:galanin 1815257A:Mammalia:galanin 701192A:Mammalia:gastric peptide 1413347A:Mammalia:gastrin 640126A:Mammalia:gastrin 660134A:Mammalia:gastrin 680320A:Mammalia:gastrin 680320B:Mammalia:gastrin 690052A:Mammalia:gastrin 690262A:Mammalia:gastrin 0802193A:Mammalia:gastrin little 0503136A:Mammalia:gastrin mini 1009184A:Mammalia:gastrin N term,pro 0510270A:Mammalia:gastrin releasing peptide 1202335A:Mammalia:gastrin,little 1405288A:Mammalia:gelsolin 1003207A:Mammalia:globin epsilonlll 1003207B:Mammalia:globin epsilonlV 1110283A:Mammalia:glucagon 1207205A:Mammalia:glucagon 1606260B:Mammalia:glucagon 1616407A:Mammalia:glucagon 570104A:Mammalia:glucagon 710325A:Mammalia:glucagon 711649A:Mammalia:glucagon 720483A:Mammalia:glucagon 720967A:Mammalia:glucagon 1512256A: Mammalia'.glucagon like peptide 1 0806160A:Mammalia:glycentin related peptide 1304348A:Mammalia:gonadotropin releasing peptide 730306A:Mammalia:growth modulating 3-peptide 1805415A:Mammalia:growth/mitosis inhibitory peptide 1805411A:Mammalia:guanylin 0711246A:Mammalia:head activator peptide 1408207C:Mammalia:histatin 5 1010306A:Mammalia:Ig C deltal fragment -143- 1101448A:Mammalia:Ig D/JH 1101448B:Mammalia:Ig D/JH 0407221A:Mammalia:Ig L-V extra peptide 0407221B:Mammalia:Ig lambda C extra peptide 770551A:Mammalia:Ig lambdal N Term extra 1011444C:Mammalia:Ig VH E4.15 1011444D:Mammalia:Ig VH E4psi 1807240A:Mammalia:indolicidin 0412251A:Mammalia:inhibitor Pl.alphal proteinase 0806130A:Mammalia:inhibitor,angiotensin converting enzyme 1312309A:Mammalia:insulin 1312309B:Mammalia:insulin 560164B:Mammalia:insulin 1506296A:Mammalia:insulin A 1506296B:Mammalia:insulin B 0510475A:Mammalia:insulin B,24/25-Leu 680495A:Mammalia:insulin C peptide 711102A:Mammalia:insulin C peptide 720389A:Mammalia:insulin C peptide 720389C:Mammalia:insulin C peptide 740063A:Mammalia:insulin C peptide 1008138A:Mammalia:insulin releasing peptide 1008138B:Mammalia:insulin releasing peptide 610207A:Mammalia:kallidin II 1301340A:Mammalia:kinase,B creatine 1103228A:Mammalia:kinase.phosphoglycerate 1107242A:Mammalia:kinase,phosphoglycerate 1206205A:Mammalia:kinetensin 0905156A:Mammalia:kinin T 0505388A:Mammalia:kyotorphin 0811241A:Mammalia:kyotorphin,neo 0905239A:Mammalia:kyotorphin,neo 710434A:Mammalia:luliberin 720377A:Mammalia:luliberin 1714374A:Mammalia:luliberin,9-Hyp 0806461A:Mammalia:lymphocyte stimulating peptide 1510156B:Mammalia:M assocd ORE 1609099A:Mammalia:macrophage chemotactic factor 1516181A:Mammalia:melanin concentrating hormone 710420A:Mammalia:melanostatin I 711502A:Mammalia:melanostatin I 720300A:Mammalia:melanostatin II 0609249A:Mammalia:melanotropin 9-18,9-Tyr 570116A:Mammalia:melanotropin alpha 590325A:Mammalia:melanotropin alpha 600123A:Mammalia:melanotropin alpha 610131A:Mammalia:melanotropin alpha 630479A:Mammalia:melanotropin alpha 560090A:Mammalia:melanotropin beta 570098A:Mammalia:melanotropin beta 590120A:Mammalia:melanotropin beta 610131B:Mammalia:melanotropin beta 610263A:Mammalia:melanotropin beta 630479B:Mammalia:melanotropin beta 0703205A:Mammalia:melanotropin beta,des-l-Asp 0810171A:Mammalia:melanotropin delta 0807304A:Mammalia:melanotropin gammal 0706201A:Mammalia:melanotropin gamma3 0807304B:Mammalia:melanotropin gamma3 0707206A:Mammalia:melanotropin like peptide,gammal 0506561A:Mammalia:melanotropin potentiating factor 0702463A:Mammalia:melanotropin,des-Ac 0512240A:Mammalia:melanotropin,N,0-(Ac)2 0909223A:Mammalia:mesotocin 1504308A:Mammalia:Met enkephalin like peptide 0512482A:Mammalia:Met enkephalin releasing peptide -144 0912288A:Mammalia:metorphamide 0904334A:Mammalia:motilin 1105286A:Mammalia:motilin 730210A:Mammalia:motilin 740052A:Mammalia:motilin 1702149A:Mammalia:myelin peptide amide pMPA14 1411251A: Mammalia:nephritogenoside 0908308A:Mammalia:neurokinin alpha 0908308B:Mammalia:neurokinin beta 0908190A:Mammalia:neuromedin B 1003162A:Mammalia:neuromedin C 1011992A:Mammalia:neuromedin L 1008139A:Mammalia:neuromedin N 1411284A:Mammalia:neuromedin U 1414195A:Mammalia:neuromedin U 1612383A:Mammalia:neuromedin U 1709387A:Mammalia:neuromedin U 25 1709387B:Mammalia:neuromedin U 8 1109148A:Mammalia:neuromedin U25 1711482A:Mammalia:neuromedin U25 1406325A:Mammalia:neuropeptide gamma 1413348A:Mammalia:neuropeptide K 1-24 1612231A:Mammalia:neuropeptide Y 12-36 1208283A:Mammalia:neurotenisin,7-Ser 0605222A:Mammalia:neurotensin 1716411A:Mammalia:neurotensin 750525A:Mammalia:neurotensin 1306331A:Mammalia:neurotensin related peptide 1809273A:Mammalia:neutral thyroliberin-like peptide 1809273B:Mammalia:neutral thyroliberin-like peptide 1811319A:Mammalia:neutrophil granule peptide HP1 0811159A:Mammalia:opioid 4-peptide 0704330A:Mammalia:opioid peptide,adrenal 0706309A:Mammalia:opioid peptide,adrenal 1512206A:Mammalia:ORF 1 520009A:Mammalia:oxytocin 530009A:Mammalia:oxytocin 580093A:Mammalia:oxytocin 580181A:Mammalia:oxytocin 590227A:Mammalia:oxytocin 600454B:Mammalia:oxytocin 640049A:Mammalia:oxytocin 690929A:Mammalia:oxytocin 730938A:Mammalia:oxytocin 1004251A:Mammalia:pancreatic 20-peptide 1301201A:Mammalia:pancreatic 20-peptide 1003237A:Mammalia:pancreatic 20-peptide C term 0801264A:Mammalia:pancreatic islet peptide 720699A:Mammalia:pancreatic polypeptide 0411198A:Mammalia:pepsinogen I/II major glycopeptide 1003823A:Mammalia:peptide 3b,Pro rich 0405230A:Mammalia:peptide A12d,glyco 0405230B:Mammalia:peptide A17c,glyco 0405230C:Mammalia:peptide B12,glyco 0610176A:Mammalia:peptide BAM12P 1707342B:Mammalia:peptide His-Ile 1109251A:Mammalia:peptide 0A24b 0712238A:Mammalia:peptide PHI 1010243A:Mammalia:peptide PHI 1011215A:Mammalia:peptide PHI 1610159A:Mammalia:peptide PHI 1-27 1109144A:Mammalia:peptide SCP3 1109144B:Mammalia:peptide SCP4 1109144C:Mammalia:peptide SCP5 1109144D:Mammalia:peptide SCP6 0405258A:Mammalia:peptide,carbohydrate carrying 145- 0405258B:Mammalia:peptide,carbohydrate carrying 711641A:Mammalia:peptide,conditioned avoidance response 711078A:Mammalia:peptide,glyco 763904A:Mammalia:peptide,inhibitory 1310217A:Mammalia:peptide,mitosis inhibiting 0712205A:Mammalia:peptide,salivary low MW 0712205B:Mammalia:peptide,salivary low MW 0909223B:Mammalia:phenypressin 1208305A:Mammalia:physalaemin like peptide 1312257A:Mammalia:physalaemin like peptide 0509287A:Mammalia:placental lactogen precursor 1617235A:Mammalia:pneumadin 720638A:Mammaliaposterior pituitary peptide 1603334A:Mammalia:prepro-tachykinin C flanking peptide 1603334B:Mammalia:prepro-tachykinin C flanking peptide 0906230A:Mammalia:preprocalcitonin derived peptide 1706329A:Mammalia:pro-bursin 14-peptide 1412264A:Mammalia:pro-enkephalin A 209-239 0501205A:Mammalia:prolactin fragment,pre 1503284A:Mammalia proliferation inhibiting 4-peptide 0905217A:Mammalia protein B,alpha2HS glyco 711659A:Mammalia protein,glyco 0409335A:Mammalia protein.myeloma,IF3 H chain fragment 1508220A:Mammalia:Pyr-Glu-Pro-NH2 0401165A:Mammalia:redox active peptide 1503186A:Mammalia:relaxin A 1010328B:Mammalia:renin precursor N terra 0705052A:Mammalia:rigin 0811244A:Mammalia:rimorphin 0811266A:Mammalia:rimorphin 1708211A:Mammalia:salivary His rich peptide 721151A:Mammalia:scotophobin 0706234A:Mammalia:secretin 1111240A:Mammalia:secretin 1311331A:Mammalia:secretin 1508173A:Mammalia:secretin 1608239A:Mammalia:secretin 1707342C:Mammalia:secretin 700579A .‘Mammalia: secretin 1009234A:Mammalia:secretin,pro 1310205A:Mammalia:seminal peptide 20 1509222A:Mammalia:seminal plasma peptide,major 1509222B:Mammalia:seminal plasma peptide,minor 710685B:Mammalia:somatoliberin 1007198A:Mammalia:somatostatin 730308A:Mammalia:somatostatin 760556A:Mammalia:somatostatin 0706466A:Mammalia:somatostatin 14 0701278A:Mammalia:somatostatin 25 0701278B:Mammalia:somatostatin 28 1101392A:Mammalia:somatostatin 28 0602201A:Mammalia:somatostatin,N term extended 710685A:Mammalia:somatotropin releasing 10-peptide 0703632A:Mammalia:spinal cord 3-peptide 0804312A:Mammalia:spinal cord 3-peptide 1305396A:Mammalia:steroidogenesis activator protein 1516350A:Mammalia:substance P 710757A:Mammalia:substance P 730714A:Mammalia:substance P 1102255B:Mammalia:superoxide dismutase N term,Cu/Zn 0612159A:Mammalia:synaptosomal 3-peptide 0612159B:Mammalia:synaptosomal 4-peptide 1109246C:Mammalia:T cell receptor alpha TA20 1106231B:Mammalia:T cell receptor beta2 1107197B:Mammalia:T cell receptor D/Jbeta 1107197C:Mammalia:! cell receptor D/Jbeta -146- 1209256R:Mammalia:T cell receptor J alpha 1209256T:Mammalia:! cell receptor J alpha 1209256V:Mammalia:T cell receptor J alpha 1209256W:Mammalia:T cell receptor J alpha 0401182A:Mammalia:thymic factor 1408159A:Mammalia:thymic humoral factor gamma2 1714203A:Mammalia:thymocyte growth peptide 0503215A:Mammalia:thymosin alphal 770552A:Mammalia:thymosin alphal 690932A:Mammalia:thyroliberin 700037A:Mammalia:thyroliberin 0608648A:Mammalia:toxin,uremic 1002226A:Mammalia:transferase Munich,HG phosphoribosyl 0311213A:Mammalia:trypsinogen 2nd activation peptide 0507223B:Mammalia:trypsinogen N term fragment,cationic 732142A:Mammalia:tuftsin 1814294A:Mammalia:tumor invasion inhibiting factor 2 0409242A:Mammalia:uremic 3-peptide 0509213A:Mammalia:uremic 5-peptide 0410178A:Mammalia:uremic 7-peptide 0901251A:Mammalia:uremic peptide 0810210A:Mammalia:urokinase A1 1111231A:Mammalia:valosin 0603334A:Mammalia:vasoactive 11-peptide 0603334B:Mammalia:vasoactive 5-peptide 0603334C:Mammalia:vasoactive 5-peptide 0601216A:Mammalia:vasoactive intestinal peptide 1007235A:Mammalia:vasoactive intestinal peptide 1012267A:Mammalia:vasoactive intestinal peptide 1106144A:Mammalia:vasoactive intestinal peptide 1109226A:Mammalia:vasoactive intestinal peptide 1301342A:Mammalia:vasoactive intestinal peptide 1301342B:Mammalia:vasoactive intestinal peptide 1616408A:Mammalia:vasoactive intestinal peptide 1707342A:Mammalia:vasoactive intestinal peptide 1809387A:Mammalia:vasoactive intestinal peptide 720473A:Mammalia:vasoactive intestinal peptide 0909628A:Mammalia:vasopressin metabolite 0611243A:Mammalia:vasopressin,2-Phe 530215A:Mammalia:vasopressin,Arg 580093B:Mammalia:vasopressin,Arg 580181B:Mammalia:vasopressin,Arg 590227B:Mammalia:vasopressin,Arg 600454C:Mammalia:vasopressin,Arg 600454D:Mammalia:vasopressin,Arg 610025A:Mammalia:vasopressin,Arg 640049B:Mammalia:vasopressin,Arg 681070A:Mammalia:vasopressin,Arg 690929B:Mammalia:vasopressin,Arg 730938B:Mammalia:vasopressin,Arg 0909223C:Mammalia:vasopressin,Lys 520042A:Mammalia:vasopressin,Lys 530011A:Mammalia:vasopressin,Lys 610024A:Mammalia:vasopressin,Lys 681070B:Mammalia:vasopressin,Lys 690929C:Mammalia:vasopressin, Lys 0603289A:Mammalia:vasopressin,N-Ala-Gly Arg 0603289B:Mammalia:vasopressin,N-Val-Asp Arg 701174A:Mammalia:vasotocin 1806498A:Mammalia:VIP 1616409A:Mammalia:xenopsin precursor 1614424A:Mammalia:xenopsin-related peptide 1817263N:Mammalia:Zn finger protein ZNF60 1508175A:Mollusca:achatin I 1712115A:Mollusca:achatin I 0910276A:Mollusca:alpha bag cell peptide -147- 1412184A:Mollusca alpha conotoxin SI 1718180A:Mollusca alpha conotoxin SIA 1605128A:Mollusca APGWamide 1711282A:Mollusca APGffamide 1411339A:Mollusca buccalin 1203283B:Mollusca califin S 1607138A:Mollusca cardio-excitatory peptide 0901287A:Mollusca cardioactive peptide 0309321A:Mollusca cardioexcitatory peptide 1312259A:Mollusca catch relaxing peptide 1806388A:Mollusca cephalotocin 1607264A:Mollusca conantokin T 1314217A:Mollusca conopressin 1314217B:Hollusca conopressin 0706261A:Mollusca conotoxin GI 0706261B:Mollusca conotoxin GIA 0706261C:Mollusca conotoxin GII 1108256A:Mollusca conotoxin GIIIA 1108256B:Mollusca conotoxin GIIIB 1108256C:Mollusca conotoxin GIIIC 1011179A:Mollusca conotoxin GVIA omega 0812169A:Mollusca conotoxin MI • 1501345A:Mollusca egg laying hormone precursor 1207245A:Mollusca egg laying peptide A-AP 630171A:Mollusca:eledoisin 1003242A:Mollusca enkephalin,Leu 1003242B:Mollusca enkephalin,Met 1006817A:Mollusca enkephalinyl-Arg-Phe.Met 0811378A:Mollusca FMRFamide like peptide 1201209A:Mollusca FMRFamide like peptide 1309313A:Mollusca FMRFamide like peptide 2 1309313B:Mollusca FMRFamide like peptide 3 1714188A:Mollusca fulicin 1504408A:Mollusca molluscivorous conus toxin 1310373A:Mollusca myomodulin 1718371A:Mollusca myomodulin B 1407114A:Mollusca mytilus inhibitory peptide 1810269A:Mollusca mytilus inhibitory peptide 1810269B:Mollusca mytilus inhibitory peptide 1810269C:Mollusca mytilus inhibitory peptide 1810269D:Mollusca mytilus inhibitory peptide 1810269E:Mollusca mytilus inhibitory peptide 1815162A:Mollusca Mytilus inhibitory peptide A1 1815162K:Mollusca Mytilus inhibitory peptide A10 1815162B:Mollusca Mytilus inhibitory peptide A2 1815162C:Mollusca Mytilus inhibitory peptide A3 1815162D:Mollusca Mytilus inhibitory peptide A4 1815162E:Mollusca Mytilus inhibitory peptide A5 1815162F:Mollusca Mytilus inhibitory peptide A6 1815162G:Mollusca Mytilus inhibitory peptide A7 1815162H:Mollusca Mytilus inhibitory peptide A8 1815162J:Mollusca Mytilus inhibitory peptide A9 1506519B:Mollusca neuropeptide RISbeta 1506427A:Mollusca pedal peptide 1602255B:Mollusca peptide I 1203403A:Mollusca peptide II- 1816140A:Mollusca peptide PYF 1101325A:Mollusca sleeper peptide 1304344A:Mollusca small cardioactive peptide A 0906220A:Mollusca toxin I,geographu 0906220B:Mollusca toxin II,geographu 1803237B:Mollusca toxin TxIA 1803237C:Mollusca toxin TxIB 1803237A:Mollusca toxin TxIIA 1610245B:Nematoda ceh-12 gene 1813281A:Nematoda FMRFamide —148 — 1611406A:Nematoda:neuropeptide AF1 1813281B:Nematoda:SADPNFLRFamide 1109265A:Nematoda:vitellogenin 1 1109265B:Nematoda:vitellogenin 2 1109265C:Nematoda:vitellogenin 4 1109265D:Nematoda:vitellogenin 6 0403176A:Osteichthyes:angiotensin I 1510162A:Osteichthyes:angiotensin-converting enzyme inhibitor 1805355A:Osteichthyes:angiotensin-converting enzyme inhibitor B-l 1805355B:Osteichthyes:angiotensin-converting enzyme inhibitor B-2 1805355C:Osteichthyes:angiotensin-converting enzyme inhibitor B-3 1717175A:Osteichthyes:ANP 1515184A:0steichthyes:atrial natriuretic factor 1613139A:Osteichthyes:BNP like natriuretic peptide 1709312A:0steichthyes:carassin 721464A:Osteichthyes:clupeine Y2 0803150A:Osteichthyes corticotropin like interm lobe peptide 0508149A:Osteichthyes endorphin 0604188A:Osteichthyes endorphin II 0708424A:Osteichthyes glucagon 1105264A:Osteichthyes glucagon 1205373A:Osteichthyes glucagon 1307182B:Osteichthyes glucagon 1716265A:Osteichthyes glucagon 1709218B:Osteichthyes glucagon-like peptide 1206278A:Osteichthyes glycoprotein 7,antifreeze 1206278B:Osteichthyes glycoprotein 7,antifreeze 1206278C:Osteichthyes glycoprotein 7R,antifreeze 1206278D:Osteichthyes glycoprotein 8R,antifreeze 690924A:Osteichthyes:insulin 1412252A:Osteichthyes insulin A 1412252B:Osteichthyes insulin B 1603264A:Osteichthyes insulin C peptide 1718169A:Osteichthyes intestinal peptide EIPP 620022A:Osteichthyes:isotocin 650767A:Osteichthyes:isotocin 681068A:Osteichthyes:isotocin 690928A:Osteichthyes:isotocin 701183A:Osteichthyes:isotocin 765600A:Osteichthyes."learning induced brain peptide 765600B:0steichthyes:learning induced brain peptide 0905260A:0steichthyes rluliberin 0910249A:Osteichthyes:melanin concentrating hormone 0612177A:Osteichthyes rmelanotropin 0511280A:Osteichthyesimelanotropin alpha 0511280B:Osteichthyes:melanotropin beta 0608228A:Osteichthyesimelanotropin II alpha 671037A:Osteichthyes:mesotocin 701188A:Osteichthyesimesotocin 1814320C:Osteichthyes:neurokinin A 1403319A:Osteichthyes:oxyntomodulin 610242A:Osteichthyes:oxytocin 1208325A:Osteichthyes:peptide,pro-aPY 1412186A:0steichthyes:polysialo glycoprotein repeat seq 1412186B:Osteichthyes:polysialo glycoprotein repeat seq 1313241A:0steichthyes:pro-GIF derived peptide SI 1313241C:Osteichthyes:pro-GIF derived peptide S2 1209204A:0steichthyes:protamine 1209204B:Osteichthyes:protamine 0601202A:Osteichthyes:somatostatin 1012240A:Osteichthyes:somatostatin 22 1210254A:Osteichthyes:somatostatin 25 1101339A:Osteichthyes:somatostatin 28 1305261A:Osteichthyes:somatostatin 28 1010245A:Osteichthyes:somatostatin 28 II 0605221A:Osteichthyes:somatostatin I 149- 1103330A:Osteichthyes:somatostatin precursor 1414253A:0steichthyes:somatostatin/glucagon related peptide 1414253B:0steichthyes:somatostatin/glucagon related peptide 0503186A:Osteichthyes:stellin A 764668A:0steichthyes:sturine B 1814320A:0steichthyes:substance P 1814320B:Osteichthyes:substance P 753677A:0steichthyes:thyroliberin 0902237A:Osteichthyes:urotensin I 1701464B:Osteichthyes:urotensln I N-terminal flanking peptide 0610250A:Osteichthyes:urotensin II 1817244A:0steichthyes:urotensin II 1007166A:0steichthyes:urotensin Ilalpha 1007166B:Osteichthyesrurotensin Ilbeta 1007166C:Osteichthyes:urotensln Ilgamma 0909285A:Osteichthyes:urotensin UIIA 0909285B:Osteichthyes:urotensln UIIB 610017A:0steichthyes:vasotocin 610023A:Osteichthyes:vasotocin 610203A:Osteichthyes:vasotocin 610242C:Osteichthyes:vasotocin 650767B:Osteichthyes:vasotocin 681068B:Osteichthyes:vasotocin 690928B:Osteichthyes:vasotocin 701183B:0steichthyes:vasotocin 1603248A:Osteichthyes:VIP related peptide 1601305A:Plantae:ACE inhibitory peptide FLP 1 1601305B:Plantae:ACE inhibitory peptide FLP 2 1601305C:Plantae:ACE inhibitory peptide FLP 3 1508168A:Plantae:agglutinin S 1810244A:Plantae:antimicrobial peptide 1 1810244B:Plantae:antimicrobial peptide 2 1709506A:Plantae:cycloleonurinin 671044A:Plantae:fibrinopeptide A 671044D:Plantae:fibrinopeptide B 1108239E:Plantae:gliadin gamma 1103178B:Plantae:inhibitor CPGTI-I,trypsin 1103178C:Plantae:inhibitor CPTI-II,trypsin 1103178F:Plantae:inhibitor CSTI-IV,trypsin 0708183A:Plantae:inhibitor III,trypsin 0903201A:Plantae:inhibitor LTD I,trypsin iso 0903201B:Plantae:inhibitor LTD III,trypsin iso 750673A:Plantae:melanotropin beta 1603356CN:Plantae:ORF 23 1603356CD:Plantae:ORF 28 1603356N:Plantae:ORF 29 1506382A:Plantae:protease inhibitor 1405344C:Plantae:ribosomal protein L2 1010307E:Plantae:ribosomal protein S19’ 1211248A:Plantae:S rich 2S protein S 1503172C:Plantae:Ser protease inhibitor MCEI-I 1503172A:Plantae:Ser protease inhibitor MCTI-I 1503172B:Plantae:Ser protease inhibitor MCTI-II 1715381A:Plantae:systemin 1314174A:Plantae:trypsin inhibitor 1314174B:Plantae:trypsin inhibitor 1702179A:Plantae:trypsin inhibitor 1801181A:Plantae:trypsin inhibitor 1 1801181B:Plantae:trypsin inhibitor 2 1817218A:Plantae:trypsin inhibitor I 1817218B:Plantae:trypsin inhibitor II 1817218C:Plantae:trypsin inhibitor III 1111187A:Plantae:URF 601 1111187C:Plantae:URF 603 1414347B:Prokaryota:aceB gene assocd ORF 1109139B:Prokaryota:amylase alpha -150- 1708222A:Prokaryota:anantin/des-Phe analog 1616302A:Prokaryota :appA assocd ORF X 1512237B:Prokaryota :bfr assocd ORF 24' 1702271B:Prokaryota:cinnamycin 1312339D:Prokaryota:colicin E9 lysis gene 1004221A:Prokaryota :dehydrogenase fragment K,glucose 1616249A:Prokaryota:delta-like toxin 1501381C :Prokaryota :dicB operon ORF 1702271A:Prokaryota:duramycin 1702270A:Prokaryota:duramycin B 1702270B:Prokaryota :duramycin C 1101217A:Prokaryota :enterotoxin ,heat stable 1111219A :Prokaryota :enterotoxin ,heat stable 1201224A:Prokaryota:enterotoxin ,heat stable 1314236C:Prokaryota :F sex factor traD 1208313B :Prokaryota:flavoprotein 1414238A :Prokaryota :gallidermin 1310302B:Prokaryota:gene cotB 1308167H:Prokaryota:gene F 1313282B:Prokaryota:gene glgA 1111234B:Prokaryota :gene glnL 1309264A:Prokaryota:gene nodD 1309261B :Prokaryota:gene orf 19 1108230B :Prokaryota :gene tmpB 1302279A:Prokaryota :gene trpE 1307194A :Prokaryota :gene trpL 1109252A :Prokaryota : germ specificity regulating peptide 1504256J:Prokaryota:H translocating ATPase beta 1506318A :Prokaryota :halo toxin 1813209A:Prokaryota:heat-stable enterotoxin 0608214A:Prokaryota:hemolysin delta 1005204A:Prokaryota:hemolysin delta 1812252A :Prokaryota :iad gene 1302189B :Prokaryota :leader peptide ermG 1302189C :Prokaryota :leader peptide ermG 1810255A:Prokaryota:marinostatin Cl 1810255B:Prokaryota:marinestatin C2 1805264A:Prokaryota:marinostatin D 1109142A:Prokaryota:microcin 7 1105311B:Prokaryota :ORF 1413245C:Prokaryota:ORF 2 1502355C:Prokaryota:ORF 2 1803344B :Prokaryota :ORF 2 in noncoding region of sra gene 1209230C :Prokaryota :ORF 3 1803344C :Prokaryota :ORF 3 in noncoding region of sra gene 1209230D :Prokaryota :ORF 4 1807289B :Prokaryota :ORF 7K 1305262A:Prokaryota :ORF E 1010255A:Prokaryota:ORF in ori 1012216A:Prokaryota:ORF N term,toluate operon 1615170D:Prokaryota :pdhA assocd ORF 1202257G:Prokaryota :peptide 1 1202257H:Prokaryota:peptide 2 1101257A:Prokaryota:pheromone cADl.bacterial sex 1502267B:Prokaryota :phoP gene 0504198A :Prokaryota :phosphorylase fragment.maltodextrin 1010260B:Prokaryota:phycobiliprotein N term 1109172A :Prokaryota :phycocyanin beta 1402269F:Prokaryota:plasmid virD 1504241B:Prokaryota :PQQ gene assocd ORF 1810428B :Prokaryota :pqqA gene 1002248A:Prokaryota:protein araE,regulatory 0812230A:Prokaryota:protein fragment ,replication initiation 1002280B:Prokaryota:protein H,reaction center 1002280C:Prokaryota:protein L,reaction center 1002280D:Prokaryota:protein M,reaction center 151 - 1501278D:Prokaryota pSPA12 gene 1501278E:Prokaryota pSPA13 gene 1807309C:Prokaryota pufK gene 1807238A :Prokaryota pyrrolo-quinoline-quinone synthesis gene 1403333B:Prokaryota repAX gene 1712183A:Prokaryota replication locus ORE 30 0907240E:Prokaryota ribosomal protein S17 1516355H:Prokaryota ribosomal protein S8 1501251B:Prokaryota rpoB gene assocd ORE 1612377A:Prokaryota rRNA methylase assocd ORE 1210251A:Prokaryota sex pheromone CAM373 1413289A:Prokaryota sex pheromone cCFlO 1708286A :Prokaryota sex pheromone inhibitor iADl 1708286B :Prokaryota sex pheromone inhibitor iADl downstream ORE 1616276A:Prokaryota shl leader peptide 1313358C:Prokaryota streptomycin syn gene aphD 1203213B:Prokaryota synthase ,enolpyruvylshikimate phosphate 1012306A:Prokaryota synthetase N term,Gin 0709232A:Prokaryota toxin,heat stable entero 0902149A :Prokaryota toxin,heat stable entero 0902181A:Prokaryota toxin,heat stable entero 0906203A:Prokaryota toxin,heat stable entero 1812252B:Prokaryota traE upstream ORE TTS1 1704313A:Prokaryota trp operon leader peptide 1513305A:Prokaryota trpEG attenuator peptide 1607231A:Prokaryota trpGDC operon leader peptide 1805362A:Reptilia:alpha MSH 0402224A:Reptilia:angiotensin 0402224B:Reptilia:angiotensin 0402224C:Reptilia:angiotensin 710784A:Reptilia:angiotensin converting enz inh V2 710784B:Reptilia:angiotensin converting enz inh V6I 710784C:Reptilia:angiotensin converting enz inh V6II 710784D:Reptilia:angiotensin converting enz inh V7 710784E:Reptilia:angiotensin converting enz inh V8 710784F:Reptilia:angiotensin converting enz inh V9 1002212A:Reptilia:angiotensin I 0903145A:Reptilia:bradykinin potentiating peptide 1005285A:Reptilia:bradykinin potentiating peptide 1208380A:Reptilia:bradykinin potentiating peptide 690149A:Reptilia:bradykinin potentiating peptide A 700017A:Reptilia:bradykinin potentiating peptide B 710067A:Reptilia:bradykinin potentiating peptide C 690149B:Reptilia:bradykinin potentiating peptide D 700471A:Reptilia:bradykinin potentiating peptide E 1617159A:Reptilia:bradykinin potentiating peptide S3-.1 1617159D:Reptilia:bradykinin potentiating peptide S4-.l-.2- 1617159B:Reptilia:bradykinin potentiating peptide S4-,3,l- 1617159C:Reptilia:bradykinin potentiating peptide S4-,3,2- 1617159H:Reptilia:bradykinin potentiating peptide S5-.1- 1617159G:Reptilia:bradykinin potentiating peptide S5-.2- 1617159F:Reptilia:bradykinin potentiating peptide S5-.3- 1617159E:Reptilia:bradykinin potentiating peptide S5-.4- 700202A:Reptilia:bradykinin potentiating peptide V3A 1607186A:Reptilia:bradykinin ,6-Thr 701176A:Reptilia:bradykinin ,6-Thr 721948B:Reptilia:fibrinopeptide A 721948E :Reptilia:fibrinopeptide B 1612382B:Reptilia:glucagon 1709419A:Reptilia:lethal toxin I 1711481A:Reptilia:luliberin I 1711481B:Reptilia:lullberin II 690927A:Reptilia:mesotocin 721912A:Reptilia:mesotocin 610522B:Reptilia:oxytocin 660031A: Reptilia .-peptide A, venom -152- 660031B:Reptilia:peptide B,venom 1208381A:Reptilia:peptide POL236 1410356A:Reptilia:sarafotoxin S6al 1410356B:Reptilia:sarafotoxin S6b 1410356C:Reptilia:sarafotoxin S6c 1515248A:Reptilia:sarafotoxin SRTXd 610522D:Reptilia:vasotocin 680069A:Reptilia:vasotocin 69092TB:Reptilia:vasotocin 721912B:Reptilia:vasotocin 1813189A :SUBUNIT=A chain:ISOTYPE=II:Mollusca:insulin-related peptide 1803251A :SUBUNIT=A chain:Reptilia:insulin 1810291A:SUBUNIT=A:ISOTYPE=III:Mollusca:insulin-related peptide 1715201A:SUBUNIT=A:Mammalia:relaxin 1712127A:SUBUNIT=A:Osteichthyes :insulin 1812293A :SUBUNIT=A:Osteichthyes :insulin 1803251B:SUBUNIT=B chain:Reptilia:insulin 1715201B:SUBUNIT=B:Mammalia:relaxin 1812293B :SUBUNIT=B:Osteichthyes :insulin 1715168M :SUBUNIT=gamma :IS0TYPE=1:Reptilia:crotoxin A1 1715168N :SUBUNIT=gamma :IS0TYPE=2:Reptilia:crotoxin A1 1715168P :SUBUNIT=gamma :IS0TYPE=3:Reptilia:crotoxin A1 1715168Q:SUBUNIT=gamma:IS0TYPE=4 :Reptilia:crotoxin A1 1716290A:SUBUNIT=heavy chain:Mammal!a:Ig J1 segment 1716290B :SUBUNIT=heavy chain:Mammalia:Ig J2 segment 1716290C:SUBUNIT=heavy chain:Mammalia:Ig J3 segment 1716290D :SUBUNIT=heavy chain:Mammalia:Ig J4 segment 1303320A:Virus art gene exon 1 1615267B:Virus capsid protein 1304195A:Virus early leader protein 1414220D:Virus G protein 1304292E:Virus gene 1.5 1604374K:Virus gene 5.1 1813170G:Virus gene J 1206374B:Virus gene L 1806326E:Virus gene VII 1712189A:Virus genome linked protein VPg 0403209A:Virus internal peptide VII 1210381A:Virus ORE 1412351B:Virus ORE 1408236A:Virus ORE 1 1710262B:Virus ORE 26 1709308B:Virus ORE 30 1301338X:Virus ORE 58.3 1112178M:Virus ORE 7 1209333D:Virus ORE IIB satellite RNA5 1309330A:Virus ORE satellite RNA 1715306B:Virus 0RF,5'-terminal 0908286L:Virus protein 1.5 1202223A:Virus protein C 1512364C:.Virus protein gp51 0902231AQ:Virus:protein orf28 0908283B:Virus:protein VPg 1105312L:Virus:protein VPg 101l278A:Virus:protein VPx N term 1009209A:Virus:RNA 5 1402304A:Virus:RNA polymerase 1403393C:Virus :stp gene ORE B 1505419B :Virus:tat gene 1412350B:Virus:terminal protein 1504354C:Virus:upstream ORE * Animal ia O -153- m.5. temper)se^itcx^xr* (7n7f-fa^STR30N) #: 1 f ; y#T%^T ; / ?' fK 93.2 strn #: 2 (DNA {c 3 - K £ ft T ^ & l' ^ X f- K 0 y 7. h ) #: 3 ##) #: 4 3:##4#®#®2:5:2>is#: #: 5 3 . D=D-T 5 smz^ts #: 6 LIST OF PEPTIDES CONTAINING NON PROTEIN AMINO ACIDS #: 7 FORMAT=LITERATURE NO:NAME:COMMENT(structure,component ,propery ) 0608217 :Aad-Ala-Val Aad-Ser-Val Aad-Ser-IsodehydroVal : 1305532 :ACE Inhibitor K13:Cyclo(Tyr-Isodityrosine ) Deriv 1305533 :ACE Inhibitor WF10129 :MeCH(OH)(CH2)2C0CH2CH(COOH)-Ala-Tyr 1306580n :Actinoidin:Glycopeptide Antibiotic 0512246n:Adenochrome :Fe Binding Peptide 0602198n:Amaninamide: 1412598n :Amphibine:Cyclopeptide Alkaloid 1103492D :Ancovenin:AVQaAXFGPLXWSADGNXK a=D-Ala 1610381 :Ancovenin:Lanthionine,Abu,DehydroAla 1815450 :Anthelmintic PF1022A:Cyclo (Lac-MeLeu-3-Ph Lac-MeLeu)2 1819804n:Antibiotic A10255B/G/J:Thiopeptide 1508428n:Antibiotic A42867:Vancomycin Family 1612612n:Antibiotic A54145:Lipopeptide 1409561n:Antibiotic A83586C:Depsipeptide 1011404 :Antibiotic BMY28160 :Cyclodepsi-10-Peptide Dbu(Ac),Val,Phe,Leu,Ser 0907210 :Antibiotic Bu2470:Dbu-8-Peptide 1612607D:Antibiotic FR112123:Lys(Gly-Glu )-D-Ala 1714591n:Antibiotic GE2270A:Thiazolyl Peptide 0906226P:Antibiotic K582:Arg-Arg(0H)-0rn-Thr-0rn-Lys-Tyr 1717651 :Antibiotic MM55266/M55268:Glyco-7-Peptide 0701171n:Antibiotic Nol907: 0701170n:Antibiotic P168: 1008327 :Antibiotic P168:9-Peptide 6-0H-4-Me-8-0xo Aminodecanoate ,3-OH Ile.Aib 1816436 :Antibiotic P168 :Aib-9-Peptide MePro,Hyl ,Aib,Leu.beta Ala.AHMOD 1403592 :Antibiotic Sch37137:Ala-Gin(X) X=Epoxy Dicarboxylic Acid Monoamide 0907171 :Antitumor Cyclo-6-Peptide: 1506745P:Aphostatin:Ile-Ile-Ser(Phospho)-Gln-Glu 1211448 :Argiopine:C6H3((OH )2)CH2C0-Arg-Polyamine-Arg 1313442 :Argiotoxin:Arg-Polyamine-Asn 1803716 :Aureobasidin:Cyclodepsi-9-Peptide 1803717 :Aureobasidin: Cyclodepsi-9-Peptide 1718512 :Aureobasidin :MeVal,Phe,MePhe,Pro,alle,MeVal,Leu beta-OH MeVal 1307361D:Avellanin:Cyclo(D-MePhe-D-Ala-Ile-o-Abe-Pro) 1803768P :Axinastatin:Cyclo(Asn-Pro-Phe-Val2-Pro-Val) 1202461D:Azinothricin:3-0H Leu,D-Thr,D-MeAla,HO-Ser(Me) D/L-Piperazic Acid 1406382 :Azotobactin :D/L-Ser,3-0H D-threo-Asp,D,G,Hse,D-Citrulline,D-Orn(Ac,OH) 1305415 :B1625 FA2betal:Thr-Val-Sar-MeVal Deriv 1201380D :Bacillomycin :beta AA,D-Tyr ,D/L-Asn,Gin,Pro,Thr 0709212D :Bacillomycin :beta AA,Asp,D-Tyr ,D-Asn,Pro,Glu D-Ser,Thr 0509590D :Beauverilide:Cyclo(Hmd-Val-Phe-D-alle) 1212537n:beta Lactam Potentiator:0 Sulfated Glycopeptide 0808325P:beta MAPI:CO(Phe)-Arg-Val-Phe-H 1903606 :Bistratamide:Cyclo(Ala-Tzl-Val-Tzl-Val-Ozl ) 1903606 :Bistratamide:Cyclo(Ozl-Val-Ozl-Val-Tzl-Val-mOzn) 1410632n:Blastolysin: 1503540 :Blue Green Algae Toxin I/II:Cyclo(Ala-Leu-MAsp-Arg-Adda-Glu-Mdha) 1815454n :Bottromycin : 0707285 :Brevigellin :Cyclodepsi-5-Peptide 1818672 :BZR Cotoxin:Depsi-9-Peptide 1705341P:Cadystin:(Glu(Cys))n-Gly n=2/3 1010441P:Cadystin :Glu(Cys-Glu (Cys-Glu (Cys-Gly ))) 1807606D:Calophycin:Cyclo(AUNQGRJPXV) U=D-Asp,J=MeAsn X=Arainopalmitate Deriv 0907212P:cAMP Phosphodiesterase Inhibitor:Acyl-Glu-Leu2-Val-Asp-Leu2 Lactone 1310405P:Caricastatin:Ac-Leu-DL-Arg-H 1803667P:Cd Binding Peptide:((Glu(Cys))4-Gly 0612201 :Celenamide:4-Peptide 1110365 :Cephabacin:Ala-Ser-0rn-Val-0rn-Val 1008320 :Cephalosporin:4-Peptide 0808184 :Cirratiomycin :7-Peptide -154- 1610297 :Citropeptin: Cyclo-Depsi-6-Peptide Antibiotic 1603866P :Cleromyrine :Cycle(Ala-Gly-Pro-Ile-Val-Phe ) 1711651n:Conagenin : 1006359 :Cortinarin:Bicyclodepsi-10-Peptide 1715556 :Cortinarin:Cyclic 10-Peptide 1403721D:Cyanogenosin :MeDehydroAla ,D-Ala,Arg,D-Asp (3-Me),Arg beta-AA,D-IsoGlu 1202488 :Cyanoginosin :Cyclo-7-Peptide 1011415D:Cyanoginosin :D-Ala,Leu,beta-Me D-Asp,Ala,Adda,D-Glu N-Me DehydroAla 1312673 :Cyanoviridin :Cyclo (Ala-Arg-Masp-Arg-Adda-Glu-Mdha ) 1805429P :Cyclo(APFWGGP ): 1805429P :Cyclo(EAEWGEVP ): 1305652P:Cyclo(Hyp-Leu ): 1505628P:Cyclo(Hyp-Phe) : 1305652P:Cyclo(Hyp-Pro) : 1805429P:Cyclo(LIATGTF) : 1805429P:Cyclo(LLPYGSP) : 1503521P:Cyclo(PNSXNYNQ) : 0706215P:Cyclo(Thr-Val2-Leu ) N-Acyl Deriv: 1306545P:Cycloaspeptide :Cyclo(Ala-Phe-Leu-Tyr-X ) 0809267D:Cyclosporin: Cyclo-10-Peptide MeVal,MeLeu,D-Ala 1212534D:Cytostatic Peptide WF3161:Cyclo(Leu-Pip-Aoe-D-Phe ) 1516458D:D-Asp(Gly) : 1502533 :Decadienoic Acid Deriv:Cyclic 7-Peptide Toxin 1616628 :Depsidomycin :Cyclodepsi-7-Peptide 1409638 :Depsipeptide FR900359 :Ala,MeAla,Leu(beta-OH),Thr(Me),MeDehydroAla 1809743D :Desferri Ferribactin Pyoverdin :D-Ala,Dbu,Glu,Lys ,Orn,D-Ser,Thr,D-Tyr 0710282 :Destruxin:Depsi-6-peptide, MeAla,MeVal,Pipecolic Acid 0710238 :Detoxin Complex Congener :lie,Val,Phe,Detoxinine 1005339P:Diprotin:Ile-Pro-Ile,Val-Pro-Leu 1212595 :Discarin:3-0H Leu,Melle,Trp,p-0H Styrylamine 1104476D:Discodermin:MeGln,t-Leu,D-Trp,D-Cys (OSH),D-Leu 1012457D:Discodermin:Thr,Asn,Sar D-Ala,D-Pro,D-Trp,D-Cys ,D-Leu D/L-tLeu,MeGln 1811428 :Discokiolide:Phe,Pro,Asn,Melle,Asp,Discokiic Acid 1407508 :Dityromycin :MeVal,Pro,Val,Gly (Ph),Me2Thr,DehydroAA ,Epoxide Deriv 1212543D:Dolastatin:Cyclo(Pro-D-Leu-Val-D-Thz-Thz) 1314416 :Dolastatin:Cyclo(Val-Pro-Leu- (gin)Thz-(gly)Thz) 1512544n:Dolastatin:Depsipeptide 1602473 :Dolastatin:Gly2 ,Ala2,MeLeu,MeTyr(Me) ,MeVal,Aminopentanoate 1604691 :Dolastatin:Me2Val-Val-MeVal-Pro2-Hiv-Dpy 0803206n:Dolastatin:Thiazole Containing Cyclopeptide 0507218 :Echinocandine:Cyclo-6-Peptide 1111420 :Edeine:Gly(Ph )-beta-Ala(OH)-Dbu-Dha-Gly-Spe 0806152n :Efrapeptin : 1811540 :Efrapeptin:15-Peptide 1706616 :Efrapeptin :Pip,Aib,beta-Ala,Iva,Ala,Leu,Gly 1010384D:Empedopeptin:Depsi-9-Peptide D/L-Pro,D-Ser,Arg,3-Hyp ,D/L-beta-OH Asp 1805543D:Endothelin Antagonist :Cyclo(D-AA-Leu-D-Trp-D-Glu-Ala ) 1804602D:Endothelin Binding Inhibitor :Cyclo(D-Glu-Ala-D-Val-Leu-D-Trp ) 1817583n:Enniatin:Cyclodepsipeptide 1804729 :Enopeptin:Cyclodepsi-5-Peptide 0901283 :Ergobutyrine :Aib,Pro,Leu 1901570 :Eurystatin :Leu,Orn,3-Amino-2-0xobutyric Acid 1806831 :Fellutamide:3-0H Decanoyl-Asn-Gln-Leu-H 1501727P:Fenestin:Cyclo(Pro2-Leu-Ile) ,Cyclo(Pro-Val-Pro-Leu-Ile) 0608265P:Feruloyl-Gly-Phe: 1112362D:Fluoroactinomycin:(MeVal-Sar-X-D-Val-Thr)2 Deriv 1301475 :Folyl Poly-Glu:Abe-(Glu)n n=2-6 1201394 :Foroxymithine:For-Orn(For ,OH)-Ser-Cyclo-2-Peptide 0906301 :Fosfazinomycin :Val-Arg-NHNMePO (OH)CH(OH)COOMe 1412598n :Frangufoline :Cyclopeptide Alkaloid 1808459n :Galacardin:Glycopeptide Antibiotic 1212564 :gamma-Glu Peptide:Glu(Cys(Me)-beta-Ala) ,Glu(Orn(Ac)) 1606469n :Geodiamolide :Cytotoxic Cyclodepsipeptide 0502218P :Globomycin :Cyclo (alle-Ser-aThr-Gly-X-MeLeu ) 0712212 :Globomycin :Cyclodepsi-6-Pept ide 1505533P:Glu(Asp): -155- 1211549 :Glu(D-Hph):Hph=3-Ph-3-Aminopropanoic Acid 1413528D:Glycopeptidolipid Antigen :X-D-Phe-aThr(X)-D-Ala-Ala-X 1801429D :Glycopeptidolipi :D-Phe-D-aThr-D-Ala-Alaninol 1007292n:Gramicidin S2/S3: 1505427P:GSH Analog:Glu(Ser-Gly) 1603557 :Halocyamine:His-Phe((OH)2)-Gly-Tryptamine Deriv 1707360 :Harmaomycin :aThr,He,3-Me Phe,4-Propenyl Pro 3-Nitrocyclopropyl Ala 0804347 :HC Toxin: Cyclo-4-Peptide 9, 10-Epoxy-8-Oxo Aminodecanoate Ala2,Pro 0901322 :HC Toxin: Cyclo-4-Peptide Ala,Pro,Aminoepoxyoxodecanoic Acid 1709567n :Helvecardin:Glycopeptide Antibiotic 1515700 :Hepatotoxin :Arg-isoAsp-Arg-Adda-isoGlu-mdhAla-Ala 1703716D:Hepatotoxin :Cyclic 7-Peptide D-Ala,Leu,D-MeAsp,D-Glu,Arg 1112392D:Herbicolin:X-Thr-D-aThr-D-Leu-Gly-D-Gln-Gly-Y-Arg 1210453P:Homophytochelatin :Glu(Cys )n-beta Ala n=2-7 1605356D:Hormaomycin :D-aThr Pro(Propenyl) threo Phe(Me) Nitro Cyclopentyl Ala 1810793D :Hormothamnin :D-Phe,D-Leu,lie,D-alle,Leu,Gly ,D-Baoa Hse,Dhha,Hyp , Hse 1104477 :Host Specific Plant Toxin :Cyclo(Pro-Ala-Gly-Epd ) 0908203 :Host Specific Toxin :Cyclo(Pro-Ala2-0ed) 1508276n:HV Toxin M:Cyclodepsipeptide 1009308 :Hypelcin:Aib-19-Peptide 1516538n :Hypeptin :Peptide Antibiotic 0802177 :Imacidin:10-Peptide Lactone 1612548 :Immunoreactive Lipopeptide WS1279:XNSGGS X=Acyl-Cys(Glyceryl) 0811220 D:Immunoreactive Peptide FK156:D-Lac-Ala-D-Glu(Dpm-Gly ) 0909205D:lsariin:Cyclo(X-Gly-Val-D-Leu-Ala-AA) 0711220D:Isariin:Cyclodepsi- 6-Peptide 1305530 :Iturin:Asp3,Glu,Pro,Ser,Tyr Heterogeneous beta-AA 0808326 :Iturin:beta-AA,Asn3,Tyr ,Gin,Ser 1006405D:Iturin:beta-AA,Pro,Ser,D-Asp,Asp,Glu,D-Tyr 1412491P :Iturin:SNPQNYNX 1614572D:Janthinocin:lie,Thr,Ser,Phe,D-Ser,D-Orn beta-OH Trp,DehydroAbu , Hyl 1207499 :Jasplakinolide :Cyclodepsipeptide Ala,MeTrp(Br).beta Tyr 1409560 :Katanosin:Cyclic 11-Depsipeptide Asp,Leu,Phe,beta-OH Deriv 1801564 :Keramamide:Cyclo(Phe-MeCht-Leu2-Lys-C0-Phe ) MeCht=MeTrp(Cl,OH) 1801515 :Keramamide:Pro,Orn,lie,Abu,Nva,Trp(2-Br,5-OH),2-0H-3-Me Pentanoate 1819805 :Keramamide:Thiazole,DehydroTrp ,IsoSer Ile,Ala,Dpr,Aminohexanoate 1716594 :Konbamide :Cyclo ((CO-Leu)-Lys-Ala-Leu-MeLeu-BhTrp ) 1708462 :L156602:3-0H Leu,D/L-Peperazic Acid,HO-Ala Tetrahydropyranylpropionate 1509414P :Lanthibiotic :TAGPAIRASVKQCQKTLKATRLFTVSCKGKNGCK 1414692 :Lantiopeptin :aRQaaXFGPFUFVadGHUX U=Abu d=3-0H Asp 1109340 :Lavendomycin :6-Peptide erythro-Dbu.Me Arg,DehydroAbu,Pipecolic Acid 1818556 :Laxaphycin :Ade,Aoc,Dhb,Hse,Hyp Leu(OH),Asn(beta-OH),MeIle 1815462 :Leualacin:Leu,MePhe,beta Ala,R/S-Leucic Acid 1302409 :Leucinostatin:9-Peptide Antibiotic 1001277 :Leucinostatin:FA-Pro(Me)-X-HyLeu-Aib-Leu2-Aib2-Y 1306583 :Leucinostatin:Pro(Me)-X-Hyl-Aib-Leu2-Aib2-beta-Ala-R 0802173 :Leucinostatin:Pro(Me)-X-HyLeu-Aib-Leu2-Aib2-beta-Ala 1308304D :Lipopeptide Antibiotic :R-WNDTGOrnD(A)DG(S)MeGluKyn ()=D-AA 1505632 :Lipopeptide: 3,4-(OH)2 HomoTyr,3-0H Gin,Hyp (Me),Hyp Thr,4,5-(0H)2 Orn 0612271P:Lipopeptin :Cyclodepsi-8-Peptide Thr,Asp,Ser,MeAsn,MePhe,HyGln ,Glu 1611703 :Lissoclinamide:Cyclo-7-Pept ide 1404691D :Lophyrotomin :Bz-(A)(F)VI(D)D(E)Q ()=D-AA 0909245D :Lophyrotoxin :Bz-Ala-Phe-Val-Ile-Asp-Asp-Glu-Gln D-AA at 1,2,5,7 1505693 :Lysobactin:3-0H Phe,3-0H Leu,3-0H Asn 1414667P:Maculosin:Cyclo(Pro-Tyr) 1413479 :Maduraferrin:beta-Ala,Gly ,Ser,MeOrn(OH) 1003303 :Majusculamide:Cyclodepsi-9-Peptide 1818673 :Malaysiatin :Cyclo-7-Peptide 1803819n:Malformin: 0606153 :Malonichrome:Cyclo-6-Peptide Ala,Gly ,Orn(OH) 1706617 :Melanostatin:MePhe-NHCH(COOH)CH2-His 1815457n:Mersacidin:Lantibiotic 1307321 :Methanofuran b:(gamma-Glu)4 Moiety 1713741 :Microcystin XY:beta-MeAsp,Asp,Glu,Ala,Leu,Arg 1903686n:Microcystin: 1903687n:Microcystin: —156 — 1903684D :Microcystin :7-Dha,3-D-Asp,7-Dha,3-D-Asp,7-Ser Deriv 1605607n:Microcystin :Cyclic Peptide Hepatotoxin 1903685 :Microcystin:Cyclo-7-Peptide 1818675D:Microcystin:D-Ala, D-3-Me Asp,Adda,D-Glu N-Me DehydroAla 1807605D :Microcystin :D-Ala,Leu,D-MeAsp,Arg,D-Glu 1801655 :Microcystin:Toxic Cyclo-7-Peptide 1207498 rMoroidin:Tricyclic 8-Peptide 1810794 :Motuporin:Cyclo-5-Peptide 1304500n:Mulundocandin:Lipopeptide Antibiotic 0909236D:Mycoplanecin: Cyclodepsi-10-Peptide Me-D-Leu MeThr 4-Me/Et Pro 1803821P :Nazumamide:Bz((OH)2)-Arg-Pro-Ile-Abu 1003314P:Neopeptin :Cyclodepsi-8-Pept ide MeAsn,beta-MeGlu,beta-OH Gln.MePhe 1203424P:Neopeptin :Ser,Asp,MeAsp,MePhe beta-Me Glu,beta-OH Glu 1003248n:Neoviridogrisein Cls:Cyclodepsipeptide 1405484 :Nephilatoxin:Lys ,Asp,Orn.Putreamine,Cadavarine 1504432 :Nephilatoxin:X-Orn-Asn-Cad-(Ptn)n-Y 1306582 :Nitropeptin :Leu-Glu(beta-N02) 1101617 :Nummularine:14/13 Membered Cyclopeptide 1606455 :Nummularine:Cyclo-13-Pept ide Alkaloid 1307439n :Nummularine:Cyclopeptide 1402611n:Nummularine:Cyclopeptide 1412598n :Nummularine:Cyclopeptide 1801514 :0rbiculamide:Pro,Ala,Abu,0rn,Trp(2-Br,5-0H).Theonalanine.Theoleucine 1409563 :P Containging Antibiotic :Gly-Leu-Aminosiopropenyl Phosphonate 1105414n:Paracelsin:Aib-Peptide 1309443n :Parvodicin:Glycopeptide Antibiotic 0806221 :Patellamide:lie,Val,Ser,Thr,Thiazole 0912210n:Peptide Alkaloi : 1506823P:Peptide Lactone:RCH(OH)CH2C0-Glu-Leu2-Val-Asp-Leu2-Leu2 0911233n:Peptide Monobactam SQ28332: 1307406 :Peptidoglycan :GlcNAc-MurNAc-Ala-Glu-Dpm-Ala 0912239 :Phomopsin:Cyclo-6-Peptide 3-OH lie,Dehydrolle ,DehydroPro 1302323P:Phytochelatin :(Glu(Cys))3-Gly 1111493P:Phytochelatin:(Glu(Cys))n-Gly n=3-7 1004352D:Phytotoxic Cy :Cyclo(D-Tyr(Me )-Ile-Pip-Aoe ) 1203426 :Phytotoxic Cy:Epoxide AA 8-0xo-9,10-Epoxy Aminodecanoate 1813497 :Phytotoxin: Cyclodepsi-10-Peptide 1311674 :Platelet Activating Factor Inhibitor:Cyclo(Met-Trp) Analog 1207405D:Plipastatin:E(0)Y(t)E(A)PQ(Y)I ()=D-AA t=aThr 0=0rn 1815456P:Plusbacin:aThr-Ala-Hyp-Arg-X-Ser-Hyp-X X=3-0H Asp 1311706P:Poly (Glu(Cys))-Gly: 1809662 :Polydiscamide :A-Phe(Br)-P-tLeu-Ile(beta-Me)-WR-Cys(03H )-T-MeGln-VPN 1718514 :Poststatin:(Val)2-NHCH(Et)COCO-D-Leu-Val 1605493 :Probestin:2-0H-4-Ph-3-Aminobutanoyl-Leu-Pro2 1211482P:Propioxatin:Acyl-Pro-Val 0503225P:Protease Inhibitor alpha-MAPI:CO(Phe)-Arg-Val-Phenylalaninal 1614500D:Pseudobactin 589A:Asp,Lys,3-OH D-Asp,D-Ser,Thr D-Ala,D-Glu,Ser,Orn(OH) 1717844D :Pseudobactin :D-Orn(OH),Ala,D-aThr,beta-OH Asp,Lya 1211430D:Pseudobactin:X-Ser-Ala-Gly-Ser-Ala-Y-aThr-Z 1819869 :Puwainaphycin :Cyclo-10-Peptide 1810795 :Pyoverdin:13 Membered Cyclic Peptide 1507670D:Pyoverdin:D-Ser-Arg-D-Ser-0rn(X)-Lys-0rn(X)-Thr2 0912294D :Pyoverdin :D-Ser-Arg-D-Ser-X-Thr2-Lys-X X=0rn(OH) 1704446D:Pyoverdin :UKSUTUXTUY U=D-Ser 1704630 :Pyoverdin :X-Ala-Lys-Gly2-H0Asp-Y-Ser-Ala-H00rn 1505634 :Quinomycin Like Antibiotic:3-0H Quinaldic Acid N-Me Me-Cyclopropane AA 0704279n :Quinoxaline Cyclodepsipeptide : 1812415 :RA XI/XII/XIII/XIV:Antitumor Bicyclic 6-Peptide 1211454 :RA-VI/III/II/I:Cyclo-6-Pept ide 1804515 :RAI-III/VI:Bicyclic 6-Peptide 1505636 :Ramoplanin:3-0H Asp,D,aThr3,G,A,L,F Gly(Ph(Cl,0H) ,(Gly(Ph(OH) ) )5 0rn2 1801513 :Ramoplanose :A,D,OH Asp,G,L,0rn2,F,N,Gly(Ph (Cl,OH)),Gly(Ph(OH) )4,aThr 1410560 :Rhizocticin:AA-Arg-APPA APPA=Amino Phosphono Pentenoic Acid 0903222P :Rhizonin:Cyclo (aIle-Val2-X-Leu-MeAla-X) 0503208n :Ristocetin:Glycopeptide 0810168n :Ristomycin :Glycopeptide -157- 1808460P :Ro091679 :Fumaryl-Arg-Leu-Arg 0510199P :Roseotoxin :Cyclo(Hpt-Pro(Me )-Ile-MeVal-beta-Ala-MeAla 1005336 .-Roseotoxin: Cyclodepsi-6-Peptide 3-Me Pro , lie, MeVal, beta Ala, Me Ala 1816438 :Rotihibin:6-Peptide 1812408 :Rotihibin:Dbu,Ser,Cit,beta-0H Asn,Asparaginol ,aThr 1501689 :Rugosanine :For-MeAla ,Val,Hyp ,Leu as Component 1603680 :Sandramycin :Cyclodepsi-10-Peptide 1713718n :Sanjoinenine :Cyclopeptide 1101618 :Sativanine:13 Membered Cyclopeptide 1207453 :Sativanine:13 Membered Cyclopeptide 1207478 :Sativanine:13 Membered Cyclopeptide 1207479n :Sativanine:N-For Cyclopeptide Alkaloid 1809578D :Serrawettin:Cyclo(Hdc-D-Leu-Ser-Thr-D-Phe-Ile ) 0701240n:Siomycin : 0702208n :Siomycin : 1309378 :Spider Toxin NSTX:Acyl-Asn-Cadaverine (Arg-Putreanyl ) 1305203 :Staccopin :Val4-Phe-H,Val4-Tyr-H 1011438 :Stenothricin :D-Cys ,D-aThr,D/L-Ser,Dpr,Val,Lys ,Sar D-DehydroAbu 1405656 :Sulfomycin :Oxazole AA.Thiazole AA,DehydroAla ,Thr 1802558n :Surfactin: 1106354n:Suzukacillin:Aib-Peptide 1607769 :Syringomycin :DehydroThr ,Thr(Cl),Asp(Hy) 1515556 :Syringomycin :Ser2-Dab2-Arg-Phe-Dhb-Thr (Cl)-X 1717623 :Syringopeptin :Dhb,Pro,Val,Ala,aThr,Ser,Dbu,Tyr Dhb=DehydroAbu 1810723 :Syringostatin :Cyclo-9-Depsipeptide 1705343 :Syringotoxin :Cyclodeps i-9-Peptide Phytotoxin 1818627 :Tawicyclamide: Cyclo-8-Peptide Thiazoline x3 1513586n:Teicoplanin Related SubstancerGlycopeptide Antibiotic 1302408n :Teicoplanin : 1506742n:Teicoplanin :Glycopeptide 1701485 :Tentoxin: Cyclo-4-Peptide N-Me DehydroPhe ,Gly ,MeAla,Leu 1210387 :Tentoxin:Cyclo-4-Pept ide 0901321 :Tentoxine: Cyclo-4-Peptide N-Me DehydroPhe ,Gly ,MeAla,Leu 1512600 :Thaxtomin:Cyclo(MeTrp-MePhe ) Deriv 1507564 :Theonellamide:Bicyclic 11-Peptide 1210553D:Theonellamine :MeOAc-Cyclo-13-Pept ide D/L-MeAA 1307362D:Theonellapeptolide :13-Peptide Lactone D-AA MeAA beta-Ala alle 1711811D :Theonellapeptolide :D-MeLeu,beta Ala,D-Leu,Melle D-alle,MeVal,D-Mealle 1713573D:Theonellapeptolide :D-MeLeu,D-Leu,beta Ala,D-alle Melle,MeAla,D-Mealle 1208453D :Theonellapeptolide :Melle,MeAla,MeVal,D-MeLeu D-Leu,D-alle,D-Mealle 0702208n:Thiostrepton: 0907225D :Toxin BE4:Cyclo(D-Ala-Leu-X-Ala-betaAA-D-iso-Glu ) 1711499 :Trialaphos :MP02HCH2CH2CH(NH2)C0-Ala3 0708199 :Trichopolyn:RC0-Pro-X-Ala-Aib2-AA-Ala-Aib2-Tcd 1304492 :Trichorzianine:Ac-XA2XYQX3SLXPVXIQ2-Pheol X=Aib,Y=Ive,Pheol=Pheol 1611798 :Trichorzianine:Ac-XAAXUQX3SLXPVXIQQ-Pheol X=Aib U=Iva 1409564 :Trichosporin: Aib-20-Peptide 1310496P:Trypsin Inhibitor:For-His-Val 1505423P:Tyrostatin:Isovaleryl-Tyr-Leu-Tyr-H 1611628P:UDP-Disaccharide 5-Peptide:Glu-Ala-Lys(gamma Glu)-Ala 1404628 :Vancomycin :Me2-Ala-Tyr (3-C1)-Gln-Tyr4 Analog 1608276n:Variapeptin:Cyclo Depsipeptide 1612608 :Variapeptin: Depsi-6-Pep tide 1301460 :Verlamelin:Cyclo-Depsi-7-Peptide 1301443 :Vietorin: Cyclo-5-Peptide 0812205 :Virginiamycin :6/7-Peptide Lactone 0812205 :Viridogrisein:6/7-Peptide Lactone 1816622n:Westiellamide:Cyclic Oxazoline Peptide 1812598 :Xanthostatin:Cyclodeps i-6-Peptide -158- m# - ma&A f - T550 ^clErffBIKM^SJ 1-8-4 M4@z&A f - TEL 06(443)5321 FAX 06(443)5319 • yn tf *-;M*&{±. it^^^EiS^'arW.b^oTS'Cv^-^, t's • yntf A-;i/«ig^3gffc^>r^>tcJ:oT